03 May 2021

Fragments in the clinic: AG-270

A promising oncology approach is to target “synthetic lethal” proteins that are required for cancer cells but not for ordinary cells. Methionine adenosyltransferase 2A (MAT2A) is a metabolic enzyme that produces S-adenosyl methionine (SAM). Cancer cells lacking another gene, methylthioadenosine phosphorylase (MTAP), appear particularly dependent on MAT2A. In a recent J. Med. Chem. paper, Zenon Konteatis and colleagues at Agios, Viva, and ChemPartner describe the first clinical inhibitor of MAT2A.
The researchers began by screening >2000 fragments in pools of 20, each at 50 µM, using ultrafiltration. The 31 hits were tested in enzymatic and SPR assays, and compound 1 confirmed in both. Testing 54 commercially available analogs led to compound 2, with low micromolar activity, and this molecule was profiled intensively.

Kinetic studies revealed compound 2 to be non-competitive with respect to substrates ATP and L-methionine, and crystallography confirmed that compound 2 binds in a previously discovered allosteric pocket. The molecule makes polar and hydrophobic contacts to MAT2A and also displaces several water molecules. SAR studies found a preference for aromatic moieties at the 2-position of the central core, but the phenyl off the 3-position could be substituted with more shapely moieties such as the piperidine in compound 9.
Closer examination of the structure of compound 2 bound to MAT2A revealed a protein-bound water molecule, and displacing this with the phenol in AGI-24512 led to a satisfying boost in biochemical potency as well as cell activity. However, the molecule has poor oral absorption and a short half-life in rats. Metabolite identification studies pinned the blame on the piperidine, with the phenol no doubt doing no favors. Medicinal chemistry ultimately led to AGI-25696, which despite its lower biochemical activity was active in cells, metabolically stable, and showed efficacy in a mouse xenograft model when dosed orally.
Despite these favorable properties, AGI-25696 has very high protein binding in human plasma (>99.9%) as well as high efflux, which would likely necessitate a high clinical dose. The researchers proposed that, due to the weakly acidic nature of the molecule, it could tautomerize, and each tautomer could bind to different plasma proteins. Simply methylating the N-H led to decreased plasma protein binding but also lower binding to MAT2A. Thus, the researchers sought to shield the N-H by forming an intramolecular hydrogen bond. After appending more than 70 heterocycles they eventually arrived at AG-270, which has a more respectable plasma protein binding of around 98.5%.
Extensive characterization of AG-270 revealed it to be potent with good pharmacokinetics and oral bioavailability. It is relatively clean in a panel of 95 potential off-targets and showed tumor growth reduction in xenograft models. But it is not without warts: low solubility necessitated a spray-dried dispersion for animal dosing, not surprising giving its high lipophilicity. Nonetheless, the molecule has entered a phase 1 clinical trial in patients with MTAP loss.
This is a lovely fragment-to-lead success story with several lessons. First, although enzymes are sometimes considered “easy,” this is not necessarily true. Indeed, at an ACS meeting in 2018 Anil Padyana mentioned that metabolic enzymes in particular often have shallow, polar active sites. Targeting allosteric sites, as done here, can be a useful alternative.
Second, it is striking how the core of the initial fragment remains intact in the clinical compound, a reminder of the power of fragments to efficiently explore chemical space.
Finally, this story is another important reminder that affinity is often just the beginning of a long journey. It took considerable effort to optimize the pharmaceutical properties from AGI-24512 to AG-270, including a decrease in ligand efficiency. In the end the team has succeeded, and Practical Fragments wishes them – and the patients – luck in the trials.

26 April 2021

STD NMR on putative SARS-CoV-2 main protease ligands

Over the past sixteen months SARS-CoV-2 has infected more than 146 million people worldwide and killed over 3 million. Highly effective vaccines are now available, but not everywhere, and how long the vaccinations will last as new variants arise remains unknown. COVID-19 will likely be with us indefinitely, necessitating drugs as well as vaccines.
More than a year ago we highlighted the COVID Moonshot effort, which began with a crystallographic screen against the essential main protease (Mpro) to find starting points for drug discovery. In a new open-access J. Biomol. NMR paper, Ioannis Vakonakis and collaborators at University of Oxford and University of Patras have attempted to characterize some of the hits using saturation transfer difference NMR (STD NMR, see here for a brief description).
The researchers had access to a 950 MHz NMR (jealous much?). Samples were screened at 10 µM protein using irradiation of a Mpro methyl group with a chemical shift at 0.5 ppm. To try to minimize differences in relaxation parameters among different ligands, only the strongest STD signals in aromatic moieties were examined.
Of 39 non-covalent ligands discovered in the crystallographic screen, five either did not produce an NMR signal or the spectra were inconsistent with the expected structures, suggesting the ligands may be insoluble or unstable in aqueous buffer. The remaining 34 compounds were nominally screened at 0.8 mM each, but the reference spectra differed in intensity by as much as 15-fold, suggesting dramatic differences in concentration. Since the strength of an STD signal is related to both affinity and concentration, this could obviously complicate interpretation of results. As we’ve written previously, careful curation of your library is essential.
Of thirteen active site ligands, only four showed strong STD signals. Dose-response titrations between 0.05 and 4 mM revealed dissociation constants of 1.6-1.7 mM for two of them, with the other two being too weak to accurately measure. Molecular dynamics simulations starting with the known structures were consistent with these results, with the tighter binders tending to maintain their positions more than the weaker binders.
The researchers also characterized 650 elaborated molecules from the COVID Moonshot, some of which had been reported to be nanomolar inhibitors. Disturbingly, 35 gave no NMR signal and another 86 yielded weak signals. Among those remaining there was a weak correlation (R2=30%) between IC50 in an enzymatic assay and the STDratio (integrated signal intensity of peaks in the STD spectrum over reference spectrum). STD NMR is not appropriate for molecules with Kd < 10 µM, so the researchers also used a competition experiment in which four putative high-affinity molecules would compete a weaker “spy” fragment. This exercise confirmed two ligands but not two others, calling into question their mechanism.
STD NMR is often used as part of an assay cascade prior to attempting crystallography, but as crystallography throughput increases there is a case for starting with crystallography, as we argued five years ago. Results from the 39 crystallographic hits perhaps gives pause to that notion, or at least emphasize the need for confirmatory assays. It is easy to be seduced by a high-resolution structure, but because of its sensitivity crystallography may identify ligands so weak as to be unadvanceable. As for the 650 elaborated molecules, it’s too early to draw conclusions, though it’s good to always be on the lookout for false positives.
Hopefully the COVID Moonshot will ultimately lead to drugs against SARS-CoV-2. But even if it doesn’t, the intensive focus of multiple techniques on a few proteins is providing useful guidance and best practices that will be applicable to other targets.

19 April 2021

Fragments vs KEAP1: deconstruction and merging

One of the more challenging protein-protein interactions targeted by drug hunters is the interface between the transcription factor NRF2 and its repressor KEAP1. This is part of the cellular defense against reactive oxygen species; increasing NRF2 activity may be useful for treating a variety of diseases. Unfortunately, the binding site on KEAP1 that interacts with NRF2 is large and has a predilection for carboxylic acids. Thus, many of the molecules reported as inhibitors tend not to be druglike. Anders Bach (University of Copenhagen) and a multinational team of collaborators sought to do better, and have just published some of their journey in J. Med. Chem.
The researchers had previously tested 19 reported small-molecule KEAP1 inhibitors, of which only nine confirmed. (This is a salutary reminder to take any individual publication with a large grain of salt.) The nine fell into six chemical series (two shown below), and the researchers decided to fragment some of these molecules into 77 fragments. The fragments were then tested in four assays: fluorescence polarization (FP), a thermal shift assay (TSA), saturation transfer difference (STD) NMR, and surface plasmon resonance (SPR).
Primary hit rates were generally high, from 25%-64%, but long-time readers will not be surprised that the overlap was not great: no fragments hit in all four assays, and only eight hit in three. As the researchers point out, this could reflect differences in sensitivity, conditions (from 3-8% DMSO and from 0.5 to 8 mM fragment), and different types of false positives and false negatives. Interestingly, and in contrast to previous work, overlap was good between STD NMR and SPR.
Crystal structures of seven hits were solved bound to the protein, and compounds 4c and 1m (from different precursor molecules) were merged to provide compound 8, with low micromolar affinity. Compound 8 was the subject of considerable medicinal chemistry, with five different vectors chosen for growing. Despite being structurally enabled, the researchers struggled; changes that improved affinity in one context did not do so in another. After considerable effort, the researchers obtained compound 77o, with mid-nanomolar activity.

Compound 77o is stable in human plasma and mouse liver microsomes. Unfortunately, and unsurprisingly given the two carboxylic acids, it has poor permeability. Indeed, a fragment-derived KEAP1 inhibitor we described previously has only a single carboxylic acid, as does precursor compound 7. As the researchers themselves acknowledge, “the physicochemical properties of our compounds are not favorable for membrane permeability.”
Nonetheless, this paper is a lovely example of fragment-based deconstruction reconstruction (FBDR) and is well worth studying for the thorough descriptions of fragment screening in orthogonal assays and structure-based design. Another lesson may be that despite considerable effort, the final molecule is far from a chemical probe, let alone a drug. Perhaps some targets truly are undruggable. Or maybe – as for other seemingly undruggable targets – a change in strategy is needed.

12 April 2021

Fragment merging on c-MET

Fragment-based inhibitors of kinases are legion, particularly those that bind in the so-called hinge region where the adenine of ATP normally sits. However, even among these there are many different flavors of inhibitors. In particular, about 10 kinases can adopt a “folded P-loop” conformation, in which the phosphate-binding loop collapses into the ATP binding site. This was the focus of a recent open-access paper in ACS Med. Chem. Lett. by Gavin Collie and colleagues at AstraZeneca.
The researchers were interested in the oncology target c-MET. A ligand-based NMR screen of 1150 fragments (in pools of 6 at 200 µM each) yielded a 6% hit rate, of which 20 confirmed by SPR. Crystallography was attempted unsuccessfully on most of these, but compound 1 was found to snuggle into the active site with the protein in the folded P-loop conformation.
A computational similarity search of AstraZeneca’s internal library identified compound 2, which crystallography revealed to bind in a similar manner, with two hydrogen bonds to the hinge region and the benzyl group buried in a hydrophobic pocket. A second similarity search of the library – this time based on compound 2 – identified compound 3. Crystallography confirmed that the core azaindole moieties of compounds 2 and 3 overlay, and thus fragment merging was attempted.

The resulting compound 5 bound as expected. This prompted yet another computational search of the internal library, and after a bit of medicinal chemistry compound 7 was identified as a mid-nanomolar inhibitor with low micromolar cell-based activity. Crystallography revealed that it too binds to the folded P-loop conformation of c-MET.
Because the folded P-loop conformation is rare among kinases, the researchers hoped that the resulting molecule would be selective. Unfortunately, when profiled against a panel of 140 kinases at the low concentration of 100 nM, 27 of them were inhibited by at least 60%. This is perhaps not surprising given the 7-azaindole core, which has been found to bind to more than 90 kinases, though some compounds containing this moiety are selective.
Nonetheless, this paper is a nice example of structure-guided fragment merging. A cynic could point out that had the researchers screened the entire AstraZeneca compound collection they likely would have identified molecules very similar to compound 7 anyway, but this may have cost more and would not be an option at smaller organizations without million-compound libraries. And the approach is useful for more difficult targets for which high-affinity molecules may not exist – yet.

05 April 2021

A general fragment-based approach to… targeting RNA?

This is taken from the title of a recent open-access paper by Matthew Disney and collaborators at Scripps Research Institute Jupiter and Florida Atlantic University in Proc. Nat. Acad. Sci. USA. RNA has long been a target of FBLD: Practical Fragments first blogged about it in 2009, and a 2002 paper reported using fragment linking to obtain a low micromolar binder. So how general is the new approach?
The researchers describe chemical cross-linking and isolation by pull-down fragment mapping (Chem-CLIP-Frag-Map). This involves using photoaffinity probes that can crosslink to biomolecules such as RNA. The probes also have an alkyne tag that can be used to isolate bound molecules using click chemistry. We’ve written previously about such “fully functionalized fragments” (FFFs).
Earlier work had resulted in the identification of compound 1, which binds to a specific site on pre-miR-21, the precursor to a non-coding microRNA linked to cancer. An FFF version of compound 1 was shown to crosslink to pre-miR-21 after irradiation with UV light, and the site of modification could be mapped using a reverse-transcriptase-mediated primer extension, which stalled at the modified bases.
Next, the researchers screened 460 FFFs at 100 µM and found 21 that crosslinked to pre-miR-21. They were ultimately looking to link fragments with compound 1, and thus competition studies were used to eliminate fragments that bound at the same site. This left three fragments, and primer extension studies confirmed that these bound near but not at the binding site of compound 1.
Next, the researchers attached these three fragments to compound 1, with or without various linkers. Some of the resulting molecules had improved affinity, and compound 9 showed the tightest binding according to microscale thermophoresis (MST). Mutational and competition studies confirmed that the molecule binds to the expected site. Importantly, compound 9 not only bound to pre-miR-21, it also blocked processing by the enzyme Dicer. Moreover, it showed activity in cell models consistent with inhibition of pre-miR-21.

This is a nice paper, but there are several limitations. First, compound 9 is still a fairly modest binder with lackluster ligand efficiency. Indeed, while potency can be overrated, I would love to see a fully synthetic low nanomolar RNA binder. Second, while the approach may be general, it is not necessarily easy, and it requires specialized fragments. And as we noted last year, there is no relation between crosslinking efficiency and affinity. I wish the researchers had tried linking some of the non-selected fragments to see whether these were false negatives. Indeed, given the complexity of the approach, I wonder if the researchers would have been better off simply making and testing an anchor library around compound 1, in a similar fashion as described here.
But whether or not Chem-CLIP-Frag-Map turns out to be the solution to targeting RNA, I wholeheartedly agree with the conclusion: “It may be time to describe biomolecules that are perceived to be challenging small molecule targets as ‘not yet drugged’ rather than ‘undruggable.’”

01 April 2021

Fragments from Mars

Four years ago today we highlighted work invalidating ligand efficiency on Venus. Recently though, the planet of war has been getting all the love. You've probably seen the stunning pictures from the Perseverance rover on Mars. One of its missions is to collect samples for later return to Earth. Many scientists are eagerly awaiting their allotment, among them Herbert George Wells of Bromley University.
There are those who believe that life here began out there - maybe even on Mars. According to this theory, Martian meteorites, or even Martians themselves, seeded life on Earth. If so, any organic fragments on Mars could be ancestral to all life and may generate particularly high hit rates, perhaps approaching those of the "universal fragments" we profiled here and here. And any molecules that can remain intact near the harsh surface of Mars for a few billion years may also have good metabolic stability.
Prof. Wells plans to separate and identify all the organic fragment-like molecules in his sample. While this will undoubtedly make an interesting publication, Prof. Wells is also dreaming of cosmic riches and has teamed up with Dr. Lyttle, Jr. to sell a custom collection of Martian Fragments.
Some people are concerned that a Martian sample may harbor dangerous organisms, but Prof. Wells is not worried. "Frankly, with so many viruses running around Earth these days, it's the Martians who should be afraid!"

29 March 2021

Fragment chemistry roundup part 5

Last week we highlighted three chemistry-focused papers, and this week we’ve got three more. And to add a third three, all these papers address “three-dimensional” fragments.
The first, in ACS Med. Chem. Lett. by Brian Cox (University of Sussex), Philip Cox (AbbVie) and colleagues, describes using photochemical [2 + 2] cycloadditions to generate bridged pyrrolidine fragments, which can be further diversified.

The researchers analyzed 54 products by principal moments of inertia (PMI) and plane of best fit (PBF), which revealed them to be quite shapely, more so than AbbVie’s Rule of Three collection. Interestingly, amide derivatives with aromatic substituents were often shapelier despite being less saturated, and the researchers thus caution against using Fsp3 as a proxy for shapeliness – a point Teddy made several years ago.
The next paper, open access in ChemComm, also deals with cyclobutane-containing molecules. However, rather than building them from scratch in cycloaddition reactions, David Spring and collaborators at University of Cambridge and California State Polytechnic University Pomona use palladium-catalyzed C-H arylation to functionalize the rings. Lactonization and further derivatization generates a range of molecules.

The researchers generated a virtual library of 90 scaffolds that were rule-of-three compliant and quite shapely. It would be interesting to explore this chemistry on the bridged pyrrolidines of the previous paper – perhaps deliberately “losing control,” as discussed last week.
Finally, in Chem. Eur. J., Peter O’Brien (University of York) and a large group of collaborators from academia and industry describe (also open access) the “design and synthesis of 56 shape diverse 3-D fragments.” Because of their prevalence in drugs, pyrrolidines and piperidines were chosen as targets. The researchers specifically set out to make diverse molecules that would be shapelier (as assessed by PMI) than members of typical libraries. In considering three-dimensionality, the researchers considered not just the lowest energy conformation, as is typically done, but also other conformations with energies up to 1.5 kcal/mol higher; these would be present roughly 8% of the time at 37 °C. Some of the molecules are shown.

A PMI analysis of these fragments revealed them to be more three-dimensional than representatives of six commercial fragment libraries. In fact, although three of the commercial libraries are touted as being “3D,” a PMI analysis revealed them to “have only a marginally better 3-D profile compared to the standard 2-D rich commercial fragment libraries.” As in the paper discussed above, there was no correlation between Fsp3 and PMI.
Despite being relatively simple, 42 of these molecules had been previously unreported. In addition to their shapeliness, they also adhered to the rule of three, with an average ClogP of just 0.54. Moreover, 52 of the fragments were stable for >6 weeks in DMSO, 48 were stable in aqueous buffer for >24 hr, and 40 of them were soluble at >0.5 mM in buffer.
Most of these fragments are available for screening at the Diamond XChem facility. It will be interesting to see what kinds of hit rates they produce, and whether they generate superior leads. As we noted last year, the majority of drugs are not particularly shapely. Still, it is fun to explore new regions of chemical space, and these three papers are good starting points.

22 March 2021

Fragment chemistry roundup part 4

Last week’s post on diversity-oriented synthesis (DOS) reminds me that it has been nearly a year since we’ve done a post on fragment chemistry. Since then, several interesting papers have appeared. These next two posts will cover them.
The point of DOS is to generate lots of analogs from a small number of starting materials in a controlled fashion. But as any chemist knows, reactions often go out of control. In ChemMedChem, John Spencer (University of Sussex) and collaborators at several institutions have decided to turn lemons into lemonade by deliberately losing control, though they are quick to emphasize “in the selectivity (not health and safety) context.”
The researchers focused on C-H bond activation, a handy class of reactions in which a carbon-hydrogen bond is broken in order to generate a new molecule. C-H bonds are of course ubiquitous in drugs, and chemists normally try to selectively activate just one. Here, the researchers focused on Ru/Pd-catalyzed photochemical arylation in the presence of alcohols, which can further react with certain substrates. For example, reacting 2-phenylpyridine with a 4-fluorophenyldiazonium salt in methanol led to five products (some of which breach the rule of three).

These products, and those of other similar reactions, were screened crystallographically against an enzyme called NUDT7, resulting in one hit.
On a related chemical subject, Quentin Lefebvre and colleagues at SpiroChem explore photoredox-nickel dual catalyzed N-arylation reactions in Beilstein J. Org. Chem. In 4 days they tested 29 combinations of amines with various aryl halides, 15 of which gave products; examples are shown to the right.
Given that SpiroChem is a chemical vendor, expect to see more of these sorts of molecules in their catalog.
Finally, in Chem Sci., Nicholas Turner (University of Manchester - corrected) and collaborators at Keele University ask whether it is “time for biocatalysis in fragment-based drug discovery.” Biocatalysis involves using enzymes to run reactions. Despite stunning advances in synthetic organic chemistry, Nature is still the master, so why not work together?
The researchers review examples where biocatalysis could be used to generate new fragments or elaborate fragment hits. Importantly, enzymes can perform selective reactions even in the presence of multiple reactive centers that would normally need to be protected in conventional synthesis. Moreover, researchers are increasingly engineering enzymes to increase their substrate scope, efficiency, or completely alter the reactions performed. Some of the products are illustrated here.
I confess I haven’t done much biocatalysis, largely due to unfamiliarity but also because enzymes for organic synthesis don’t seem to be as widely offered by vendors as – for example – transition metal catalysts. Perhaps there is a market opportunity for fragment libraries designed for enzyme-mediated elaboration?
A decade ago, the main challenge of fragment-based drug discovery was finding fragments. Now it is elaborating them. It is nice to see solutions accumulating.

15 March 2021

Fragments from DOS, advanced

DOS – diversity-oriented synthesis – intentionally generates a disparate set of compounds from a small number of starting materials in just a few synthetic steps. The idea is that if any of these turn out to be hits, it will be straightforward to make analogs. Since figuring out what to do with a fragment is a common bottleneck, DOS-derived fragments could help. An open-access paper published in Chem. Sci. by David Spring (University of Cambridge) and collaborators from several institutions demonstrates how to use DOS to move fragment hits forward.
The researchers had previously disclosed (also open access) a rule-of-three-compliant DOS library of 40 compounds derived from racemic α-methyl propargylglycine. In the current paper, these molecules were screened crystallographically at 500 mM on the Diamond Light Source XChem platform against three protein targets.
The first, penicillin binding protein 3 (PBP3) from P. aeruginosa, is a classic antibiotic target. A single hit, compound 1, was identified. Interestingly, this turned out to be a covalent modifier, with the catalytic serine opening up the lactone. The researchers made 10 analogs exploring four different vectors, each in five synthetic steps using cheap reagents (< £3 per gram). These were screened crystallographically and six bound; one example is compound 6.
The next protein screened, cleavage factor 25kDa (CFI25), is a subunit of the pre-mRNA cleavage factor Im. (No, I hadn’t heard of it either.) A crystallographic screen yielded two hits, one of which – compound 19 – was elaborated into 14 analogs. This provided some preliminary SAR around the phenyl ring as well as a surprise: compound 31 bound to a different region of the protein.
Finally, the researchers screened activin A, a member of the transforming growth factor β superfamily. Compound 40 was elaborated into 14 analogs exploring four vectors, and compound 42 was found to bind in a similar manner.
There are several take-aways from this paper. First, DOS libraries can be remarkably diverse: compounds 1, 19, and 40 are all quite different from one another. Second, although I hesitate to discuss hit rates from such a small library, it is encouraging that hits were found at all even from fairly shapely fragments. These are also the first reported small molecule binders for CFI25 and activin A. Laudably, all the structures have been deposited in the protein data bank, extensive details are provided in 185 pages of supplementary material, and the library itself is available for screening at XChem.
One downside to crystallographic screening is that affinities are not part of the package, and some hits may be so weak as to be difficult to advance. But the researchers note they are further characterizing the compounds in the hope of producing more potent analogs. Although Teddy’s deadline for demonstrating a highly ligand-efficient molecule from DOS has long passed, hopefully the Safran Zunft Challenge will soon be met.

08 March 2021

Fragments in the clinic: LYS006

Practical Fragments’ first list of fragment-derived clinical compounds, published in 2009, listed just 17 molecules. The current count is approaching 50. One of the original compounds, DG-051, targeted the enzyme leukotriene A4 hydrolase (LTA4H). That molecule did not advance, but in a recently published J. Med. Chem. paper Christian Markert and colleagues at Novartis describe the discovery of a superior clinical compound.
LTA4H catalyzes the rate-determining step in the biosynthesis of leukotriene B4, a pro-inflammatory lipid implicated in multiple diseases. The researchers performed a differential scanning fluorimetry (DSF) screen of the enzyme against a library of 1800 fragments, 350 of which had been selected by in silico screening. This exercise yielded 14 hits that stabilized the protein against thermal denaturation, including compounds 1 and 2. Crystallography revealed that they both bind in the hydrophobic substrate pocket.

Merging compounds 1 and 2 led to compound 3, with low nanomolar potency. However, this lipophilic amine had poor stability in rat liver microsomes and also inhibited hERG and a couple CYP450s. Adding a carboxylic acid (compound 13) fixed these problems, though the molecule did bind to the dopamine transporter and had low solubility. Interestingly, the two enantiomers of compound 13 have very similar affinities, and crystallography revealed they could each bind in a similar fashion. Further optimization of the lipophilic tail ultimately led to LYS006. The crystal structure of this molecule overlays nicely with the initial fragments.
Much of the paper is devoted to characterization of LYS006, which appears to be a remarkably selective molecule. It does not bind or inhibit > 150 GPCRs, hERG, CYP450s, or a panel of metalloproteases. Oral bioavailability and pharmacokinetics are good in mouse, rat, and dog, and the molecule achieves essentially complete target inhibition at low nanomolar plasma levels. Moreover, LYS006 showed efficacy in preclinical efficacy studies. The drug is currently in four phase 2 clinical trials for ulcerative colitis, inflammatory acne, NASH, and hidradenitis suppurativa. (One of these began in 2018, yet only now are we finding out the origins of LYS006; this illustrates the difficulty of maintaining an up-to-date list of fragment-derived drugs.)
This is a beautiful drug discovery story with several lessons. First, like AZD5363 and many other examples, enzymatic potency was achieved relatively quickly; the bulk of the effort was focused on improving other properties. Second, the final molecule is not necessarily “surprising”: it contains the same biaryl-ether pharmacophore found in previous clinical compounds. Yet fragments along with careful medicinal chemistry allowed the researchers to obtain a best-in-class inhibitor.
Finally, this effort is a useful reminder that persistence can pay off: although LTA4H has been the target of drug discovery for decades, no inhibitors have yet been approved. Hopefully LYS006 will succeed.

01 March 2021

Fragments vs MEK1: allosteric binders

MEK1 is a central player in the MAP kinase signaling cascade, which is often dysregulated in cancer. As such the enzyme has been the focus of considerable research and the target of four approved drugs. Interestingly, these drugs bind not to the hinge region targeted by most kinase inhibitors but rather to an allosteric pocket adjacent to the ATP binding site. The drugs also look somewhat alike. Seeking something completely different, Paolo Di Fruscia, Fredrik Edfeldt, Helena Käck, and colleagues at AstraZeneca turned to fragments. They have recently published their results in ACS Med. Chem. Lett.
As we discussed in 2016, the AstraZeneca fragment library is quite large at 15,000 molecules. The researchers used a computational screen to narrow this down to a more manageable 1000 compounds for ligand-detected NMR screening. AMP-PNP, a nonhydrolyzable version of ATP, was included to block the hinge region, biasing the screen for fragments that bind the allosteric site. (See here for earlier work looking for ATP-competitive molecules.) A total of 142 fragments were identified and further characterized by SPR, and 46 showed dissociation constants better than 1 mM and similar affinities in both the presence and absence of AMP-PNP, suggesting they do indeed bind in the allosteric site.
Crystallography was attempted on all the fragments, but only two produced structures. Reassuringly, both bound in the allosteric site. But with only limited structural information, the researchers tested analogs of the fragment hits within their corporate collection. This identified compound 10, which is more potent than initial fragment 3. Moreover, compound 10 lends itself well to library synthesis.

All library members were initially made and tested as racemates. When the two enantiomers of the best hit were separated, compound 23 was found to be a sub-micromolar binder, roughly 100-fold better than the other enantiomer. At this point the researchers finally obtained a crystal structure of compound 23, confirming that it did bind in the allosteric pocket. Compound 23 is also still fragment-sized, just three heavy atoms larger than compound 3.
The astute reader will notice that the word “inhibitor” has not appeared until now, and indeed despite the encouraging affinity no mention is made in the paper of inhibition – a rather important feature! At a conference in 2019 Paolo did describe further optimization to a functional molecule, so hopefully we will see a second publication detailing this work.
Like the NPBWR1 story last month, this is another nice example of advancing fragments in the absence of structural information. It is also a good case study of fragments yielding completely different chemical matter in a crowded field.

22 February 2021

Antifreeze opens cryptic pockets, experimentally and computationally

Imagine an alien species sees a single photo of a human. They would have no idea how our arms and legs move, or that our mouths can open and close. So it is with protein crystal structures: even multiple static images often fail to show possible conformations. Pockets open and close in unexpected places, and these can be critical for drug discovery. But how do you find these “cryptic” pockets? Harsh Bansia, Suryanarayanarao Ramakumar, and collaborators at Indian Institute of Science, Bengaluru and Pennsylvania State University provide a new approach in J. Chem. Inf. Mod.
The researchers were studying a bacterial xylanase called RBSX and had mutated a tryptophan residue to an alanine. When they solved the crystal structure, they found that the mutation had created a surface pocket that was filled with a molecule of 1,2-ethanediol (EDO). EDO is an ingredient in antifreeze because of its ability to prevent ice formation, and this property also makes it a common cryoprotectant in crystallography. The EDO molecule was making both van der Waals contacts as well as a hydrogen bond with the protein. The researchers found similar results when they used propylene glycol. (See here for a related discussion of MiniFrags, the smallest of which are the size of propylene glycol.)
To see whether water could make these same interactions, the researchers determined another crystal structure in the absence of EDO. Surprisingly, a phenylalanine side chain rotated and closed the pocket. Had this been the only structure solved, the possibility of pocket formation would not have been suspected.
Next, the researchers conducted molecular dynamics simulations. Starting from the closed state, the pocket remained occluded by the phenylalanine, giving no hint of its potential presence. Starting from the open state and removing EDO, the pocket also rapidly closed. In other words, in the absence of a ligand, the pocket appears to collapse in upon itself.
Importantly, these observations are not limited to a mutant bacterial protein. The researchers looked at published crystal structures of four unrelated proteins with known cryptic pockets and found that EDO could bind in all of them. They also ran molecular dynamic simulations on two proteins in which EDO was included as a virtual cosolvent. For both NPC-2 and IL-2, addition of EDO was able to open up cryptic pockets that had been previously found using other molecules; we’ve discussed earlier computational work on IL-2 here.
This is a nice example of following up on an unexpected observation, and is well-suited for further study. For example, it would be interesting to do a systematic study of EDO and propylene glycol binding sites throughout the entire protein data bank. For those of you doing molecular dynamics or crystallography, it may be worth adding EDO – virtually or experimentally – to see if it reveals any surprises in your favorite proteins.

15 February 2021

Fragments vs Notum, three ways

The Wnt signaling pathway has been implicated in multiple diseases, from Alzheimer’s to cancer to osteoporosis. To be able to bind receptors, Wnt proteins must be post-translationally modified with a palmitoleate group. The carboxylesterase Notum removes this group, shutting of signaling. Thus, inhibitors of Notum could maintain Wnt activity. In two J. Med. Chem. papers, E. Yvonne Jones (University of Oxford), Paul Fish (University College London), and collaborators describe three series of inhibitors derived from fragments.
The palmitoleoyl group is highly lipophilic, and previous work with Notum had revealed a predilection for hydrophobic carboxylic acids. Thus, in the first paper, the researchers assembled a library of 250 diverse carboxylic acids, all rule-of-three compliant. Each was tested (10-point dose-response) in a biochemical screen up to 100 µM. Twenty compounds had IC50 < 25 µM, and all of these were soaked into crystals of Notum, resulting in 14 structures. Two series were pursued.
Compound 5 was one of three pyrroles with low micromolar potency. Fragment growing led to compound 20, and further SAR ultimately led to compound 20z, with high nanomolar activity. Unfortunately, this is a fairly lipophilic molecule, with clogP = 5.5. Indeed, despite best efforts, including paying close attention to lipophilic ligand efficiency (LLE), potency tracked closely with clogP for this series.
The second series, as represented by compound 8, was less potent but also less lipophilic. Walking various substituents around the phenyl increased both properties (compound 25n) and further tweaking led to compound 26. Although this molecule had promising in vitro ADME properties, it was deprioritized in favor of another series described in the second paper

In addition to the biochemical screen, the researchers also conducted a crystallographic fragment screen at the Diamond Light Source XChem platform. This yielded a whopping 60 hits of the 768 fragments screened, with compound 7 being notable for its high potency, ligand efficiency, and LLE. Iterative structure-based design led ultimately to low nanomolar compound 23dd.
Compound 23dd was active in cell-based assays and had acceptable pharmacokinetic properties in mice. The researchers were particularly interested in modulating Wnt signaling in the brain, and in vitro studies suggested that compound 23dd would have good blood-brain permeability. Unfortunately, this turned out not to be the case, for reasons that are still not clear. However, another molecule derived from compound 7 was superior – hopefully the subject of a future paper.

These are nice structurally-enabled fragment to lead stories, and the medicinal chemistry strategies are particularly well described. Notably, the researchers were able to optimize fragment hits to nanomolar binders while maintaining low molecular weights and (in the second and third cases) reasonable lipophilicity. In addition to clear examples of property-driven medicinal chemistry, these papers illustrate that fragment-based methods can yield a variety of starting points, which can be useful when one lead series runs into trouble.

08 February 2021

Fragments in the clinic: PF-06835919

A year into the COVID-19 pandemic more than 2.3 million people have died, with deaths in the US approaching 500,000. These are staggering numbers, and the scientific community has rapidly responded. Amidst this disaster, it is easy to lose sight of longstanding, even more deadly threats, such as heart disease.
A leading cause of metabolic disease is overconsumption of fructose. Because it is sweeter than other natural sugars and cheap to produce, fructose is widely used in processed foods. Fructose is not subject to the negative feedback regulation of other sugars, and overconsumption has been linked to nonalcoholic fatty liver disease (NAFLD), insulin resistance, and cardiovascular disease. The first step in fructose metabolism is mediated by the enzyme ketohexokinase (KHK), so blocking it seems like a reasonable approach.
More than three years ago we highlighted a paper from Pfizer describing the fragment-based effort which led to compound 1, an inhibitor of KHK. That post ended by noting that there was “still some way to go” to reach a drug. A paper published late last year in J. Med. Chem. by Kentaro Futatsugi and colleagues from Pfizer describes the journey to the clinic.

Compound 1 was well-suited to SAR by parallel synthesis, and a variety of replacements for the methylpyrrolidine (on top) led to compound 3. Although this molecule had similar affinity as compound 1, a crystal structure revealed that it had shifted its binding mode such that the other pyrrolidine ring was pointing towards an important arginine residue. Exploring a diverse range of replacements led to compound 4, with improved affinity driven in part due to interactions between the hydroxyl and the arginine side chain. Replacing this hydroxyl with a carboxylic acid led at last to a low nanomolar lead.
Compound 6 was unstable when incubated with human hepatocytes, and various studies revealed that glucuronidation at the remaining hydroxyl was responsible. Removing the hydroxyl and lowering lipophilicity by removing the nitrile ultimately led to PF-06835919. This compound is potent, orally bioavailable, and clean in a variety of off-target assays.
This is a beautiful example of lead optimization guided by structure with a keen focus on molecular and pharmaceutical properties. The initial fragments are difficult to discern in the final molecule, which is not a bad thing: the whole point of fragment-based discovery is giving multiple options for creative medicinal chemistry. In contrast to last week’s post, crystallography was essential for the program; it also benefited from the applied serendipity of parallel synthesis.
Often these sorts of publications are the valediction of a halted program, but not here: PF-06835919 is moving forward in three clinical trials, including a phase 2 trial for NAFLD. Interestingly, the compound was first dosed in humans in 2016 – a year before the initial paper. This gap between clinical efforts and publications is a reminder that our list of fragment-derived clinical compounds will always be incomplete. I look forward to watching PF-06835919 advance.

01 February 2021

Advancing fragments without structures: NPBWR1

Last week’s post highlighted how biophysical methods, and in particular structural insights, can be critical for advancing fragments to leads. But while everyone likes a structure, one quarter of respondents to our 2017 poll said they were comfortable optimizing fragments on the basis of SAR alone. (See also a recent review.) A new example of structure-free optimization has been published in Bioorg. Med. Chem. Lett. by Remond Moningka and colleagues at Merck.
The researchers were interested in the GPCR neuropeptide B/W receptor subtype 1 (NPBWR1, also known as GPR7), a potential target for obesity. Although impressive advances have been made towards obtaining structural information on membrane-bound proteins such as GPCRs, especially using cryo-EM, routine structure-based design is generally not an option.
The researchers started with a 30,000 member library of fragments between 200-350 Da. Both the size of the library and the size of the fragments are on the large side compared to what is typically used. A cell-based screen (cAMP assay) at 100 µM yielded 500 hits that inhibited at least 30%. Counter-screening against an unrelated GPCR whittled down the number to 20, of which just 3 provided dose-responses. The low confirmed hit rate illustrates both the utility of a larger library as well as the number of false positives likely to arise in a cell assay.
SAR by catalog on compound 1 led to compound 2, and further SAR led to compound 3c, with low micromolar activity and good ligand efficiency. Replacing the nitro group with a more pharmaceutically acceptable trifluoromethyl group produced compound 10. It is worth noting that compound 10 is still fragment-sized yet is >300-fold more active than the initial hit. This is a useful reminder that one can often make significant improvements even before fragment growing. Finally, extensive SAR studies around the phenyl ring ultimately led to compound 21a, with low nanomolar activity.

The pharmacology around GPCRs can be complicated, and compound 21a turned out not to be a simple competitive (orthosteric) antagonist of NPBWR1. Rather, it seems to act as a negative allosteric modulator: it reduces the affinity of the natural ligand.

This is a concise success story of advancing a fragment in the absence of structural information. Does this mean we should not strive for structures? Heck no! Not only would structures likely facilitate faster and further improvements, they might explain the mechanism of action of the compounds. I, for one, would love to know where and how they bind.
But this paper is another reminder that you do not always need crystallography - or even a model -  to take a fragment to a lead.

25 January 2021

Fragments vs TNFα advanced with biophysics, linking, and growing

The cytokine tumor necrosis factor α (TNFα) is a key mediator of inflammation and has long been a target for rheumatoid arthritis, Crohn’s disease, psoriasis, and a host of other inflammatory diseases. Several biologic drugs, such as adalimumab, are approved but these monoclonal antibodies and fusion proteins can be immunogenic or induce neutralizing antibodies. Small molecules could avoid these pitfalls and also reach organs, such as the brain, less accessible to biologic agents. Impressive efforts towards this goal have just been reported in J. Med. Chem. by Justin Dietrich, Chaohong Sun, and colleagues at AbbVie. (Andrew Petros presented this work at the CHI DoT meeting last September.)
The researchers began by screening 18,000 fragments using two-dimensional (13C-HSQC) NMR against TNFα in which the methyl groups of isoleucine, valine, leucine, and methionine were isotopically labeled. Only 11 fragments caused significant perturbations, an 0.06% hit rate reflecting the difficulty of finding hits against this target. All the fragments were characterized by SPR, and compound 1 turned out to have reasonable affinity and ligand efficiency. Synthesis of a few dozen analogs led to compound 2, with improved activity.

TNFα forms a homotrimer, and a crystal structure of compound 2 bound to TNFα revealed that two copies of the fragment bind within a large hydrophobic cavity at the interface of the three protein monomers. Not present in the apo-form of the protein, this central pocket is formed by the movement of tyrosine side chains, causing desymmetrization of the protein trimer. The researchers linked the two nearby fragments to produce compound 3 with improved affinity but decreased ligand efficiency. Further optimization led to compound 4, which was active in cells. But perhaps not surprisingly given its size and lipophilicity, this molecule had high clearance and poor oral bioavailability in mice. 
A second fragment, compound 6, had lower affinity than fragment 1, and parallel chemistry efforts generated only flat SAR. A crystal structure revealed that compound 6 bound in a similar manner as compound 1, with two copies in the central cavity. Surprisingly, a crystal structure of compound 8, which differs from compound 6 only by a single methyl group and actually has slightly lower affinity, revealed a singly copy bound in the central void. Scaffold hopping led to compound 9, which was ultimately optimized through structure-based design and careful attention to drug-like properties to compound 12. This molecule is orally bioavailable and showed activity in a mouse arthritis model.
This is a lovely paper that illustrates several important lessons. First, as the researchers note, “we have learned from multiple programs, including this one, aimed at developing small-molecule inhibitors of protein-protein interactions, that biophysical methods, when used to drive a fragment-based approach, offer the greatest chance of success.” NMR was essential for finding the initial fragments, SPR provided necessary thermodynamic and kinetic information, and crystallography led to the breakthrough discovery of the binding mode of compound 8.
Second, although the initial series generated by fragment linking ultimately did not advance, it proved critical for developing chemical tools, validating assays, and providing structural insights.
And finally, this paper is a paean to persistence for difficult targets. As the researchers note, scientists have been seeking small molecule inhibitors of TNFα for decades, and although compounds were reported as early as 2005, most of these have had poor physicochemical properties. Seemingly undruggable targets can sometimes be unlocked. But it usually takes time.

18 January 2021

Does configurational entropy explain why fragment linking is so hard?

Linking two weak fragments to get a potent binder is something many of us hope for. Unfortunately, as a poll taken a few years back indicates, it often doesn’t work. But why? This is the question tackled by Lingle Wang and collaborators at Schrödinger and D. E. Shaw in a recent J. Chem. Theory Comput. paper.
When a ligand binds to a protein it pays a thermodynamic cost in terms of lost translational and orientational entropy. By linking two fragments, this cost is paid only once instead of twice. In theory this should lead to an improvement of 3.5-4.8 kcal/mol in binding energy, resulting in a 400-3000-fold improvement in affinity over what would be expected from simple additivity. As we noted here, this is possible, though rare. Linker strain often takes the blame as a primary villain. But is there more to the story?
The researchers computationally examined published examples of fragment linking (most of which we’ve covered on Practical Fragments) using free energy perturbation (FEP) to try to understand why the linked molecules bound more or less tightly than expected. Impressively, they were able to computationally reproduce experimentally derived numbers, and by building a thermodynamic cycle they could extract the various components of the “connection Gibbs free energy.” These included changes in binding mode or tautomerization, linker strain or linker interactions with the protein, and the previously mentioned entropic benefits of fragment linking.
The analysis also identified two additional components. If two fragments favorably interact with each other, covalently linking them may not give as much of a boost. This concept had been considered decades ago, though the current work provides a more general understanding.
The more important factor appears to be what the researchers refer to as “configurational entropy.” The notion is that even when a fragment is bound to a protein, both the ligand and protein retain considerable flexibility, which is entropically favorable. Linking two fragments reduces the configurational entropy of each component fragment, and the linked molecule binds less tightly than would be expected. The researchers argue that this previously unrecognized “unfavorable change in the relative configurational entropy of two fragments in the protein pocket upon linkage is the primary reason most fragment linking strategies fail.” They advise that maintaining a bit of flexibility in the linker can help, as has been previously suggested.
This is an interesting analysis, and explicitly considering configurational entropy is likely to improve our understanding of molecular interactions. But is it really the main barrier to successful fragment linking? The researchers explore only nine different protein-ligand systems, though they did consider multiple linked molecules for three of these (pantothenate synthetase, RPA, and LDHA). Still, these represent just a fraction of the 45 examples collected in a recent review, and they only considered one somewhat contrived case (avidin) in which especially strong superadditivity was observed. It will be interesting to see whether the analysis holds true for more examples of fragment linking.

11 January 2021

Hundreds of fragments hits for the SARS-CoV-2 Nsp3 Macrodomain

COVID-19 will be with us for some time. Despite the unprecedented speed of vaccine development, it is worth remembering that humanity has only truly eradicated two widespread viral diseases, smallpox and rinderpest. Thus, the long march of small molecule drug discovery against SARS-CoV-2 is justified. In a paper recently posted on bioRxiv, Ivan Ahel and more than 50 multinational collaborators take the first steps.
Last year we highlighted two independent crystallographic screens against the main protease of SARS-CoV-2. Another potential viral target is the macrodomain (Mac1) portion of non-structural protein 3 (Nsp3), an enzyme which clips ADP-ribose from modified proteins, thus helping the virus evade the immune response.
The researchers soaked crystals of Mac1 against a total of 2683 fragments curated from several collections. This yielded 214 hits, and most of the structures were solved at high resolution (better than 1.35 Å). About 80% of the fragments bound in the active site, with many binding in the adenosine sub-pocket. Two different crystal forms were used for soaking, and one set of 320 fragments was soaked against both. Interestingly, this yielded a hit rate of 21% for one crystal form and just 1.3% for the other. Even more surprising, of the five hits found in both crystal forms, only two bound in the same manner in both. This is a clear demonstration that it is worth investing up-front effort to develop a suitable crystal form of a protein before rushing into soaking experiments.
Independently, the researchers computationally screened more than 20 million fragments (mostly from ZINC15) against the protein using DOCK3.7, a process which took just under 5 hours on a 500-core computer cluster. Of 60 top hits chosen for crystallographic soaking, 20 yielded structures, all at high resolution (0.94-1.01 Å). The ultra-high resolution structures revealed that four fragments had misassigned structures (wrong isomers), which long-time readers may not find surprising. Importantly, most of the 20 experimentally determined structures confirmed the docking predictions.
A strength and weakness of crystallographic screening is that it can find extraordinarily weak binders, which may be difficult to optimize. To see whether they could independently verify binding, the researchers tested 54 of the docking hits in a differential scanning fluorimetry (DSF) assay. Ten increased thermal stability, and all of these had yielded crystal structures. Only four of 19 fragments tested yielded reliable data in isothermal titration calorimetry (ITC) assays, but encouragingly these four also gave among the most significant thermal shifts in the DSF assay. Finally, 57 of the docking hits and 18 of the crystallographic hits were tested in a homogenous time-resolved fluorescence (HTRF) based peptide-displacement assay, yielding 8 and 3 hits respectively, the best with an IC50 of 180 µM.
This paper is a tour de force, and may represent the largest collection of high-resolution crystallographic fragment hits against any target. Laudably, all 234 of the crystal structures have been released in the public domain, and the researchers have already suggested ideas for merging and linking. As they point out, many of the fragments bind in the adenine pocket, so selectivity will be an issue not just against human macrodomains but also against kinases and other ATP-dependent enzymes. Still, as the dozens of approved kinases inhibitors demonstrate, achieving selectivity is possible. 
From a technology perspective, this publication affirms the rising power of both crystallographic and computational screening. Indeed, the hundreds of crystal structures will themselves be useful input for training new computational methods. And from a drug discovery perspective, each of these fragments represents a potential starting point for SARS-CoV-2 leads.
Let’s get busy!

04 January 2021

Fragment events in 2021

Gotten vaccinated yet? Don't worry - the first few conferences of the year will be virtual, but hopefully we'll be meeting in person later this year.

March 9-12:  While not exclusively fragment-focused, the Second NovAliX Virtual Conference on Biophysics in Drug Discovery will have several relevant talks. You can read my impressions of the 2018 event here, the 2017 Strasbourg event here, and Teddy's impressions of the 2013 event herehere, and here.
May 18-19: CHI’s Sixteenth Annual Fragment-Based Drug Discovery, the longest-running fragment event, will again be held virtually. This is part of the larger Drug Discovery Chemistry meeting, running May 18-20. You can read impressions of the 2020 virtual meeting here, the 2019 meeting here, the 2018 meeting here, the 2017 meeting here, the 2016 meeting here; the 2015 meeting herehere, and here; the 2014 meeting here and here; the 2013 meeting here and here; the 2012 meeting here; the 2011 meeting here; and 2010 here.
September 27-30: CHI’s Nineteenth Annual Discovery on Target returns to the real world - or at least Boston. 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 2020 virtual event here, the 2019 event here, and the 2018 event here.

December 16-21: What better place to say goodbye to COVID than Hawaii? Postponed from last year, the second Pacifichem Symposium devoted to fragments will be held in Honolulu. Pacifichem conferences are normally held every 5 years and are designed to bring together scientists from Pacific Rim countries including Australia, Canada, China, Japan, Korea, New Zealand, and the US. Here are my impressions of the 2015 event.
Know of anything else? Please leave a comment or drop me a note!