Review of 2022 reviews

The winter solstice is behind us in the Northern Hemisphere, which means 2022 is rapidly drawing to a close. As we have done for the past decade, Practical Fragments will spend this last post of the year summarizing conferences and reviews.

 

The remarkable progress in vaccines against SARS-CoV-2 allowed the full return of in-person conferences, and it was nice to see folks at CHI’s Discovery on Target in Boston and Drug Discovery Chemistry in San Diego. Nearly twenty reviews of interest to this readership were published, and these are covered thematically.

 

Targets

Several reviews cover the use of FBLD to target antiviral and antibacterial targets. Sangeeta Tiwari and colleagues at University of Texas El Paso cover both in an open access Pharmaceuticals review, focusing on tuberculosis and HIV, which often afflict the same individuals, leading to worse outcomes. The paper includes several tables with chemical structures, though the fragment origins of some molecules are not apparent.

 

Tuberculosis is caused by Mycobacterium tuberculosis, but there are more than 170 known members of the Mycobacteriaceae family. In an open access Int J. Mol. Sci. paper, the Tiwari group describes fragment-based approaches against these bugs. In addition to multiple examples, the review provides summaries of fragment finding methods and some of the challenges the field faces.

 

Another organism, Pseudomonas aeruginosa, infects the lungs of people with cystic fibrosis. In an open access Front. Mol. Biosci. paper, Tom Blundell and collaborators at University of Cambridge summarize fragment-based campaigns against this organism and its enzymes. The authors focus on structure-guided methods and note that the work is “at an early stage” but encouraging.

 

Switching to mammalian targets, Katrin Rittinger and colleagues at The Francis Crick Institute review (open access) applications of FBLD for targeting the ubiquitin system in Front. Mol. Biosci. The paper includes a nice table summarizing 15 examples that includes target, enzyme class, fragment binding mode, detection methods, and chemical structures of the fragment hit and optimized compound where applicable. Many of these are covalent modifiers; more on that topic below.

 

Finally, Tarun Jha, Shovanlal Gayen, and collaborators at Jadavpur University discuss “recent trends in fragment-based anticancer drug design strategies” in Biochem. Pharm. In addition to case studies (with chemical structures) of FBLD approaches against 18 oncology targets, the review covers fragment libraries, screening methods, optimization, and challenges.

 

Methods

Many of the targets above are challenging, and it’s always nice to be able to assess how challenging a project might be at the outset. In Curr. Opin. Struct. Biol., Sandor Vajda and collaborators at Boston University and Stony Brook University discuss (open access) “mapping the binding sites of challenging drug targets.” This is a brief, readable account of computational methods to identify hot spots, including allosteric ones. The authors examine the various small-molecule binding sites on KRAS and conclude that, due to “limited druggability,” the “other G12 oncogenic mutants will be very challenging.” Perhaps, but not impossible, as researchers at Mirati demonstrated earlier this year with the (open access) publication of a low (or sub) nanomolar KRAS^G12D inhibitor.

 

Among experimental methods used in FBDD, NMR is a mainstay, as demonstrated by Luca Mureddu and Geerten Vuister (University of Leicester) in Front. Mol. Biosci. (open access). The paper covers methods, successes, and challenges, focusing on three compounds that reached the clinic: AZD3839, venetoclax, and S64315.

 

In contrast to NMR, dynamic combinatorial chemistry (DCC) and DNA-encoded libraries (DEL) are used less frequently in FBLD. In RSC Chem. Biol., Xiaoyu Li and collaborators at University of Hong Kong and Jining Medical University discuss “recent advances in DNA-encoded dynamic libraries.” This concise paper covers lots of ground and does not understate the challenges.

 

Libraries

“The importance of high-quality molecule libraries” is emphasized by Justin Bower and colleagues at the Beatson Institute in Mol. Oncol. This highly readable and wide-ranging open access review covers all aspects of library design and use and includes comparisons of some of the major commercial vendors. An important point is that the “hit rate does not define the success of a library as it is more important to identify ligand-efficient and chemically tractable start points.”

 

Thus, even though shapely fragments may have lower hit rates than more planar aromatic fragments, they may still be worth including – if you can make them. In Drug Discov. Today (open access), Peter O’Brien and collaborators at University of York and Vrije Universiteit Amsterdam review synthetic strategies behind 25 “3D” fragment libraries. The tabular summary showing all the scaffolds emphasizes that most of these libraries are modest in size, with the largest being 102 members. Chemists will particularly enjoy the multiple synthetic schemes. The authors note the importance of “fragment sociability” to facilitate SAR and elaboration.

 

Covalent fragments

Special libraries are required for covalent fragment-based drug discovery, the most notable feature being the “warhead” that reacts with the protein target. These are the focus of a chapter in Adv. Chem. Prot. by Péter Ábrányi-Balogh and György Keserű of the Hungarian Research Centre for Natural Sciences. The review includes a table containing more than 100 warheads with associated mechanisms and amino acid selectivity.

 

The “reactivity of covalent fragments and their role in fragment-based drug design” is the focus in an (open access) Pharmaceuticals review by Kirsten McAulay and colleagues at the Beatson Institute. This is a nice overview of the field and contains several case studies. The authors conclude that “striking a balance between reactivity, potency and selectivity is key to identifying potential candidates.”

 

“Advances in covalent drug discovery” are reviewed (open access) by Dan Nomura and colleagues at University of California Berkeley in Nat. Rev. Drug Disc. This is a highly readable and comprehensive overview of the field. The authors differentiate between “ligand-first” approaches, in which a covalent warhead is appended to a known binder (such as here) and “electrophile-first,” in which “the initial discovery process is rooted in finding a covalent ligand from the outset,” such as for KRAS^G12C inhibitors.

 

Another broad overview of covalent inhibitors is provided by Juswinder Singh (Ankaa Therapeutics) in J. Med. Chem. Jus is a pioneer in the field, having published the first targeted covalent inhibitor in 1997. Of 1673 small molecules approved as drugs by the US FDA, only about 7% are covalent, and it wasn’t until recently that these have been intensively pursued. Part of the reluctance has been concerns over toxicity, but the paper suggests that – at least among kinase inhibitors – covalent drugs may actually be safer, perhaps due to conjugation of glutathione to the warhead and rapid clearance rather than formation of reactive metabolites.

 

Other

Whether covalent or not, thermodynamics plays a fundamental role in protein-ligand interactions, and this is the topic of an (open access) review in Life by Conceição Minetti and David Remeta of the State University of New Jersey. The paper covers a lot of ground, including drug discovery approaches, metrics (such as LE, LLE, etc.), isothermal titration calorimetry, case studies, and more. Importantly, the authors acknowledge the many challenges of applying thermodynamics to drug discovery, some of which we highlighted here.

 

Thermodynamics explains the potency increases longed for when doing fragment-linking, the subject of two reviews. In Chem. Biol. Drug Des. Anthony Coyne and colleagues at University of Cambridge provide a broad overview, starting with the historical theoretical background and newer developments. The bulk of the paper surveys published examples of fragment linking, with structure-based methods (whether X-ray, NMR, or computational) separated from target-guided methods such as DCC.

 

The second review, published in Bioorg. Chem. by Junmei Peng and colleagues at University of South China, is broader in scope, encompassing not just FBLD but also linkers used in PROTACs and even antibody-drug conjugates. The paper is organized by chemical structure of the linker.

 

Finally, in J. Med. Chem., Peter Dragovich, Wolfgang Happ, and colleagues at Genentech and Roche examine “small-molecule lead-finding trends” at their organizations between 2009 and 2020. (Although Genentech is fully owned by Roche, its research organization operates independently.) Fragment-based approaches led to only a small fraction of chemical series at Genentech and none at Roche. The authors note that leads derived from public sources such as patent applications were often found and pursued earlier, and that “purposeful dedication” of resources to fragment approaches may be necessary. Another major source of leads at Genentech is in-licensing, and some of these are fragment-derived.

 

And that’s it for 2022, year three of COVID-19. Thanks for reading and special thanks for commenting. May the coming year bring health, peace, and significant scientific progress.

Fragments win in a virtual screen against the 5-HT2A receptor

Virtual screening is continuing to make impressive strides. The latest example, in Nature, comes from William Wetsel (Duke), John Irwin (UCSF), Georgios Skiniotis (Stanford), Brian Shoichet (UCSF), Bryan Roth (UNC Chapel Hill), Jonathan Ellman (Yale), and a large group of collaborators. The paper has received considerable attention (for example In the Pipeline), but in my opinion the connection to FBLD has been understated.

 

The researchers were interested in finding new agonists for the 5-HT_2A receptor (5-HT_2AR). This GPCR is the target for LSD and psilocybin, both of which have been shown to reduce depression and anxiety. Is it possible to find molecules with similar therapeutic activity but without the accompanying psychedelic properties?

 

LSD contains a tetrahydropyridine (THP) moiety, which is relatively rare in screening libraries. The researchers developed convergent routes to THPs in which they could independently and efficiently vary multiple substituents. Using this chemistry, they constructed a virtual library of 4.3 billion compounds, all with molecular weights ≤ 400 Da and cLogP ≤ 3.5.

 

At the time the research began, there were no structures of 5-HT_2AR, so the researchers built a homology model based on the closely related 5-HT_2BR, which differs by only four amino acid residues in the orthosteric pocket where LSD binds. This model was then screened against a subset of the THP library, those ≤ 350 Da. Despite screening some 7.45 trillion complexes (sampling an average of 92 conformations and 23,000 orientations per molecule), the process took only nine hours on a 1000-core CPU cluster. The result was 300,000 hits in nearly 15,000 families. To ensure novelty, only compounds quite different from known ligands were further considered, and 17 “richly functionalized” THPs were synthesized and tested in radioligand assays. Four were active, including racemic compound 28. Searching the 4.3 billion compound library for analogs ultimately led to compound 70 and a related, slightly more potent molecule lacking the methyl substituent on the amine. A cryo-EM structure subsequently validated the predicted binding mode.

 

The paper spends considerable time characterizing these two compounds. Both are agonists and somewhat selective for 5-HT_2AR over 5-HT_2BR and 5-HT_2CR. They are highly selective over 318 other GPCRs and 45 off-targets. GPCRs can signal through arrestin and/or G-protein, and while LSD works (mainly) through the arrestin pathway, the new molecules work (mainly) through the G-protein route. Importantly, the compounds showed anti-depressive and anti-anxiety effects in mouse models. Although you can’t ask mice if they are tripping, the molecules did not cause “head-twitch responses” and other behavioral effects seen with LSD, suggesting that they may not have hallucinogenic properties.

 

This is a lovely piece of work, and a few observations relevant to FBLD stand out. First, the best molecules are actually rule-of-three compliant, despite the fact that larger molecules were included in the virtual screen. Indeed, the top two molecules are actually smaller than the initial hits. This suggests that choosing more richly functionalized molecules may not have been the most efficient approach. We’ve written previously about V-SYNTHES, which entails stepwise selection and growing of fragments; it would be interesting to retroactively test whether this type of approach would have more quickly gotten to compound 70.

 

Finally, this approach can easily be extended to other scaffolds for which syntheses are readily available. Six years ago we wrote about the synthetic accessibility of dihydroisoquinolines, and last year Practical Fragments published our fifth “fragment library roundup.” The marriage of clever chemistry with virtual screening seems to have a bright future.

Fragments vs SETD2: a chemical probe

Among the various epigenetic “writers,” only one is capable of trimethylating lysine 36 of histone H3. SET domain-containing protein 2 (SETD2) is thought to be a tumor suppressor, but some evidence suggests it may have the opposite effect in certain cancers. A chemical probe would be useful to resolve these conflicting ideas, and in an (open access) ACS Med. Chem. Lett. paper Neil Farrow and colleagues at Epizyme describe one.

 

Epizyme has been pursuing epigenetic targets for years and has built a methyltransfersase-biased compound collection. A radiometric screen of this library yielded compound 1 and a related molecule. Both were weak inhibitors, but a co-crystal structure with the enzyme revealed the indole buried deep in the substrate binding pocket. Tweaking this led to compound 4, with low micromolar activity.

 

 

Substitution off the indole and phenyl moieties ultimately led to compound 25, with low nanomolar biochemical and cell activity. However, this molecule also had low aqueous solubility and poor pharmacokinetics in mice. Recognizing that the lipophilic and aromatic nature of the molecule were likely responsible, the researchers returned to the initial hit. Replacing the phenyl with a cyclohexyl moiety and making a few more modifications ultimately led to EPX-719.

 

The pharmacokinetics of EPX-719 in mice are reasonable, and the molecule is >8000-fold selective against a panel of 14 other histone methyltransferases. It is also fairly clean against a panel of 47 off-targets and 45 kinases. EPX-719 showed antiproliferative activity in two multiple myeloma cell lines, and more detailed biological studies are promised in a future paper.

 

This is a nice hit to lead story. As the researchers note, “close attention to the physical chemical properties of the inhibitors, in particular basicity, lipophilicity, and aromatic character, led to compounds with attractive cellular activities and in vivo exposures.” Interestingly though, the word “fragment” does not appear once in the paper. Although compounds 1 and 4 venture a bit beyond the rule of three, I would argue that starting with small, low affinity binders and focusing closely on molecular properties is the very definition of fragment-based lead discovery.

 

A quarter-century of FBLD has influenced the scientific zeitgeist, and a fragment by any other name is still as sweet.

Success in drug discovery is not necessarily fast or inevitable

The biotech industry rightly prizes speed: every day people die of diseases we are trying to prevent or cure. And developments can indeed happen quickly. Just eight years elapsed from the demonstration that a mutant form of KRAS was druggable to the approval of sotorasib, with less than three of those years spent in the clinic. Even more dramatically, it took less than a year from the first reports of SARS-CoV-2 to develop effective vaccines. But as two recent pieces in Nature Rev. Drug Disc. demonstrate, such speed is not necessarily the norm.

 

The first, by Asher Mullard, is entitled “FDA approves 100^th monoclonal antibody product.” This is a nice review of a remarkably successful therapeutic approach. But this triumph was not a foregone conclusion. Mullard traces the field’s origin to the mid-1970s, and while the first drug was approved in 1986, it took another eight years for the second. The article includes a timeline showing approvals by year, and it is interesting to compare this with FBDD-derived drug approvals since the 1996 publication of the seminal SAR by NMR paper. In the chart below, the first year on the x-axis is for antibody drugs; the second is for FBDD-derived drugs.

 

 

A quarter century after work began, new antibody approvals were still uncommon; Mullard notes that “antibody approvals have only been an annual event since 2006.”

 

Antibody-drug conjugates (ADCs) are an interesting subset that – as their name suggests – comprise an antibody linked to a small molecule, usually a toxin intended to kill cancer cells. Ten of these have been approved in the US, but while the first (gemtuzumab ozogamicin) was approved in 2000 most of the rest are recent, with six of them coming since the beginning of 2019.

 

By these standards the fact that only five fragment-derived drugs have been approved thus far isn’t surprising. Indeed, antibodies have some advantages: “whereas medicinal chemists can toil for years to find small molecules with activity against a given target, antibody discovery can take a matter of months.” Moreover, as the article continues, success in the clinic is roughly double that of small molecules.

 

The second article is by Christopher Austin, until recently Director of the National Center for Advancing Translational Sciences at the US National Institutes of Health. Titled “Translational Misconceptions,” it briefly enumerates and debunks false beliefs about translating new discoveries into drugs, which include:

 

- Translation does not exist 

 

- Translation is a “thermodynamically favored” process 

 

- Translation is straightforward and does not qualify as science 

 

- Translation is a unidirectional process 

 

- Once an investigational therapy gets into humans for the first time, regulatory approval

  and marketing are all but assured 

 

- Regulatory approval is the end of the translational process

 

Those of us in industry would probably dismiss these statements as naïve, but such perceptions are widespread. Indeed, Austin himself acknowledges that he “once believed unquestioningly in all of them.”

 

Each of these misconceptions invites discussion. To take just the last, the first approved ADC was pulled from the US market in 2010 when confirmatory trials showed that patients on the drug actually did worse than those on placebo. It was reapproved in 2017 after a better dosing schedule was established. In other words, it took 17 years after initial approval to figure out how to effectively use gemtuzumab ozogamicin, and 26 years from the beginning of the project.

 

Returning to the two successes mentioned at the top of this post reveals that their apparent rapidity does not tell the full story. The Tethering technology that eventually led to sotorasib was initially published more than twenty years ago, and researchers first used mRNA packaged in liposomes to transfect cells way back in 1989.

 

Amidst rapid visible progress it is easy to lose sight of the fact that much research goes nowhere very slowly. Even when successful, it might take decades to help patients. As Austin concludes, “only by advancing our common understanding of the complexity of translation, translational research and translational science will translational gaps be narrowed and eventually eliminated.”

Review of 2020 reviews

An old curse runs, "may you live in interesting times." And 2020 has been interesting indeed. Amid all the tumult, Practical Fragments will maintain its tradition of ending the year with a post highlighting conferences and reviews.

 

Despite the travel restrictions caused by COVID-19, some conferences did go ahead, adapted to online formats: I highlighted CHI’s Fifteenth Annual Fragment-based Drug Discovery and their Eighteenth Annual Discovery on Target. Although these were quite successful, I think most of us are looking forward to returning to in-person events sometime in the coming year.

 

Perhaps because so many people were stuck working from home, the number of reviews of potential interest to fragment fans has soared to a record number of more than twenty. I’ve tried to group these thematically.

 

General

If you’re looking for a concise yet thorough review, Harren Jhoti and colleagues at Astex provide one in Biochem. Soc. Trans. Harren is one of the pioneers of FBDD, and the review touches on library design, detection of fragment binding, and fragment to lead strategies. A review in Front. Mol. Biosci. by Qingxin Li (Guangzhou Sugarcane Industry Research Institute) goes into more detail on fragment screening, optimization, and biological targets.

 

For the past five years a few fragment fanciers (myself included) have been writing annual reviews in J. Med. Chem. covering fragment-to-lead success stories from the previous year, each with a handy table showing fragment, lead, and key parameters. The 2018 edition, led by yours truly (Frontier Medicines), was published at the beginning of the year, while the 2019 edition, led by Wolfgang Jahnke (Novartis), just came out a few weeks ago. At the risk of self-promotion, both are well worth perusing to see the growing diversity of targets and emerging trends, such as covalent fragments.

 

Biophysics

Biophysical methods are by far the most commonly used for finding fragments, and an excellent overview of thermal shift, SPR, and NMR by Joe Coyle and Reto Walser (Astex) appears in SLAS Discovery. The goal is “to help the anxious biophysicist withstand the relentless unforeseen,” and the paper provides loads of practical advice. For example, over more than 50 thermal shift screens, “we have never derived anything useful from negative Tm shifts.” The researchers note that “SPR is particularly user-friendly and particularly prone to artifact, overinterpretation, and varying degrees of frustration.” As for validating ligand-observed NMR hits crystallographically, rates range from 5% to 80%.

 

As we noted earlier this year, crystallography is becoming increasingly dominant in fragment screening, and in Molecules Laurent Maveyraud and Lionel Mourey (Université de Toulouse) provide an overview of the process, covering theory, workflow, practical aspects, pitfalls, examples, and other emerging methods. David Stuart and colleagues at Diamond Light Source discuss structural efforts on SARS-CoV-2 proteins in an open-access paper in Biochem. Biophys. Res. Commun. As of late October this included more than 500 released structures of 16 different proteins. Efforts against the main protease (which I reviewed in Nat. Commun.) have led to molecules with mid-nanomolar activity, and the researchers rightly highlight the worldwide collaboration that has led to such rapid progress.

 

NMR

NMR is of course a biophysical technique, but there are so many papers this year that it makes sense to group them into their own section. Ray Norton (Monash Institute of Pharmaceutical Sciences) and Wolfgang Jahnke (Novartis) introduce a special issue of J. Biomol. NMR focused on “NMR in pharmaceutical discovery and development” by briefly summarizing the state of the art and introducing 13 articles, one of which we covered previously and three of which are highlighted below.

 

“NMR in target driven drug discovery, why not?” ask Gregg Siegal and collaborators at ZoBio and Gotham in an (open access) J. Biomol. NMR review. In addition to characterizing small molecules, proteins, and their interactions, the researchers present cases studies in which NMR data has helped clarify a crystallographic protein-ligand structure, or even suggested that the crystal structure represented at most a minor conformation in solution.

 

In other words, NMR is “the swiss army knife of drug discovery,” as Reto Horst and colleagues at Pfizer put it in another J. Biomol. NMR review. The researchers describe successful NMR fragment screens against difficult targets such as an ion channel and a large (145 kDa) trimeric enzyme. They also make a good case for using NMR to determine the solution conformations of small molecules early in a project, a strategy that has paid off in more than 15 Pfizer projects over the past six years.

 

Benjamin Diethelm-Varela (University of Maryland) focuses on using NMR for “fragment-based drug discovery of small-molecule anti-cancer targeted therapies” in ChemMedChem. This is a thorough yet accessible overview of FBDD, ligand- and protein-based NMR methods, plus ten case studies. “A practical perspective on the roles of solution NMR spectroscopy in drug discovery” is provided by Qinxin Li and CongBao Kang (A*STAR) in Molecules. As the title suggests, this review is fairly broad, and includes an interesting section on NMR screening in cells.

 

All these papers might have you thinking that NMR is a “Gold Standard,” and that phrase does indeed appear in the title of another Molecules review by Abdul-Hamid Emwas (King Abdullah University of Science and Technology) and a multinational group of collaborators. This is a large (66 page) monograph with 455 references and is particularly detailed on various NMR techniques; if you want to see the pulse sequence of the HSQC experiment or review the Einstein-Stokes equation this is the place to turn.

 

In addition to the six reviews on NMR above, two specifically cover ¹⁹F NMR. The first, from the J. Biomol. NMR special issue by Claudio Dalvit (Lavis) and colleagues, focuses on fluorine NMR functional screening, or n-FABS. This paper provides an excellent theoretical and practical overview of the technique, and includes a handy table of 17 published case studies. And in Prog. Nuc. Mag. Res. Spect. Peter Howe (Syngenta) reviews “recent developments in the use of fluorine NMR in synthesis and characterization.” As the title suggests, much ground is covered, from spectrometer technology to quantum chemistry calculations, and there is a short section on fragment-based screening.

 

Computational

Turning to in silico techniques, Floriano Paes Silva Jr. and collaborators at LaBECFar and several other (mostly) Brazilian institutes provide an open-access overview in Front. Chem. After summarizing FBDD they describe how computational techniques can help along the way, from druggability prediction to docking, de novo design, and assessment of ADMET properties and synthetic accessibility. The review ends with several case studies.

 

In an open-access article in Drug Disc. Today, Stefano Moro and colleagues at University of Padova focus on “the rise of molecular simulations in fragment-based drug design.” This accessible overview covers hotspot identification, hit identification and characterization, and hit to lead optimization, and includes a nice section on free energy perturbation.

 

Other topics

Molecular properties are critical for developing good drugs, and in J. Med. Chem. Christopher Tinworth (GlaxoSmithKline) and Robert Young (Blue Burgundy) “appraise the rule of 5 with measured physicochemical data.” This is packed full of good stuff including a supplementary table with calculated and measured data for hundreds of compounds. The summary is that molecular weight is much less important than (measured) lipophilicity and hydrogen bond donors. “Good practice is all about compromise, aiming to maximize efficacy and efficiency while navigating many potential pitfalls in molecular optimization.” People sometimes obsess over rules vs guidelines, and the researchers close by stating that “rules are for the obedience of fools and guidance of the wise.”

 

As a poll from several years ago suggested, fragment linking tends to be less common than fragment growing, though it can work spectacularly. In J. Med. Chem. Isabelle Krimm and colleagues mostly at Université de Lyon review 45 successful fragment linking case studies (though it would have been appropriate for them to acknowledge Practical Fragments for the clearly borrowed table of clinical compounds). While by no means exhaustive, this is a useful resource. Interestingly, only 20% of the examples display superadditivity.

 

Target-guided synthesis (TGS) can be thought of as a special case of fragment linking. In J. Med. Chem., Rebecca Deprez-Poulain and colleagues at Université de Lille review kinetic TGS, in which two components react irreversibly with one another in the context of a protein to form a higher-affinity binder. Kinetic TGS may have some practical advantages over reversible TGS (or dynamic combinatorial chemistry), but as the researchers note most examples start with compounds larger than fragments, and thus only 38% of examples lead to products with a molecular weight less than 500 Da. This could partly explain why only 6 of the 50 reported examples have gone into animal studies.

 

Finally, György Keserű and collaborators at the Hungarian Research Centre for Natural Sciences review covalent fragment-based drug discovery in Drug Discovery Today (open access). Library design and validation is well-covered, as are various methods for screening covalent fragments, and there is a handy table of some four-dozen published examples. Given the increasing popularity of covalent FBLD, this contribution should be of wide interest.

 

When I wrote my concluding post for 2019, COVID-19 was an obscure and nameless disease, and SARS-CoV-2 had not even been identified. I ended with, "may 2020 bring wisdom, and progress." We've gained both, though the cost has been incalculable. So I'll just close this post by thanking you for reading and commenting.

From noncovalent fragment to reversible covalent CatS inhibitor

We noted just a couple weeks ago that covalent fragment-based approaches have been on a tear. Much of the recent focus has been on irreversible inhibitors, but as we discussed back in 2013 there is much to be said for reversible covalent molecules too. These are the subject of a new paper in J. Med. Chem. by Markus Schade and colleagues at Grünenthal GmbH.

 

The researchers were interested in cathepsin S (CatS), one of 11 members of a family of cysteine proteases. The enzyme has been implicated in a laundry list of diseases, from arthritis to neuropathic pain to Sjögren’s Syndrome, and indeed a few inhibitors entered clinical trials in the early twenty-first century. However, selectivity turns out to be essential: inhibiting the related cathepsin K can lead to cardiovascular problems and stroke. Molecules that appear selective often  contain a basic nitrogen and so can accumulate in lysosomes, achieving sufficiently high local concentrations to inhibit CatK.

 

CatS is a small (24 kDa) enzyme, ideal for protein-observed NMR. An ¹⁵N-HSQC screen of 1858 noncovalent fragments yielded 18 hits, all of which showed similar chemical shift perturbations (CSPs) suggesting binding in the S2 pocket. X-ray crystallography was successful for three fragments, confirming that they do indeed bind in the S2 pocket. Appealingly, this region of the protein is structurally different from the other cathepsins, suggesting a route to selectivity.

 

The sulfonamide moiety of compound 1 (blue) binds in a very similar fashion to the sulfone of a previously reported reversible covalent inhibitor, compound 16 (red). Growing compound 1 to compound 37 led to a significant boost in potency, and crystallography revealed that the binding mode remained the same.

At this point the researchers sought to remove a few heteroatoms as well as introduce the nitrile warhead from compound 16, yielding compound 39b. Surprisingly, this molecule was no more potent than the non-covalent precursor. However, fragment growing into the S3 pocket yielded a massive boost in potency in the form of compound 44. Further SAR and crystallography revealed that much of the increased affinity is due not to specific interactions in the S3 pocket but rather to a hydrogen bond between the newly introduced amide proton and a main chain carbonyl of CatS. Compound 44 is also highly selective against CatK and CatB, showing negligible inhibition of either at 10 µM. Unfortunately, cellular potencies of representative compounds were down by more than three orders of magnitude, likely due to low permeability.

 

While there is still some way to go to establish whether these molecules will succeed where others have failed, this is nonetheless a nice case of fragment-assisted lead discovery And while one can certainly argue that it would have been possible to derive compound 44 from compound 16 through classical medicinal chemistry, fragments clearly helped.

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.