Fragments vs PRMT5/MTA: the runners-up

In January this year we highlighted the discovery of MRTX1719, Mirati’s clinical-stage inhibitor of the PRMT5/MTA complex which is being tested in patients with solid tumors bearing a homozygous MTAP deletion. In a recent article, Chris Smith, Svitlana Kulyk, and collaborators at Mirati and ZoBio discuss some of the other series that came from this campaign. (This article is part of a RSC Med. Chem. special issue on FBDD; more on that early next year.)

 

The researchers note that they chose FBLD “based on timelines” and the fact that they had the capability to “rapidly run a fragment screen.” FBLD is sometimes relegated to second place after other approaches fail, so it is refreshing to see the technique, pushed to the forefront, succeed. Details of the fragment screen are described in the earlier paper; this paper focuses on fragment optimization and elaboration.

 

Of the top 24 fragment hits, five yielded co-crystal structures with PRMT5/MTA. All bound in a similar region and participated in a hydrogen-bond network with the protein as well as van der Waals interactions with MTA. Fragment 2 was structurally unique and was ultimately advanced to MRTX1719, while the other four fragments contained a 2-amino substituent next to an aromatic nitrogen, and these are the focus of this paper. The researchers paid close attention to lipophilic ligand efficiency (LLE) to ensure that increases in potency were being driven by polar interactions rather than hydrophobic interactions that might negatively impact the physicochemical properties of the molecules.

 

Fragment 1 was the most potent, with high nanomolar affinity. Unfortunately, the molecule was not very synthetically tractable. Nonetheless, by merging this fragment with a previously reported PRMT5 inhibitor the researchers were able to obtain low nanomolar compound 9. Interestingly, crystallography revealed that while the fragment maintained its binding mode, the bit taken from the previous molecule bound quite differently than expected.

Fragment 3 was the most lipophilic of the hits, so before diving into serious chemistry the researchers sought to optimize the fragment. This led to compound 13, with lower clogP and improved LLE (as well as LE). Fragment growing quickly led to 150 analogs, with compound 27 showing low nanomolar potency.

 

Fragments 4 and 5, which differ only in the position of a methyl group, were the weakest of the five hits. Like fragment 3 they were also synthetically tractable, and the researchers were able to make 50 analogs, with compound 36 coming in at mid-nanomolar with improved LLE.

 

The paper is a nice case study in fragment- and structure-based design. The use of LLE as an explicit SAR driver is notable, as is the optimization of fragments before beginning growing efforts. The importance of chemical tractability is reflected in the fact that the most potent fragment did not ultimately lead to the clinical compound. It would have been nice to see more discussion on what factors led to the prioritization of the series derived from fragment 2: cell activity, DMPK properties, or other considerations. But at the end of the day the message is that fragments can provide multiple starting points for lead optimization.

From covalent fragment to lead against TEAD

As noted just last month, covalent fragment-based drug discovery is becoming ever more popular. However, many papers report relatively weak hits with little or no optimization. A new preprint posted to bioRxiv (HT Covalent Modifiers) by Tinghu Zhang, Nathanael Gray, and collaborators at Stanford and elsewhere describes a fragment-to-lead story for the TEAD family of transcription factors.

 

The four highly homologous members of the TEAD family play a role in the Hippo signaling pathway. When spurred by the coactivator YAP they cause gene expression that has been implicated in certain cancers, particularly mesothelioma. To bind YAP, TEAD needs to be palmitoylated on a specific cysteine residue. A covalent inhibitor that binds to this cysteine could prevent palmitoylation and thus block Hippo signaling.

 

Multiple academic and industrial groups have been pursuing this target, and one previously reported inhibitor is flufenamic acid. This molecule was used in the new paper to design a small library of analogs each functionalized with an acrylamide moiety. These were screened against TEAD2 and analyzed by mass spectrometry; MYF-01-37 modified the protein (though unfortunately time and exact concentrations are not specified). Proteolysis and tandem mass spectrometry confirmed that the molecule binds to C380, the site of palmitoylation.

 

Analysis of previously published crystal structures revealed a side pocket off the main hydrophobic channel that normally binds the palmitoyl group. The researchers created a focused library of analogs to try to access this pocket, which led to molecules such as MYF-03-69. This compound was active in a biochemical assay and showed rapid labeling of the protein as assessed by mass spectrometry. A crystal structure of the compound bound to TEAD1 confirmed the molecule forms a covalent bond to the target cysteine and does in fact bind in both pockets. 

 

MYF-03-69 inhibited palmitoylation of all four TEAD paralogs in biochemical assays. More importantly, it showed activity in several cell assays, including blocking palmitoylation and disrupting the interaction between TEAD and YAP. The molecule downregulated YAP-TEAD transcription in reporter gene assays as well as RNA sequencing assays. Finally, MYF-03-69 showed mid-nanomolar antiproliferative activity in mesothelioma cells but not in non-cancerous cell lines.

 

Despite this promising activity, MYF-03-69 lacked acceptable oral bioavailability. Further medicinal chemistry led to MYF-03-176, which has improved bioavailability and showed even better activity in reporter gene assays and better antiproliferative activity in mesothelioma cell lines. The molecule also led to tumor regression in a mouse xenograft model when dosed orally.

 

This is a nice story with lots of information, though were I a reviewer I would ask for the k_inact/K_I values for the molecules. This ratio describes the rate of covalent modification and is time and concentration independent, which makes comparisons with other molecules more straightforward (see this 2017 open-access paper for a good discussion). Since this is a preprint hopefully the final published paper will include these values. 

 

Regardless, MYF-03-176 looks like an excellent chemical probe for studying the effect of irreversible inhibition of Hippo signaling.

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.

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.

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.

Fragments vs JAK1, a sequel from LEO

Four years ago we highlighted a paper from LEO Pharma describing inhibitors of the kinase JAK1, which is implicated in a host of inflammatory conditions. Although they developed low nanomolar inhibitors, these showed phototoxicity, which was unacceptable for the topical applications the researchers had in mind. The molecules were also not selective against closely related JAK2, whose inhibition can cause neutropenia and anemia. A recent paper in J. Med. Chem from Andrea Ritźen and collaborators at LEO and GVK Biosciences describes a more selective series.

As mentioned previously, hits came from about 500 fragments screened against JAK2 using SPR and validated in a biochemical assay against JAK1. Most fragments had similar activities against both proteins, but compound 1 was moderately selective for the latter. Initial SAR around the fragment revealed that the methyl group was essential to activity and that methylating the pyrazole nitrogen atoms also obliterated binding. The molecule looks like a hinge-binder, but because it can assume four different tautomers docking was difficult. Fortunately, replacing the difluoromethyl substituent with a phenyl ring in compound 6 improved affinity and led to a crystal structure, which showed the methyl group making lipophilic interactions with the protein.

The crystal structure also revealed that the phenyl ring didn’t quite fill the lipophilic ribose-binding pocket, so the researchers replaced this with the more three-dimensional cyclohexyl substituent in compound 7, which yielded a ten-fold improvement in biochemical potency as well as the first cellular activity. The philosophy behind further optimization is described eloquently: “in the spirit of fragment-based drug design – start small and make every added atom count – small substituents with balanced polarity were added to the cyclohexyl analogue 7.” Unfortunately, although several polar substituents introduced onto the cyclohexyl ring improved biochemical potency, they did not do much for cell activity.

Replacing the cyclohexane moiety of compound 7 with a norbornane led to compound 11, which was not only more potent against JAK1 but also less lipophilic and more soluble. The researchers then borrowed a nitrile from an approved pan-JAK inhibitor, leading to compound 40. This molecule has low nanomolar activity against JAK1 and is somewhat selective against closely related JAK2, JAK3, and TYK2. It is quite selective against a panel of 50 other kinases and does not inhibit several cytochrome P450 enzymes or bind to hERG. Oral bioavailability in rats is fairly low, but this should not be a problem for topical indications.

As noted in the paper, the researchers were able to benefit from published work from multiple other companies that has led to five approved JAK inhibitors plus several more in clinical development. While another JAK inhibitor may not be the most pressing medical need, this paper is still a nice example of structure-based design that illustrates several points. First, ligand efficiency was improved during the optimization process, in contrast to common perceptions. Second, fragment selectivity was also improved during optimization. And finally, although this sounds banal, it matters what you have in your fragment library: had the des-methyl version of compound 1 been the only representative of this core in the LEO library the researchers would not have discovered it. In other words, while simplifying your fragments will decrease molecular complexity, sometimes a single methyl group can make all the difference.

Fragments vs Mycobacterium tuberculosis InhA

Though this could change in a bad way, tuberculosis is currently the deadliest infectious disease worldwide, causing nearly 1.7 million deaths per year. Multidrug-resistant and extensively drug-resistant strains are widespread. Some approved drugs work by blocking the mycolic acid pathway essential for mycobacterial envelope formation. One member of the pathway, the enzyme InhA, is the target of isoniazid and ethionamide. Both of these molecules are prodrugs, and a major mechanism of resistance shuts down their bioactivation. To sidestep this problem, Mohamad Sabbah, Chris Abell, and collaborators at University of Cambridge and Comenius University in Bratislava have targeted InhA directly, as they describe in a recent open-access J. Med. Chem. paper. (See here for a previous FBLD effort against this target.)

The researchers began with a differential scanning fluorimetry (DSF) screen of 800 fragments, each at 5 mM. Forty-two fragments stabilized InhA by at least 3 °C and were tested at 1 mM in three ligand-based NMR assays: CPMG, WaterLOGSY, and STD. All 18 fragments that hit in at least two of these confirmatory assays were soaked into InhA crystals at 20 mM, yielding 5 hits.

None of the fragments inhibited enzymatic activity at 2 mM, but compound 1 was chosen for optimization based on an attractive growth vector into a hydrophobic region of the binding pocket. The carboxylic acid was replaced with an isosteric sulfonamide to yield compound 6, which has measurable activity. Various substituents were tested around the new phenyl ring, with a significant boost in activity caused by an aminomethyl moiety. Cyclizing the molecule and further medicinal chemistry ultimately led to compound 23, with high nanomolar activity. A crystal structure revealed that compound 23 bound as expected and that the primary amine was making interactions with the enzyme cofactor as well as an ordered water molecule.

Unfortunately, although compound 23 slightly inhibited the growth of M. tuberculosis, it did not inhibit synthesis of mycolic acids, suggesting that activity was through a different mechanism. The researchers suggest that the molecules may not be sufficiently cell permeable or that they are effluxed or metabolized. However, it may be that they just aren’t potent enough. Perhaps further medical chemistry will improve affinity by another couple orders of magnitude and achieve pathway inhibition. Regardless, this is a nice example of a robust biophysical assay cascade followed by fragment growing and structure-based design.

Fragments towards the clinic: ERK1/2

The first fragment-based drug to reach the market, vemurafenib, targets a mutant form of the kinase BRAF. Initial responses can be miraculous, but metastatic melanoma is an implacable foe, and patients often relapse. One mechanism of resistance involves upregulation of the kinases ERK1 and EKR2, which are downstream of BRAF. These are the subject of a paper just published in J. Med. Chem. by Tom Heightman and collaborators at Astex and Sygnature.

Most kinase drugs bind to the so-called hinge region of the protein, where the adenine moiety of ATP binds. Previously reported ERK1/2 inhibitors do indeed bind here, but one molecule from Merck (Schering) also binds in a second pocket some distance away. This molecule both inhibits the kinase and also blocks it from becoming phosphorylated itself, thereby preventing it from becoming activated.

Unfortunately this molecule did not have good pharmacokinetic properties, so the researchers sought a new series. They began with virtual, crystallographic, and thermal-shift fragment screens against ERK2. Compound 5 was active in a biochemical assay with impressive ligand efficiency. The bound structure showed multiple interactions with the protein as well as good vectors for further growth. Recognizing that spanning from the hinge region to the second pocket would require a large molecule, the researchers first sought to increase the sp³ character of the fragment to maximize solubility by replacing the pyrazole with a tetrahydropyran (compound 7), which also provided a nice boost in potency. 

Next, the researchers started growing towards the second pocket, guided by docking. This led to another large jump in potency, to the low nanomolar compound 11. Further growth to compound 16 led to marginal improvements in biochemical potency but did show antiproliferative activity in the Colo205 cell line, which contains the oncogenic BRAF mutation. Building into the second pocket, again guided by both modeling and crystallography, significantly improved the cell activity, ultimately leading to compound 27. Consistent with the design, the molecule blocks ERK activity as well as phosphorylation of ERK. It is also orally bioavailable, has good pharmacokinetics, and causes tumor regression in mouse xenograft models. Moreover, Compound 27 is quite selective for ERK1/2 in a panel of 429 kinases.

This is a lovely example of fragment-based and structure-based design. Although the final molecule is on the large side, careful attention to molecular properties maintained acceptable pharmacokinetics. The paper ends by noting that “further pharmacological characterization of 27 will be published elsewhere.” Indeed, Astex has taken an ERK1/2 inhibitor called ASTX029 into the clinic. Practical Fragments wishes everyone involved the best of luck.

Essays in Biochemistry special issue: Structure-based drug design

Structure-based drug design is often an integral part of fragment-based drug discovery. Indeed, a majority of respondents in a recent poll would not work on a fragment without experimental structural information. Given the close relationship between SBDD and FBDD, I was pleased to learn that a recent issue of Essays in Biochemistry is completely devoted to SBDD.

The collection begins with an editorial by issue editors Rob van Montfort and Paul Workman, both at the Institute of Cancer Research. It briefly introduces SBDD and FBDD and provides an overview of the rest of the issue. It also contains a laudable call for rigor, awareness of artifacts, and making data publicly available.

The first full review, by Martin Noble and collaborators at Newcastle University, discusses the role of SBDD in discovering inhibitors of cyclin-dependent protein kinases (CDKs), with a particular focus on selectivity. Several small molecules are discussed, though I do wish the paper included the fragment-derived compound AT7519, which made it to phase 2 clinical trials.

The following paper, by Bas Lamoree and Rod Hubbard (University of York), is completely devoted to FBLD. This is a concise and self-contained review of the field, and is also sufficiently up to date that it provides a good primer on the state of the art.

Chris Abell and collaborators at the University of Cambridge discuss mass spectrometry for fragment screening in the next paper, including ultrafiltration, WAC, HDX-MS, and native mass spectrometry (though not Tethering). The review also includes a handy table summarizing the advantages and limitations of commonly used fragment-finding methods.

Next up is another review devoted to FBDD, this one from Benjamin Cons and his Astex colleagues. The focus is on challenging drug targets such as BCL-family proteins and KEAP1 where SBDD was pivotal, and the researchers particularly emphasize the utility of X-ray crystallography.

NMR was the first experimental technique used for FBDD, and this is the topic of a paper by Gregg Siegal and colleagues at ZoBio. The review includes examples where NMR revealed that crystallographically-determined binding sites were not biologically relevant. Newer techniques, such as NMR², are also discussed.

Frank von Delft and collaborators describe the fourth funding phase of the Structural Genomics Consortium (SGC), which includes generating a couple dozen “target enabling packages” around new genetic targets. The ten year goals are certainly ambitious: “no crystal structure is complete without a careful analysis of the target’s disease linkage, a fully analysed fragment screen, and a series of follow-up compounds with demonstrated potency and rationalized SAR.” Given the tools and partnerships they have already established, I wouldn’t bet against them.

Hitting a single protein target can be difficult enough, but Scott Hughes and Alessio Ciulli (University of Dundee) focus on ternary interactions, in which a small molecule acts as a “molecular glue” to bring proteins together. PROTACS, molecules designed to target proteins for degradation, comprise one class that has garnered significant attention recently, and as we’ve noted previously FBDD could play a role in discovering and optimizing them. Targeted protein degradation is also the subject of the next paper, by Honorine Lebraud and Tom Heightman (Astex). In particular, the researchers focus on the use of click chemistry to rapidly build chemical probes that degrade specific target proteins.

Crystallographers have steadily been shrinking how big a crystal must be for analysis, in part due to brighter X-ray beams. Michael Hennig and collaborators at leadXpro discuss X-ray free electron lasers, which were experimentally realized less than a decade ago. The energy of these photons is more than a billion times higher than in the newest synchrotrons – so powerful that they destroy the crystals almost instantaneously, but not before producing a diffraction pattern. This means that tens of thousands of individual crystals need to be studied in order to obtain a full dataset. Needless to say the technical and computational demands are intense and still being optimized. The rewards include being able to use weakly-diffracting microcrystals, such as those of membrane proteins, and the ability to collect data at physiological temperatures, as opposed to the cryogenic temperatures typically used.

The last paper, by David Barford and collaborators at the MRC Laboratory, discusses the use of cryo-electron microscopy – which was recognized by a Nobel Prize this year. Single particle cryo-EM does not require a crystal at all, and recent advances have made near-atomic resolution possible. The idea is to image thousands of individual proteins and then computationally reconstruct them. The review discusses multiple protein-ligand complexes, and although none of these are from fragment programs, some of the ligands are approaching the size of fragments.

This collection of papers nicely captures where SBDD currently stands and illuminates the path ahead. For at least a while all the articles are free to download – so check them out now!

Uninteresting GPCR Fragment Work...meant as a Compliment!!

There are certain movies that when they are on TV, I can't not watch.  I call these Broken Leg Movies (as in if I were laid up with a broken leg what would I watch).  As I have said, Road House is one, Apollo 13 another.  Its about America's blase attitude towards the amazing feat of putting men on the moon.  It takes a potential horrific tragedy (For those off you who haven't seen it, let me say (**Spoiler Alert**) don't worry it has a happy ending.) in order for America to care about men in space.  Which of course is in direct contrast to Pigs in Space (with Swedish Subtitles)! 

One of the field changing technologies is Heptares' STAR technology (for creating stabilized, soluble GPCRs).   We have discussed it often on this blog.  Well, they are back with another paper, this time working the voodoo they do so well on a Class C GPCR.  Negative allosteric modulation of the mGluR has the potential for significant medical impact in a variety of diseases.  In a relatively well trod drug space (there have been several molecules in late stage trials), an issue appears to be the acetylenic moiety in these drugs (which appears to be manageable).  So, non-acetylenic molecules would be desireable.  

To attempt to ligand this molecule, they screened 3600 non-acetylenic fragments using a radio-labeled assay.  This is in contrast to previous work where they used SPR. From this screen, 178 fragments were tested in concentration-response curves leading to "a number of promising" hits, including the compound shown below. 

Cpd 5.  pKi=5.6, LE=0.36

This compound was advanced using the tools you would expect (especially from Heptares): modeling, X-ray crystallography, medchem, and so on.  The final molecule is an advanced lead with excellent mGluR selectivity and in vivo activity, clean tox, and so on. 

This is excellent work, but "yawn".  I think it might be interesting to hear why they went with the radioligand approach, as opposed to SPR.  You could quibble that 5 is too big to be a fragment, but really?  Papers like this are uninteresting, we know its going to work.  The science is excellent, but I want to see the triumph out of tragedy.  Not here.  I want to congratulate Heptares for making an achievement like this paper perfectly uninteresting.  And I mean uninteresting as the very best of compliments. 

When a Lead is a Lead

As we keep on saying, epigenetics is big.  So, today we present another paper on an old friend, BRD4.  This paper is a follow up from previous work where they used docking and X-ray to find the thiazolidinone fragment hit that was elaborated as shown below (Figure 1), but with potency in the single digit micromolar in vitro and double digit in cellulo. 

Figure 1.  Previous work from these authors.

In this work, they continue developing this scaffold investigating the reversed sulfonamide(Figure 2)

Figure 2.  Reversed Sulfonamide

which had significantly improved activity.  Cyclo-aliphatic rings showed increases in potency, but with larger rings also decreasing ligand efficiency.  Aromatic rings decreased potency and larger groups (rings with linkers) were not tolerated at all.  

The crystal structure of the cyclopentyl derivative was solved and was seen to have a different binding mode from the original fragment.  In this case, the WPF shelf is NOT the major binding site for the compound.  In the end, they ended up with

Figure 3.  End Result of this study.  

This is compound is potent (albeit not super potent), ligand efficient, with cell-based activity,  selectivity, and good PK properties.   What I really like is that final sentence of the conclusion:

a promising BRD4inhibitor and a useful lead for further anticancer drug development.

Huge Library + Tiny Hit Rate = Novel Chemotype

As Dan recently pointed out that I pointed out, epigenetics is big.  Bromodomains get a lot of play on this blog.  One bromodomain that is not mentioned a lot in the literature is ATAD2 (because everyone is actually working on it?).  It is promising because of the diverse cellular activities it is involved in.  However, its bromodomain is quite dissimilar from to "druggable" bromodomains.  [Just an aside, can't we get away from druggable already?] Only 3 of seven residues lining the KAc pocket that interact with peptide are similar (compared to Brd4) (Figure 1)

Figure 1.  View of residues within the KAc binding site of BRD4 that interact with diacetylated residues.  Residues from the peptide are shown in teal.

So, in this paper from the Fesik lab at Vanderbilt, the use fragments to discover chemical matter against this tough target.  They utilized 15N-HSQC, like the previous post on bromodomains, because it can detect millimolar binders and (with resonance assignments) determine where on the protein it is binding.  They screened 13800 fragments (NOT a typo!) as mixtures of 12, or 1150 individual experiments.  Using the SO-FAST pulse sequence allows each experiment to be acquired in 7 minutes (6 days of acquisition).  This required more than 2 grams of labeled material.  Hits were then deconvoluted as singletons, resulting in 65 actives with Kds from 350uM to more than 2 mM (determined by HSQC titration).  12 had affinities of less than 1 mM.  This hit rate of 0.1% is low, especially for a fragment based screen, even against a PPI.  While it may ligandable, a hit rate this low still indicates this will be a very tough nut to crack. 

The assignments of ATAD2 are NOT known, but they observed a consistent cluster of resonances being perturbed (Figure 2).

Figure 2.  A. Fragment 1, B, Fragment 5, C Fragment 12.  Green Circles represent resonances which may report on ligand binding.

They discovered several novel chemotypes, never seen against bromodomains, albeit with a very low hit rate, that could be put in three clusters (Figure 3).  Cluster 1 represents known bromodomain inhibitors, while cluster 2 and cluster 3 are unique to ATAD2.  Interestingly, the Kds only differ by 2-fold, but are still more potent than other recently published ATAD2 compounds.

Figure 3.

One representative from each cluster was crystallized (1, 5, and 12).  All three fragments occupy the same pocket and make a critical contact to the conserved N1064.  They also compare their fragments to work from the SGC that scooped them. 

Figure 4. A. Fragment 1, B, Fragment 5, C Fragment 12.

In the end, this is an unsatisfying paper.  There is speculation as to how these fragments can be progressed and made more potent.  But, this entire paper is about the novel chemotypes for ATAD2.  There is no chemistry in a journal that has Chemistry in its title.  I expect more from this journal and this group.  To summarize, if you throw enough fragments at a target you can find a few that bind.