tag:blogger.com,1999:blog-11361534394512245842024-03-19T01:47:54.037-07:00Practical FragmentsThis blog is meant to allow Fragment-based Drug Design Practitioners to get together and discuss NON-CONFIDENTIAL issues regarding fragments.Dr. Teddy Zhttp://www.blogger.com/profile/07288045760981372367noreply@blogger.comBlogger956125tag:blogger.com,1999:blog-1136153439451224584.post-91581030477879224542024-03-18T06:05:00.000-07:002024-03-18T06:05:48.798-07:00Fragments vs SHP2<div class="MsoNormal" style="text-align: justify;">One of the success stories we
<a href="https://practicalfragments.blogspot.com/2024/03/fragments-2024.html">highlighted</a> in last week’s summary of Fragments 2024 was the discovery of a
potent inhibitor of SH2 domain-containing protein tyrosine phosphatase 2
(SHP2). James Day and colleagues at Astex and Taiho have just <a href="https://pubs.acs.org/doi/10.1021/acs.jmedchem.3c02118">published</a> the full
account in <i>J. Med. Chem</i>.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Previous studies had shown that blocking
SHP2 might be effective in certain cancers, particularly those dependent on
mutant KRAS. As its name suggests, however, SHP2 is a phosphatase. This class
of enzymes has highly charged active sites, which makes drug discovery
notoriously difficult (see <a href="https://practicalfragments.blogspot.com/2023/07/fragments-vs-ve-ptp-biophysics-in-action.html">here</a> for example). Indeed, a crystallographic fragment
screen of the isolated phosphatase domain produced just one hit.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Simultaneously, the researchers performed NMR and crystallographic
screens of the full-length protein, which contains two SH2 domains. This campaign
was much more successful, with 88 crystallographically validated fragment hits.
(Interestingly, a <a href="https://practicalfragments.blogspot.com/2017/08/assessing-ligandability-by-thermal.html">thermal shift</a> assay of the same construct came up empty.) As
Astex has previously <a href="https://practicalfragments.blogspot.com/2016/01/secondary-ligand-binding-sites-are.html">reported</a>, secondary binding sites on proteins are common,
and SHP2 is no exception, with fragments binding to five sites. However, the vast
majority – 83 of 88 – bound to what is called the tunnel region between the
phosphatase domain and one of the SH2 domains. </div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The researchers note that “following
completion of our Pyramid fragment screen, Novartis independently reported
several SHP2 inhibitors” binding to the same site, which must have been both validating
and irritating. Indeed, the Astex researchers did work on fragments binding to other
sites, advancing one to a low micromolar inhibitor. But it’s hard to ignore a
<a href="https://practicalfragments.blogspot.com/2024/02/hot-spots-real-and-imagined.html">hot spot</a> with dozens of bound fragments, and the tunnel region became their primary
focus. One fragment was optimized to a low micromolar inhibitor. Another, fragment
3, had measurable affinity by <a href="https://practicalfragments.blogspot.com/2010/02/isothermal-titration-calorimetry-itc.html">ITC</a> and respectable <a href="https://practicalfragments.blogspot.com/2017/04/ligand-efficiency-invalidated.html">ligand-efficiency</a>, and this
was taken the furthest.</div><div class="MsoNormal" style="text-align: justify;"> </div>
<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi__u3uVSR9HTWGJQD3lJBcgriPIlv40zHpI8rpuUeifGyJYDtgirNmA1aiVHmmX81V09EzpjoU0imDN_ws9mpV48gJKeTr6FAn17G_pCcTKyxFGI4KIrZ1xgOjNjdudWtpEcA6FjMvHjQQpmnJrjXCSWWC4-L5j1gB_5xOsn9NEZOv7Gl6HyC5oo3mXdCn/s1423/Day-Astex-SHP2-240318.bmp" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="363" data-original-width="1423" height="164" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi__u3uVSR9HTWGJQD3lJBcgriPIlv40zHpI8rpuUeifGyJYDtgirNmA1aiVHmmX81V09EzpjoU0imDN_ws9mpV48gJKeTr6FAn17G_pCcTKyxFGI4KIrZ1xgOjNjdudWtpEcA6FjMvHjQQpmnJrjXCSWWC4-L5j1gB_5xOsn9NEZOv7Gl6HyC5oo3mXdCn/w640-h164/Day-Astex-SHP2-240318.bmp" width="640" /></a></div><br />
<div class="MsoNormal" style="text-align: justify;">We’ve <a href="https://practicalfragments.blogspot.com/2014/09/from-fragment-to-lead-just-remove-high.html">written</a> previously about
the importance of water in molecular interactions, and here the researchers performed
solvent mapping molecular dynamics to identify water molecules that could be advantageously
engaged. Scaffold hopping led to compound 15, and crystallography confirmed that
the pyridine nitrogen forms a hydrogen bond to a water molecule. Increasing the
lipophilicity around the phenyl ring and adding a basic amine to engage an
electronegative region of the protein led to compound 18, with nanomolar biochemical
activity and low micromolar activity in cells. Further structure-based design
ultimately led to compound 28, with sub-micromolar cell activity. This compound
has low efflux, low clearance and excellent oral bioavailability. When dosed
orally in mouse xenograft models the molecule significantly inhibited tumor growth.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The exo-diastereomer of compound
28, in which the primary amine is facing down instead of up, shows interesting
differences. It has a similar pKa as well as similar biochemical and cell-based
activity but is plagued by high efflux and poor oral bioavailability. The researchers
suggest that “steric shielding of the tropane bridge or pharmacophoric differences
in efflux transporter recognition” may be responsible. There was considerable
discussion at Fragments 2024 as to the precise source of the differences, but whatever
the cause, this pair serves as a useful reminder that pharmacokinetics may vary dramatically
even between nearly identical molecules.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Clinical development of SHP2
inhibitors has slowed due to a variety of reasons, including apparent on-target
toxicity, but this is still a nice fragment-to-lead success story. Perhaps, as
with <a href="https://practicalfragments.blogspot.com/2023/11/capivasertib-seventh-approved-fragment.html">capivasertib</a>, it will just take time to find the right clinical
strategy and patients who can benefit from these molecules.</div>
Dan Erlansonhttp://www.blogger.com/profile/07927082337051189270noreply@blogger.com0tag:blogger.com,1999:blog-1136153439451224584.post-48746512762494289772024-03-11T06:08:00.000-07:002024-03-11T06:08:00.827-07:00Fragments 2024<div class="MsoNormal" style="text-align: justify;">Last week saw the first of four
dedicated <a href="https://practicalfragments.blogspot.com/2024/01/fragment-events-in-2024.html">fragment meetings</a> this year: <a href="https://www.rscbmcs.org/events/fragments24/">Fragments 2024</a>, the 9<sup>th</sup> RSC-BMCS
Fragment-based Drug Discovery Meeting, was held in historic Hinxton Hall,
Cambridge, UK. I won’t attempt to cover the 17 talks, 40+ posters, and 20
exhibitors in detail but just try to hit on some broad themes.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">One highlight was a talk by Chris
Swain, whose <a href="https://www.cambridgemedchemconsulting.com/">Cambridge MedChem Consulting</a> has come up <a href="https://practicalfragments.blogspot.com/2019/04/help-develop-new-antibiotics-from.html">several times</a> at <i>Practical
Fragments</i>. Chris has been systematically cataloging fragment hits reported
in the literature, and his database now includes >2500 fragments from
>300 papers that hit 265 targets. This has not been easy: as we’ve noted in
our annual F2L <a href="https://practicalfragments.blogspot.com/2023/12/review-of-2023-reviews.html">reviews</a>, papers don’t always mention fragments in the title or
abstract; sometimes you need to dig deep into the experimental methods to find
out the origin of the initial hits, and even then there are questions of interpretation.
Chris noted that the the drug <a href="https://en.wikipedia.org/wiki/Aprepitant">aprepitat</a> originated from a fragment-like
pharamacophore extracted from a more complex literature compound. That story was <a href="https://pubs.acs.org/doi/abs/10.1021/jm980299k">published</a> in 1998,
predating the term “fragment-based drug discovery,” but perhaps it would be
considered FBDD today.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The fragments themselves are a
diverse bunch, with an average <a href="https://en.wikipedia.org/wiki/Jaccard_index#Tanimoto_similarity_and_distance">Tanimoto similarity</a> of just 0.09, but there are
small clusters. Looking at them in
more detail, the ten most common scaffolds are aromatic (benzene, indole), which is a <a href="https://practicalfragments.blogspot.com/2023/10/spacial-scores-new-metrics-for.html">departure</a> from approved drugs. There is also
a significant fraction of charged molecules, including 298 acids and 348 basic
groups. About 10% of the fragments hit more than one target, exactly what
you would expect from the theory of <a href="https://practicalfragments.blogspot.com/2023/10/spacial-scores-new-metrics-for.html">molecular complexity</a>.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Chris’s talk was followed by a
wide-ranging panel discussion that expanded on some of these themes. Solubility
was recognized as important, though with different techniques being more persnickety:
Justin Dietrich (AbbVie) noted that pre-screening is critical for <a href="https://practicalfragments.blogspot.com/2010/02/surface-plasmon-resonance-spr.html">SPR</a>, but for
protein-detected NMR the protein is present at high enough concentrations to
act as a “phase transfer reagent.” </div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The topic of <a href="https://practicalfragments.blogspot.com/2019/06/is-thermodynamic-data-useful-for-drug.html">thermodynamics</a> also
came up, with Chris Murray noting that Astex collects lots of <a href="https://practicalfragments.blogspot.com/2010/02/isothermal-titration-calorimetry-itc.html">ITC</a> data but uses
it for assessing free energy (ΔG) values rather than enthalpic energy (ΔH) values.
Helena Danielson (Uppsala University) noted that the <a href="https://practicalfragments.blogspot.com/2012/12/entropy-enthalpy-transduction-time-to.html">early correlation</a> between
compound quality and enthalpy found with HIV protease inhibitors did not seem to
apply to other targets despite significant investment in collecting data at
multiple companies, as also noted by Chris Smith (Mirati) and Mike Hann (GSK).
Rod Hubbard (Vernalis) puckishly suggested that the study of ΔH had produced “more
heat than light.”</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The topic of <a href="https://practicalfragments.blogspot.com/2019/03/tiny-fragments-at-high-concentrations.html">MiniFrags</a> also came
up during the panel discussion. Chris Murray noted that they had been tried on
quite a few targets but, as Rod Hubbard confirmed, were more helpful in
identifying binding sites than providing starting points. But Chris Smith pronounced
himself a “complete convert” after a MiniFrag identified an induced pocket on a
previously intractable target where fragments (and other techniques) had
failed.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Covalent fragments also made
several appearances, with Jonathan Pettinger describing a phenotypic screen at
GSK looking for compounds that block the pro-inflammatory M1 polarization of
macrophages. After screening some 2000 covalent fragments they used chemoproteomics
to determine that one of the best compounds acted by modifying cysteine 817 of
the kinase JAK1. Interestingly, this is the same cysteine <a href="https://practicalfragments.blogspot.com/2022/04/seventeenth-annual-fragment-based-drug.html">identified</a> independently
by researchers from Vividion, which could speak to the centrality of this
target, the reactivity of this particular cysteine, or both.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Pursuing residues other than
cysteine is seen as difficult, with Mike Hann noting in the panel discussion
that these may require more extensive non-covalent interactions and
Chris Murray noting that the warheads themselves were less attractive. But these
challenges have not dissuaded Peter Cossar (Eindoven University of Technology),
who has introduced cysteine-reactive disulfide and lysine-reactive aldehyde
moieties into the <i>same</i> fragment to crosslink a 14-3-3 protein to substrate
ERRγ.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Another theme was screening crude
reaction mixtures in a “direct to biology” approach. Vernalis was an early adopter
with their <a href="https://practicalfragments.blogspot.com/2014/03/off-rate-screening-ors.html">off-rate screening</a>, and a talk by Lucie Guetzoyan confirmed
that they are continuing to invest here not just with SPR but also with
affinity-selection mass spectrometry and <a href="https://practicalfragments.blogspot.com/2020/09/crude-reaction-screening-by.html">X-ray crystallography</a>. Lucie also
described using flow chemistry to enable sensitive organometallic chemistries such
as Grignard and Negishi couplings. John Spencer (University of Sussex) is also
using crude reaction screening by crystallography and thought the approach can compress
ten years worth of work into a few months. </div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">As with most conferences these days
there were plenty of success stories. Martina Schaefer (Nuvisan) described the discovery
of the Bayer SOS1 inhibitor BAY-293, which we wrote about <a href="https://practicalfragments.blogspot.com/2019/03/stabilizing-and-destabilizing-sos1-ras.html">here</a>. Anna Vulpetti
(Novartis) described the discovery of IL-1β inhibitors, which we wrote about
<a href="https://practicalfragments.blogspot.com/2023/09/fragments-vs-hil-1-growing-into-cryptic.html">here</a>. Nicola Wilsher (Astex) described the discovery of potent SHP2 inhibitors;
I’ll write more about these later. And Matthew Calabrese described the
discovery of allosteric activators selective for the γ3 subunit of <a href="https://practicalfragments.blogspot.com/2017/12/fragments-activators-of-ampk.html">AMPK</a>, which
could avoid the cardiotoxicity seen with less selective molecules. Three HTS
screens had failed but fragments ultimately led to a potent tool molecule.
Interestingly, some of the HTS compounds were later found to be hits but had
been overlooked because they were so weak that they did not rise above the
noise of the assay.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Finally, Justin Dietrich described
several success stories, including against TNFα (which we wrote about <a href="https://practicalfragments.blogspot.com/2021/01/fragments-vs-tnf-advanced-with.html">here</a>) as
well as CD40 ligand. Justin noted that FBLD is used alongside HTS and DEL at
AbbVie, and that the techniques can be complementary – a theme noted by several
others.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Despite being so intimately integrated
with other discovery approaches, FBLD continues to innovate and evolve and remain
sufficiently quirky that stand-alone meetings are still valuable and rewarding.
I’m looking forward to seeing what the next several meetings reveal.</div>
Dan Erlansonhttp://www.blogger.com/profile/07927082337051189270noreply@blogger.com1tag:blogger.com,1999:blog-1136153439451224584.post-18208455762702707002024-03-03T23:17:00.000-08:002024-03-03T23:17:55.972-08:00The EU-OPENSCREEN fragment library<div class="MsoNormal" style="text-align: justify;">A well-curated fragment library
is usually the starting point for fragment-based lead discovery, and not an
insignificant investment. If you are just starting out you may want to use an
existing library. One such option is described in an (open-access) <a href="https://pubs.rsc.org/en/Content/ArticleLanding/2024/MD/D3MD00724C">paper</a> in <i>RSC
Med. Chem.</i> by Jordi Mestres and collaborators at IMIM Hospital del Mar
Medical Research Institute and across Europe.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The EU-OPENSCREEN European
Research Infrastructure Consortium (ERIC) allows researchers to access early
lead discovery and chemistry resources. Among other components, it includes a
set of more than 96,000 compounds for high-throughput screening, the European
Chemical Biology Library, or ECBL. To complement this, the researchers have
developed what they call the European Fragment Screening Library, or EFSL.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Recognizing that rapid follow-up
is a critical next step in fragment-based lead discovery, the researchers
designed EFSL based on ECBL. They did this by choosing fragments commercially
available from <a href="https://practicalfragments.blogspot.com/2018/12/poll-results-library-vendors.html">Enamine</a> that were sub-structures of ECBL members.
Fragments were chosen to represent as much of the ECBL as possible, as well as
for rule-of-three compliance. Fragments with multiple vectors for growing were
also prioritized, similar to the “sociable fragments” concept we wrote about
<a href="https://practicalfragments.blogspot.com/2021/06/sociable-vs-unsociable-fragments-and.html">here</a>. Finally, a set of 88 very small “<a href="https://practicalfragments.blogspot.com/2019/03/tiny-fragments-at-high-concentrations.html">minifrags</a>” were also
included.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Fragments were dissolved in
deuterated DMSO at 100 mM (or 1000 mM for minifrags). Solubility and integrity
were assessed at 1 mM (or 10 mM for minifrags) in PBS using <sup>1</sup>H-NMR
using an internal standard; those with solubility < 0.2 mM were rejected, as
were those with missing or extra peaks in the NMR spectra. Of 1056 compounds
tested, 913 passed these QC criteria.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The EFSL is available for
screening (via grant applications), and the paper
summarizes the results of eight screens performed over two years using a range
of detection technologies including crystallography, ligand-detected NMR,
small-angle X-ray scattering, thermal shift, and <a href="https://practicalfragments.blogspot.com/2011/07/biolayer-interferometry-bli.html">BLI</a>. Hit rates ranged
form just 0.1% to 31.3%, though in the last case
only a small subset of the library was tested.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">After fragment screening and
confirmation, four of the projects tested larger compounds from the ECBL in
follow-up studies, and two were able to identify hits. One project targeting a
bacterial beta-ketoacyl-ACP synthase 2 (FabF) used BLI to identify a fragment
with a dissociation constant of 35 µM. Of the 147 compounds related compounds
from the ECBL, two had slightly higher affinity, albeit at the expense of lower
<a href="https://practicalfragments.blogspot.com/2017/04/ligand-efficiency-invalidated.html">ligand efficiency</a>. Perhaps exploring Enamine REAL Space as in this
<a href="https://practicalfragments.blogspot.com/2022/01/virtually-screening-11-billion.html">example</a> would be more effective at finding significantly more potent
molecules.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">In summary, the EFSL seems to be
a useful resource, particularly for academic labs. If you’ve got a target and
no internal fragment-screening capabilities, it might be worth putting in an
<a href="https://apply.eu-openscreen.eu/submit-call/eu-openscreen-fragment-library-">application</a>.<span style="mso-spacerun: yes;"> </span></div>
Dan Erlansonhttp://www.blogger.com/profile/07927082337051189270noreply@blogger.com0tag:blogger.com,1999:blog-1136153439451224584.post-17966387183931330192024-02-26T05:59:00.000-08:002024-02-26T05:59:45.139-08:00Fragments in the clinic: 2024 edition<div class="MsoNormal" style="text-align: justify;">It has been more than a year since our last <a href="https://practicalfragments.blogspot.com/2022/11/fragments-in-clinic-2022-edition.html">list</a> of fragment-derived clinical compounds. Since then <a href="https://practicalfragments.blogspot.com/2023/11/capivasertib-seventh-approved-fragment.html">capivasertib</a> has been approved, bringing the number of marketed drugs to seven. There have also been a few other changes.<br /></div><div class="MsoNormal" style="text-align: justify;"><span style="background-color: white;"> </span></div><div class="MsoNormal" style="text-align: justify;"><span style="background-color: white;">As always, this table includes compounds <i>whether or
not
they are still in development</i></span> (indeed, some of the companies no longer
even exist). Because of this, the Phase 1 section contains a higher
proportion of compounds that are no longer progressing. The full list contains 59 molecules, up slightly from 2022, with just under 40% approved or in active trials<span style="background-color: white;">.<br /></span></div><div class="MsoNormal" style="text-align: justify;"> </div><div class="MsoNormal" style="text-align: justify;">Drugs reported
as still active in <a href="http://clinicaltrials.gov/">clinicaltrials.gov</a>, company websites, or other sources are in <b>bold</b>, and the 37 that have been discussed on <i style="mso-bidi-font-style: normal;">Practical Fragments</i> are <span style="color: #2b00fe;"><span><span style="background-color: white;"><span>hyperlinked</span></span></span></span> to the most relevant post. The list is almost certainly
incomplete, particularly for Phase 1 compounds. If you know of others please leave a comment.</div>
<div class="nobrtable">
<span style="font-family: inherit;"><span style="font-size: large;">
</span></span>
<br />
<table border="2" bordercolor="#0033FF" cellpadding="3" cellspacing="3" style="background-color: white; width: 649px;"><tbody>
<tr><th><span style="font-size: x-large;">Drug</span></th><th><span style="font-size: x-large;">Company</span></th><th><span style="font-size: x-large;">Target</span></th></tr>
<tr></tr>
<tr class="alt"><td><b style="mso-bidi-font-weight: normal;"><span style="font-size: large;">Approved!</span></b></td><td><br /></td><td><br /></td></tr>
<tr><td><b><a href="http://practicalfragments.blogspot.com/2021/10/asciminib-sixth-fragment-derived-drug.html">Asciminib</a></b></td><td><b>Novartis</b></td><td><b>BCR-ABL1</b></td></tr>
<tr><td><a href="https://practicalfragments.blogspot.com/2023/11/capivasertib-seventh-approved-fragment.html"><b>Capivasertib</b></a><br /></td><td><b style="mso-bidi-font-weight: normal;">AstraZeneca/Astex/CR-UK</b></td><td><b style="mso-bidi-font-weight: normal;">AKT</b></td></tr>
<tr><td><b><a href="http://practicalfragments.blogspot.com/2019/10/the-story-of-erdafitinib-abridged.html">Erdafitinib</a></b></td><td><b>Astex/J&J</b></td><td><b>FGFR1-4</b></td></tr>
<tr><td><b><a href="http://practicalfragments.blogspot.com/2015/10/fragments-in-clinic-plx3397.html">Pexidartinib</a></b></td><td><b>Plexxikon</b></td><td><b>CSF1R, KIT</b></td></tr>
<tr><td><a href="http://practicalfragments.blogspot.com/2021/05/sotorasib-fifth-fragment-derived-drug.html"><b>Sotorasib</b></a><br /></td><td><b style="mso-bidi-font-weight: normal;">Amgen</b></td><td><b style="mso-bidi-font-weight: normal;">
KRAS<sup>G12C</sup></b></td></tr>
<tr><td><b style="mso-bidi-font-weight: normal;"><a href="http://practicalfragments.blogspot.com/2011/08/first-fragment-based-drug-approved.html">Vemurafenib</a></b></td><td><b style="mso-bidi-font-weight: normal;">Plexxikon</b></td><td><b style="mso-bidi-font-weight: normal;">B-RAF<sup>V600E</sup></b></td></tr>
<tr><td><b style="mso-bidi-font-weight: normal;"><a href="http://practicalfragments.blogspot.com/2016/04/second-fragment-based-drug-approved.html">Venetoclax</a></b></td><td><b style="mso-bidi-font-weight: normal;">AbbVie/Genentech</b></td><td><b style="mso-bidi-font-weight: normal;"><span style="mso-tab-count: 2;">Selective </span>BCL-2</b></td></tr>
<tr></tr>
<tr class="alt"><td><b><span style="font-size: large;">Phase 3</span></b></td><td><br /></td><td><br /></td></tr>
<tr><td>Lanabecestat</td><td>Astex/AstraZeneca/Lilly</td><td>BACE1</td></tr>
<tr><td><a href="https://practicalfragments.blogspot.com/2016/04/second-fragment-based-drug-approved.html"><b style="mso-bidi-font-weight: normal;">Navitoclax (ABT-263)</b></a></td><td><b style="mso-bidi-font-weight: normal;">Abbott</b></td><td><b style="mso-bidi-font-weight: normal;">BCL-2/BCL<sub>xL</sub></b></td></tr>
<tr><td><a href="https://practicalfragments.blogspot.com/2013/11/fragment-to-lead.html"><b>Pelabresib (CP-0610)</b></a><b> </b><br /></td><td><b style="mso-bidi-font-weight: normal;">Constellation</b></td><td><b style="mso-bidi-font-weight: normal;">BET</b></td></tr>
<tr><td><span style="mso-bidi-font-weight: normal;"><a href="http://practicalfragments.blogspot.com/2017/01/fragments-in-clinic-verubecestat.html">Verubecestat</a></span></td><td><span style="mso-bidi-font-weight: normal;">Merck</span></td><td><span style="mso-bidi-font-weight: normal;">BACE1</span></td></tr>
<tr></tr>
<tr class="alt"><td><b><span style="font-size: large;">Phase 2</span></b></td><td><br /></td><td><br /></td></tr>
<tr><td><b style="mso-bidi-font-weight: normal;"><a href="http://practicalfragments.blogspot.com/2018/06/fragments-towards-clinic-erk12.html">ASTX029</a></b></td><td><b style="mso-bidi-font-weight: normal;">Astex</b></td><td><b style="mso-bidi-font-weight: normal;">ERK1,2</b></td></tr>
<tr><td><b style="mso-bidi-font-weight: normal;"><a href="http://practicalfragments.blogspot.com/2018/09/fragments-in-clinic-astx660.html">ASTX660</a></b></td><td><b style="mso-bidi-font-weight: normal;">Astex</b></td><td><b style="mso-bidi-font-weight: normal;">XIAP/cIAP1</b></td></tr>
<tr><td><a href="http://practicalfragments.blogspot.com/2008/08/clear-thorough-account-of-fragment.html">AT7519</a></td><td>Astex</td><td>CDK1,2,4,5,9</td></tr>
<tr><td><span style="mso-bidi-font-weight: normal;"><a href="http://practicalfragments.blogspot.com/2009/01/fragments-in-clinic-at9283.html">AT9283</a> </span></td><td><span style="mso-bidi-font-weight: normal;">Astex</span></td><td><span style="mso-bidi-font-weight: normal;">Aurora, JAK2</span></td></tr>
<tr><td>AUY-922</td><td>Vernalis/Novartis</td><td>HSP90</td></tr>
<tr><td><a href="http://practicalfragments.blogspot.com/2019/05/fragments-in-clinic-azd5991.html">AZD5991</a></td><td>AstraZeneca</td><td>MCL1</td></tr>
<tr><td><a href="http://practicalfragments.blogspot.com/2010/01/fragments-in-clinic-dg-051.html">DG-051</a></td><td>deCODE</td><td>LTA4H</td></tr>
<tr><td><a href="http://practicalfragments.blogspot.com/2018/04/fragments-in-clinic-eft508.html"><b>eFT508</b></a></td><td><b>eFFECTOR</b></td><td><b>MNK1/2</b></td></tr>
<tr><td><a href="http://practicalfragments.blogspot.com/2009/01/fragments-in-clinic-indeglitazar.html">Indeglitazar</a></td><td>Plexxikon</td><td>pan-PPAR agonist</td></tr>
<tr><td>LY2886721</td><td>Lilly</td><td>BACE1</td></tr>
<tr><td>LY3202626</td><td>Lilly</td><td>BACE1</td></tr>
<tr><td><b>LY3372689</b></td><td><b>Lilly</b></td><td><b>OGA </b></td></tr>
<tr><td>LY517717</td><td>Lilly/Protherics</td><td>FXa</td></tr>
<tr><td><b><a href="https://practicalfragments.blogspot.com/2021/03/fragments-in-clinic-lys006.html">LYS006</a></b></td><td><b>Novartis</b><br /></td><td><b>LTA4H</b></td></tr>
<tr><td><b>MAK683</b></td><td><b>Novartis</b></td><td><b>PRC2 EED</b></td></tr>
<tr><td><b><a href="https://practicalfragments.blogspot.com/2023/01/fragments-in-clinic-mk-8189.html">MK-8189</a></b><br /></td><td><b>Merck</b><br /></td><td><b>PDE10A</b><br /></td></tr>
<tr><td><b><a href="http://practicalfragments.blogspot.com/2010/09/fragments-in-clinic-at13387.html">Onalespib</a></b></td><td><b>Astex</b></td><td><b>HSP90</b></td></tr>
<tr><td><span style="mso-bidi-font-weight: normal;"><a href="http://practicalfragments.blogspot.com/2017/07/fragments-in-clinic-pf-06650833.html">PF-06650833</a></span></td><td><span style="mso-bidi-font-weight: normal;">Pfizer</span></td><td><span style="mso-bidi-font-weight: normal;">IRAK4</span></td></tr>
<tr><td><a href="https://practicalfragments.blogspot.com/2021/02/fragments-in-clinic-pf-06835919.html">PF-06835919</a></td><td>Pfizer</td><td>KHK</td></tr>
<tr></tr>
<tr><td>PLX51107</td><td>Plexxikon</td><td>BET<b><br /></b></td></tr>
<tr><td><b><a href="http://practicalfragments.blogspot.com/2019/12/fragments-in-clinic-s64315-mik665.html">S64315</a></b></td><td><b>Vernalis/Servier/Novartis</b></td><td><b>MCL1</b></td></tr>
<tr><td><a href="http://practicalfragments.blogspot.com/2019/07/fragments-vs-viral-protein-ebna1.html"><b>VK-2019</b></a><br /></td><td><b>Cullinan Oncology / Wistar</b><br /></td><td><b>EBNA1</b><br /></td></tr>
<tr class="alt"><td><b><span style="font-size: large;">Phase 1</span></b></td><td><br /></td><td><br /></td></tr>
<tr><td><a href="https://practicalfragments.blogspot.com/2021/05/fragments-in-clinic-ag-270.html">AG-270</a><br /></td><td>Agios/Servier<br /></td><td>MAT2A<br /></td></tr>
<tr><td><a href="http://practicalfragments.blogspot.com/2020/02/fragments-in-clinic-abbv-744.html"><b style="mso-bidi-font-weight: normal;">ABBV-744</b></a></td><td><b style="mso-bidi-font-weight: normal;">Abbott</b></td><td><b style="mso-bidi-font-weight: normal;">BD2-selective BET</b></td></tr>
<tr><td>ABT-518</td><td>Abbott</td><td>MMP-2 & 9</td></tr>
<tr><td>ABT-737</td><td>Abbott</td><td>BCL-2/BCL<sub>xL</sub></td></tr>
<tr><td>AT13148</td><td>Astex</td><td>AKT, p70S6K, ROCK</td></tr>
<tr><td>AZD3839</td><td>AstraZeneca</td><td>BACE1</td></tr>
<tr><td><a href="http://practicalfragments.blogspot.com/2014/01/kill-them-bugs.html">AZD5099</a></td><td>AstraZeneca</td><td>Bacterial topoisomerase II</td></tr>
<tr><td><a href="https://practicalfragments.blogspot.com/2022/10/from-noncovalent-fragment-to-covalent.html"><b>BI 1823911</b></a></td><td><b>Boehringer Ingelheim</b></td><td><b>KRAS<sup>G12C</sup></b></td></tr>
<tr><td>BI 691751</td><td>Boehringer Ingelheim</td><td>LTA4H</td></tr>
<tr><td><b>CFTX-1554</b></td><td><b>Confo Therapeutics</b></td><td><b>AT<sub>2</sub> receptor </b></td></tr>
<tr><td><a href="http://practicalfragments.blogspot.com/2018/06/fragments-in-clinic-etc-206.html">ETC-206</a></td><td>D3</td><td>MNK1/2</td></tr>
<tr><td>GDC-0994</td><td>Genentech/Array</td><td>ERK2</td></tr>
<tr><td><b>HTL0014242</b></td><td><b>Sosei Heptares</b></td><td><b>mGlu5 NAM</b></td></tr>
<tr><td><a href="https://practicalfragments.blogspot.com/2022/07/fragments-in-clinic-htl9936.html">HTL0018318</a></td><td>Sosei Heptares</td><td>M1-receptor partial agonist</td></tr>
<tr><td><a href="https://practicalfragments.blogspot.com/2022/07/fragments-in-clinic-htl9936.html">HTL9936</a></td><td>Sosei Heptares</td><td>M1-receptor partial agonist</td></tr>
<tr><td>IC-776</td><td>Lilly/ICOS</td><td>LFA-1</td></tr>
<tr><td>LP-261</td><td>Locus</td><td>Tubulin</td></tr>
<tr><td><a href="http://practicalfragments.blogspot.com/2011/11/and-once-more-into-breach.html">LY2811376</a></td><td>Lilly</td><td>BACE1</td></tr>
<tr><td><a href="http://practicalfragments.blogspot.com/2018/02/fragments-in-clinic-abbv-075-mivebresib.html">Mivebresib</a></td><td>AbbVie</td><td>BRD2-4</td></tr>
<tr><td><a href="https://practicalfragments.blogspot.com/2022/01/fragments-almost-in-clinic-mrtx1719.html"><b>MRTX1719</b></a></td><td><b>Mirati</b></td><td><b>PRMT5•MTA </b></td></tr>
<tr><td><a href="http://practicalfragments.blogspot.com/2019/08/fragments-in-clinic-navoximod.html">Navoximod</a></td><td>New Link/Genentech</td><td>IDO1</td></tr>
<tr><td>PLX5568</td><td>Plexxikon</td><td>RAF</td></tr>
<tr><td>SGX-393</td><td>SGX</td><td>BCR-ABL</td></tr>
<tr><td>SGX-523</td><td>SGX</td><td>MET</td></tr>
<tr><td>SNS-314</td><td>Sunesis</td><td>Aurora</td></tr>
<tr><td><a href="https://practicalfragments.blogspot.com/2021/09/fragments-in-clinic-tak-020.html">TAK-020</a><br /></td><td>Takeda<br /></td><td>BTK<br /></td></tr>
</tbody></table>
</div>
<br /><br />Dan Erlansonhttp://www.blogger.com/profile/07927082337051189270noreply@blogger.com0tag:blogger.com,1999:blog-1136153439451224584.post-1919267333683166562024-02-19T07:20:00.000-08:002024-02-19T07:20:18.677-08:00Hot spots real and imagined<div class="MsoNormal" style="text-align: justify;"><i>Practical Fragments</i> has written
several times about “<a href="https://practicalfragments.blogspot.com/2012/08/two-types-of-hot-spots.html">hot spots</a>”: regions on proteins where small molecules and
fragments readily bind. Knowing whether your target protein has a hot spot can help
you decide whether to pursue the target in the first place. A <a href="https://practicalfragments.blogspot.com/2016/05/calculating-hotspots-in-detail.html">variety</a> of computational
approaches have been developed for finding hot spots, most of which start with
a crystallographically determined structure. In a new <i>J. Chem. Inf. Mod.</i>
<a href="https://pubs.acs.org/doi/10.1021/acs.jcim.3c01761">paper</a>, Sandor Vajda and collaborators at Boston University and Stony Brook
University ask whether computational models of proteins can also be used for one of the more popular methods, <a href="https://practicalfragments.blogspot.com/2015/05/predicting-protein-ligandability-and.html">FTMap</a>.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The researchers started with a
set of 62 proteins, each of which had a published crystal structure bound to a
fragment (MW < 200 Da) as well as to a larger molecule. The predicted
structures of these proteins were then downloaded from the AlphaFold2 (<a href="https://alphafold.ebi.ac.uk/">AF2</a>) site,
and these models were truncated to correspond to the residues seen in the
crystal structures to facilitate comparisons. The computational models were
quite similar to the experimental models, particularly when comparing the positions
of the peptide backbone atoms which define the overall shape of the proteins. </div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Next, the researchers applied the
program FTMap, which computationally explores the surface of proteins using a
set of 16 very small probes such as ethanol. Hot spots are regions
where lots of probes bind, and the “<a href="https://practicalfragments.blogspot.com/2015/05/predicting-protein-ligandability-and.html">hotness</a>” of these spots correlates with the
number of bound probes. FTMap assessed hotness on the AF2 structures and the
crystallographicaly determined structures. (Before running FTMap, the bound
ligands in the crystal structures were computationally removed.) Additionally, the
researchers ran FTMap on unliganded crystal structures for the 47 proteins where
these had been reported. </div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">FTMap was broadly successful at
finding the hotspots defined by bound fragments, succeeding 77% of the time starting
with either the fragment-bound or unliganded structures and 71% starting with
the AF2 models. Implementing stricter criteria (demanding the experimental fragment
binding site be the top hot spot, for example) reduced the success to 56% for
the crystallographic starting points and 47% for the AF2 models.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The paper discusses several examples
in detail, in particular the two where the AF2 models were most different from
the experimental models. Both of these were large, multidomain proteins. When AF2
models of just the ligand-binding domains were used, the models were significantly
improved. This seems to be a generally useful hack: generating truncated AF2 models for other proteins also improved
the performance of FTMap.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The utility of AF2 models for
docking has been the subject of some debate, with some arguing that even though
the overall protein folds may be accurate, local side chain conformations may
be wrong, and a single side chain rotation may make the difference between ligand
binding or not. This paper suggests that hot spots are not too sensitive to these
subtleties, and that AF2 models can be used for finding hot spots.</div>
Dan Erlansonhttp://www.blogger.com/profile/07927082337051189270noreply@blogger.com0tag:blogger.com,1999:blog-1136153439451224584.post-57766208756467833902024-02-12T06:10:00.000-08:002024-02-12T06:10:01.577-08:00Fragment screening across the proteome, noncovalently<div class="MsoNormal" style="text-align: justify;">Last week we <a href="https://practicalfragments.blogspot.com/2024/02/fragment-screening-across-proteome.html">discussed</a> methodological
improvements to industrialize covalent fragment screening across the proteome.
While I’m a huge fan of covalent binders, their noncovalent counterparts are
the vanilla ice cream of FBLD: also tasty and much more common. Back in 2017 we
<a href="https://practicalfragments.blogspot.com/2017/02/fragments-in-cells.html">described</a> how “fully functionalized fragments,” or FFFs, could be used to
screen noncovalent fragments in cells. A new <a href="https://pubmed.ncbi.nlm.nih.gov/38167919/">paper</a> in <i>Nat. Chem. Biol.</i>
by Christopher Parker and collaborators at Scripps and BMS further optimizes
the approach.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">FFFs contain, in addition to the
variable fragment, a photoreactive group (often a diazirine) and an alkyne tag.
When exposed to light the photoreactive group can react with nearby proteins
and the alkyne tag can be used to fish out the proteins. In the new paper the
researchers started with a dozen FFFs.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">One challenge, which we <a href="https://practicalfragments.blogspot.com/2021/05/understanding-fully-functionalized.html">discussed</a>
in 2021, is that the FFFs may react with many sites on a given protein. During
analysis, a protein is typically digested into peptides for mass spectrometry.
If a FFF reacts at several sites on a peptide the resulting spectra will be “chimeric”
and more difficult to characterize.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The researchers developed methods
to take these chimeric spectra into account when searching for sites of
modification. The approach, called Dizco (for diazirine probe-labeled peptide
discoverer) can identify three times as many peptides as standard approaches,
as well as more detailed information on sites of modifications. </div><div class="MsoNormal" style="text-align: justify;"> </div><div class="MsoNormal" style="text-align: justify;">Two pairs of
FFF probes consisted of <a href="https://practicalfragments.blogspot.com/2018/08/256th-american-chemical-society.html">enantiomers</a>, and these showed differential labeling across
the proteome, consistent with specific molecular recognition. The researchers
also confirmed binding of a few FFF probes to several proteins using a cellular thermal shift assay
(<a href="https://en.wikipedia.org/wiki/Cellular_thermal_shift_assay">CETSA</a>).</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">In all, the probes modified 3603
peptides on 1669 proteins. The sites of modification were then mapped onto
predicted or modeled three dimensional structures of the proteins. Importantly,
and consistent with the 2017 work, most of the labeled sites were near predicted
pockets. The researchers confirmed this for four proteins by showing that FFF probe
binding could be competed by adding ligands known to bind to the pockets. </div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Next, the researchers docked
(using <a href="https://practicalfragments.blogspot.com/2020/12/benchmarking-docking-methods-new-public.html">AutoDock</a>) their FFF probes onto 175 proteins (108 from structures in the <a href="https://www.rcsb.org/">Protein Data Bank</a>
and 67 from <a href="https://en.wikipedia.org/wiki/AlphaFold">AlphaFold</a> structures). They found that the docking experiments
recapitulated the experimental data, and in fact often placed the diazirine tag
near the protein residues found to react. Strikingly, and in another step
forward for in silico approaches, docking against structures from AlphaFold was
nearly as effective as those from the protein data bank.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">As the researchers conclude, “we
identified many binding pockets that have no reported ligands… these probes may
serve as leads for further optimization.” It will be fun to see how far they
go.</div>
Dan Erlansonhttp://www.blogger.com/profile/07927082337051189270noreply@blogger.com0tag:blogger.com,1999:blog-1136153439451224584.post-16572436900542187412024-02-05T06:52:00.000-08:002024-02-05T06:52:43.240-08:00Fragment screening across the proteome, industrialized<div class="MsoNormal" style="text-align: justify;">Last week we <a href="https://practicalfragments.blogspot.com/2024/01/covalent-fragments-vs-sars-cov-2.html">discussed</a> covalent
fragment screens against isolated enzymes, which can be very effective. But
screening in cells or cell lysates preserves proteins in a more physiological environment
and allows many proteins across the proteome to be screened simultaneously. In 2016
we <a href="https://practicalfragments.blogspot.com/2016/06/covalent-fragments-writ-large.html">wrote</a> about covalent screens in human cell lysates which identified fragment
hits for 758 cysteine residues in 637 proteins. Mass spectrometry techniques have
improved since then in terms of both speed and sensitivity, as illustrated in a
new <i>Cell Chem. Biol.</i> <a href="https://pubmed.ncbi.nlm.nih.gov/38118439/">paper</a> from Steve Gygi, Qing Yu, and collaborators
at Harvard Medical School and Biogen. (Disclosure: Steve Gygi is on the Scientific
Advisory Board of my current company, Frontier Medicines.)</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The approach is called TMT-ABPP, or
tandem mass tag activity-based protein profiling, and it involves multiple improvements
to previous methods, some of which Steve discussed at the Discovery on Target
<a href="https://practicalfragments.blogspot.com/2023/10/discovery-on-target-2023.html">meeting</a> last year. Covalent fragments are added separately to cell lysate
aliquots, after which a desthiobiotin iodacetamide (DBIA) probe is introduced.
If a given site on a protein has reacted with a fragment, it will not be available
to react with the DBIA probe. </div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Next, proteins are digested to
peptides and labeled with TMT (tandem mass tag) reagents, which allow multiple samples
(18 in this case, either individual fragments or DMSO-only controls) to be combined
for simultaneous analysis. Peptides functionalized with the DBIA probe are
captured on streptavidin resin while those that had previously reacted with a
covalent fragment will not stick to the resin and be lost. Peptides eluted from
the resin are then analyzed by mass spectrometry. The “competition ratio”
between treated and untreated lysate gives a measure of how strongly a given
site on a given protein is labeled by a fragment.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Multiple other tweaks, such as
capturing proteins using magnetic beads and using a special type of mass-spectrometry
(high-field asymmetric waveform ion mobility spectrometry, or <a href="https://en.wikipedia.org/wiki/High-field_asymmetric-waveform_ion-mobility_spectrometry">FAIMS</a>), further streamline
the process to a 96-well plate format, with each well containing a mere 10-20 µg
of cell lysate, as much as 100-fold less than earlier approaches. </div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The researchers benchmarked TMT-ABPP
using three reactive “scout fragments,” including compound 1 from last week’s
<a href="https://practicalfragments.blogspot.com/2024/01/covalent-fragments-vs-sars-cov-2.html">post</a>. Collectively they identified 6813 cysteine residues hit by one or more of
the scouts.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">To demonstrate throughput, the
researchers next screened 192 fragments, a third of which were acrylamides
while the rest were chloroacetamides. Even with two controls for every 16 samples,
this only required 12 injections on a mass spectrometer and resulted in hits against
38,450 cysteines, about 50-fold more than the 2016 paper. Proteins that were more
highly expressed were better represented, as were proteins with known reactive
cysteine residues, such as thioredoxins. Surprisingly though, surface-exposed
cysteine residues were only slightly enriched over more buried cysteines. </div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The researchers also applied TMT-ABPP
to five well-characterized covalent molecules, including the mutant KRAS<sup>G12C</sup>
inhibitor ARS-1620, which we wrote about <a href="https://practicalfragments.blogspot.com/2020/01/fragments-in-clinic-amg-510.html">here</a>. In addition to the G12C site of
KRAS, several other proteins were also liganded, including adenosine kinase
(ADK). The researchers confirmed that ARS-1620 inhibited ADK in an enzymatic assay.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">As the researchers note, “proteome-wide
profiling of thousands of compounds remains a formidable challenge, both
technically and financially.” This paper reveals how to significantly reduce
the costs. By using such approaches, it is possible to build a catalog of fragment
ligands for thousands of proteins. Doing so with a well-curated library could
enable rapid fragment-to-lead campaigns.</div>
Dan Erlansonhttp://www.blogger.com/profile/07927082337051189270noreply@blogger.com0tag:blogger.com,1999:blog-1136153439451224584.post-90272823531627743722024-01-29T05:34:00.000-08:002024-01-29T05:34:02.450-08:00Covalent fragments vs a SARS-CoV-2 helicase<div class="MsoNormal" style="text-align: justify;">Last week we <a href="https://practicalfragments.blogspot.com/2024/01/covalent-complexities-for-kinase.html">wrote</a> about the difficulties
of trying to understand even well-characterized covalent inhibitors of well-characterized
targets. Most projects have far less information, as illustrated in a recent
<a href="https://pubs.acs.org/doi/10.1021/jacs.3c10581">paper</a> in <i>J. Am. Chem. Soc.</i> by Ekaterina Vinogradova, Tarun Kapoor, and
collaborators at Rockefeller University and Sanders Tri-Institutional Therapeutics
Discovery Institute, who report the first inhibitors of a particular SARS-CoV-2
enzyme.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The researchers were interested
in helicases, enzymes that unwind DNA, RNA, or both. To do so, helicases cycle
between “open” and “closed” forms, with conformational changes of as much as 15
Å. That dynamism complicates structure-based drug design, and many screens have
yielded false positives. An irreversible covalent inhibitor that remained bound
to the enzyme through its gyrations would potentially be easier to optimize.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The protein nsp13 from SARS-CoV-2
is essential for viral replication and thus an attractive drug target. The researchers
started by testing previously reported and reactive “scout fragments” in a
functional assay. Compound 1 inhibited the enzyme, and mass-spectrometry (MS)
assays revealed that it modified three sites on the protein. Although multiple
modifications are not desirable, the enzyme does contain 26 cysteine residues,
so it could be worse. Peptide mapping and mutagenesis experiments revealed that
modification of cysteine 556 (C556) is responsible for the inhibitory activity
of compound 1. </div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;"><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjJl5lRsRNN8lGlpi8NqNFpzJULean9jusznea97ZirCt4ahvilKCvvT6sIAKnoYxFbAfknCnWEPIgvAD74-ytHMmkZAkRifCyRj3-r6DDDhsYWFA41u7AbrH-mTR4D8QmPDriXH6WaZUs3dJV1hTyqp3AdSQkMKMk-jNX-8RyIcw3nuI8aop0FeknmMjQV/s743/Kapoor-covalent-240129.bmp" style="clear: right; float: right; margin-bottom: 1em; margin-left: 1em;"><img border="0" data-original-height="235" data-original-width="743" height="126" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjJl5lRsRNN8lGlpi8NqNFpzJULean9jusznea97ZirCt4ahvilKCvvT6sIAKnoYxFbAfknCnWEPIgvAD74-ytHMmkZAkRifCyRj3-r6DDDhsYWFA41u7AbrH-mTR4D8QmPDriXH6WaZUs3dJV1hTyqp3AdSQkMKMk-jNX-8RyIcw3nuI8aop0FeknmMjQV/w400-h126/Kapoor-covalent-240129.bmp" width="400" /></a></div>A series of analogs culminated in
compound 3b, which had low micromolar activity after a four hour incubation and
also seemed more selective than compound 1, with less modification of other cysteine residues. The enantiomer of compound 3b was at least 6-fold less
potent, suggesting molecular recognition rather than simple reactivity. In addition
to nsp13, the researchers examined two mammalian helicases with disease relevance,
WRN and BLM, and found that compound 3b was modestly selective for nsp13. (The
researchers find different inhibitors for these two enzymes, though these are
weaker and not as extensively characterized as those for nsp13.)</div>
<div class="MsoNormal" style="text-align: justify;"><br /></div>
<div class="MsoNormal" style="text-align: justify;">Cysteine 556 is not in the
ATP-binding site and does not seem to be involved with RNA binding, and the researchers
suggest that compound 3b may act allosterically. It seems to be highly conserved
too, which might mean mutational resistance is less likely to evolve.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">As the researchers acknowledge, compound
3b contains a <a href="https://practicalfragments.blogspot.com/2016/06/covalent-fragments-writ-large.html">chloroacetamide</a> warhead, which is likely too reactive and
unstable to move forward into in vivo studies, let alone the clinic. Also, had
I reviewed the manuscript I would have requested the researchers to provide <i>k<sub>inact</sub>/K<sub>I</sub></i>
values rather than merely IC<sub>50</sub> values; a rough calculation using the
methodology in <a href="https://pubs.acs.org/doi/10.1021/acsmedchemlett.9b00258">this paper</a> suggests a modest 10 M<sup>-</sup>1s<sup>-1</sup> for
compound 3b. That said, the discovery that liganding C556 inhibits nsp13 means that
working to develop more potent and selective molecules may be worth the effort.</div>
Dan Erlansonhttp://www.blogger.com/profile/07927082337051189270noreply@blogger.com0tag:blogger.com,1999:blog-1136153439451224584.post-76209526605873999272024-01-22T06:07:00.000-08:002024-01-22T06:07:54.689-08:00Covalent complexities for kinase inhibitors<div class="MsoNormal" style="text-align: justify;">Covalent drugs are becoming
increasingly <a href="https://practicalfragments.blogspot.com/2022/10/from-noncovalent-fragment-to-covalent.html">popular</a>. But as more researchers search for them,
they may encounter pitfalls. A new <a href="https://pubs.acs.org/doi/10.1021/acs.jmedchem.3c01502">paper</a> in <i>J. Med. Chem.</i>
by <span style="mso-spacerun: yes;"> </span>David Heppner and collaborators at
the State University of New York Buffalo, AssayQuant Technologies, and Eberhard
Karls Universität Tübingen provides a nice roadmap for avoiding them.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The researchers focus on covalent
inhibitors of epidermal growth factor receptor (EGFR), a kinase that is frequently
mutated in cancer. The first drugs against this target, such as <a href="https://en.wikipedia.org/wiki/Erlotinib">erlotinib</a>, were
non-covalent, and these have been largely displaced by more effective
covalent molecules such as <a href="https://en.wikipedia.org/wiki/Afatinib">afatinib</a>. Unfortunately, these earlier drugs are not
effective against a common mutant (T790M), spurring the development of third
generation molecules such as <a href="https://en.wikipedia.org/wiki/Osimertinib">osimertinib</a>, which was approved by the FDA in
2015. Osimertinib has been extensively studied, with more than 2800 <a href="https://pubmed.ncbi.nlm.nih.gov/?term=osimertinib+&sort=date">references</a>
in PubMed. Yet it is not as well understood as you might expect.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The team uses this system to demonstrate
how characterizing irreversible inhibitors is not simple. For reversible enzyme
inhibitors, researchers frequently discuss IC<sub>50</sub> values or, when
they are being more precise, inhibition constants (<i>K<sub>i</sub></i>). The
latter are in theory absolute values that do not depend on concentrations of
cofactors such as ATP. But for irreversible inhibitors, the IC<sub>50</sub>
values change depending on how long (and at what concentration) incubation
occurs. The proper assessment of an irreversible inhibitor is <i>k<sub>inact</sub>/K<sub>I</sub></i>,
which takes into account both the irreversible inactivation step (<i>k<sub>inact</sub></i>)
as well as the inhibition constant (<i>K<sub>I</sub></i>). Note that <i>K<sub>i</sub></i>
is not the same as <i>K<sub>I </sub></i>; the former describes only the initial
<i>reversible</i> association between protein and inhibitor, while <i>K<sub>I</sub></i>
incorporates the irreversible step. Told you it was complicated!</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">And it gets worse. The researchers
examined three irreversible covalent inhibitors under various conditions. In
one condition, the inhibitors were pre-dissolved in 10% DMSO before being added
to the assay mixture to give a final DMSO concentration of 1%. In another
condition, the inhibitors were dissolved in pure DMSO before being added to the
assay. Despite the final concentration of DMSO being the same (1%), the second
condition gave <i>k<sub>inact</sub>/K<sub>I</sub></i> values up to 11-times greater (more potent).</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">If subtle experimental variations
in one lab can change values by more than an order of magnitude, you might
expect the literature to vary even more, and you’d be right. In the case of
osimertinib, the reported values of <i>k<sub>inact</sub>/K<sub>I</sub></i> vary
by nearly 500-fold. Some of the experimental parameters the researchers
consider are concentrations of reducing agents such as DTT, which can react
with covalent inhibitors, and serum albumin, which also contains a free
cysteine residue. Although these did not seem to be problematic for osimertinib
itself, they could affect other molecules.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Another consideration for kinases
in particular is the concentration of the cofactor ATP. The value of <i>k<sub>inact</sub>/K<sub>I</sub></i>
itself will vary depending on [ATP], and the researchers describe how to calculate
a “true” <i>k<sub>inact</sub>/K<sub>I</sub></i> which could be used to compare the
potency of a given inhibitor against the wild-type vs mutant forms of the
enzyme. But while this is more theoretically rigorous, it may be less biologically
relevant, since physiological ATP concentrations are less variable than
differences in the <a href="https://en.wikipedia.org/wiki/Michaelis%E2%80%93Menten_kinetics">Michaelis constant</a> (<i>K<sub>M</sub></i>) for ATP for
different kinases and mutants.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">There is lots more to digest in this
paper, including analyses of structure-kinetic relationships (SKR, akin to structure-activity
relationships, or SAR) for different inhibitors and thorough experimental descriptions.
The take-home message is that, due in part to different and often incomplete
details, “potency measurements are generally difficult to compare among
literature studies,” and “any potency assessments should include appropriate controls
under the same conditions as the experimental inhibitors.”</div>
Dan Erlansonhttp://www.blogger.com/profile/07927082337051189270noreply@blogger.com1tag:blogger.com,1999:blog-1136153439451224584.post-40303457429375856812024-01-15T06:56:00.000-08:002024-01-15T06:56:54.678-08:00What makes molecules aggregate?<div class="MsoNormal" style="text-align: justify;">The propensity for some small
molecules to form <a href="https://practicalfragments.blogspot.com/2009/08/avoiding-will-o-wisps-aggregation.html">aggregates</a> in water has bedeviled fragment-finding efforts
for decades. Indeed, the phenomenon was not fully recognized until early this
century. Although plenty of <a href="https://practicalfragments.blogspot.com/2019/11/a-new-tool-for-detecting-aggregation_10.html">tools</a> are available for detecting aggregates, I
still see too many <a href="https://practicalfragments.blogspot.com/2022/01/an-epidemic-of-aggregators-and.html">papers</a> that omit these crucial quality controls. As annoying
as aggregation can be in activity assays, in certain cases it could actually be
useful for formulating drugs. There has been speculation that the good oral bioavailability
of <a href="https://practicalfragments.blogspot.com/2016/04/second-fragment-based-drug-approved.html">venetoclax</a> is due to aggregation. But despite <a href="https://practicalfragments.blogspot.com/2015/10/aggregation-alert.html">computational methods</a> to
predict aggregation, the structural features of molecules that cause them to
aggregate are still not well understood. In a new open-access <i>Nature Comm.</i>
<a href="https://pubmed.ncbi.nlm.nih.gov/38097573/">paper</a>, Daniel Heller and collaborators at Memorial Sloan Kettering Cancer
Center and elsewhere provide some answers.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The researchers had previously published
an <a href="https://pubmed.ncbi.nlm.nih.gov/29403054/">article</a> describing how indocyanine green (<a href="https://en.wikipedia.org/wiki/Indocyanine_green">ICG</a>) could be used to stabilize
and visualize aggregates, and they applied the same technique to examine the
aggregation potential of a small set of fragments. Benzoic acid and 2-napthoic
acid did not aggregate, while 4-phenylbenzoic acid did. Intrigued, the
researchers tested a set of 14 4-substituted biphenyl fragments and found that
those containing both a hydrogen bond donor and acceptor, such as acids,
sulfonamides, amides, and ureas, could aggregate, while those containing only
donors (aniline) or acceptors (nitrile) did not.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Fourier transform infrared
spectroscopy was used to examine the stretching region of the carbonyl of
4-phenylbenzoic acid in various states: in an aqueous aggregate, in solution in
either t-butanol or DMSO, or in the solid state. Interestingly, the aggregate
most resembled the solid state, consistent with close-packed self-assembly as
opposed to free in solution.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">From all this, the researchers hypothesized
that a combination of aromatic groups and hydrogen bond donors and acceptors
was necessary for aggregation. However, having these features does not mean
aggregation is inevitable. Neither 3-phenylbenzoic acid nor 2-phenylbenzoic
acid formed aggregates, with the former precipitating while the latter remained
completely soluble. These three phenylbenzoic acid isomers behave very differently despite the fact that they have the same calculated logP values,
and the suggestion is that the latter two molecules are less able to form pi-pi
stacking interactions that lead to stable aggregation.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Next the researchers examined the
approved drug <a href="https://en.wikipedia.org/wiki/Sorafenib">sorafenib</a>, which had previously been shown to aggregate. This was
confirmed, and the aggregates were characterized with a battery of biophysical
methods including dynamic light scattering, transmission electron microscopy,
and X-ray scattering, along with molecular dynamics simulations. The conclusion
is that sorafenib forms amorphous aggregates whose assembly is driven by a
combination of pi-pi stacking and hydrogen-bonding. A series of sorafenib
analogs was synthesized, and those that could not form strong intermolecular
hydrogen bonds were less prone to aggregation.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">All of this is fascinating from a
molecular assembly viewpoint and will help to explain and predict which
compounds are likely to aggregate, for better or for worse. But as of now, experimental
assessment is still best practice for any new compound. </div>
Dan Erlansonhttp://www.blogger.com/profile/07927082337051189270noreply@blogger.com1tag:blogger.com,1999:blog-1136153439451224584.post-70585488707560205932024-01-08T06:22:00.000-08:002024-01-08T06:22:06.931-08:00Electrophilic MiniFrags vs HDAC8<div class="MsoNormal" style="text-align: justify;">In fragment-based lead discovery,
<a href="https://practicalfragments.blogspot.com/2014/11/plenty-of-room-at-bottom-of-chemical.html">small is good</a> – at least down to a certain point. While <a href="https://practicalfragments.blogspot.com/2023/05/poll-results-fragment-libraries-in-2023.html">most</a> fragments consist
of between 7 and 20 non-hydrogen atoms, some investigators have built libraries
of much smaller fragments with at <i>most</i> 7 or 8 heavy atoms. We’ve written
about <a href="https://practicalfragments.blogspot.com/2019/03/tiny-fragments-at-high-concentrations.html">MiniFrags</a> and <a href="https://practicalfragments.blogspot.com/2023/04/rsc-medicinal-chemistry-special-fbdd.html">MicroFrags</a>, which are typically screened
crystallographically at high concentrations to find <a href="https://practicalfragments.blogspot.com/2015/05/predicting-protein-ligandability-and.html">hot spots</a>. In a new
open-access <i>J. Med. Chem.</i> <a href="https://pubs.acs.org/doi/10.1021/acs.jmedchem.3c01779">paper</a>, Franz-Josef Meyer-Almes, György Keserű,
and collaborators at the Budapest University of Technology and Economics, the
University of Applied Sciences Darmstadt, and the University of Veterinary Medicine
Vienna have applied the concept to covalent fragments.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The researchers started with a
set of 84 fragments, all heterocycles functionalized with one of six warheads,
which we wrote about <a href="https://practicalfragments.blogspot.com/2018/12/new-types-of-covalent-fragments.html">here</a>. They systematically methylated nitrogen atoms on some
of these to generate 58 more fragments containing obligate positive charges,
such as compound B6+ below. The intrinsic reactivity of the fragments was assessed
by reacting them with the biologically relevant thiol glutathione (GSH). </div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Methylating the heterocycles made them more electrophilic
and thus more reactive. For example, only 16 of the 84 non-methylated fragments
had a half-life (t<sub>1/2</sub>) < 48 hours against GSH, in contrast with
30 of the 58 methylated fragments. In fact, 17 of the methylated fragments had t<sub>1/2</sub>
< 10 minutes.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Next, all 142 fragments were
screened at 250 µM for 2 hours at 30 ºC in a biochemical assay against histone
deacetylase 8 (HDAC8), an enzyme important for cell cycle progression. Hits
were confirmed in dose-response experiments after 1 hour pre-incubation.
Consistent with the glutathione data, only 12 of the non-methylated compounds
showed IC<sub>50</sub> < 50 µM, while 54 of the 58 methylated compounds were
active. One of the fragments, B6+, had a k<sub>inact</sub>/K<sub>I</sub> value
of 4006 M<sup>-1</sup>s<sup>-1</sup>, not far from that found in approved
<a href="https://practicalfragments.blogspot.com/2021/05/sotorasib-fifth-fragment-derived-drug.html">covalent drugs</a>.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">HDAC8 contains ten cysteine
residues, and sites of modification were determined using both site-directed
mutagenesis as well as tryptic digestion followed by mass spectrometry. In
total, seven residues could be labeled by one or more fragments. The most
reactive cysteine, C153, is close to the binding site of a previously reported
inhibitor (compound 1), and the researchers tried merging reactive fragments
such as B6+ onto this molecule. The best molecule, compound 3, had a k<sub>inact</sub>/K<sub>I</sub>
value of 1566 M<sup>-1</sup>s<sup>-1</sup>. However, the drop from B6+ alone
suggests that the non-covalent affinity component of compound 1 may have been
lost.</div><div class="MsoNormal" style="text-align: justify;"> </div>
<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhdTbxOeXQ8XyM0UfJW45lrPUGCFtGwJlhuDP_jyJ4qi5nB_PlwwPEL_wGAApy0FLTHtwRvzhB1blIO_dg20SY2iVjDbh0f2eBPoKI3GdaOdeBs99oOqjjjuhrhz-UVDxmENGtHiXcqOFky73WXF2QjgnW2obv6iPChUJw4rp0fGaoC-bJlLlLMoqoy-LAr/s1017/Keseru-covalent-MiniFrag-240108.bmp" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="404" data-original-width="1017" height="254" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhdTbxOeXQ8XyM0UfJW45lrPUGCFtGwJlhuDP_jyJ4qi5nB_PlwwPEL_wGAApy0FLTHtwRvzhB1blIO_dg20SY2iVjDbh0f2eBPoKI3GdaOdeBs99oOqjjjuhrhz-UVDxmENGtHiXcqOFky73WXF2QjgnW2obv6iPChUJw4rp0fGaoC-bJlLlLMoqoy-LAr/w640-h254/Keseru-covalent-MiniFrag-240108.bmp" width="640" /></a></div><br />
<div class="MsoNormal" style="text-align: justify;">This is an interesting approach,
and as the researchers note, activity assays available for covalent fragments
are higher-throughput than the crystallographic screens required for MiniFrags
and MicroFrags. On the other hand, there are limitations. For one thing, the obligate
positive charge on the methylated fragments could overwhelm other properties,
and could even lead to denaturation of proteins at high concentrations,
rendering screens uninformative. These fragments are also less likely to be cell
permeable. </div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Finally, as we <a href="https://practicalfragments.blogspot.com/2014/06/irreversible-fragments.html">wrote</a> ten years
ago, characterizing irreversible covalent fragments presents a challenge in deconvoluting
intrinsic reactivity from specific binding. Computational mapping of <a href="https://practicalfragments.blogspot.com/2012/08/two-types-of-hot-spots.html">hot spots</a>
on HDAC8 using <a href="https://practicalfragments.blogspot.com/2015/05/predicting-protein-ligandability-and.html">FTMap</a> revealed that some correlate
with modified cysteine residues. But other modified cysteine residues are in
surface-exposed flexible loops with no nearby pockets, and hits against these
are likely not advanceable. The fact that some of the fragments modify as many
as five cysteine residues on HDAC8 suggests they may be too reactive.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Still, the systematic characterization
of this library is useful experimentally and for training models. It will be interesting
to see it deployed against additional protein targets.</div>
Dan Erlansonhttp://www.blogger.com/profile/07927082337051189270noreply@blogger.com2tag:blogger.com,1999:blog-1136153439451224584.post-20797696597692523682024-01-02T06:09:00.000-08:002024-01-02T16:46:49.843-08:00Fragment events in 2024<div style="text-align: justify;">We don't know for sure what 2024 has in store for us, but barring <a href="https://practicalfragments.blogspot.com/2020/01/fragment-events-in-2020.html">pandemics</a> or other disasters, the year is shaping up to be an annus mirabilis for fragments. For the first time ever, all four of the recurring fragment meetings are scheduled for the same year, and other conferences also look exciting. I hope to see you at one. <br /></div><br /><div style="text-align: justify;"><div style="text-align: justify;"><b>March 3-5: </b>RSC-BMCS Ninth <a href="https://www.rscbmcs.org/events/fragments24/">Fragment-based Drug Discovery Meeting</a>
will be held in Cambridge, UK. This venerable biannual event will be
particularly focused on case studies "that have delivered compounds to
late stage medicinal chemistry, preclinical, or clinical programmes."
You can read my impressions of the 2013 meeting <a href="https://practicalfragments.blogspot.com/2013/03/fragments-2013.html">here</a> and the 2009 event <a href="https://practicalfragments.blogspot.com/2009/03/fragments-2009.html">here</a>.<br /></div><div style="text-align: justify;"><b> </b></div><div style="text-align: justify;"><b>April 1-4: </b>CHI’s Nineteenth
Annual <a href="https://www.drugdiscoverychemistry.com/fragment-based-drug-discovery">Fragment-Based Drug Discovery</a>, the longest-running fragment event, returns as always to San Diego. This is part of the larger <a href="https://www.drugdiscoverychemistry.com/">Drug Discovery Chemistry</a> meeting. You can read impressions of the 2023 meeting <a href="https://practicalfragments.blogspot.com/2023/04/eighteenth-annual-fragment-based-drug.html">here</a>, the 2022 event <a href="https://practicalfragments.blogspot.com/2022/04/seventeenth-annual-fragment-based-drug.html">here</a>, the 2021
virtual meeting <a href="http://practicalfragments.blogspot.com/2021/05/sixteenth-annual-fragment-based-drug.html">here</a>, the 2020 virtual meeting <a href="http://practicalfragments.blogspot.com/2020/08/fifteenth-annual-fragment-based-drug.html">here</a>, the 2019
meeting <a href="http://practicalfragments.blogspot.com/2019/04/fourteenth-annual-fragment-based-drug.html">here</a>, the 2018 meeting <a href="http://practicalfragments.blogspot.com/2018/04/thirteenth-annual-fragment-based-drug.html">here</a>, the 2017 meeting <a href="http://practicalfragments.blogspot.com/2017/05/twelfth-annual-fragment-based-drug.html">here</a>, the 2016 meeting <a href="http://practicalfragments.blogspot.com/2016/04/eleventh-annual-fragment-based-drug.html">here</a>; the 2015 meeting <a href="http://practicalfragments.blogspot.com/2015/04/tenth-annual-fragment-based-drug.html">here</a>, <a href="http://practicalfragments.blogspot.com/2015/04/tenth-annual-fragment-based-drug_29.html">here</a>, and <a href="http://practicalfragments.blogspot.com/2015/05/more-notes-from-ddc-2015.html">here</a>; the 2014 meeting <a href="http://practicalfragments.blogspot.com/2014/04/ninth-annual-fragment-based-drug.html">here</a> and <a href="http://practicalfragments.blogspot.com/2014/04/drug-discovery-chemistry-conference.html">here</a>; the 2013 meeting <a href="http://practicalfragments.blogspot.com/2013/04/eight-annual-fragment-based-drug.html">here</a> and <a href="http://practicalfragments.blogspot.com/2013/04/whats-fire-behind-smoke.html">here</a>; the 2012 meeting <a href="http://practicalfragments.blogspot.com/2012/04/seventh-annual-fragment-based-drug.html">here</a>; the 2011 meeting <a href="http://practicalfragments.blogspot.com/2011/04/sixth-annual-fragment-based-drug.html" target="_blank">here</a>; and 2010 <a href="http://practicalfragments.blogspot.com/2010/04/fifth-annual-fragment-based-drug.html" target="_blank">here</a>. </div><div style="text-align: justify;"> </div><div style="text-align: justify;"><div style="text-align: justify;"><b>June 2-4</b>: The theme of the <a href="https://www.novalix-conferences.org/">Tenth NovAliX Conference</a>, to be held in the Swiss resort town of Brunnen, is "reinventing drug discovery." You can read my impressions
of the 2018 Boston event <a href="http://practicalfragments.blogspot.com/2018/06/fifth-novalix-biophysics-in-drug.html">here</a>, the 2017 Strasbourg event <a href="http://practicalfragments.blogspot.com/2017/06/fourth-novalix-biophysics-in-drug.html">here</a>, and Teddy's impressions of the 2013 event <a href="http://practicalfragments.blogspot.com/2013/10/biophysics-in-alsace.html">here</a>, <a href="http://practicalfragments.blogspot.com/2013/10/biophysics-with-white-wine-pt-2.html">here</a>, and <a href="http://practicalfragments.blogspot.com/2013/11/biophysics-conference-pt-3.html">here</a>. <br /></div><div style="text-align: justify;"></div><div style="text-align: justify;"> <br /></div></div><div style="text-align: justify;"><b>June 25-27</b>: <a href="https://fbdddownunder.com.au/">FBDD Down Under 2024</a> will take place in beautiful Brisbane. I believe this is the fifth FBDD DU event and the first to be held outside Melbourne. You can read my impressions of <a href="https://practicalfragments.blogspot.com/2019/11/fragment-based-drug-design-down-under.html">FBDD DU 2019</a> and <a href="https://practicalfragments.blogspot.com/2012/11/fbdd-down-under.html">FBDD DU 2012</a>.<br /> <br />
<b>September 22-25</b>: After a six year hiatus, <a href="https://fbldconference.org/">FBLD 2024</a> will be held in Boston. This will
mark the eighth in an illustrious series of conferences organized by
scientists for scientists. You can read impressions of <a href="http://practicalfragments.blogspot.com/2018/10/fbld-2018.html">FBLD 2018</a>, <a href="http://practicalfragments.blogspot.com/2016/10/fbld-2016.html">FBLD 2016</a>, <a href="http://practicalfragments.blogspot.com/2014/09/fbld-2014.html">FBLD 2014</a>, <a href="http://practicalfragments.blogspot.com/2012/09/fbld-2012.html">FBLD 2012</a>, <a href="http://practicalfragments.blogspot.com/2010/10/fbld-2010.html" target="_blank">FBLD 2010</a>, and <a href="http://practicalfragments.blogspot.com/2009/09/fbld-2009.html" target="_blank">FBLD 2009</a>.</div><div style="text-align: justify;"> </div><div style="text-align: justify;"><div style="text-align: justify;"><b>September 30 to Oct 3: </b>Autumn is usually a nice time of year in Boston, so why not stick around to attend CHI’s Twenty-Second
Annual <a href="https://www.discoveryontarget.com/">Discovery on Target</a>. As the name implies
this event is more target-focused than chemistry-focused, but there are
always plenty of FBDD-related talks. You can read my impressions of the 2023 meeting <a href="https://practicalfragments.blogspot.com/2023/10/discovery-on-target-2023.html">here</a>, the
2022 meeting <a href="https://practicalfragments.blogspot.com/2022/10/twentieth-annual-discovery-on-target.html">here</a>, the 2021 event <a href="https://practicalfragments.blogspot.com/2021/10/nineteenth-annual-discovery-on-target.html">here</a>, the 2020 virtual event <a href="http://practicalfragments.blogspot.com/2020/09/eighteenth-annual-discovery-on-target.html">here</a>, the 2019 event <a href="http://practicalfragments.blogspot.com/2019/09/seventeenth-annual-discovery-on-target.html">here</a>, and the 2018 event <a href="http://practicalfragments.blogspot.com/2018/10/sixteenth-annual-discovery-on-target.html">here</a>.</div><div style="text-align: justify;"> <br /></div></div><div style="text-align: justify;">Know of anything else? Please leave a comment or drop me a note. </div></div>Dan Erlansonhttp://www.blogger.com/profile/07927082337051189270noreply@blogger.com0tag:blogger.com,1999:blog-1136153439451224584.post-31032742808916529622023-12-18T06:28:00.000-08:002023-12-18T06:46:44.934-08:00Review of 2023 reviews<div class="MsoNormal" style="text-align: justify;">The annual <i>Practical Fragments</i> <a href="https://practicalfragments.blogspot.com/2022/12/review-of-2022-reviews.html">look-back</a> on the preceding year may not be the most highly anticipated year-end
tradition, but I hope you find something of interest in this twelfth edition.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">I was fortunate to attend several
conferences and wrote about CHI’s <a href="https://practicalfragments.blogspot.com/2023/10/discovery-on-target-2023.html">Discovery on Target</a> in Boston and <a href="https://practicalfragments.blogspot.com/2023/04/eighteenth-annual-fragment-based-drug.html">Drug Discovery Chemistry</a> in San Diego. As for reviews, Louise Walsh and
collaborators at Astex, Vrije Universiteit Amsterdam, Novartis, and Frontier
Medicines (me!) <a href="https://pubs.acs.org/doi/10.1021/acs.jmedchem.2c01827">published</a> our annual analysis of fragment-to-lead success
stories in <i>J. Med. Chem.</i>, this one covering the year 2021. Some twenty other reviews of interest
to this readership were also published. I’ll cover them thematically below. </div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;"><b>Methods</b></div>
<div class="MsoNormal" style="text-align: justify;"><a href="https://practicalfragments.blogspot.com/2020/06/crystallographic-fragment-screening.html">Crystallography</a> is the most
<a href="https://practicalfragments.blogspot.com/2019/12/poll-results-affiliation-and-fragment.html">popular</a> fragment-finding technique, and in <i>Expert Opin. Drug Disc.</i>
Wladek Minor and collaborators at University of Virginia and Jagiellonian
University <a href="https://pubmed.ncbi.nlm.nih.gov/37592849/">examine</a> “the current role and evolution of X-ray crystallography in
drug discovery and development.” At the start of 2023 the Protein Data Bank
(<a href="https://www.rcsb.org/">PDB</a>) contained more than 200,000 structures, which sounds impressive until you
learn that the <a href="https://alphafold.ebi.ac.uk/">AlphaFold</a> database contains more than 200 <i>million</i> predicted
protein structures. But this is not experimentalist vs machine: the researchers
note how machine learning approaches can be used to more rapidly refine and
improve experimental data with resources such as <a href="https://checkmyblob.bioreproducibility.org/server/">CheckMyBlob</a> and <a href="https://pdb-redo.eu/">PDB-REDO</a>.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">For those wishing to dig deeper,
two papers in <i>Methods Enzymol.</i> go into experimental detail. In the
<a href="https://pubmed.ncbi.nlm.nih.gov/37858530/">first</a>, Natalie Tatum and colleagues at Newcastle University describe
“crystallographic fragment screening in academic drug discovery.” May Sharpe
and collaborators at the Swiss Light Source and University of Hohenheim describe
their fast fragment-screening pipeline in a comprehensive (49 page) <a href="https://pubmed.ncbi.nlm.nih.gov/37858531/">guide</a>. The
focus is on reproducibility, and there is plenty of practical advice. For
example, “the authors have even been successful in flying with crystal plates,”
though getting these through airport security may be easier in some countries than others.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;"><a href="https://practicalfragments.blogspot.com/2015/11/nmr-poll-results.html">Protein-detected NMR</a> was the
first truly practical fragment-based approach, and another <a href="https://pubmed.ncbi.nlm.nih.gov/37858532/">paper</a> in <i>Methods
Enzymol.</i> by Brian Volkman, Brian Smith, and colleagues at Medical College
of Wisconsin describes “fragment-screening by protein-detected NMR.” This
distills eight years of effort building their internal protein-detected NMR fragment
screening platform that has been applied to 16 proteins thus far. The chapter
is particularly detailed on protein and library preparation and screening.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Compared with crystallography and
NMR, virtual screening can be dramatically faster; we’ve <a href="https://practicalfragments.blogspot.com/2022/01/virtually-screening-11-billion.html">highlighted</a> multibillion-compound screens. In <i>WIREs Comput. Mol. Sci.</i>, Artem
Cherkasov, Francesco Gentile, and colleagues at University of British Columbia
and University of Ottawa <a href="https://wires.onlinelibrary.wiley.com/doi/full/10.1002/wcms.1678">discuss</a> (open access) how computational methods are
“keeping pace with the explosive growth of chemical libraries.” They cover
brute force methods, fragment-based virtual screening, and machine-learning
based methods, all while avoiding hype, and conclude that it will take time for
these methods to “have a real impact on practical drug discovery.”</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Finally, Marianne Fillet and
collaborators at University of Liege and University of Namur provide a general
<a href="https://www.sciencedirect.com/science/article/abs/pii/S0165993623002480">review</a> in <i>Trends Anal. Chem.</i> covering multiple <a href="https://practicalfragments.blogspot.com/2019/12/poll-results-affiliation-and-fragment.html">methods</a> to detect
non-covalent fragments. These include established techniques such as
biochemical assays, ligand-observed NMR, crystallography, thermal shifts, and
<a href="https://practicalfragments.blogspot.com/2010/02/surface-plasmon-resonance-spr.html">SPR</a>, as well as less common ones such as <a href="https://practicalfragments.blogspot.com/2023/11/finding-weak-fragments-for-membrane.html">WAC</a>, <a href="https://practicalfragments.blogspot.com/2016/01/microscale-thermophoresis-revisited.html">microscale thermophoresis</a>, <a href="https://practicalfragments.blogspot.com/2018/10/capillary-electrophoresis-revisited.html">ACE</a>,
and <a href="https://practicalfragments.blogspot.com/2023/07/dna-encoded-fragment-growing.html">DEL</a>. The paper includes several nice tables and even a decision tree to
help choose among the various approaches.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;"><b>Covalent fragments</b></div>
<div class="MsoNormal" style="text-align: justify;">Many techniques to detect
noncovalent interactions also apply to reversible covalent inhibitors, the
subject of a <a href="https://pubmed.ncbi.nlm.nih.gov/37305209/">review</a> in <i>Med. Chem. Res.</i> by Faridoon and collaborators at
Genhouse Bio and Olema Oncology. The researchers focus on various warheads
including <a href="https://practicalfragments.blogspot.com/2013/05/reversibly-covalent-fragments-vs-kinases.html">cyanoacrylamides</a>, <a href="https://practicalfragments.blogspot.com/2020/11/from-noncovalent-fragment-to-reversible.html">nitriles</a>, ketones and aldehydes, <a href="https://practicalfragments.blogspot.com/2017/07/reagents-as-covalent-fragments.html">boronic acids</a>, and
others, and provide multiple examples for each.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">In contrast, an open-access
<a href="https://pubmed.ncbi.nlm.nih.gov/37111304/">review</a> in <i>Pharmaceuticals</i> by Monique Multeder and collaborators at
Leiden University Medical Center discusses methods to detect both reversible as
well as irreversible covalent protein-drug adducts. Crystallography is the most
informative, but the researchers also delve into various mass-spectrometry
techniques including <a href="https://practicalfragments.blogspot.com/2011/03/fragments-vs-pdk1-again-into-adaptive.html">top-down</a> (with intact proteins) and <a href="https://practicalfragments.blogspot.com/2022/03/nucleophilic-fragments-other-kind-of.html">bottom-up</a> (after
digestion of modified proteins). Also covered are activity-based protein profiling
(<a href="https://practicalfragments.blogspot.com/2016/06/covalent-fragments-writ-large.html">ABPP</a>) methods, NMR, and fluorescence-based approaches. The nearly 300
references make a useful compendium.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">One of the most exciting recent
developments is “proteome-wide fragment-based ligand and target discovery,” the
subject of an open-access <a href="https://onlinelibrary.wiley.com/doi/full/10.1002/ijch.202200098">review</a> in <i>Isr. J. Chem.</i> by Ines Forrest and
Christopher Parker, both at Scripps. This concise, highly readable account
covers a lot of ground, from <a href="https://practicalfragments.blogspot.com/2016/06/covalent-fragments-writ-large.html">ABPP</a> to fully functionalized fragments (<a href="https://practicalfragments.blogspot.com/2021/05/understanding-fully-functionalized.html">FFFs</a>) to
<a href="https://practicalfragments.blogspot.com/2022/09/is-phenotypic-fragment-screening.html">phenotypic screening</a>.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">If you’re doing covalent FBLD
you’ll need a library of covalent fragments, and if you’re building one, I’d
recommend a <a href="https://pubmed.ncbi.nlm.nih.gov/37981350/">review</a> in <i>Prog. Med. Chem.</i> by David Mann and colleagues at
Imperial College London. The paper nicely summarizes design principles such as
choice of warhead and the fact that <a href="https://practicalfragments.blogspot.com/2014/06/irreversible-fragments.html">reactivity can vary</a> considerably even among
compounds with the same warhead. Synthetic methods and screening approaches are
also well covered, along with methods to distinguish specific binding from
nonspecific reactivity.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Most covalent fragments target
cysteine residues, but there at least nine other potentially reactive amino
acids, and these are the subject of an open-access <a href="https://pubmed.ncbi.nlm.nih.gov/37770315/">review</a> by György Keserű and
colleagues at Budapest University of Technology and Economics in <i>Trends
Pharm. Sci</i>. Lysine, serine, threonine, tyrosine, and histidine are the most
common targets, though some of the warheads are so reactive that specificity
will be challenging, let alone reasonable pharmacokinetic properties. This is
especially true for aspartic and glutamic acids, methionine, and tryptophan.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Finally, another <a href="https://pubmed.ncbi.nlm.nih.gov/37263826/">article</a> in <i>Trends
Pharm. Sci.</i> by Carlo Ballatore and colleagues at University of California
San Diego describes using covalent strategies to develop stabilizers and
inhibitors of protein-protein interactions (PPIs). Site-directed fragment
<a href="https://practicalfragments.blogspot.com/2023/05/from-reversible-to-irreversible.html">tethering</a> with disulfide and imine chemistry is a focus, particularly in the
context of <a href="https://practicalfragments.blogspot.com/2023/08/stabilizing-protein-protein.html">14-3-3 proteins</a>. Proximity-enabled covalent strategies, in which
warheads are grafted onto non-covalent molecules, are also covered. There is also
a short section on covalent PROTACs – more on that topic below. </div>
<div class="MsoNormal" style="text-align: justify;"><b> </b></div>
<div class="MsoNormal" style="text-align: justify;"><b>Targets</b></div>
<div class="MsoNormal" style="text-align: justify;">Keeping with the theme of
protein-protein interactions, Ge-Fei Hao, Guang-Fu Yang, and collaborators at
Central China Normal University and Guizhou University <a href="https://pubmed.ncbi.nlm.nih.gov/36841635/">discuss</a> fragment-based
approaches against “undruggable” PPIs in <i>Trends Biochem. Sci</i>. After
describing why protein-protein interactions can be difficult, the paper presents several successful case studies, including <a href="https://practicalfragments.blogspot.com/2016/04/second-fragment-based-drug-approved.html">venetoclax</a>, <a href="https://practicalfragments.blogspot.com/2021/05/sotorasib-fifth-fragment-derived-drug.html">sotorasib</a>, and targeting
<a href="https://practicalfragments.blogspot.com/2023/08/stabilizing-protein-protein.html">14-3-3 proteins</a>.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Targeted protein degradation
continues to be a major <a href="https://practicalfragments.blogspot.com/2023/10/discovery-on-target-2023.html">focus</a> for drug discovery, and this is commonly achieved
by hijacking E3 ligases to cause them to ubiquitinate a target of interest. Iacovos
Michaelides and Gavin Collie (AstraZeneca) describe how FBLD has been used to
find ligands against E3s in an open-access <i>J. Med. Chem.</i> <a href="https://pubs.acs.org/doi/10.1021/acs.jmedchem.2c01882">paper</a>. There
are more than 600 E3s, and because their biology relies on protein-protein
interactions they are often tough targets. Fragment hits can be weak and
difficult to advance, though the researchers do describe several success
stories including against <a href="https://practicalfragments.blogspot.com/2021/04/fragments-vs-keap1-deconstruction-and.html">KEAP1</a> and <a href="https://practicalfragments.blogspot.com/2018/09/fragments-in-clinic-astx660.html">XIAP/cIAP</a>. <a href="https://practicalfragments.blogspot.com/2019/06/screening-irreversible-covalent.html">Covalent fragments</a> have the
potential to permanently reprogram E3 ligases, and these are covered well too.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Another difficult type of target
is RNA, the topic of two reviews. In an open-access <i>Curr. Opin. Struct.
Biol.</i> <a href="https://pubmed.ncbi.nlm.nih.gov/36863268/">paper</a> Kevin Weeks and colleagues at University of North Carolina
Chapel Hill provide a concise and beautifully illustrated overview of the
field. They note that “RNA-targeted FBLD is in its infancy,” but given that the
first report dates to 2002 it is a long childhood, and the paper does a good
job of describing the challenges.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">A more extensive <a href="https://pubs.acs.org/doi/10.1021/acs.jmedchem.3c00034">treatment</a> of “fragment-based
approaches to identify RNA binders” is provided by Matthew Disney and
colleagues at UF Scripps in <i>J. Med. Chem.</i> The paper describes many case
studies, some of which we’ve <a href="https://practicalfragments.blogspot.com/2023/03/fragment-linking-on-bacterial-tpp.html">covered</a>, and also contains a handy table comparing
the pros and cons of a dozen different methods for finding RNA-binding
fragments.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Tuberculosis kills more than 1.5
million people each year, and fragment-based approaches have been applied
against <a href="https://practicalfragments.blogspot.com/2021/06/twenty-seven-hits-against-four.html">multiple targets</a> within the pathogen, as <a href="https://pubmed.ncbi.nlm.nih.gov/37423127/">reviewed</a> by Baptiste
Villemagne and colleagues at University Lille in <i>Eur. J. Med. Chem.</i> We’ve
<a href="https://practicalfragments.blogspot.com/2020/06/fragments-vs-mycobacterium-tuberculosis.html">covered</a> many of these studies on <i>Practical Fragments</i>, but as the paper
notes none have advanced to the clinic. This is attributed in part to cell
permeability, and the researchers suggest turning to <a href="https://practicalfragments.blogspot.com/2022/09/is-phenotypic-fragment-screening.html">phenotypic screens</a> (see
below).</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;"><b>Other</b></div>
<div class="MsoNormal" style="text-align: justify;">Fragment linking can be <a href="https://practicalfragments.blogspot.com/2014/09/fragment-growing-vs-fragment-linking.html">difficult</a>
but highly effective, especially for difficult targets. An <a href="https://pubmed.ncbi.nlm.nih.gov/37466331/">overview</a> of
published linkers is provided by Isabelle Krimm and collaborators at Université
Claude Bernard Lyon and Université Montpellier in <i>Expert Opin. Drug Disc.</i>
The paper includes a table summarizing 40 fragment linking stories, noting that
most linkers are short and flexible. Another table summarizes 19 examples of <a href="https://practicalfragments.blogspot.com/2017/03/fragment-linking-on-proteins-amide.html">target-guided synthesis</a>, including <a href="https://practicalfragments.blogspot.com/2017/10/dynamic-combinatorial-chemistry.html">dynamic combinatorial chemistry</a>. As the paper notes, all
of these are small model studies based on known compounds. In silico
approaches, the last topic covered, will probably prove more practical.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">And on the subject of practical, Dean
Brown (Jnana Therapeutics) provides an “<a href="https://pubs.acs.org/doi/10.1021/acs.jmedchem.3c00521">analysis</a> of successful hit-to-clinical
candidate pairs” in <i>J. Med. Chem.</i> This is an update to his 2018 <a href="https://practicalfragments.blogspot.com/2018/12/review-of-2018-reviews.html">article</a>
and captures 156 clinical candidates reported in the journal between 2018 and
2021. Of these, 14 had fragments in their lineage. Most of these drugs appear
in our <a href="https://practicalfragments.blogspot.com/2022/11/fragments-in-clinic-2022-edition.html">list</a> of fragment-derived clinical candidates (though <a href="https://en.wikipedia.org/wiki/Berotralstat">berotralstat</a>
does not – I’ll need to look closer). The paper contains lots of interesting analyses.
For example, of the 138 oral drugs, 39 had a molecular weight > 500 Da, 24
had Clog > 5, and 17 had more than 10 hydrogen bond acceptors (HBA). On the
other hand, none had more than 5 <a href="https://practicalfragments.blogspot.com/2023/06/a-rule-of-1-hydrogen-bond-donor-library.html">HBD</a>, emphasizing that you should be parsimonious
with hydrogen bond donors.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Finally, veteran drug hunter Nicholas Meanwell provides
“<a href="https://link.springer.com/article/10.1007/s00044-023-03070-6">reflections</a> on a 40-year career in drug design and discovery” (open access) in <i>Med. Chem.
Rev.</i> Those of you who saw his talk earlier this year at the <a href="https://practicalfragments.blogspot.com/2023/04/eighteenth-annual-fragment-based-drug.html">CHI DDC</a> meeting
will know what to expect, and those of you who didn’t will be in for a treat. A
personal and entertaining romp through pharma starting in the early 1980s, the
paper is full of surprises, such as the pursuit of minor impurities in a
phenotypic screen that ultimately led to the hepatitis C drug <a href="https://en.wikipedia.org/wiki/Daclatasvir">daclatasvir</a>. Nicholas
notes that “you discover what you screen for, so screen design is of paramount
importance.”</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The paper also reveals a passion
for medicinal chemistry: “In a search for inspiration for design concepts, I
sat down one Saturday afternoon in early October of 1987 and perused every
molecule in the United States Adopted Names (USAN) dictionary.” And, as he
notes near the end, “Decision making in drug discovery and development is a
delicate balancing act, inherently flawed based on absence of predictive accuracy,
and knowing when to conclude a discovery program with grace is also an
important trait.” That said, he provides examples of successful programs
that were almost killed multiple times – and others that <i>were</i> killed at Bristol
Myers Squibb but subsequently succeeded elsewhere. While this is frustrating on
one level, Nicholas takes satisfaction in the fact that “the science that we
conducted and the molecules and pharmacophores that we defined have been of
benefit to mankind.”</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">There are still a couple weeks
left in the year, but that’s it for <i>Practical Fragments</i> for 2023. Thanks
for reading, and special thanks for commenting. And if you live in one of the
70+ countries with elections in 2024, please vote.</div>
Dan Erlansonhttp://www.blogger.com/profile/07927082337051189270noreply@blogger.com1tag:blogger.com,1999:blog-1136153439451224584.post-91420707271603110502023-12-11T06:03:00.000-08:002023-12-11T06:03:57.913-08:00Fragments vs IP6K1 without using structural information<div class="MsoNormal" style="text-align: justify;">The three members of the inositol
hexakisphophate kinase family are potential targets for a wide range of
diseases, from Alzheimer’s to cancer to metabolic disease. However, current
inhibitors are not specific for individual isoforms. Also, the most potent
compounds contain a carboxylic acid moiety, which is usually at odds with brain
penetration. In a new <i>ACS Med. Chem. Lett.</i> <a href="https://pubs.acs.org/doi/abs/10.1021/acsmedchemlett.3c00409">paper</a>, James Barrow and collaborators
at Johns Hopkins School of Medicine, the Lieber Institute for Brain
Development, and AstraZeneca describe neutral, selective inhibitors.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The researchers started with a
high-throughput biochemical screen of 17,000 fragments, each at 100 µM, against
IP6K1. The library itself is available <a href="https://openinnovation.astrazeneca.com/">here</a>. After dose-response follow-up
studies, 90 hits confirmed, with IC<sub>50</sub> values as good as 2 µM. Most
of the hits contained carboxylic acids, but compound 5 did not, and also had
good <a href="https://practicalfragments.blogspot.com/2017/04/ligand-efficiency-invalidated.html">ligand efficiency</a>.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;"><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgYaZTYCoAYaGRQ2xabIcQ0fHTCyT_gzTS_ekDUPwjH-_1rmmN723N0rBnmB1s1pu3qSNjWkmnEM5eCrzh-ikdUxrQtcTHQMzOgSjiXn_OHHAfJGPuRbM1aCVh4p76h1dgIrGCep1LXMLPUeWRPih8eCuodiOtEVnOqg0jj_dPwot83Wuxfuxx6iJgJl85o/s742/Barrow-IP6K1-231211.bmp" style="clear: right; float: right; margin-bottom: 1em; margin-left: 1em;"><img border="0" data-original-height="274" data-original-width="742" height="148" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgYaZTYCoAYaGRQ2xabIcQ0fHTCyT_gzTS_ekDUPwjH-_1rmmN723N0rBnmB1s1pu3qSNjWkmnEM5eCrzh-ikdUxrQtcTHQMzOgSjiXn_OHHAfJGPuRbM1aCVh4p76h1dgIrGCep1LXMLPUeWRPih8eCuodiOtEVnOqg0jj_dPwot83Wuxfuxx6iJgJl85o/w400-h148/Barrow-IP6K1-231211.bmp" width="400" /></a></div>No crystal structures of IP6K1
have been reported, so the researchers used an AlphaFold model for docking
compound 5. This suggested a fragment growing approach. A variety of
replacements for the pyrrolidine were attempted, and while some of these had
improved activity many also proved to be <a href="https://practicalfragments.blogspot.com/2023/11/beware-of-fused-tetrahydroquinolines.html">chemically unstable</a>. Removing the nitrogen
and growing led to compound 24, which was both chemically stable and had sub-micromolar
activity.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The quinazolinone core itself was
associated with poor solubility, and the researchers made multiple attempts to
modify it, such as introducing additional nitrogen atoms or methylating to
remove a hydrogen-bond donor. Unfortunately, all these modifications led to significant
losses in potency.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Compound 24 is highly selective
for IP6K1 over IP6K2 and somewhat selective over IP6K3. Unfortunately, it
showed no cellular activity, possibly due to modest biochemical potency and
solubility. Nonetheless, this brief paper illustrates that starting with a
<a href="https://practicalfragments.blogspot.com/2023/05/poll-results-fragment-libraries-in-2023.html">larger</a> than normal fragment library can lead to new chemotypes. Screening the
larger library gave the researchers more chances to find fragments that did not
contain carboxylic acids. Indeed, the difficulty of modifying the quinazolinone
moiety demonstrates the utility of screening more molecules. Had the closely
related molecules been in the library, they might not have turned up as hits, but their
presence would suggest that relevant chemical space had been interrogated. </div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The paper is also a nice example
of optimizing hits in the <a href="https://practicalfragments.blogspot.com/2017/06/poll-results-what-structural.html">absence</a> of structural information. Although much
needs to be done to turn compound 24 into a <a href="https://practicalfragments.blogspot.com/2023/01/the-chemical-probes-portal-at-eight.html">chemical probe</a>, the fact that it is
still so small (almost <a href="https://practicalfragments.blogspot.com/2013/07/the-rule-of-three-at-ten.html">rule-of-three</a> compliant) provides hope that this can be done.</div>
<p></p>Dan Erlansonhttp://www.blogger.com/profile/07927082337051189270noreply@blogger.com0tag:blogger.com,1999:blog-1136153439451224584.post-16717228291900618902023-12-04T06:44:00.000-08:002023-12-04T06:44:43.969-08:00Screening tough proteins by SPR<div class="MsoNormal" style="text-align: justify;">Surface-plasmon resonance (<a href="https://practicalfragments.blogspot.com/2010/02/surface-plasmon-resonance-spr.html">SPR</a>)
is among the most popular <a href="https://practicalfragments.blogspot.com/2019/12/poll-results-affiliation-and-fragment.html">methods</a> for finding fragments. However, as we have
noted, SPR can be very prone to operator error and misinterpretation. In a recent (open
access) <i>SLAS Discovery</i> <a href="https://pubmed.ncbi.nlm.nih.gov/37714432/">paper</a>, U. Helena Danielson (Uppsala University)
and a who’s-who team of biophysicists from across Europe provide experimental
strategies for screening difficult proteins.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The researchers chose five
different proteins, some of which were screened in two or three different forms
for a total of nine protein constructs. Six of these were screened against
their FL1056 library, a custom-built 1056-member library which include molecules
from the <a href="https://practicalfragments.blogspot.com/2018/01/fragnet-next-generation.html">FragNet</a> program. The library includes a number of “<a href="https://practicalfragments.blogspot.com/2020/01/three-dimensional-fragments-revisited.html">three dimensional</a>” molecules
as assessed by principal moment of inertia (<a href="https://practicalfragments.blogspot.com/2020/07/flatland-nice-place-to-be.html">PMI</a>). The other library, FL90, is
a small set of commercially available fragments we highlighted <a href="https://practicalfragments.blogspot.com/2020/06/crystallographic-fragment-screening.html">here</a>.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Before screening compounds
against proteins, the researchers conducted a “clean screen.” This involved
injecting fragments (at 500 µM each) over the sensor surface using the same
buffer that would be used in the actual screen to pre-identify fragments that
stick to the surface. This typically disqualified about 1% of fragments, though
for one set of conditions the number was closer to 3%.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">That work done, the researchers
turned to the actual screens. After proteins were immobilized on the sensor
chips, the fragments were typically screened at a single concentration of 250 µM
each. The threshold for the initial hit cutoff was set low,
often around 10% of the library, to minimize false negatives. Subsequent follow-up studies at varying
concentrations were used for confirmation. This led to a significant winnowing,
with the final number of confirmed hits between 0.5 and 7% of the library.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The proteins themselves were intentionally
chosen to present various difficulties. Acetylcholine binding protein (AChBP, which
we wrote about <a href="https://practicalfragments.blogspot.com/2011/03/growing-into-closed-pockets.html">here</a>) forms a large (125 kDa) pentameric complex with multiple
binding sites. Lysine demethylase 1 (LSD1) is a multidomain, cofactor-dependent
protein that requires a partner protein, CoREST, for activity. LSD1 was
screened in the presence or absence of CoREST. Farnesyl pyrophosphate synthase
(FPPS, which we wrote about <a href="https://practicalfragments.blogspot.com/2010/09/allosteric-fpps-inhibitors-not-so.html">here</a>) is a target for cancer and osteoporosis, and
the microbial forms are targets for trypanosomiasis drugs. Human as well as <i>Trypanosoma
cruzi</i> and <i>Trypanosoma brucei</i> proteins were screened. Protein
tyrosine phosphatase 1B (PTP1B, which we recently wrote about <a href="https://practicalfragments.blogspot.com/2023/03/crystallography-heats-up-it-seems-for.html">here</a>) is a difficult
enzyme with a couple allosteric sites. The C-terminal region is intrinsically
disordered, and the protein was screened with or without this region. Finally,
human tau is both intrinsically disordered and prone to aggregation. As we
<a href="https://practicalfragments.blogspot.com/2023/04/eighteenth-annual-fragment-based-drug.html">noted</a> earlier this year it is of interest due to its potential
role in Alzheimer’s disease.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Happily, hits were identified
against all the proteins, some with <a href="https://practicalfragments.blogspot.com/2017/04/ligand-efficiency-invalidated.html">ligand efficiency</a> values above 0.5 kcal/mol
per heavy atom. The chemical structures for selected hits are shown, and the researchers
do appropriately caution that validating them using orthogonal (non-SPR)
methods is essential before further studies. </div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">I do wish the researchers had
noted whether shapely hits were enriched or depleted among the confirmed hits.
To my eye most seemed fairly flat, and some seemed dubiously <a href="https://practicalfragments.blogspot.com/2014/09/pains-in-nature.html">PAINS</a>-like,
including an eyebrow-raising dinitro-<a href="https://practicalfragments.blogspot.com/2013/02/fragmenting-natural-products-sometimes.html">catechol</a>. Nonetheless, the paper is a nice
summary of multiple SPR campaigns. If you’re about to embark on one yourself,
it is worth carefully perusing. </div>
<p></p>Dan Erlansonhttp://www.blogger.com/profile/07927082337051189270noreply@blogger.com0tag:blogger.com,1999:blog-1136153439451224584.post-51014470880395998332023-11-27T06:15:00.000-08:002023-11-27T06:15:37.513-08:00Beware of fused tetrahydroquinolines<div class="MsoNormal" style="text-align: justify;"><i>Practical Fragments</i> has
written frequently about pan-assay interference compounds, or <a href="https://practicalfragments.blogspot.com/2014/09/pains-in-nature.html">PAINS</a>. These molecules
contain substructures that frequently show up in hits that tend not to be
advanceable, often wasting considerable effort. One <a href="https://practicalfragments.blogspot.com/2016/04/an-interview-with-dr-saysno.html">criticism</a> of the PAINS
concept is that the original definitions were based on a limited number of
screens in one assay format. In a new (open-access) <i>J. Med. Chem.</i> <a href="https://pubs.acs.org/doi/10.1021/acs.jmedchem.3c01277">paper</a>, Alison Axtman
and collaborators at University of North Carolina Chapel Hill, Emory University,
and Oxford University characterize one class of PAINS in more detail.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;"><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEizRYA9nhN8JoaR_9eMqJgwei1MCIfjgri7hM7YvQeAexx5WgRFg0xwp2WHQAc9XJkDgIWbvINwCORkSHQH_pzaDco3JwiEz5BcHfyR02bAQ9fcYOvg-LO3TmOmZIJAkveusQSeiedj5TDSVOW6pG8y1R8pC5-oZHi0NIYeNNpAfXJiCf1ZHlBShpzYLfPL/s191/Axtman-THQs-231127.bmp" style="clear: right; float: right; margin-bottom: 1em; margin-left: 1em;"><img border="0" data-original-height="155" data-original-width="191" height="106" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEizRYA9nhN8JoaR_9eMqJgwei1MCIfjgri7hM7YvQeAexx5WgRFg0xwp2WHQAc9XJkDgIWbvINwCORkSHQH_pzaDco3JwiEz5BcHfyR02bAQ9fcYOvg-LO3TmOmZIJAkveusQSeiedj5TDSVOW6pG8y1R8pC5-oZHi0NIYeNNpAfXJiCf1ZHlBShpzYLfPL/w130-h106/Axtman-THQs-231127.bmp" width="130" /></a></div>The researchers focused on fused
tetrahydroquinolines, or THQs. Of the 51 molecules containing this substructure
in the original 2010 PAINS <a href="https://pubs.acs.org/doi/10.1021/jm901137j">paper</a>, 34 hit in at least one of the assays, and one
hit all six. At the time Jonathan Baell and Georgina Holloway noted that “it is
not clear for some PAINS, such as the fused tetrahydroquinolines, what the
relevant mechanisms of interference may be.” </div>
<div class="MsoNormal" style="text-align: justify;"><br /> </div>
<div class="MsoNormal" style="text-align: justify;"> </div><div class="MsoNormal" style="text-align: justify;">The new paper notes that fused THQs are
common in screening libraries, with more than 15,000 commercially available. They
also frequently show up as hits: the researchers summarize more than two dozen
examples against a wide variety of targets including phosphatases, kinases,
protein-protein-interactions, and more. In most cases the hits are modestly
active, with low to mid micromolar IC<sub>50</sub> values, though a few are high
nanomolar. </div><div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Promiscuity per se is not
necessarily bad. Just last week we <a href="https://practicalfragments.blogspot.com/2023/11/capivasertib-seventh-approved-fragment.html">noted</a> that the <a href="https://practicalfragments.blogspot.com/2018/12/review-of-2018-reviews.html">7-azaindole</a> fragment was the
starting point for three approved drugs. However, despite showing up as hits in
so many screens, only one peer-reviewed paper reports a crystal structure of a fused
THQ bound to a protein, and the researchers note that “no optimized <a href="https://practicalfragments.blogspot.com/2023/01/the-chemical-probes-portal-at-eight.html">chemical probes</a> or approved drugs contain the chemotype.”</div><div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Importantly, fused THQs hit in a
variety of different assay formats, including spectrophotometric,
chemiluminescent, SPR, and radiochemical. Thus, these are not merely
problematic in the AlphaScreen format studied in the original PAINS paper.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">So what’s going on? The researchers
found that, while molecules containing the fused THQ core were initially colorless,
they darkened when dissolved in DMSO or chloroform, turning purple within 72
hours. Interestingly, the reaction seems to be light-dependent: solutions
stored in the dark remained colorless. Thin layer chromatography and NMR showed
new species forming, and mass spectrometry revealed oxidation with loss of two
or four hydrogen atoms. The isolated double bond in the cyclopentene ring seemed
to be the culprit, as the saturated analog was stable. Indeed, all of the hits
shown in the paper contain the double bond, so fused THQs that lack this feature may be
fine – if they ever show up in your assay.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">It is still not clear exactly how
the decomposition products light up so many assays, but in general it’s a good
idea to steer clear of molecules that fall apart in ambient light, unless you’re
trying to make a photosensitizer.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The researchers conclude that “it
is tragic to continue to watch groups invest time and resources in dead-end
hit-to-lead campaigns, and the medicinal chemistry community will benefit
everyone if the cycle stops.” </div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">This concludes our public service
announcement.</div>
<p></p>Dan Erlansonhttp://www.blogger.com/profile/07927082337051189270noreply@blogger.com4tag:blogger.com,1999:blog-1136153439451224584.post-27369129612404851992023-11-20T06:13:00.000-08:002023-11-20T06:13:11.922-08:00Capivasertib: the seventh approved fragment-derived drug<div class="MsoNormal" style="text-align: justify;">On Thursday last week the FDA
<a href="https://www.fda.gov/drugs/resources-information-approved-drugs/fda-approves-capivasertib-fulvestrant-breast-cancer">approved</a> capivasertib for certain breast cancer patients. This marks the <a href="https://practicalfragments.blogspot.com/2022/11/fragments-in-clinic-2022-edition.html">seventh</a>
fragment-derived drug to be approved. It is also the first approved drug targeting
the kinase AKT.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;"><i>Practical Fragments</i> first
<a href="https://practicalfragments.blogspot.com/2013/04/fragments-in-clinic-azd5363.html">wrote</a> about capivasertib, then called AZD5363, way back in 2013, where we described
the decade-long odyssey from fragment to drug. Interestingly that fragment, <a href="https://practicalfragments.blogspot.com/2018/12/review-of-2018-reviews.html">7-azaindole</a>,
was also the starting point for two other approved drugs, <a href="https://practicalfragments.blogspot.com/2015/10/fragments-in-clinic-plx3397.html">pexidartinib</a> and
<a href="https://practicalfragments.blogspot.com/2011/08/first-fragment-based-drug-approved.html">vemurafenib</a>. As we noted at the time, “high-affinity molecules were obtained
relatively quickly, but these still required a huge amount of effort to achieve
selectivity, oral bioavailability, and other properties.”</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">What happened next is a poster
child to counter one of the <a href="https://practicalfragments.blogspot.com/2021/09/success-in-drug-discovery-is-not.html">false beliefs</a> Christopher Austin noted as being widespread
outside industry: “Once an investigational therapy gets into humans for the
first time, regulatory approval and marketing are all but assured.”</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Capivasertib entered the clinic
in 2010 in the first of more than 30 studies listed on <a href="https://clinicaltrials.gov/search?term=AZD5363">ClinicalTrials.gov</a> to
date. One challenge was finding patients that would benefit sufficiently to
offset a long list of side effects, including diarrhea and glucose fluctuations.
In the end, the current approval is in combination with <a href="https://en.wikipedia.org/wiki/Fulvestrant">fulvestrant</a> for “adult
patients with hormone receptor (HR)-positive, human epidermal growth factor
receptor 2 (HER2)-negative locally advanced or metastatic breast cancer with
one or more PIK3CA/AKT1/PTEN-alterations, as detected by an FDA-approved test,
following progression on at least one endocrine-based regimen in the metastatic
setting or recurrence on or within 12 months of completing adjuvant therapy.”</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Needless to say, these were not
the first patients tested. Use of genetic testing to match patients with a drug
likely to help them is not routine even today, let alone in 2010. Managing side
effects also <a href="https://endpts.com/fda-approves-astrazenecas-akt-inhibitor-truqap-for-subtype-of-breast-cancer/">required</a> figuring out how much of the drug to dose and how often. But
additional combination trials are <a href="https://www.fiercepharma.com/pharma/astrazenecas-first-class-truqap-approval-breast-cancer-marred-surprise-restriction">ongoing</a>. Perhaps, as with <a href="https://practicalfragments.blogspot.com/2016/04/second-fragment-based-drug-approved.html">venetoclax</a>, capivasertib
will eventually prove to be useful for a wider range of patients.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The first marketed fragment-derived
drug, <a href="https://practicalfragments.blogspot.com/2011/08/first-fragment-based-drug-approved.html">vemurafenib</a>, sprinted from program initiation to approval in just six
years. Capivasertib took twenty. As we previously <a href="https://practicalfragments.blogspot.com/2021/09/success-in-drug-discovery-is-not.html">noted</a>, success in drug
discovery is not necessarily fast or inevitable. Every year more than 40,000
people die of breast cancer in the US alone, but the death rate has slowly been
<a href="https://gis.cdc.gov/Cancer/USCS/#/Trends/">declining</a>. Hopefully the introduction of capivasertib will continue to reduce
this. </div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Congratulations to all the
researchers at AstraZeneca, Astex, and the Institute for Cancer Research for participating
and persisting in this 20-year marathon to bring a new treatment to people with
cancer.</div>
Dan Erlansonhttp://www.blogger.com/profile/07927082337051189270noreply@blogger.com0tag:blogger.com,1999:blog-1136153439451224584.post-308229208271139352023-11-13T06:06:00.000-08:002023-11-13T06:06:35.681-08:00An update on the COVID Moonshot<div class="MsoNormal" style="text-align: justify;">On March 18, 2020, a group called
the <a href="https://dndi.org/research-development/portfolio/covid-moonshot/">COVID Moonshot</a> released crystal structures of 71 fragments bound to the SARS-CoV2
Mpro protein. The same day, they <a href="https://practicalfragments.blogspot.com/2020/03/a-crowdsourcing-call-to-action-fbld-vs.html">launched</a> an online crowdsourcing initiative seeking
ideas for how to advance these fragments, none of which had activity in an
enzymatic assay. The results of this experiment in open science have just been
<a href="https://www.science.org/doi/10.1126/science.abo7201">published</a> in <i>Science</i>, appropriately open-access.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Within the first week, the group
received more than 2000 submissions. Ultimately more than 20,000 molecules were
submitted, and all of these were evaluated in “alchemical free-energy calculations.”
These are computationally intensive, requiring ~80 GPU hours per compound, so
the consortium used the volunteer-based distributed computing network
Folding@home. Compounds were evaluated not just for potency but also synthetic
accessibility, and those that passed were synthesized at <a href="https://practicalfragments.blogspot.com/2018/12/poll-results-library-vendors.html">Enamine</a> and tested in
various functional assays.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">In addition to accepting submissions
for how to advance fragments, a core group of researchers proposed their own ideas.
Interestingly, at least in the early stages of the project, this elite group
did no better at coming up with more potent or synthetically accessible molecules,
despite being intimately involved with the project. This finding validates the open-sourcing
of ideas from the larger scientific community.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Ultimately more than 2400 compounds
were synthesized, and more than 500 crystal structures were determined. All experimental
results were posted online to help guide the synthesis of additional compounds.
Speed was consistently prioritized, not just with high-throughput
crystallography but also high-throughput chemistry and "<a href="https://practicalfragments.blogspot.com/2022/02/photoaffinity-fragment-phabits-faster.html">direct-to-biology</a>"
screening of crude reaction mixtures. </div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The paper highlights one lead series,
which originated from a community submission (TRY-UNI-714a760b-6, itself
fragment-sized) inspired by merging overlapping fragments. This mid micromolar inhibitor
was ultimately optimized to MAT-POS-e194df51-1, with mid-nanomolar activity in
both biochemical and cell assays. (Despite a chloroacetamide in one of the
original fragments and a nitrile in the final molecule, which is the warhead found
in the approved covalent Mpro inhibitor <a href="https://en.wikipedia.org/wiki/Nirmatrelvir">nirmatrelvir</a>, MAT-POS-e194df51-1 is
non-covalent.) </div><div class="MsoNormal" style="text-align: justify;"> <div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjjSZDYCNuh1EI3MXDvQG-D_bP5xiUR9Q9CzFkxNCLYW4HMsuWj8qDARc017rxAvrvP1DjzpKqZ5TBqo44b2trpEfYIWVpEctadqOZOiSyNlVxjwyhSJP-6QqBRHcHkpAxe3gn88Fg7QZ4imF707ZmbVpFwME3gBpLlw-FyxiZhVk3v_7cXUcsQvIAFH7nc/s1449/Delft-Moonshot-231113.bmp" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="396" data-original-width="1449" height="174" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjjSZDYCNuh1EI3MXDvQG-D_bP5xiUR9Q9CzFkxNCLYW4HMsuWj8qDARc017rxAvrvP1DjzpKqZ5TBqo44b2trpEfYIWVpEctadqOZOiSyNlVxjwyhSJP-6QqBRHcHkpAxe3gn88Fg7QZ4imF707ZmbVpFwME3gBpLlw-FyxiZhVk3v_7cXUcsQvIAFH7nc/w640-h174/Delft-Moonshot-231113.bmp" width="640" /></a></div><br /></div><div class="MsoNormal" style="text-align: justify;"><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgQefTNA04jm8VGOOI43UivbOUmVMLuyXxoDttiFQNFLX5z0CB3-u-IMRhbYv22i7lFOSSmejeFLLQ8SE8a29N5DZccS2PAX2XdoXX1sXcK6-h4iCigChXdwqrBwzLJ1wj_N1GnyGNOfQk09So4K0PXkLsR47lIrfHQyl3Wh_EZpqkBB53763tjJ-AEOLIN/s653/Delft-Moonshot-231113.png" style="clear: right; float: right; margin-bottom: 1em; margin-left: 1em;"><img border="0" data-original-height="653" data-original-width="587" height="200" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgQefTNA04jm8VGOOI43UivbOUmVMLuyXxoDttiFQNFLX5z0CB3-u-IMRhbYv22i7lFOSSmejeFLLQ8SE8a29N5DZccS2PAX2XdoXX1sXcK6-h4iCigChXdwqrBwzLJ1wj_N1GnyGNOfQk09So4K0PXkLsR47lIrfHQyl3Wh_EZpqkBB53763tjJ-AEOLIN/w180-h200/Delft-Moonshot-231113.png" width="180" /></a></div>The molecule is potent against known SARS-CoV-2 variants, including
recent ones such as Omicron. A crystal structure of the final molecule also
overlays remarkably well onto the initial fragments.</div><div class="MsoNormal" style="text-align: justify;"> <br /></div>
<div class="MsoNormal" style="text-align: justify;">The paper notes that there is
still considerable work to do, particularly optimizing the pharmacokinetics to
lower clearance and improve bioavailability. These efforts can take vast sums
of <a href="https://practicalfragments.blogspot.com/2013/04/fragments-in-clinic-azd5363.html">time</a> and money, and the lead series has been adopted by the Drugs for Neglected
Diseases initiative for further development. Although a handful of drugs are
already approved against SARS-CoV-2, there is room for improvement: Derek Lowe posted a vivid personal account of his experience on nirmatrelvir <a href="https://www.science.org/content/blog-post/paxlovid-personally">here</a>.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">When we <a href="https://practicalfragments.blogspot.com/2020/03/a-crowdsourcing-call-to-action-fbld-vs.html">wrote</a> about the COVID Moonshot
in March of 2020, we correctly predicted that vaccines would be approved before
drugs from this effort emerged. Fortunately, our warning that “there <i>will</i>
be a SARS-CoV-3” has not proven correct – yet. But open science endeavors such
as the COVID Moonshot will help us prepare for this eventuality. We may not have
made it to the moon yet, but perhaps we’ve learned how to leave Earth’s orbit.</div>
<p></p>Dan Erlansonhttp://www.blogger.com/profile/07927082337051189270noreply@blogger.com0tag:blogger.com,1999:blog-1136153439451224584.post-32766163613918664432023-11-06T05:47:00.000-08:002023-11-06T05:47:44.712-08:00Finding weak fragments for membrane proteins with WAC<div class="MsoNormal" style="text-align: justify;">Last week we <a href="https://practicalfragments.blogspot.com/2023/10/nmr-for-sar-all-about-ligand.html">wrote</a> about NMR, one
of the most <a href="https://practicalfragments.blogspot.com/2019/12/poll-results-affiliation-and-fragment.html">popular</a> fragment-finding methods. This week we turn to a less common
technique: weak affinity chromatography, or WAC. As we’ve <a href="https://practicalfragments.blogspot.com/2017/04/fragment-optimization-without.html">written</a> previously, WAC
involves immobilizing a protein of interest in a chromatography column and
flowing a ligand-containing solution through the column. If the ligand
interacts with the protein, its elution time will be delayed in proportion to its
affinity. In a new (open-access) <i>Molecules</i> <a href="https://pubmed.ncbi.nlm.nih.gov/37894592/">paper</a>, Claire Demesmay and collaborators
at Universite Claude Bernard Lyon and Ecole Supérieure de Biotechnologie de
Strasbourg extend the technique to membrane proteins.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Membrane proteins are themselves
tricky to study, since removing them from their membranes often denatures them.
One trick is to use <a href="https://en.wikipedia.org/wiki/Nanodisc">nanodiscs</a>, which are tiny lipid bilayer islands surrounded
by proteins that keep them soluble in water. These scaffolding proteins can
also be biotinylated so that the nanondiscs can be attached to streptavidin,
which itself can be linked to a surface or matrix. Each nanodisc holds one or
at most a few membrane proteins.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">When we first <a href="https://practicalfragments.blogspot.com/2011/03/weak-affinity-chromatography-wac.html">wrote</a> about WAC in
2011 the technique used standard HPLC columns, which required non-negligible
amounts of protein. Here, the technique has been miniaturized to use glass
capillaries with volumes of less than 1 microliter, requiring only a few tens
of picomoles of protein. The researchers fill the capillaries with a
bio-compatible polymer, functionalize it with streptavidin, and then capture
biotinylated nanodiscs containing the membrane protein of interest. </div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">A long-recognized challenge with
WAC is nonspecific binding of the fragments to the column or matrix. Here, the
researchers chose a filling (or monolith) that is more hydrophilic (for
aficionados, they picked poly(DHPMA-co-MBA)) and found it superior to the
previous polymer both with regards to capacity and non-specific binding.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Another challenge with WAC is
detecting low-affinity binders: because interactions with the protein are weak,
the shift in retention time is harder to detect. One solution is to pack more
protein in the column, and the researchers develop a clever way of doing this with
a “multilayer grafting” approach in which successive injections of streptavidin
and nanodiscs more effectively fill the capillary. The combination of a more
hydrophilic filling and multilayer grafting increased the column capacity for nanodiscs
by three-fold.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The researchers tested their approach
on the adenosine-A2A receptor (AA<sub>2A</sub>R), which has frequently been
used as a model GPCR. Two previously reported weak ligands, both with
affinities around 0.2 mM, could be detected, and competition with an
orthosteric binder revealed that they were binding specifically.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">This is a nice, how-to guide for
performing WAC on membrane proteins, and the paper includes detailed equations for
calculating affinities from differences in retention times. I look forward to
seeing the technique used in de novo screens.</div>
<p></p>Dan Erlansonhttp://www.blogger.com/profile/07927082337051189270noreply@blogger.com0tag:blogger.com,1999:blog-1136153439451224584.post-86507151927331522192023-10-30T06:09:00.000-07:002023-10-30T06:09:12.224-07:00NMR for SAR: All about the ligand<div class="MsoNormal" style="text-align: justify;">In last week’s <a href="https://practicalfragments.blogspot.com/2023/10/a-liability-predictor-for-avoiding.html">post</a> we described
a free online tool for predicting bad behavior of compounds in various assays.
But as we noted, you often get what you pay for, and computational methods can’t
(yet) take the place of experimentation. In a new (open-access) <i>J. Med.
Chem.</i> <a href="https://pubs.acs.org/doi/10.1021/acs.jmedchem.3c00656">paper</a>, Steven LaPlante and collaborators at NMX and INRS describe a
roadmap for discovering, validating, and advancing weak fragments. They call it
NMR by SAR</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Unlike <a href="https://practicalfragments.blogspot.com/2014/10/fragments-vs-bcl-xl-selectively_27.html">SAR by NMR</a>, the grand-daddy of fragment-finding techniques which involves
<a href="https://practicalfragments.blogspot.com/2015/11/nmr-poll-results.html">protein-detected</a> NMR, NMR for SAR focuses heavily on the ligand. The
researchers illustrate the process by finding ligands for the protein HRAS, for
which drug discovery has lagged in comparison to its sibling <a href="https://practicalfragments.blogspot.com/2021/05/sotorasib-fifth-fragment-derived-drug.html">KRAS</a>.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The researchers started by
screening the G12V mutant form of HRAS in its inactive (GDP-bound) state. They screened
their internal library of 461 fluorinated fragments in pools of 11-15 compounds
(each at ~0.24 mM) using <sup>19</sup>F NMR. An initial screen at 15 µM protein
produced a very low hit rate, so the protein concentration was increased to 50 µM.
After deconvolution, two hits confirmed, one of which was NMX-10001.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The affinity of the compound was found
to be so low that <sup>1</sup>H NMR experiments could not detect binding. Thus,
the researchers kept to fluorine NMR to screen for commercial analogs. They used
<sup>19</sup>F-detected versions of differential line width (DLW) and CPMG
experiments to rank affinities, and the latter technique was also used to test
for compound aggregation using methodology we <a href="https://practicalfragments.blogspot.com/2019/11/a-new-tool-for-detecting-aggregation_10.html">highlighted</a> in 2019. Indeed, the researchers
have developed multiple tools for detecting aggregators, such as those we <a href="https://practicalfragments.blogspot.com/2022/01/an-epidemic-of-aggregators-and.html">wrote</a>
about in 2022.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Ligand concentrations were measured
by NMR, which sometimes differed from the assumed concentrations. As the
researchers note, these differences, which are normally not measured
experimentally, can lead to errors in ranking the affinities of compounds. The
researchers also examined the 1D spectra of the proteins to assess whether compounds
caused dramatic changes via pathological mechanisms, such as precipitation.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;"><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj7B1-0LNBhZY6Rkqrbfnbgphf3Ec9v63TfXp_5I_WHMcS2dI0pntsgNrf0agz3E6W5-pSMayZKiQ1pIH28KUnX81Y4knMNR7MhVvq_-1aI3jTbt82kM693t9mtRWPxxGbLkcNm8JyrJH9j5r-6wlNIcRJpx8C6ytxO-c4QMbxXhdjMj6g4bMVrG7lOqFW4/s724/LaPlante-NMR-for-SAR--231030.bmp" style="clear: right; float: right; margin-bottom: 1em; margin-left: 1em;"><img border="0" data-original-height="314" data-original-width="724" height="174" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj7B1-0LNBhZY6Rkqrbfnbgphf3Ec9v63TfXp_5I_WHMcS2dI0pntsgNrf0agz3E6W5-pSMayZKiQ1pIH28KUnX81Y4knMNR7MhVvq_-1aI3jTbt82kM693t9mtRWPxxGbLkcNm8JyrJH9j5r-6wlNIcRJpx8C6ytxO-c4QMbxXhdjMj6g4bMVrG7lOqFW4/w400-h174/LaPlante-NMR-for-SAR--231030.bmp" width="400" /></a></div>The researchers turned to
protein-detected 2D NMR for orthogonal validation and to determine the binding
sites of their ligands. These experiments revealed that the compounds bind in a
shallow pocket that has previously been targeted by several groups (see <a href="https://practicalfragments.blogspot.com/2019/08/fragments-vs-ras-family-proteins.html">here</a> for
example). Optimization of their initial hit ultimately led to NMX-10095, which
binds to the protein with low double digit micromolar affinity. This compound
also blocked SOS-mediated nucleotide exchange and was cytotoxic, albeit at high
concentrations.</div><div class="MsoNormal" style="text-align: justify;"><br /></div>
<div class="MsoNormal" style="text-align: justify;">I do wish the researchers had
measured the affinity of their molecules towards other RAS isoforms as this
binding pocket is conserved, and inhibiting all RAS activity in cells is
generally toxic. Moreover, the best compound is reminiscent of a <a href="https://practicalfragments.blogspot.com/2012/06/fragments-versus-ras-part-2.html">series</a> reported
by Steve Fesik back in 2012. </div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">But this specific example is less important
than the clear description of an NMR-heavy assay cascade that weeds out
artifacts in the quest for true binders. The strategy is reminiscent of the “<a href="https://practicalfragments.blogspot.com/2016/12/review-of-2016-reviews.html">validation cross</a>” we mentioned back in 2016. Perhaps someday computational methods will
advance to the point where “wet” experiments become an afterthought. But in the
meantime, this paper provides a nice set of tools to find and rigorously
validate even weak binders.</div>
<p></p>Dan Erlansonhttp://www.blogger.com/profile/07927082337051189270noreply@blogger.com3tag:blogger.com,1999:blog-1136153439451224584.post-63360513581208389242023-10-23T06:15:00.000-07:002023-10-23T06:15:08.034-07:00A Liability Predictor for avoiding artifacts?<div class="MsoNormal" style="text-align: justify;">False positives and artifacts are
a constant source of irritation – and <a href="https://practicalfragments.blogspot.com/2023/01/the-chemical-probes-portal-at-eight.html">worse</a> – in compound screening. We’ve written
frequently about small molecule <a href="https://practicalfragments.blogspot.com/2022/01/an-epidemic-of-aggregators-and.html">aggregation</a> as well as generically <a href="https://practicalfragments.blogspot.com/2015/02/new-pains-and-their-painful-mechanisms.html">reactive</a>
molecules that repeatedly come up as screening hits. It is possible to weed
these out experimentally, but this can entail considerable effort, and for particularly
difficult targets, false positives may dominate. Indeed, there may be no true
hits at all, as we noted in this <a href="https://practicalfragments.blogspot.com/2020/07/failing-honorably-and-openly-on-prp.html">account</a> of a five-year and ultimately fruitless
hunt for prion protein binders.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">A computational screen to rapidly
assess small molecule hits as possible artifacts would be nice, and in fact
several have been developed. Among the most popular are computational filters
for pan-assay interference compounds, or <a href="https://practicalfragments.blogspot.com/2014/09/pains-in-nature.html">PAINs</a>. However, as Pete Kenny and
others have <a href="https://practicalfragments.blogspot.com/2016/04/an-interview-with-dr-saysno.html">pointed out</a>, these were developed using data from a limited number
of screens in one particular assay format. Now Alexander Tropsha and collaborators
at University of North Carolina Chapel Hill and the National Center for
Advancing Translational Science (NCATS) at the NIH have provided a broader resource
in a new <i>J. Med. Chem.</i> <a href="https://pubs.acs.org/doi/10.1021/acs.jmedchem.3c00482">paper</a>.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The researchers experimentally
screened around 5000 compounds, taken from the NCATS Pharmacologically Active
Chemical Toolbox, in four different assays: a fluorescence-based thiol
reactivity assay, an assay for redox activity, a firefly luciferase (FLuc)
assay, and a nanoluciferase (NLux) assay. The latter two assays are commonly
used in cell-based screens to measure gene transcription. The thiol reactivity
assay yielded around 1000 interfering compounds, while the other three assays
each produced from 97 to 142. Interestingly, there was little overlap among
the problematic compounds.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">These data were used to develop
quantitative structure-interference relationship (QSIR) models. The NCATS
library of nearly 64,000 compounds was virtually screened, and around 200
compounds were tested experimentally for interference in the four assays, with
around half predicted to interfere and the other half predicted not to
interfere. The researchers had also previously built a computational model to
predict aggregation, and this – along with the four models discussed here – have
been combined into a free web-based “<a href="https://liability.mml.unc.edu/">Liability Predictor</a>.”</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">So how well does it work? The researchers
calculated the sensitivity, specificity, and balanced accuracy for each of the
models and state that “they can detect around 55%-80% of interfering compounds.”</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">This sounded encouraging, so naturally
I took it for a spin. Unfortunately, my mileage varied. Or, to pile on the
metaphors, lots of wolves successfully passed themselves off as sheep. <a href="https://www.chemicalprobes.org/unsuitables/iniparib">Iniparib</a> was recognized correctly as a possible thiol interference compound. On the other hand, the known
redox cycler <a href="https://practicalfragments.blogspot.com/2012/04/universal-fragments.html">toxoflavin</a> was predicted not to be a redox
cycler – with 97.12% confidence. Similarly, <a href="https://practicalfragments.blogspot.com/2017/01/cussed-curcumin.html">curcumin</a>, which can form adducts
with thiols as well as aggregate and redox cycle, was pronounced innocent. <a href="https://advisor.docking.org/search/">Quercetin</a> was recognized as possibly thiol-reactive, but its known propensity to aggregate was not. Weirdly, Walrycin B, which the researchers note interferes with all the
assays, got a clean bill of health. Perhaps the online tool is still being optimized.<br /></div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">At this point, perhaps the
Liability Predictor is best treated as a cautionary tool: molecules that come
up with a warning should be singled out for particular interrogation, but
passing does not mean the molecule is innocent. Laudably, the researchers have
made all the underlying data and models publicly available for others to build
on, and I hope this happens. But for now, it seems that no computational tool can
substitute for experimental (in)validation of hits.</div>
<p></p>Dan Erlansonhttp://www.blogger.com/profile/07927082337051189270noreply@blogger.com2tag:blogger.com,1999:blog-1136153439451224584.post-76666183223560669032023-10-16T06:16:00.000-07:002023-10-16T06:16:10.784-07:00Spacial Scores: new metrics for measuring molecular complexity<div class="MsoNormal" style="text-align: justify;">Molecular complexity is one of the
theoretical underpinnings for fragment-based drug discovery. Mike Hann and
colleagues <a href="https://practicalfragments.blogspot.com/2011/09/decade-of-molecular-complexity.html">proposed</a> two decades ago that very simple molecules may not have
enough features to bind tightly to any proteins, whereas highly functionalized
molecules may have extraneous spinach that keeps them from binding to any
proteins. Fragments, being small and thus less complex, are in a <a href="https://practicalfragments.blogspot.com/2014/11/plenty-of-room-at-bottom-of-chemical.html">sweet spot</a>:
just complex enough.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">But what does it mean for one
molecule to be more complex than another? Most chemists would agree that pyridine
is more complex than methane, but is it more complex than benzene? To decide,
you need a numerical metric, and there are plenty to choose from. The problem,
as we <a href="https://practicalfragments.blogspot.com/2017/02/many-measures-of-molecular-complexity.html">discussed</a> in 2017, is that they don’t correlate with one another, so it
is not clear which one(s) to choose. In a new (open access) <i>J. Med. Chem.</i>
<a href="https://pubs.acs.org/doi/10.1021/acs.jmedchem.3c00689">paper</a>, Adrian Krzyzanowski, Herbert Waldmann and colleagues at the Max Planck Institute
Dortmund have provided another. (Derek Lowe also recently <a href="https://www.science.org/content/blog-post/measuring-molecular-complexity">covered</a> this paper.)</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The researchers propose the Spacial
Score, or SPS. This is calculated based on four molecular parameters for each
atom in a given molecule. The term <i>h</i> is dependent on atom hybridization:
1 for sp-, 2 for sp<sup>2</sup>-, 3 for sp<sup>3</sup>-hybrized atoms, and 4
for all others. Stereogenic centers are assigned an <i>s</i> value of 2, while
all other atoms are assigned a value of 1. Atoms that are part of non-aromatic
rings are also assigned an <i>r</i> value of 2; those that are part of an aromatic
ring or linear chain are set to 1. Finally, the <i>n</i> score is set to the
number of heavy-atom neighbors.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">For each atom in a molecule, <i>h</i>
is multiplied by <i>s</i>, <i>r</i>, and <i>n</i><sup>2</sup>. The SPS is calculated
by summing the individual scores for all the atoms in a molecule. Because there
is no upper limit, and because it is nice to be able to compare molecules of
the same size, the researchers also define the nSPS, or normalized SPS, which
is simply the SPS divided by the number of non-hydrogen atoms in the molecule. Although SPS can
be calculated manually, the process is tedious and the researchers have kindly
provided <a href="https://github.com/frog2000/Spacial-Score">code</a> to automate the process. Having defined SPS, the researchers
compare it to other molecular complexity metrics, including the simple fraction
<span style="mso-spacerun: yes;"> </span>of sp<sup>3</sup> carbons in a molecule,
Fsp<sup>3</sup>, which we <a href="https://practicalfragments.blogspot.com/2009/11/too-many-aromatics-stink.html">wrote</a> about in 2009. </div><div class="MsoNormal" style="text-align: justify;"> </div><div class="MsoNormal" style="text-align: justify;">The researchers next calculated nSPS for four sets of molecules including drugs, a screening library from <a href="https://practicalfragments.blogspot.com/2018/12/poll-results-library-vendors.html">Enamine</a>, natural
products, and so-called “dark chemical matter,” library compounds that have not
hit in numerous screens. The results are equivocal. For
example, the nSPS for dark chemical matter is very similar to that for drugs.
On the other hand, natural products tend to have higher nSPS scores than drugs,
as expected. Interestingly, the average nSPS score for compounds in the <a href="https://practicalfragments.blogspot.com/2012/12/gdb-17-166-billion-fragments-and.html">GDB-17</a>
database, consisting of theoretical molecules having up to 17 atoms, is also quite
high.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The researchers assessed whether nSPS
correlated with biological properties, and found that compounds with lower nSPS
tended to have lower potencies against fewer proteins, as predicted by theory.
That said, this analysis was based on binning compounds into a small number of
categories, and as Pete Kenny has repeatedly <a href="https://practicalfragments.blogspot.com/2013/02/beware-correlation-inflation.html">warned</a>, this can lead to spurious
trends.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The same issue of <i>J. Med.
Chem.</i> carries an analysis of the paper by Tudor Oprea and Cristian Bologa,
both at University of New Mexico. This contextualizes the work and confirms
that drugs do not seem to be getting more complex over time, as measured by nSPS.
This may seem odd, though Oprea and Cristian note that by “normalizing” for
size, nSPS misses the increasing molecular weight of drugs.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">This observation also raises
other questions, such as the fact that SPS explicitly excludes element identity.
Coming back to benzene and pyridine, both have identical SPS and nSPS, which
does not seem chemically intuitive. One could quibble more: why square the
value of <i>n</i> in the calculation of SPS? Why allow <i>s</i> to be only 1
and 2, as opposed to 1 and 5?</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">In the end I did enjoy reading
this paper, and I do think having some metric of molecular complexity might be
valuable. I’m just not sure where SPS will fit in with all the existing and conflicting
metrics, and how such metrics can lead to practical applications.</div>
<p></p>Dan Erlansonhttp://www.blogger.com/profile/07927082337051189270noreply@blogger.com5tag:blogger.com,1999:blog-1136153439451224584.post-84877719361217291352023-10-09T06:00:00.000-07:002023-10-09T06:00:11.522-07:00Fragments finger the BPTF PHD Finger<div class="MsoNormal" style="text-align: justify;">Plant homeodomain (PHD) fingers,
despite their name, are found in nearly 300 human proteins. They are small (50-80
amino acid) domains that typically recognize post-translational modifications
such as trimethylated lysine residues in histones. The PHD finger in BPTF is
implicated in certain types of acute myeloid leukemia. However, because of the
large number of PHD fingers as well as their small binding sites, few attempts
have been made to develop corresponding <a href="https://practicalfragments.blogspot.com/2023/07/a-rule-of-two-for-using-chemical-probes.html">chemical probes</a>. (Indeed, the only <a href="https://practicalfragments.blogspot.com/2014/12/fragments-finger-phd-finger.html">mention</a>
of them on <i>Practical Fragments</i> was in 2014.) In a just-published <i>ACS
Med. Chem. Lett.</i> <a href="https://pubs.acs.org/doi/10.1021/acsmedchemlett.3c00343">paper</a>, William Pomerantz and collaborators at University
of Minnesota and St. Jude Children’s Research Hospital report the first steps.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The researchers started by
screening a library of 1056 fragments (from <a href="https://practicalfragments.blogspot.com/2018/12/poll-results-library-vendors.html">Life Chemicals</a>) against the BPTF
PHD finger using ligand-observed (<sup>1</sup>H <a href="https://practicalfragments.blogspot.com/2019/11/a-new-tool-for-detecting-aggregation_10.html">CPMG</a>) NMR. Fragments were at
100 µM in pools of up to five. This gave a preliminary hit rate of 5.7%, but
only ten compounds (<1%) reproduced when compounds were repurchased and retested
individually.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">These ten fragments were next
tested by SPR (at 400 µM), which confirmed six of them. Also, all ten CPMG hits
were tested in an AlphaScreen assay in which they competed with a known peptide
binder. This confirmed nine, including the six that confirmed by SPR.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Interestingly, the most potent fragment
in the AlphaScreen assay was the starting point for the KRAS inhibitor we
<a href="https://practicalfragments.blogspot.com/2022/10/from-noncovalent-fragment-to-covalent.html">highlighted</a> last year. However, this fragment did not show binding to the BPTF
PHD finger by SPR, and the researchers had identified the 2-aminothophene substructure
as a hit against an unrelated protein. Whether this fragment is <a href="https://practicalfragments.blogspot.com/2023/03/versatile-fragments-from-protein-data.html">privileged</a> or <a href="https://practicalfragments.blogspot.com/2019/12/poll-results-affiliation-and-fragment.html">pathological</a>
may be context dependent.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">This and the top three fragments
that confirmed in all assays were used as starting points for SAR by catalog,
and a handful of analogs were purchased. The researchers also resynthesized two
of the compounds. Oddly, resynthesized F2 turned out to be three-fold more
active in the AlphaScreen assay than the commercial material. One analog, compound
F2.7, showed mid-micromolar activity.</div><p>
</p><div class="MsoNormal" style="text-align: justify;"> <div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhJQ7EwYp0ycOHjJMXPKQRVvAf8mMsjR9J5yNnS1ZTqHDe3Zj5kf0a_SRb8LNFog2nvAUbkTD58fZbq7T0GCpRDlO_0oLIt-X-jWVq7g7ReoL4uxkIwqjKEALDHs0pKcSx4W5pfAkPTx4iGLZSzwYg46c4wO2lkgSlC0u1GJl2fDd8UJf561Zv0CR2uSO2R/s1307/Pomerantz-BPTF-231009.bmp" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="265" data-original-width="1307" height="130" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhJQ7EwYp0ycOHjJMXPKQRVvAf8mMsjR9J5yNnS1ZTqHDe3Zj5kf0a_SRb8LNFog2nvAUbkTD58fZbq7T0GCpRDlO_0oLIt-X-jWVq7g7ReoL4uxkIwqjKEALDHs0pKcSx4W5pfAkPTx4iGLZSzwYg46c4wO2lkgSlC0u1GJl2fDd8UJf561Zv0CR2uSO2R/w640-h130/Pomerantz-BPTF-231009.bmp" width="640" /></a></div> </div><div class="MsoNormal" style="text-align: justify;">Docking and two-dimensional
protein-observed (<sup>1</sup>H,<sup>15</sup>N HSQC) NMR experiments suggest
that most of the fragments bind in the “aromatic cage” which normally recognizes
methylated lysine residues, but F2 may bind in an adjacent region. Both subpockets
were also identified as being <a href="https://practicalfragments.blogspot.com/2017/08/assessing-ligandability-by-thermal.html">ligandable</a> using the program <a href="https://practicalfragments.blogspot.com/2015/05/predicting-protein-ligandability-and.html">FTMap</a>.</div><p></p><p>
</p>
<div class="MsoNormal" style="text-align: justify;">This paper is a nice example of using
<a href="https://practicalfragments.blogspot.com/2019/12/poll-results-affiliation-and-fragment.html">orthogonal methods</a> to find and carefully validate fragments against an underexplored
class of targets. The researchers conclude by stating that “these hits are
suitable for further SAR optimization and development into future methyl lysine
reader chemical probes.” I look forward to seeing more publications. </div>
<p></p>Dan Erlansonhttp://www.blogger.com/profile/07927082337051189270noreply@blogger.com0tag:blogger.com,1999:blog-1136153439451224584.post-4569733917913900452023-10-02T06:18:00.003-07:002023-10-02T06:18:43.864-07:00Discovery on Target 2023<div class="MsoNormal" style="text-align: justify;">Last week CHI’s <a href="https://www.discoveryontarget.com/">Discovery on Target</a> was held in Boston. This was the Twentieth Anniversary edition, though oddly last year also <a href="https://practicalfragments.blogspot.com/2022/10/twentieth-annual-discovery-on-target.html">claimed</a> to be the twentieth. Regardless, attendance surpassed <a href="https://practicalfragments.blogspot.com/2019/09/seventeenth-annual-discovery-on-target.html">pre-pandemic</a> levels, with some
1200 attendees, 90% of them in person. Eight or nine concurrent tracks over the
course of three days competed with one another, while a couple pre-conference
symposia and a handful of short courses were held before the main event. Outside
obligations kept me from seeing many talks, including plenary keynotes by Jay
Bradner (Novartis), Anne Carpenter, and Shantanu Singh (both at the Broad
Institute), but most of these were recorded and will be made available for a
year, and I look forward to watching them. Here I’ll just touch on a few of the
fragment-relevant talks I was able to attend.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">“Protein Degraders and Molecular
Glues” was a popular track during all three days of the main conference, and in
a featured presentation Steve Fesik (Vanderbilt) described how he is using NMR-based
FBLD to identify tissue-specific E3 ligases and β-catenin degraders. In the
case of β-catenin, a difficult oncology target, a fragment screen identified a
500 µM hit that was optimized to 10-20 nM. This has no functional activity on its
own, but combining it with a ligand for an E3 ligase to generate a bivalent
<a href="https://en.wikipedia.org/wiki/Proteolysis_targeting_chimera">PROTAC</a> causes degradation of the protein. Steve is currently optimizing the pharmaceutical
properties of these molecules.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">One exciting application for PROTACs
is tissue-specific targeted protein degradation, which could avoid systemic toxicity
for proteins such as <a href="https://practicalfragments.blogspot.com/2016/04/second-fragment-based-drug-approved.html">Bcl-x<sub>L</sub></a>. Steve said that for the past five years
he has been pursuing ligands against E3 ligases preferentially expressed in
certain tissues, and he presented brief vignettes for three of them. These came
from an initial list of 20 E3 targets, but many of them turned out to be too
difficult to express.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Steve typically screens a library
of nearly 14,000 fragments, large according to our recent <a href="https://practicalfragments.blogspot.com/2023/05/poll-results-fragment-libraries-in-2023.html">poll</a>, but this has
proven fruitful as only about 10% of proteins he has screened have turned out
to be “teflon.” He noted the odd little fragment hit that proved so impactful to
the KRAS program we <a href="https://practicalfragments.blogspot.com/2022/10/from-noncovalent-fragment-to-covalent.html">highlighted</a> last year as being something that might have
been excluded from a smaller library.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">We <a href="https://practicalfragments.blogspot.com/2023/09/fragments-vs-dcaf1-new-tool-for.html">wrote</a> last week about ligands
for the E3 ligase DCAF1, and Rima Al-Awar (Ontario Institute for Cancer
Research) described another series. She also described ligands against the oncology
target WDR5, a target Steve Fesik has <a href="https://practicalfragments.blogspot.com/2020/05/fragment-merging-on-wbm-site-of.html">pursued</a> as well.<br /></div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Continuing the theme of targeted
protein degradation, Jing Liu described Cullgen’s discovery of fragment-sized ligands
for a broadly-expressed E3 ligase which could be an alternative to
CRBN-targeting ligands when resistance (inevitably) arises. Although he did not
specify the E3 ligase, Cullgen has filed a patent application for ligands
targeting DCAF1.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Rounding out targeted protein degradation,
Kevin Webster, my colleague at Frontier Medicines, described the discovery of covalent
ligands for the E3 ligase DCAF2 (or DTL) using chemoproteomics and a variety of
other techniques including cryo-electron microscopy. Consistent with Steve’s
comments, considerable effort went into successfully obtaining a soluble,
well-behaved protein.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The late Nobel laureate Sydney
Brenner said that “progress in science depends on new techniques, new discoveries,
and new ideas, probably in that order.” Harvard’s Steve Gygi, one of Frontier’s
Scientific Advisory Board members, described multiple new techniques in a featured
presentation focused on cysteine-based profiling. These included multiplexed
methods to more rapidly find covalent ligands for targets across the proteome.
A just-released mass spectrometry instrument made by Thermo Fisher called the
Astral further accelerates the process with order-of-magnitude improvements in
both speed and sensitivity compared to existing machines.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">The <a href="https://practicalfragments.blogspot.com/2016/06/covalent-fragments-writ-large.html">cell-based covalent screening</a>
described by Steve Gygi is very powerful, but so is investigating a single
protein, as demonstrated by the discovery of <a href="https://practicalfragments.blogspot.com/2021/05/sotorasib-fifth-fragment-derived-drug.html">sotorasib</a>. AstraZeneca did early
work on covalent screening (which Teddy <a href="https://practicalfragments.blogspot.com/2015/07/covalent-inhibitor-of-kras.html">noted</a> in 2015), and they have continued
to build their platform, as described by Simon Lucas. The company has around
12,000 covalent fragments, some beyond the <a href="https://practicalfragments.blogspot.com/2013/07/the-rule-of-three-at-ten.html">rule of three</a>, with molecular
weights between 200 and 400 Da and logP between 0 and 4. More than 90% are acrylamides,
a clinically validated warhead, and the researchers are careful to avoid particularly
reactive molecules that would be non-specific. </div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">In contrast to the electrophilic fragments
that comprise most covalent libraries, Megan Matthews (University of Pennsylvania)
is exploring nucleophilic fragments for “reverse polarity activity-based protein
profiling,” as we <a href="https://practicalfragments.blogspot.com/2022/03/nucleophilic-fragments-other-kind-of.html">highlighted</a> last year. This has led to the discovery of unusual
post-translational modifications. For example, the sequence of the protein
SCRN3 suggests that it should be a cysteine hydrolase, but the purified protein
has no cysteine hydrolase activity, and in cells the N-terminal cysteine is
processed to form a glyoxylyl moiety.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Finally, Alex Shaginian provided
an overview of DNA-encoded library screening (DEL) at HitGen. The company currently
has 1.2 trillion compounds spread across more than 1500 libraries, and an
obvious question is whether this is overkill. Alex noted that one protein has
been screened three times over the course of several years. In the original
screen, a modest (30 µM) hit was found from 4.2 billion compounds screened. A later
screen of 130 billion compounds produced nothing new, but a more recent screen
of 1 trillion compounds led to four mid-nanomolar series. As Steve Fesik noted,
screening larger libraries, whether experimentally or <a href="https://practicalfragments.blogspot.com/2022/01/virtually-screening-11-billion.html">computationally</a>, really
can be helpful, especially for the hardest targets.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Despite only attending half the conference
this post is getting long, but for those of you who were there, which talks would
you recommend watching?</div>
<p></p>Dan Erlansonhttp://www.blogger.com/profile/07927082337051189270noreply@blogger.com1tag:blogger.com,1999:blog-1136153439451224584.post-61832003269899312832023-09-25T05:21:00.000-07:002023-09-25T05:21:44.534-07:00Fragments vs DCAF1: a new tool for targeted protein degradation<div class="MsoNormal" style="text-align: justify;">Targeted protein degradation (TPD)
goes beyond merely inhibiting a protein; it takes a protein out of commission
entirely. This is frequently done using a bivalent ligand: one part binds to the protein
of interest, while the other part binds to an E3 ligase, which ubiquitinates
the protein of interest, targeting it for destruction in the proteasome. Human
cells have hundreds of E3 ligase proteins, some of which may work better in
certain situations, such as specific cell compartments or tissues. In a recent
<i>ACS Med. Chem. Lett.</i> <a href="https://pubs.acs.org/doi/10.1021/acsmedchemlett.3c00104">paper</a>, Anna Vulpetti and colleagues at Novartis describe
progress against DCAF1.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">DCAF1 is one component of the
Cullin4-RING E3 ubiquitin ligase complex. The C-terminus of the protein
contains a WD40 repeat (WDR) domain, which in this case consists of seven “blades”
arranged around a central cavity, or “donut hole”. WDR domains are relatively common,
and indeed we <a href="https://practicalfragments.blogspot.com/2017/04/fragments-vs-prc2-revisited-chemical.html">wrote</a> about a previous Novartis effort that identified chemical
probes against another WDR domain in the protein EED. In the new work, the
researchers took 21 EED binders and screened them using both protein-detected
and ligand-detected NMR against DCAF1, identifying two hits. Crystallography
revealed that compound 1 binds in the central cavity, which previous computational
screening had suggested would be <a href="https://practicalfragments.blogspot.com/2017/08/assessing-ligandability-by-thermal.html">ligandable</a>. </div><div class="MsoNormal" style="text-align: justify;"> </div>
<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj7sXO_JMjEejzRCxO9R0WssfDZ_nPqAKPGOpAOSYSr-las6qWlAezLUMIyYJcyZRP-FBYlj-7K4MHgQFEna7R0TCOYn-m8a7xyHct6piq8VFccY0XJtHDzyeyqN11tKFct6lhITzEkhsvBMuP1QD0qmB-Y6_tLXGH-CZjmsH1FO5x_LDzYeD-beVX-ejuW/s1449/Vulpetti-DCAF1-230925.bmp" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="340" data-original-width="1449" height="150" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj7sXO_JMjEejzRCxO9R0WssfDZ_nPqAKPGOpAOSYSr-las6qWlAezLUMIyYJcyZRP-FBYlj-7K4MHgQFEna7R0TCOYn-m8a7xyHct6piq8VFccY0XJtHDzyeyqN11tKFct6lhITzEkhsvBMuP1QD0qmB-Y6_tLXGH-CZjmsH1FO5x_LDzYeD-beVX-ejuW/w640-h150/Vulpetti-DCAF1-230925.bmp" width="640" /></a></div><div class="MsoNormal" style="text-align: justify;"> </div><div class="MsoNormal" style="text-align: justify;">Next, the researchers screened 30
related compounds from within Novartis. Two of them, including compound 4, had improved affinity (as
assessed both by NMR and <a href="https://practicalfragments.blogspot.com/2010/02/surface-plasmon-resonance-spr.html">SPR</a>) and could be characterized crystallographically.
In addition to binding in the central cavity, these compounds also bound to a
site in the blade region, which the researchers wanted to avoid. Adding a piperazine to compound 4 both improved affinity and disrupted
binding to the blade region; further optimization and growing to better fill
the central cavity led to compound 13, the most potent molecule in the paper.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">A crystal structure of a closely
related molecule reveals that the acetyl group is near the entrance to the
donut hole, providing an easy synthetic attachment point to construct bivalent
degraders. A separately published <a href="https://www.biorxiv.org/content/10.1101/2023.04.09.536153v2">preprint</a> revealed this to be successful, with
degraders of BRD9, multiple tyrosine kinases, and BTK.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">There are several takeaways from
this nice fragment to lead story. First, despite the fact that compound 1 is
clearly <a href="https://practicalfragments.blogspot.com/2023/05/poll-results-fragment-libraries-in-2023.html">fragment-sized</a> (albeit a bit too lipophilic to be fully <a href="https://practicalfragments.blogspot.com/2013/07/the-rule-of-three-at-ten.html">rule-of-three</a>
compliant), the word fragment never appears in the article. FBLD has become so routine
that researchers may not even mention it, which does mean that our <a href="https://practicalfragments.blogspot.com/2022/11/fragments-in-clinic-2022-edition.html">list</a> of
fragment-derived drugs is destined to be incomplete.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Second, although DCAF1 and EED
share less than 25% sequence similarity, screening EED hits turned out to be
successful, which could argue for screening specific subsets of fragments (for example
<a href="https://practicalfragments.blogspot.com/2022/06/kinafrag-free-searchable-database-of.html">kinase-focused</a> or, in this case, WDR-focused). On the other hand, compound 1 binds
in a different manner to DCAF1 than it does to EED. Indeed, compound 1 actually
binds in two different orientations to DCAF1, consistent with its low
affinity. The researchers mention a <a href="https://pubs.acs.org/doi/10.1021/acs.jmedchem.2c02132">paper</a> published earlier this year that
reports a successful DEL screen against the target. Perhaps DCAF1 is just very
ligandable, and a naïve fragment screen would have worked just as well as
the pre-selected set.</div>
<div class="MsoNormal" style="text-align: justify;"> </div>
<div class="MsoNormal" style="text-align: justify;">Finally, the fact that this
program yielded bivalent degraders suggests that many E3 ligases might be
coopted for drug discovery. The field of targeted protein degradation is just
getting started.</div>
<p></p>Dan Erlansonhttp://www.blogger.com/profile/07927082337051189270noreply@blogger.com0