01 July 2015

Updated: fragment events in 2015 and 2016

It is hard to believe that the year is already half over, but there are still important events coming up, and 2016 is already starting to take shape!

2015

August 11-13: The OMICS Group is holding a conference entitled Drug Discovery & Designing in Frankfurt, Germany, with FBDD listed as a conference highlight.

December 15-17: More than 40 presentations. 8 countries. 3 days. One event:
The first-ever Pacifichem Symposium devoted to fragments.

The Pacifichem conferences are held only once every 5 years in Honolulu, Hawaii to bring together scientists from Pacific Rim countries including Australia, Canada, China, Japan, New Zealand, and the US. Registration is now open!

2016

February 21-24: Zing conferences is holding its inaugural Structure Based Drug Design Conference in Carlsbad, California. This looks like a cousin of last year's Caribbean meeting, so it should be a lot of fun.

April 19-22: CHI’s Eleventh Annual Fragment-Based Drug Discovery, the longest-running fragment event, will be held in San Diego. You can read impressions of this year's meeting here, here, and here; last year's meeting here and here; the 2013 meeting here and here; the 2012 meeting here; the 2011 meeting here; and 2010 here.

October 9-12: Finally, FBLD 2016 will be held in Boston, MA. This marks the sixth in an illustrious series of conferences organized by scientists for scientists, the last of which was in Basel in 2014. Surprisingly, this also seems to be the first dedicated fragment conference in Boston. You can read impressions of FBLD 2012FBLD 2010, and FBLD 2009.

Know of anything else? Add it to the comments or let us know!

29 June 2015

Crystallographic screening of soluble epoxide hydrolase

Last year we highlighted a paper from Yasushi Amano and colleagues at Astellas in which they performed fragment-screening on soluble epoxide hydrolase (sEH), a potential target for inflammation and hypertension. A new paper from the same group in Bioorg. Med. Chem. builds on that work and provides some interesting comparisons.

In the first paper, the researchers performed an enzymatic screen with fragments at high concentrations, resulting in a hit rate of around 7.3%, of which 126 of the 307 hits resulted in crystal structures. However, despite this bounty of hits, only 2 new scaffolds were found that bind to the catalytic triad of the enzyme.

Given the success rate with crystallography, the new paper focused on crystallographic screening as the primary fragment-finding method. The researchers chose 800 fragments from their in-house collection with molecular weights between 151-250 Da. To identify new scaffolds, fragments containing amide or urea moieties – known catalytic-site binders – were excluded. The fragments were then pooled into cocktails of 10 and soaked into crystals of sEH, with each fragment at a final concentration of 1 mM. X-ray diffraction data of the soaked crystals resulted in 8 hits. To ensure that nothing was missed, cocktails of the remaining 9 fragments from pools with a hit were retested, but nothing new came up. Although the researchers do not comment on the lower hit rate compared with the original screen, this could be because they were looking specifically for new scaffolds.

Despite the 1% hit rate, the fragments identified were quite interesting, with IC50 values ranging from 52 to 2200 µM. Most of the fragments formed hydrogen bonds to the catalytic triad, but the details differed from reported inhibitors. For example, several fragments contained secondary amines. Fragment 1 (cyan) in particular was well-positioned to reach into two sub-pockets on either side of the catalytic center, so 14 analogs were chosen for screening, resulting in molecules with significantly increased activity, such as compound 9 (magenta).
The crystal structure of compound 9 bound to sEH reveals that it binds in a similar manner as fragment 1. However, the added hydroxyl group is able to make new interactions that were unavailable to fragment 1, and the larger adamantyl group of compound 9 is able to make more hydrophobic interactions than the smaller phenyl ring.

This is a lovely illustration of the gains in both affinity and ligand efficiency that can be had by scaffold-hopping. It is also a nice example of using fragments to explore new chemical space. Finally, it is laudable that all the structural information is deposited in the protein data bank.

24 June 2015

One Fragment to Rule them All

Recently, I have been riffing on the ontology of FBDD.  FBDD has become so popular that we are now seeing appropriation of the term in many papers that don't really mean it.  So, I came across this paper.  Now, don't be fooled by the title, this is about fragments, the abstract promises me so.  Let me skip the science, which to my eyes is actually quite boring, and get right to the heart of their fragment case.  
How is this paper fragments you ask?  Well, this is not about scaffold hopping or innovative uses of fragments to develop SAR.  This is not about interesting approaches to screening.  It is most certainly not about in silico approaches.  This is most certainly about fragment library design.  We often discuss here the sizes of fragment libraries and what they should look like.  One important concept we often tackle here is how big should the libraries be and what size should fragments be.  More importantly we often discuss how much of chemical space a fragment library should cover.  This paper takes an anti-Reymond approach to address that question. 
The Reymond approach tries to determine how big chemical space is, what it looks like, and what portion of it is available.  The Anti-Reymond approach identifies what is available and validates its inclusion in a fragment library.  Here is the last sentence of this paper:
"These findings...verify the value of the benzamide fragment in drug design."
Now, I was worried that benzamidine was not a valuable fragment.  This paper has removed all doubt in my mind.  Now that is settled, we can go on an validate the other 165, 999, 999,999 other possible fragments. 

22 June 2015

Fragments vs P2X1

Four years ago we highlighted a paper in which researchers performed a fragment screen against ion channels. There have been other occasional reports, but for the most part this has been a quiet area. A new open-access paper in Neuropharmacology by Andrew Thompson and collaborators at Cambridge University, University of Bern, VU University Amsterdam, and Washington State University provides another case study.

The researchers were interested in the P2X1 purinergic receptor, which allows calcium ions to pass into cells when ATP binds. An antagonist could be a safe anti-clotting agent as well as a potential male contraceptive. However, the only reported inhibitors are freakish molecules like suramin.

The paper is heavily focused on assay development and validation, in this case using cells stably transfected with P2X1. These were loaded with a voltage-sensitive fluorescent dye: when the channel opens, fluorescence increases. (Control cells not expressing P2X1 do not behave this way.) By adding potential ligands first and then adding ATP, both agonists and antagonists could be identified.

The researchers screened 1443 fragments (from IOTA) at 300 µM each. Cell-based fragment screens are rare but not unprecedented. In this case, 46 hits were obtained, and these were retested at multiple concentrations; 39 hits showed dose responses. These were both agonists and antagonists, with EC50 values ranging from low micromolar to above 1 millimolar.

For confirmation, the researchers used a fluorescently labeled analog of ATP that binds to the P2X1 on transfected cells but not to cells that don’t express P2X1; the increased fluorescence of the cells could be visualized using confocal microscopy. Most of the fragment hits reduced the fluorescent signal, suggesting that they block ATP binding.

A structural analysis suggested that the hits are quite diverse, though annoyingly only a single fragment structure is provided. Still, these do look like useful assays, and the paper provides another successful example of fragment screening in a complicated cellular system.

17 June 2015

Fragments vs HIV Reverse Transcriptase - again

Some targets are so heavily studied that you would think there is nothing left to discover. HIV-1 Reverse Transcriptase (HIV-1 RT) is one of these, with 13 marketed drugs against it: half of all anti-HIV drugs. But as Gilda Tachedjian and collaborators at Burnet Institute, Monash University, the University of Pittsburgh, and the University of Melbourne show in a recent (and open-access) paper in Proc. Nat. Acad. USA, there are still new insights to be learned about this target.

The researchers started with an STD NMR screen of 630 Maybridge fragments, each at ~350 µM in pools of up to five. This gave 84 hits – a healthy 13% hit rate. However, when these were tested in a functional assay (RNA-dependent DNA polymerase activity, or RDDP) only 12 showed significant inhibition, of which 6 were better than 1 mM. Testing 14 related compounds led to 2 more hits, for a total of 8 fragments with IC50s from ~70-750 µM. However, one showed signs of aggregation in dynamic light scattering and was not further pursued.

Since HIV-1 RT has been the object of such intensive research, the team looked at the similarity of their fragments to known binders, including those from previous fragment screening. Surprisingly, their hits turned out to be quite distinct.

Next, the researchers looked at the effect of their fragments on the DNA-dependent DNA polymerase activity of HIV-1 RT, and happily found results similar to the RDDP assay above. The 5 most potent fragments were also tested against three clinically important mutants of HIV-1 RT, and while two of them showed reduced activity, the other three were either as potent or even more so. Testing these against unrelated polymerases revealed that they are not merely promiscuous inhibitors.

Of course, functional activity at high concentrations can have all sorts of causes, so the researchers performed a battery of careful enzyme kinetics experiments to ascertain the mechanisms. One fragment turned out to be competitive with respect to deoxynucleotide triphosphate substrate, even though it looks nothing like a nucleotide. Another is competitive with the DNA substrate. In other words, both these fragments operate through different mechanisms of action from clinically approved HIV-1 RT inhibitors.

One of the most potent fragments is a p-hydroxyaniline, which the researchers recognized as a PAINS compound (it can form reactive quinones). However, freshly prepared samples of this fragment were just as active as samples that had been stored in DMSO for months. Also, an analog without the ability to form a quinone was still active, albeit less so.

The p-hydroxyaniline fragment also showed activity in a cell-based assay. Just as with biochemical assays, cell-based assays are also susceptible to false positives, but the kinetics of viral inhibition were consistent with inhibition of HIV-1 RT rather than other other mechanisms. Further work on the compound may be merited; these are exactly the kinds of investigations necessary to decide if an interesting PAINS molecule is worth pursuing.

Unfortunately there is no crystallographic or detailed NMR structural information as to how these molecules actually bind. Previous work has identified multiple fragment binding sites on HIV-1 RT, so further work should eventually reveal how these molecules interact with the protein.

In the end this paper shows that, even in the absence of structure, it is possible to learn a great deal about how fragments inhibit an enzyme. It is also a useful reminder that fragment-based approaches can identify new types of inhibitors even for a target that has been intensively – and successfully – studied for decades.

15 June 2015

Natural Product Derived Fragments against MMP-13

I have been lucky to work on a lot of systems that very much interest me.  I, in particular, love metallo-proteins.  I worked on rubredoxin as a post-doc and when I moved into industry I worked on a slew of metalloproteins.  So, I love it now when I see papers on targets I used to work on.  This paper does exactly that while also letting me riff (later) on Natural-Product-Derived Fragments (NPDF). 

NPDF has a long history in FBDD having been discussed here, here, here, and so on.  Many vendors and some companies have NPDF libraries (whether they call them that or not).  However, these libraries have yet to be proven to be an efficient route for "discovering clinical drug candidates".  Lanz and Riedl set out to do this against MMP-13 (how many of your just said, yeah I worked on that target?).  All MMP-13 clinical candidates with strong ZBG (Zinc-binding groups) have failed.  They are aiming to develop a MMP-13 without a strong ZBG.  Of course, we have seen a LOT of work towards this goal: here, here, and here for example.  The authors propose that the use of NPDF prevents the problem of using fragments with "debatable biological properties".  This seems to the be the argument used by the NPDF people, since these fragments are found in nature they have desirable properties.  I have never bought this line of reasoning for a variety of reasons.  

To their end, the authors selected uracil as their starting NPDF for these reasons: good synthetic starting points, cis amide bonds, and its found in a variety of natural products (nucleic acids).  They docked it in the S1' non-zinc binding site and found a strongly conserved binding site. [For me, and I would imagine a whole lot of people, this fits in the "things you already knew" category.]  The uracil interacted with the NH an CO of Met232 via its cis amide bonds and "addresses" Lys228.  Several compounds were made from the uracil starting point (Figure 1):
Figure 1.  2: 5 uM vs. MMP-13, < 50% Inhib against 1,2,3,7,8,9,12, and 14 at 20 uM. 3: 10 nM vs. MMP-13, < 50% Inhib against 1,2,3,7,8,9,12, and 14 at 20 uM2: 5 nM vs. MMP-13, < 50% Inhib against 1,2,3,7,8,9,12, and 14 at 10 uM
So, in the end, they have created a potent and selective compound.  They did use a NPDF as a starting point.  Making these compounds is not something that bowls me over either for a Technical Difficulty score or Artistic Merit.   However, I would not go so far as to say that they have validated the NPDF approach.  I think to show that a generic approach works you need more than one (relatively well known) target with more than one (relatively well known) fragment. 

08 June 2015

Benchmarking native mass spectrometry

Mass spectrometry (MS) is one of the less common tools to find fragments. In the conceptually simplest approach (native mass spectrometry), you incubate your protein with a putative ligand and ionize the mixture. Fragment binding is detected by an increased mass for the complex, and the strength of binding by the ratio of heavier bound complex peak to protein peak. However, the liquid to gas phase transition is a big step, and often the complex does not survive. Aside from more specialized applications of MS (such as herehere, and here) there aren’t many published examples. A recent paper from Federico Sirtori and colleagues at Nerviano and Università degli Studi di Milano in Eur. J. Pharm. Sci. describes fragment screening by native MS in detail.

The researchers used the reliable model protein Hsp90, which was also used in a previous MS study and in benchmarking other techniques. One of the many benefits of Hsp90 is a wealth of well-characterized inhibitors with a range of affinities, and these were used to calibrate the technique. This turned out to be critical: beyond sample preparation itself (beware non-volatile buffer components), all kinds of parameters can be adjusted including various voltages, temperatures, vacuum strength, and ion source. Get one of these wrong and your non-covalent complex either fails to ionize or blows apart.

In addition to using published data on known compounds, the researchers ran both fluorescence polarization (FP) and surface plasmon resonance (SPR) assays to independently determine dissociation constants. Initially the results from MS (a Q-TOF) were quite different, but after optimization the team was ultimately able to find conditions that gave qualitatively as well as quantitatively similar results for ligands with affinities ranging from picomolar to ~100 micromolar.

Thus encouraged, the team embarked on a fragment screening campaign. The Nerviano fragment library consists of 1914 molecules mostly following the rule of 3, though halogenated fragments up to 380 Da are allowed as are compounds with up to 6 hydrogen bond acceptors. The fragments were run in mixtures of 5, with protein at 2.5 µM and each compound at the low concentration of 10 µM. Sample injection and data processing were automated, and the entire screen took 2 days and 2 mg of protein.

Given the low concentration of fragments, the researchers lowered the bar for potential hits, yielding 282 compounds. These were retested individually, yielding 146 confirmed hits that gave signals of 5.2-29.7% bound protein. This is a high hit-rate, particularly given that these binding levels suggest affinities in the 20-179 µM range. Indeed, only 5 fragments could be competed by a high-affinity binder, suggesting either that the others bind outside the active site or are non-specific (false positives). Regarding false negatives, Nerviano reported the results of an NMR fragment screen against Hsp90 last year, and 12 of 14 hits identified there could also be detected by MS. The other two were likely below the detection limit of the MS assay.

Unfortunately, the researchers do not discuss thermodynamics. In theory enthalpic interactions dominate over entropic interactions in the gas phase, but it is unclear whether any of the observed binders were strongly entropy-driven.

In the end, it appears that fragment screening by native MS is workable, but the sensitivity is probably lower than other techniques. Of course, increasing the ligand concentration would increase the sensitivity to weaker binders, but at the cost of more non-specific binding – which is already considerable. Also, Hsp90 is about the friendliest protein one can imagine. I would be reluctant to try this with a more challenging target that lacks good tool ligands. But if you want to give it a go, this paper provides a wealth of information for getting started. And if you have experience with native MS, please share it in the comments.

03 June 2015

Fragment Ontology

We here at Practical Fragments look for papers in the literature about fragments.  Typically, it is a Web of Science search, or I see something come in an alert that has "fragment" in the title.  Well, not everything with fragment in the title is not really about fragments as we typically think of them. So, I recently came across this paper titled : "Genetically Encoded Fragment-Based Discovery of Glycopeptide Ligands for Carbohydrate-Binding Proteins".  I decided to give the paper a good perusal, largely because one of the authors is from where I did my post-doc.

The authors are interested in making competitive inhibitors of carbohydrate recognition domains for the treatment of a variety of diseases.  The challenge with lectin inhibitors is that the native carbohydrate has relatively low affinity and are synthetically complex.  As you would think, you can use the carbohydrate for binding specificity and then add something more "drug-like" to increase affinity through other interactions.  This approach has been successful but require complex multistep syntheses.  In this paper, they decided to search for peptides which can synergize with carbohydrates rather than serving solely as a linker or standalone recognition element.  To do this, and increase throughput they used a genetically encoded library, phage display.  In short, they created a glycopeptide library of 10^8  molecules through derivatization of a peptide library with carbohydrate.  This approach allows the addition of different carbohydrates (targeting different lectins) with the same peptide library.  
Figure 1.  Library Screening Approach for Genetically Encoded Glycopeptide Libraries.
In this approach, the first library (Man-X7) is screened against the target and anti-target.  The second library (methyl-X7) and the third library (Ser-X7) are screened only against the target.  After the first round of panning, they identified a weak consensus of Man-[WYF]Y[SDEA].  These peptides were made and able to compete with ConA for ligand in SPR, the mannose was shown to be essential to activity, the specific peptide sequence was required for synergistic binding.  Further work showed that the final four residues of the peptide contributed minimally to binding, so they lopped them off.  

They then performed two more rounds of panning with Man-WY[D/E]-X7.  All of the hits from these rounds had single digit micromolar affinity and the glycan-proximal ligands are responsible for most of the affinity.  How did they know if this is actuallly binding to where they want it to?
Figure 2.  Man-WYD co-crystalllized with ConA. 
Figure 2. shows the crystal structure of Man-WYD.  The mannose moiety binds where it is expected.  However, the peptide is not binding in the remainder of the trisachharide binding site, but instead in a somewhat deeper cavity near Y12.  Additionally, a latent hydrophobic site is opened up through induced fit (asterisk), filled by the Y residue of the glycopeptide.  

This approach led to the discovery of a novel class of compounds which would not have been discoverable by "standard" approaches.  But, is this fragments?  In my eyes, fragments takes simple compounds and screen them against the target.  It then optimizes the actives as quickly as possible and does iterations.  A key component to FBDD is SBDD and identification of how the actives/hits bind.  To me, this approach adheres to all the tenets of FBDD.  We have seen super huge screening molecules before, so that should not be an issue. As I have said, FBDD is about small little things being screened effectively.  I think this paper shows it is more about how you think about your system. 

01 June 2015

Fragments vs MCL-1 revisited: on to low nanomolar potency

The protein MCL-1 binds to other proteins to protect cancer cells from apoptosis. Protein-protein interactions have historically been considered difficult, but as we’ve noted previously (herehere, here, and here, for example) fragments have been successfully deployed against this target. A recent paper in J. Med. Chem. provides the latest update from Stephen Fesik and co-workers at Vanderbilt University.

We last highlighted this program in early 2013, when the Fesik lab disclosed a series of mid-nanomolar inhibitors, such as compound 1, derived from fragment merging. In the new paper, they report compound 2 as another fragment identified in the original NMR screen.

NMR-based structural information of this molecule bound to 15N, 13C double labeled MCL-1 revealed a similar binding mode as the previous series, and merging the molecules led to the low nanomolar compound 34, with impressive ligand efficiency. This compound was also >1700-fold selective for MCL-1 over the related protein BCL-xL and >250-fold selective over BCL-2.

Although compound 34 did show activity in cell lysates, the authors note that it is unlikely to be potent enough to show unambiguous activity in cellular assays. Indeed, researchers at AbbVie and Genentech have recently reported MCL-1 inhibitors that show picomolar activity in biochemical assays but only high nanomolar to low micromolar activity in cells.

Still, this is another nice illustration of the power of fragments – combined with a healthy dose of medicinal chemistry – to tackle a difficult target. Notably, the researchers didn’t have to turn to super-sized fragments. Moreover, the best molecule shown is well within Lipinski space, and there are plenty of avenues for further optimization. It will be fun to watch this story progress.

27 May 2015

Stopping Virulence...One Fragment at a Time.

The best way to not get an infectious disease is vaccinate.   Streptococcus pneumoniae is repsonsible for a million deaths world-wide every year.  For Streptococcus pneumoniae, there are a numbers of vaccines on the market.  These vaccines are bacterial polysaccharides either naked or conjugated to a protein.  They are highly effective, but don't cover all serotypes (there are ~100).  And sometimes a novel serotype arises.  So, if you do get infected treatment is key.  Beta-lactams are the first line of defense, but multi-drug resistance is on the rise, so alternate forms of treatment are needed. Targeting virulence factors has become a recent line of research.  Pneumococcal surface antigen A (PsaA) is strictly conserved surface-exposed lipoprotein expressed by all known pneumococcal serotypes and is essential for colonization and pathogenesis.  PsaA is an integral part of an ATP-binding cassette(ABC) transporter protein complex known as the PsaBCA permease, which is involved in manganese (Mn2+) transport across the bacterial cell membrane. (See there's always a metal involved in cool biology.)  This makes PsaA a good target for pneumococcal infections.  In this paper, a group from down under presents their results using fragments to target PsaA.

They custom built a fragment library (via outsourcing) ~1500 fragments.  This struck me as unusual, if not unique.  Typically, academics make their own or just buy one off the shelf.  I would love to hear why this path was chosen.  In the SI, they do say they used "relaxed" Ro3, but the only relaxation seems to be on the MW.  Have other academics gone this route?  I would love to know more (you can be anonymous in the comments, hint hint).  These were docked into the PsaA metal binding site (Figure 1) based on 3D shape and electrostatic similarity. These were then scored using FlexX. 
Figure 1.  Structure of PsaA. 
The top 300 fragments were manually inspected and then subjected to a cluster analysis.  The 60 most diverse fragments were then tested in a competitive Zn-binding assay.  Zn is a irreversible inhibitor of PsaA and the assay uses this to test for compound binding.  10 of the 60 fragments exhibited greater than 15% inhibition at 100 microM.  Two of these compounds showed greater than 50% inhibition at 1mM (Cpd 15 and 58, Figure 2.)
Figure 2.  Fragments with greater than 50% activity at 1 mM.  Hydrogen bond acceptors are shown in red, H-bond donors in Blue.
So, with crystal structures available, the authors decided to inspect the docked poses rather than actually trying to obtain a structure of the fragments bound to the protein. So even though docked fragments can, and do tend to, keep their original locations, experimental data is key to confirming in silico predictions.  The made 31 compounds around 15, and one that replaced the p-nitro, o-methoxy phenyl with o-hydroxypphenyl was the best 15h (28 microM, pIC50/HAC=0.37).  To that end, they tried to soak apo-crystals with cpd 15h and were unsuccessful due to limited compound solubility and affinity for the target.  They did not attempt soaking compound 58, which they was unable to be further "optimized" with simple SAR.  Cpd 15h did have antimicrobial activity: significant growth inhibition at 180 ug/ml and total growth inhibition at ~800 ug/ml.  They did a further round of optimization.

This is an example of real FBDD approach, in contrast to just using the words.  However, I think this is really a MPU (minimal publishable unit).  If we are lucky, we can expect to see future papers coming out describing their success (or failure) against this target. 

25 May 2015

Charting new chemical space for kinase inhibitors

Since the advent of imatinib, kinase inhibitors have become a thing in drug discovery, with more than two dozen already approved. Indeed, kinases are the targets of more than a third of reported fragment-derived compounds to reach the clinic. Given that all 500+ human kinases bind ATP, you would think that the chemical space would be pretty well picked over by now. As Hongtao Zhao and Amedeo Caflisch at the University of Zurich show in a recent Bioorg. Med. Chem. Lett. paper, this is not the case.

The researchers started by extracting all 26,668 kinase inhibitors with MW < 600 Da and IC50 or Ki < 10 µM from the ChEMBL database; three quarters of these were better than 1 µM. These have been tested in aggregate against 367 kinases, of which 88 have more than 100 reported inhibitors!

The molecules were then deconstructed into 10,302 ring-containing fragments, such as benzene (7.1% of kinase inhibitors), 2-methylaminopyrimidine (3.5%) and N-methylmorpholine (2.3%), as well as more obscure structures. In fact, more than half (53%) of these fragments were not found within 7.5 million commercial compounds in the ZINC database. In other words, many fragments that form a part of known kinase inhibitors are not represented among commercial compounds, despite many vendors offering “kinase inhibitor libraries”.

What about the reverse question, analyzing commercial molecules for new kinase inhibitors? The researchers focused on possible “hinge-binding” fragments – those that have at least one hydrogen bond donor and one acceptor in close proximity to one another so as to be able to interact with a conserved region of kinases. Not surprisingly, more than half of the fragments (5681) found by deconstructing the kinase inhibitors fit this description. More interestingly, 196,904 potential hinge binders resulted from deconstructing the ZINC compounds, of which only 1% had been reported as kinase inhibitors.

Digging into the data more deeply, the researchers classified hinge binders as monocyclic, bicyclic, and multicyclic. This analysis revealed that the overlap between kinase inhibitors and commercial compounds was particularly low for multicyclic fragments. This intuitively makes sense: medicinal chemists often turn to ring construction to fix all manner of problems, both pharmaceutical and IP-related, so the under-representation in commercial compounds is likely because medicinal chemists introduce rings into simpler starting molecules. Also, from a molecular complexity standpoint, multicyclic ring systems may be less likely to bind to a wide variety of proteins than simpler monocyclic fragments.

More than five years ago Practical Fragments highlighted a paper from Abbott describing their efforts to generate novel hinge binders. As this and related analyses show, there is still plenty of chemical space left to explore and exploit.

21 May 2015

Just Because its called "Fragment-Based"...

When my parents were young and just starting out (the late 60s) they needed a vacuum cleaner.  So a vacuum cleaner salesman came to the house eager to make the sale.  This was the era of the Space Race, plastics, and so on.  So, it was cool to be associated with this.  The eager young vacuum cleaner salesman showed my parents the fine, sleek design of the vacuum cleaner (it was a ELECTROLUX).  It was long and sleek, looking like a rocketship (or a dachshund).  It came with a lot of nozzle attachments.  One in particular was shaped to be very narrow, and get in between the couch and wall for example.   He was particularly proud of this piece: the AEROspace tool.  He even wrote it down as such.  You must have a "AEROspace" tool for your vacuum.  It was an example of great marketing, associate yourself with something very popular to make something mundane appear special.     

So, this paper comes along from Moffatt Cancer Center and USF targeting ACK1 (aka TNK2).  This paper purports to have a "innovative fragment approach" (mix and match).  I love novel approaches to libraries.  So, let's dive in. There is a good deal of work that has been done with ACK1 by Amgen, OSI/Astellas, and others.  Dasatinib and Bosutinib also show activity against ACK1 also.  Based upon all of this previous work and the knowledge of the pyrimidine core they decided to approach the target as laid out in Figure 1.
Figure 1.  Library Design Approach
So, this paper doesn't interest me, although they do come up with some potent compounds, from a what they discovered aspect, rather from a more philosophical aspect. What does it mean to do fragments?  This harkens back to the Safran Zunft challenge.  To me, FBDD is about using simple, small molecules.  Pyrimidine series 9 does not fit any definition of a fragment (Cpd 8 would, but it was never tested AFAIK).  What they did was identify a variety of fragments which would be inputs for creating a small library of lead-like compounds.  However, for this to be "Fragment-based" I would think that they would tested each individual component and prioritized chemistry based upon that.  Or maybe they could have made R3=H.  They don't report Ligand Efficiencies (cue Pete Kenny).  This is simply not "Fragment-based" anything.  Nor, do I think this approach is novel.  Nor do they explain how this is novel.  
So, I think we have entered the time when anything that uses a fragment in the design process is fragment based.  Based on this line of thinking, Nicolaou's total synthesis of Taxol is "Fragment-based". Beware those talking the talk, but not walking the walk.

18 May 2015

Predicting protein ligandability and conservation of fragment binding modes

Say you have a protein target, and you want to know whether you will be able to find small molecules that bind to it. A fragment screen can give you a good idea as to the likelihood of success: if you find lots of different fragments with high affinities (say, better than < 0.1 mM), your protein is likely to be highly “ligandable.” On the other hand, if you get very few fragments, and most of them are weak (> 1mM), be prepared for a slog.

Of course, it would be even better if you didn’t have to do a physical screen at all, and two recent papers show how a computational approach may be sufficient. The first, by Dima Kozakov, Sandor Vajda, and their collaborators at Boston University and Acpharis is a detailed how-to guide in Nature Protocols. The second, in Proc. Nat. Acad. Sci. USA by Dima Kozakov, Adrian Whitty, and Sandor Vajda and their collaborators at Boston University, Northeastern University, and Acpharis, addresses some interesting questions about fragment binding.

The main program is called FTMap (also highlighted here); it and several related programs are accessible through a free web server. It is remarkably easy to use: just provide a protein data bank (PDB) ID or upload your own structure and away it goes.

The program works by docking a set of 16 virtual probes (such as ethanol, acetonitrile, acetamide – the largest molecule is benzaldehyde) against a protein and looking for “hot spots” where many fragments cluster. Previously the researchers demonstrated that known ligand-binding sites in proteins tend to be computational hot spots, where at least 16 probes bind. (Note that due to their small size, multiple probes of the same type – acetone, for example – can bind within the same hot spot simultaneously.) In other words,

The strongest hot spot tends to bind many different fragment structures, acting as a general “attractor.”

On the other hand, a hot spot with fewer probe molecules is unlikely to have enough inherent binding affinity to bind to ligands with low micromolar or better affinity.

A related program is called FTSite, which focuses on more thoroughly characterizing the best binding sites. Other programs allow for protein side chain flexibility, docking custom probes, or docking against ensembles of protein models such as generated by NMR structural methods.

The PNAS paper goes further to ask about ligand deconstruction. Specifically, why is it that when a larger ligand is dissected into component fragments, sometimes the fragments recapitulate the binding modes seen in the larger molecule, and sometimes they do not? The answer:

Because a substantial fraction of the binding free energy is due to protein-ligand interactions within the main hot spot, a fragment that overlaps well with this hot spot and retains the interacting functional groups will retain its binding mode when the rest of the ligand is removed.

The researchers support this assertion by examining eight literature examples in which structural information was available for fragments and larger ligands (some of which we’ve covered here, here, and here). In cases where the isolated fragments overlapped with 80% of atoms in probe molecules within a given hot spot, the fragment binding mode remained conserved. Also, these fragments tended to have high ligand efficiency values.

This is neat stuff, and it will be fun to see how general it is. I’m especially happy to see that all of the software is free and open access. Even though I’m hardly a computational chemist, I tried playing around with it and found it remarkably fast and easy to use. So if you have a protein with no known ligands, FTMap can find hot spots, and if they’re particularly promising, this should embolden experimental work.

13 May 2015

When Fragments don't deliver...

In the olden days (1980s), during the cold war, Russia was "a riddle wrapped in a mystery inside an enigma".  Kremlin Watching was serious and important thing. When I write up papers, I do the same thing but trying to figure out what the actual story is.  We all know a lot more happened than is written down in 10-20 pages of an article.  This paper has me really doing it; so follow along.

Tuberculosis is a scourge caused by a mighty nasty bug.  People have been using fragments to try to combat it for a long time: 2009 and 2014: targeting pantothenate synthesis and biotin synthesis. AstraZeneca join the party (just as Entasis spins out) with this paper.  In it, they describe their NMR fragment screen combined with a HTS biochemical screen targeting thymidine synthesis.  All the TK inhibitors are TMP or thymidine analogs.  The HTS of 120,000 compounds lead to multiple 1-30 uM active site binding (confirmed by HSQC NMR) inhibitors.  Compound 1
Cpd 1.  3.6 uM, 0.46 LE, 3.54 LLE.  
Figure 2.
was chosen as the basis for the hit to lead campaign.  Modeling suggested that the pyridone core is a thymidine mimic (Figure 2). This novel core allowed to reach sub micromolar potency within 10 compounds of the original hit.  The pyrimidine core was also potent, but not as much as the pyridone.  Pyranones were inactive, as was any other group but the cyano at the 2 position. Crystallography was a key to verifying the binding mode of the compounds.  One point of this is that verified means within 1 A of the predicted pose.  SAR led to the fused pyridinone, a 2 nM inhibitor, which nonetheless had no cellular activity.  The propose that this is due to the ionic nature of the compound, but ureas, amides, and sulfonamides did not afford the desired activity. 
Figure 3.  Fused Pyridinone showing X-ray Contacts

So, as is becoming a very common theme in fragments, they decided to use fragments to try to discover an alternate scaffold.  Using TROSY (HSQC for big proteins), they screen 1200 fragments in pools of 6.  Those fragment hits, termed FRITs which is a first for me (I think I like it.), with a LE greater than 0.25 were followed up by X-ray crystallography.
Figure 4.  Napthyridinone FRIT.  590 uM, LE=0.3. 
Figure 4. shows the best FRIT and its crystal contacts.  Combining this with the knowledge from the cyanopyridinone series, a virtual library was created and docked.  Hidden in their description, it appears that the library was passed by real chemists to prioritize the cpds.  Kudos.  With very limited SAR, they achieved significant potency (Figure 5), but still without cellular potency. 
Figure 5. 200 nM, LE=0.34.  

But, WAIT, this series wasn't advanced any further because the cyanopyridinone was in "advanced lead generation".  Why, you ask?  Well, the oxidized form of Cpd 1 had exhibited moderate cellular activity.  While they don't say it, I would imagine that this means that in doing the analytical work on the compound they found a portion that had oxidized, cleaned it up, and then tested the "bad" part.  I would love to know if this is how it happened.  I would hate to learn they had planned on an oxidized compound all along.

So, on to sulfone and sulfoxides of Cpd 1.  Knowledge from the cyanopyridinone series was used to select appropriate substituents, which seems to indicate a timeline of how things happened or a "we've got nothing left to try" issue.  Again, I would love to know which.  Both the sulfones and sulfoxides showed cellular activity with increase in IC50.  And again X-ray showed that the binding mode was retained, with the sulfoxide adjacent to Arg95.  This then caused them to go back and look at the cyanopyridinones again and realize that the sulfone/sulfoxides might have just the right physicochemical properties.

I think this is a really good paper, and hopefully indicates that more work on this target and with these series are coming.So, I don't know if the fragments failed, or if something better came along.  I would think the latter, but it could be the former.  Again, I would love to know.

11 May 2015

Fragments vs Factor VIIa

The blood coagulation cascade involves several serine proteases, many with an appetite for arginine-containing peptides. The polar, basic guanidine moiety of arginine tends to wreak havoc on the pharmacokinetic properties of small molecules, sparking an intensive search for replacements. A few months ago we described how researchers were able to use fragment screening to find an alternative moiety for one member of the blood coagulation cascade. In a recent paper in J. Med. Chem., Daniel Cheney and colleagues at Bristol-Myers Squibb report their work on another, factor VIIa.

The researchers started by filtering commercially available small molecules to look for those with ≤ 17 non-hydrogen atoms, ≤ 3 rotatable bonds, and without anything nasty. This computational work left them with 18,000 fragments. These were then clustered based on similarity, and 200 compounds were chosen by chemists as having the potential to bind in the deep S1 pocket, where the guanidine normally binds.

At the same time, the 18,000 fragments were computationally docked (using Glide) against several different crystal structures of factor VIIa; this “ensemble docking” was used to account for the protein flexibility observed in various structures. This led to a further 250 fragments being chosen.

The 450 fragments were then assessed in biochemical and STD NMR-based assays, and 41 were soaked into crystals of factor VIIa, resulting in 27 structures with fragments bound in the S1 pocket. Happily, 12 of these fragments were – unlike guanidine – neutral. All of them were quite weak (even by fragment standards), with Ki values ranging from 8-19 mM, though searching for related fragments led to some with slightly improved affinities. However, when examining the binding mode of fragment 7, the researchers realized they could use it to replace a more basic moiety in their existing lead series (17), yielding compound 18. Although this reduced the potency, it dramatically improved the permeability. Also, the researchers stated that they were able to subsequently improve the potency, with details to come in a subsequent paper.


This is another nice example of using fragments to fix part of a larger molecule, though it is not necessarily easy. The researchers note that other attempts to append new fragments onto their existing scaffold were unsuccessful, likely due to geometric incompatibilities. This paper is also an illustration of how long it can take to get things published. One of the authors gave a nice presentation on some of this work at an ACS meeting in 2012, and there’s a line in the paper referring to a publication that came out “shortly after completion of this work” – in 2006! Still, late or not, it is nice to see the story in print, with a promise of more to come.

06 May 2015

More Notes from DDC 2015

Dan and I both gave our thoughts on the conference last week.  But there was more than just the talks.  There were roundtables.  I chaired one on using kinetics and thermodynamics to drive medchem for the PPI track.  It was a lively discussion.  It was agreed that dyed in the wool enzymologists are priceless.  Kinetics is useless if clearance is the driving force, so this becomes a PK/PD issue.  But does it always have to be?  Paul Belcher from GE shared the On/off rate map (Figure 1) that shows what realm of binding you are in depending on your reates.  Paul also mentioned that Tony Gianetti, formerly of Genentech, used HSA and SPR to assess a more realistic picture of how compounds interact in plasma.  In terms of earlier phase uses, one of the round table attendees mentioned that she had seen talks of people using kinetic data to drive medchem.  She couldn't recollect who, so if any of our astute readers have references please share.  We also discussed using kinetic data to rank compounds with similar IC50.  A question was raised whether or not kinetics can be a good surrogate for receptor occupancy? 

Figure 1.  On/off Rate Map: A = affinity limited efficacy, B= on rate limited efficacy, C= rapid off rate limited, D= slow off protected efficacy
So, what about thermodynamics?  By and large, this was viewed as retrospective only.  Paul from GE did share that they have an app note of using SPR to generate thermodynamic data (I can't figure out how to link it, so if you want it contact me (or Paul) and we can send it). 

The main thrust was that neither kinetics nor thermodynamics are used to make prospective medchem decisions, rather they are used to justify in retrospection. Specifically for PPIs, the consensus was that the focus should be on on rate because you have to the compound in there when you can (i.e. when the complex is "open" enough). 

Derek Cole of Takeda led one of the FBDD round tables: Practical Aspects of Fragment Screening. Here is a picture, courtesy of Bjorn Walse of Saromics.  
His notes are replicated below:
Round table became figure 8 with two tables, with 2-3 deep seats and 40 -50 participants. FBDD expertise from novice to experts, including Teddy Zartler, Dan Erlanson, Gregg Siegal, and Andrew Petros. Large attendance highlights the number of newcomers to FBDD, confirmed by Dan Erlanson during opening when 2/3 of attendees indicated this was their first CHI FBDD meeting. Very lively debate/discussion covering 4 primary targets.

1. Designing and building and storing libraries. Discussed size of library i.e. 1000 or 40K. Agreed that a good library of 1000 should yield lots of high quality hits. Best to keep HA low, 10 - 16 (majority in 12 - 14 range). Discussed 3D vs. flat fragments. Flat give higher hit rate and should be major part of library. 3D likely give lower hit rate but may yield very exciting hits. Discussed complexity and the need for fragments to have enough complexity, but not two pharmacophores. (ref. Astex work). IF just starting out, best to buy a vendor library, e.g. Maybridge or others, which are fully characterized.

2. Screening techniques. NMR and SPR most common. Both very good. Tm - fast, inexpensive and can correlate with x-ray. What to do if no biochemical activity. Might be fine if below sensitivity of biochemical assay, i.e. very small fragment, however if larger fragment, need to understand why not being detected.

3. Potential pitfalls. Make sure library is soluble above assay conditions, i.e. > 1 mM in aqueous buffer (1 - 2% DMSO). Check for aggregation. Run SPR clean screen.

4. Fragment hit follow up. Think of fragments as seeds to identify protein compatible pharmacophore. SAR by catalog of similar fragments or fragments which present a similar pharmacophore is of great value. May find fragments which are much more potent, efficient, or which crystallize (if original was unsuccessful). Good to design diverse library, but similarity in fragments is different than similarity in large molecules, small 1-atom changes can have profound effect on binding mode, potency, etc.
 
If I missed any other highlights, please add them in the comments, or email me and I can add it in.  

04 May 2015

Sloppy science

As regular readers may have discerned, I’m favorably disposed to most of the papers I highlight. They may have flaws or inconsistencies, but, with rare exceptions, I generally just ignore particularly problematic publications. Last year Teddy introduced the term PAINS-shaming to draw attention to – how shall we phrase it? – less salubrious specimens. Building on this alliterative theme, today’s post is about sloppy science. A fundamental tenant of sound science is to consider alternate explanations for results. Ignore this at your peril.

An example was published in J. Cancer Prev. The researchers were interested in a mutant of isocitrate dehydrogenase 1 (IDH1), a hot cancer metabolism target. They screened 500 fragments in a functional spectrophotometric assay, with each fragment present at the very low concentration of 5-10 µM. One of these inhibited the mutant protein by 80% – pretty impressive for a fragment. Until you look at the structure: 2-(3-trifluoromethylphenyl)isothioazol-3(2H)-one (shown below).

Fifty years ago, researchers showed that this chemical class (isothiazolinones, also called isothiazolones) could react with thiols, like this:

Isothiazolinones have been categorized as PAINS, though they do not show up in the original computational filters. However, Pete Kenny has (repeatedly) stated that having a dubious structure should not automatically disqualify a compound from further investigation, so what else do we know about isothiazolinones?

Well, there’s this paper, which concludes a discussion of isothiazolinones by stating:
We could not develop these into useful compounds and ultimately the structure–activity relationship (SAR) was uninterpretable. Most insidiously, there were encouraging aspects of sharp SAR as there always are with these PAINS, but this is eventually overwhelmed by flat and nonsensical SAR. Unpredictable nonspecific cytotoxicity was manifest. We found our compounds to be rapidly reactive with thiols under assay conditions.
Of course, one could argue that this is anecdotal. But then there’s this paper, with the unambigious title “Isothiazolones; thiol-reactive inhibitors of cysteine protease cathepsin B and histone acetyltransferase PCA”. The first line of the abstract states:
Isothiazolones and 5-chloroisothiazolones react chemoselectively with thiols by cleavage of the weak nitrogen-sulfur bond to form disulfides.
The researchers go on to demonstrate this using both small molecules and proteins, and some of the compounds they investigate are structurally quite similar to the hit here.

So in all likelihood the fragment described in the most recent paper reacts with one or more cysteine residues in IDH1, of which there are several. It is notable that the researchers conducted their assay in the absence of added thiol reducing agents, so modification of the cysteines would effectively be irreversible under their assay conditions.

What we have here is the re-identification of a known thiol-reactive molecule without any acknowledgement or apparent awareness that the molecule is reactive. I have no problem with covalent inhibitors, but I do have a problem with a generically reactive molecule being touted “for a future lead development”, as the researchers state in the abstract. It took me just minutes to track down the references above, and the fact that neither the researchers nor the reviewers did so is inexcusable.

Granted, this paper is not published in a high profile journal, and the easiest response would be to ignore it. It is certainly not the only one of its kind. Doing so, however, implicitly endorses sloppy science. This paper will undoubtedly pad the resumes of the authors. Highlighting its problems will hopefully make others wary of wasting time with this new "selective inhibitor."

29 April 2015

Tenth Annual Fragment-based Drug Discovery Meeting...Teddy's Thoughts

Dan posted his thoughts here.  Like Dan, CHI put me to work: I chaired the first session in PPIs, co-taught (with Dan) our award winning FBDD course to 22 attendees (a new high which I think shows that interest in FBDD is still growing), moderated a breakfast roundtable on kinetics and thermodynamics,  judged posters.  All of this during weather which made the natives shiver, and me feel like spring is really here (64F and cloudy).  

First off, there was live tweeting of talks by me and a few others: @iceobar, @moleculesmith, and others.  Beware that the Dubai Diamond Conference was also going on that week.  

In the PPI track, just as last year, fragments were a key component to various projects.  Mark McCoy, Merck (and he taught me more about NMR than just about anyone, whether he will admit it or not) gave a great talk on HDM2-p53.  I took away a few things from his talk which I really liked.  Merck (legacy S-P) really relies on NMR structural information: HSQC-based screening, NMR-based Ki,  and NMR-driven docking.  I was particularly intrigued with the NMR-driven docking because they were able to generate 80+ models with a 75% success rate that was confirmed by X-ray (once that was enabled).  They were forced down this path because they went 2 years without a X-ray structure. 

Joe Patel of AZ talked on SOS-RAS.  What I liked was that AZ uses a modified Voldemort Rule (which Harren Jhoti incorrectly attributes to me; I am the Boswell to Rod Hubbard's Johnson): HAC less than/equal20, cLogP less than5, less than3 rings, less than5 rot bonds, less than 3 HBD, and less than 5 HBA.  Their initial X-ray screening ended up at a wall, so they went to a covalent approach. 

Troy Messick of the Wistar gave a nice talk on using fragments and SBDD to drug an "undruggable" target.  I think this is exactly this is exactly the kind of success that has led FBDD to be ubiquitous these days.  I have to admit I have and am working with the Wistar on the NMR component of their screening, so I may be biased. 

I won't go into the various talks from the FBDD track,  However, echoing Dan this is really a great conference.  My main take home themes is that FBDD is really mainstream.  It's no longer the red headed stepchild to other hit generation processes (apologies to my ginger friends).  Biophysics is also seeing a huge growth, having grown up with FBDD, but really finding a lot more uptake outside of that space.  Next week I will post summaries of roundtables and some useful information. 

27 April 2015

Tenth Annual Fragment-based Drug Discovery Meeting

Last week marked the tenth anniversary of CHI’s three-day Drug Discovery Chemistry conference in San Diego. The conference consists of six tracks, with three happening simultaneously. The FBDD track is the only one which dates all the way back to the beginning in 2006. In fact, this is the oldest recurring fragment conference, predating both the Royal Society Fragments meetings as well as the independent FBLD meetings.

It’s worth reflecting on how far fragments have come since 2006. Back then, as Rod Hubbard (Vernalis and University of York) noted, most of the talks were prospective and methodological. Even as late as 2010 there were talks describing how dedicated fragment groups needed to be shielded from the larger organization. Now fragments are mainstream: a large fraction of the talks in the protein-protein interaction track involved fragments, as did both plenary keynote addresses to the entire conference.

Harren Jhoti’s keynote focused on lessons learned at Astex over the past 15 years. There has been some debate in the literature over ligand efficiency (LE), and one slide that struck me was a summary of 782 dissociation constants (measured by ITC) against 20 projects. The vast majority of these compounds had LE > 0.3 kcal/mol/atom. Given that Astex has put multiple fragment-derived drugs into the clinic and was acquired by Otsuka in one of the largest M&A events of 2013, the metric appears to have some utility.

Still, it’s important not to be dogmatic, particularly for difficult targets. Harren described a program for XIAP/cIAP where they started with an extremely weak fragment with LE < 0.2, but its binding mode was sufficiently interesting that they were willing to work on it. This program also revealed the importance of biophysical measurements, as functional activity was uninterpretable and even misleading until higher affinity compounds were discovered.

One theme throughout the conference was the observation that fragments bind at multiple sites on proteins. Harren noted that Astex researchers have found fragments bound (crystallographically) to 54 sites on 25 targets – an average of 2.2 sites per target. Some targets are even more site-rich: Joe Patel (AstraZeneca) performed a crystallographic screen on a complex of Ras and SOS and found four binding sites, including one previously discussed here. In this effort, 1200 fragments were screened in pools of 4, and in one case two fragments from the same pool each bound only when they were both present at the same time – each fragment alone showed no binding by NMR or crystallography.

Troy Messick (Wistar) described his work against the EBNA1 protein from Epstein-Barr virus. An HTS screen of 600,000 compounds came up with at best marginal hits, but soaking 100 different Maybridge fragments into protein crystals led to 20 structures, with fragments bound to four different sites. Some of these fragments were then merged to give cell-active compounds with good oral bioavailability.

Rather than exploring different ligands binding at different sites, Ravi Kurumbail (Pfizer) described an interesting case of the same ligand binding at different sites. A screen against the kinase ITK identified a (large) fragment that could bind both in the adenine binding pocket as well as a nearby pocket, as determined crystallographically. Determining the affinities of the same fragment for the two sites necessitated some clever SPR and enzymology, but did lead to a highly selective series.

In terms of targets, BCL-family proteins were certainly well-represented, featuring heavily in talks by Chudi Ndubaku (Genentech, selective Bcl-xL inhibitors), Mike Serrano-Wu (Broad Institute, MCL-1 inhibitors), Zaneta Nikolovska-Coleska (University of Michigan, MCL-1), Roman Manetsch (Northeastern, Bcl-xL and MCL-1), and Andrew Petros (AbbVie, BCL-2 and MCL-1). Of course, it was AbbVie (neé Abbott) that pioneered BCL inhibitors as well as FBLD in general, and I was happy to hear that there is a renaissance occurring there, with fragment approaches being applied to all targets, even those undergoing HTS.

Finally, there were some interesting practical lessons on library design. Peter Kutchukian described how the Merck fragment library was rebuilt to incorporate more attractive molecules that chemists would be excited to pursue. There is an ongoing debate as to whether a fragment library should be maximally diverse or contain related compounds to provide some SAR directly out of the screen, and in the case of the Merck library the decision was to target roughly five analogs in the primary library, with a secondary set of available fragments for follow-up studies.

The utility of having related fragments in a library was illustrated in a talk by Mark Hixon (Takeda) about their COMT program. A HTS screen had failed, and even a screen of 11,000 fragments came up with only 3 hits (with an additional close analog found by catalog screening). Remarkably, all of these are extremely closely related, but other analogs in the library didn’t show up; had they not had multiple representatives of this chemotype in their library they would have come up empty-handed.

In the interest of space I’ll close here. Teddy will post his thoughts later this week, and please share your own. CHI has announced that next year’s meeting will be held in San Diego the week of April 19. And there are still several great events on the calendar for this year!

20 April 2015

Tethering versus RNA

Last week we highlighted one of the less common fragment-finding methods, and today we turn to another. Tethering uses reversible disulfide exchange chemistry to trap thiol-containing fragments near binding sites. Back when we developed the technology at Sunesis we used cysteine residues in proteins. We occasionally discussed applying it to nucleic acids, but at the time it was hard to make a good business case. Now that microRNAs (miRNAs) have become hot, there is more interest in going after nucleic acid targets, and in a recent paper in Molecules Kiet Tran and Peter Beal (UC Davis) and Michelle Arkin (UC San Francisco) have done just that.

The researchers were interested in an RNA sequence that is cleaved in cells to generate miR-21, a potential cancer target. The idea is to find small molecules that bind to pre-miR-21 and prevent its processing to the mature miRNA. To perform Tethering, the researchers first introduced a thiol group into adenosine and incorporated this into RNA. They made two separate versions of pre-miR-21, with the modified adenosine at a different site in each, and also made a control RNA with a completely different sequence.

Next, they incubated the modified RNAs with 30 different disulfide-containing small molecules under partially reducing conditions and used mass spectrometry to identify those that covalently bound. As expected most showed minimal binding, but there were a couple hits. One of these, a 2-phenylquinoline, bound to both modified versions of the pre-mR-21 as well the control RNA, suggesting non-specific binding. In fact, 2-phenylquinoline is a known intercalator, so while its identification is not surprising, it does validate the ability of Tethering to identify binders. The other hit, however, appeared to be specific for one of the two pre-mR-21 sequences.

Of course, there is still a long way to go; it is unclear how much affinity the hit has for the RNA, or how specific it would prove if tested against a large panel of decoy RNAs. A key challenge for Tethering – as with many fragment-finding methods – is figuring out what to do with a hit. This is all the more true with RNA, about which we’ve written several times over the years. Still, one nice feature of Tethering is that it allows one to target a specific site of interest. Also, the covalent (disulfide) bond helps with both crystallography and modeling. It will be fun to watch this story develop.

13 April 2015

Substrate activity screening for irreversible PAD3 inhibitors

Of all the ways to find fragments, one of the more unusual is substrate activity screening, or SAS, which we first discussed here. The idea is to make and screen libraries of potential enzyme substrates and transform the best ones into inhibitors. In a new paper in J. Am. Chem. Soc., Jon Ellman and coworkers at Yale University describe how they used SAS to discover irreversible inhibitors of protein arginine deiminase 3 (PAD3), a potential target for neurodegenerative diseases.

The four human PADs (conveniently named PAD1-4) transform arginine residues in proteins to citrulline residues, with subtypes distributed differently across different tissues. The researchers started by making a library of more than 200 fragment-sized guanidines (the unique side-chain moiety in arginine) as potential substrates. These were then screened in a colorimetric assay. Several compounds were found to be processed by the enzyme, though all were very weak substrates (Km > 10 mM).

Next, the best substrates from three different chemical series were optimized for activity. For example, substrate 4a was grown to substrate 15a.


Finally, the substrates were converted to irreversible inhibitors by replacing the guanidine with a known chloroacetamide warhead. This coopts the natural mechanism of the enzyme, which relies on covalent bond formation between an active-site cysteine residue and the substrate. Within a given series, the better the substrate, the better the resulting inhibitor (for example, inhibitor 15b is more potent than inhibitor 4b). However, these correlations did not hold across series.

The best compounds were also tested for selectivity, and some of them were at least 10-fold selective for PAD3 over the other three PADs.

Last year we highlighted a paper that described several difficulties encountered (and overcome) using SAS to find inhibitors of the protease urokinase. (The comments to that post are well worth reading as they include contributions from the corresponding author of the paper as well as a former Ellman postdoc who is using SAS.) However, according to the current paper, SAS was relatively straightforward for PAD3 – another confirmation that different targets require different approaches.