23 January 2023

The Chemical Probes Portal at Eight

Back in 2015, Practical Fragments highlighted a new resource calling itself “The Chemical Probes Portal.” At the time it included just seven probes, and my post concluded, “I hope this takes off. Understanding the natural world is hard enough even with well-behaved reagents and carefully controlled experiments.”
Well, take off it has, as illustrated by a new (open access) paper in Nucleic Acids Res. by Susanne Müller (Goethe University Frankfurt), Bissan Al-Lazikani (MD Anderson Cancer Center), Paul Workman (Institute of Cancer Research), and collaborators.
The paper notes that “the widespread use of small molecule compounds that are claimed as chemical probes but are lacking sufficient quality, especially being inadequately selective for the desired target or even broadly promiscuous in behavior, has resulted in many erroneous conclusions in the biomedical literature.” As an antidote, the Portal is an “expert review-based public resource to empower chemical probe assessment, selection, and use.”
Any scientist can suggest a potential probe, and these are then internally reviewed and curated. Assuming enough public information is available about the molecule, probes are then sent to three members of a Scientific Expert Review Panel for further vetting. Reviewers rate probes from one to four stars for use in cellular and/or animal models and recommend relevant concentration ranges. Importantly, reviewers can also include comments to highlight off-targets, lack of certain data, oral bioavailability, or anything else.
From a mere seven probes in 2015 the Portal has grown to include more than 500 molecules covering more than 400 protein targets in about 100 protein families. About two thirds of the probes have three or more stars, meaning they are recommended. The Portal is very easy to use and can be searched by probe or protein. Laudably, all the data can also be easily downloaded in bulk.
In addition to the chemical probes, the Portal also contains around 250 “Historical Compounds” that have been described in the literature but “are not recommended to be used to study the function of specific proteins as they are seriously flawed.” These include molecules such as gossypol, a known aggregator that has been reported as an inhibitor of multiple proteins, and curcumin. If you see a molecule used as a probe in the literature, it’s worth checking to see whether it shows up in the Portal.
The Chemical Probes Portal features heavily in a Conversation between Cheryl Arrowsmith (Structural Genomics Consortium) and Paul Workman published (open access) last year in Nat. Commun. The researchers concisely define chemical probes as “small-molecule modulators to interrogate the functions of their target proteins, as opposed to protein location, or other physical properties.” Importantly, they differentiate chemical probes from drugs. “Drugs don’t necessarily need to be as selective as high-quality chemical probes. They just need to get the job done on the disease and be safe to use. In fact, many drugs act on multiple targets as part of their therapeutic mechanism.” I have frequently heard people make comments such as, “this is just a probe, not a drug,” but a good probe should actually be more selective than many drugs.
That said, you do want a drug to actually hit the target of interest. The researchers highlight iniparib, a putative PARP inhibitor that made it all the way to phase 3 clinical trials for breast cancer and was tested in >2500 cancer patients. It failed. Moreover, that failure cast a pall over the field which likely delayed the development of actual PARP inhibitor drugs.
The researchers also discuss aggregators, which are still being reported uncritically in the literature, along with PAINS. “Such compounds should never be considered further or used as chemical probes. They should be excluded from compound libraries. Yet many are sold by commercial vendors as chemical probes and widely used.”
This statement raised the hackles of Pete Kenny. In a recently published critique, he states: “it is asserted in the conversation that commercial vendors are selling compounds as chemical probes that are unfit for purpose and I strongly recommend that anybody making such assertions should carefully examine the supporting evidence.”
Dear reader, please try the following experiment. Enter “iniparib supplier” in your favorite search engine and see what comes up. For me, the first 10 results include several that describe it as a PARP inhibitor. I won’t link to them here because I don’t want to encourage traffic to their sites. (This is also part of the reason Practical Fragments has discontinued PAINS shaming, as it only increases the profile of sloppy or harmful papers.)
Pete goes on to write: “I would strongly advise against making statements that a compound is unfit for use as a chemical probe unless the assertion is supported by measured data in the public domain for the compound in question.”
Frankly, I don’t understand Pete’s position, which I parodied here. Life is short and biology is complicated, so why waste time with dirty or inadequately characterized reagents? For me, everything is an artifact until proven otherwise. And the Chemical Probes Portal goes a long way towards demonstrating whether a particular probe is fit for purpose.

16 January 2023

Fragments vs SARS-CoV-2: the whole proteome

More than three years have passed since SARS-CoV-2 first entered human airways, and it looks set to stay despite the rapid development of remarkably protective vaccines. Drugs such as nirmatrelvir are effective, but as with any infectious agent we’ll need lots more to counteract inevitable mutational resistance. Practical Fragments has previously discussed virtual and experimental screens against individual SARS-CoV-2 viral proteins and RNA. In Angew. Chem., Harald Schwalbe at Goethe University Frankfurt and more than six dozen collaborators in ten countries describe (open access) the results of NMR screens against most of the viral proteome.
The researchers, all part of the COVID19-NMR project, used ligand-detected NMR methods to screen the DSI-PL library against 25 of the 28 viral proteins. As we’ve written previously, the DSI-PL library consists of 768 diverse fragments designed for rapid chemistry follow-up. Fragments were mostly screened in mixtures of twelve, with spectra visually inspected to identify hits for confirmation.
Fragments were classified as binders if they passed any of these four criteria: “chemical shift perturbations (CSPs) or severe line broadening, sign change in the waterLOGSY (wLOGSY), STD signal or significant decrease of signal intensity in a T2-relaxation experiment.”
A total of 311 hits were identified, with between 2 and 154 hits per protein. In some cases multiple forms of the protein were screened. For example, three forms of the main protease (called by various groups nsp5, Mpro, and CLpro) were screened, yielding from 12 to 78 binders, only 8 of which were common to all three screens. One of the protein constructs forms the biologically relevant dimer (the others are monomers), and the researchers suggest this could account for the differences. True, but I suspect many of the “hits” against some of these proteins are artifacts or non-specific binders: researchers at Vernalis, for example, prioritize fragments that hit in two or three different NMR assays over those that hit in just one.
Crystal structures were available for 18 of the proteins screened, and these were computationally analyzed using FTMap to identify between one and three potential small-molecule binding hot spots on each protein. FTMap uses 16 very small probe molecules (such as benzene and urea) to interrogate the protein surface, and a comparison between the NMR hits with those from FTMap was comfortingly good. For example, a protein with a hot spot preferring benzene and urea also bound a fragment containing those moieties. While this by no means proves that the fragments are binding at a given hot spot, it is suggestive.
Not surprisingly, most of the fragments are weak binders: titration experiments revealed that five of ten tested had dissociation constants > 5 mM, though one came in at double-digit micromolar. This result is consistent with work last year that found that most of the crystallographic hits against Mpro were also weak binders, and also consistent with an independent NMR study of Mpro.
Despite these limitations, this campaign provides multiple starting points to develop chemical probes. Laudably, the chemical structures of all the DSI-PL library compounds and the targets hit by each are provided in the supporting information. Last week we highlighted how fragment hits against the SARS-CoV-2 Nsp3 macrodomain were advanced to sub-micromolar inhibitors. The Angew. Chem. work provides fragment starting points against two dozen more targets.

09 January 2023

From fragments to inhibitors of the SARS-CoV-2 Nsp3 macrodomain

Two years ago we highlighted what was likely the largest crystallographic fragment screen against any target, the macrodomain (Mac1) of the nonstructural protein 3 (Nsp3) of SARS-CoV-2. Mac1 dampens the cellular immune response to viral infection by removing ADP-ribose from various proteins. Separate mutational studies suggested this enzyme could be a good target for treating COVID-19.
The 234 fragment hits identified in 2021 could serve as good starting points. This has proven to be true, as demonstrated in a paper just published (open access) in Proc. Nat. Acad. Sci. USA by Brian Shoichet, James Fraser, and collaborators at University of California San Francisco, University of Oxford, Diamond Light Source, Enamine, and Chemspace. Despite the wealth of fragment hits, none of them were particularly potent; the best had an IC50 value of 180 µM in a homogenous time-resolved fluorescence (HTRF) competition assay. In the new paper, the researchers leveraged computational methods to advance these fragments.
First, they explored a fragment-linking approach termed Fragmentstein. This entailed choosing pairs of fragments that bound in close proximity to one another, merging or linking them, docking them to ensure the new molecule would bind in a similar manner to the component fragments, and then searching make-on-demand libraries in Enamine’s REAL database. Four pairs of fragments were evaluated, and 13 of 16 designed compounds were synthesized. Eight of these confirmed crystallographically, and two showed low micromolar activity in the HTRF assay. Interestingly, both of these came from the same fragment pair, ZINC922 and ZINC337835. The best molecule was a mixture of diastereomers, and one of the pure stereoisomers turned out to be submicromolar.
The potency of this fragment is all the more impressive given the low affinity of the initial fragments, which could only be crystallographically characterized using PanDDA, a method to find low-occupancy ligands that we wrote about here. Unfortunately, the compound has low cell permeability, likely due to the carboxylic acid moiety.
In addition to the linking approach via Fragmentstein, the researchers also conducted two virtual docking campaigns with more than 400 million molecules with molecular weights between 250-350 amu. Of 124 molecules purchased and tested, 47 confirmed crystallographically and 13 confirmed by HTRF, with IC50 values from 42 to 504 µM. (Ten of the 13 HTRF hits were also crystallographic hits. The researchers suggest the difference in confirmation rate is due at least in part to compound concentrations, which were 10-40 higher in the crystallographic screens.) In general the crystal structures confirmed the computationally predicted binding modes, particularly for fragments with measurable activity. Structure-based optimization of some of these fragments led to multiple low micromolar inhibitors, such as LRH-0021. Despite the carboxylic acid, this molecule is cell permeable.

This paper nicely illustrates how even very weak fragments can lead to multiple and very different series of inhibitors. The researchers acknowledge that the molecules are still at an early stage of development; indeed, they note that there are currently no good cellular assays to even assess the effect of Mac1 inhibition. Laudably, all the structures are deposited in the Protein Data Bank, which should provide a useful resource not just for further efforts on this protein but for understanding molecular interactions more generally.

02 January 2023

Fragment events in 2023

Happy New Year!

Several good conferences are scheduled for this year, and while the organizers are hoping for robust in-person attendance there will still be virtual options. Hope to see you at one.

April 11-12: CHI’s Eighteenth Annual Fragment-Based Drug Discovery, the longest-running fragment event, is set for sunny San Diego. This is part of the larger Drug Discovery Chemistry meeting. You can read impressions of the 2022 event here, the 2021 virtual meeting here, 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
April 26-28:  While not exclusively fragment-focused, the Ninth NovAliX Conference on Biophysics in Drug Discovery will have several relevant talks. For the first time in five years the event returns to the US (Philadelphia) and and will also offer virtual participation. You can read my impressions of the 2018 Boston event here, the 2017 Strasbourg event here, and Teddy's impressions of the 2013 event herehere, and here.

September 25-28: CHI’s Twenty-first Annual Discovery on Target will be held in Boston, as it was last year. 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 2021 event here, the 2020 virtual event here, the 2019 event here, and the 2018 event here.
Know of anything else? Please leave a comment or drop me a note.

22 December 2022

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.
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.
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 KRASG12D 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.
“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 KRASG12C 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.
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.

12 December 2022

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.

05 December 2022

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-HT2A receptor (5-HT2AR). 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-HT2AR, so the researchers built a homology model based on the closely related 5-HT2BR, 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-HT2AR over 5-HT2BR and 5-HT2CR. 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.

29 November 2022

Fragments vs HDAC2: new metal chelators

Histone deacetylases (HDACs) are epigenetic writers that – as their name suggests – remove acetyl groups from lysine residues in histones. They have been pursued as anticancer targets for decades; vorinostat was approved back in 2006 (and arguably has fragment origins). The catalytic site of HDACs contains a zinc ion, and many inhibitors include zinc-binding moieties, often hydroxamic acids. However, the high affinity of these metallophilic fragments leads to inhibition of other zinc-containing enzymes, causing toxicity. In a new ACS Med. Chem. Lett. paper, Emiliano Tamanini, Shin Miyamura, and colleagues at Astex and Otsuka provide alternative chemistries. (Emiliano presented part of this story at Pacifichem last year.)
The researchers were specifically interested in HDAC2, which has been implicated in neurodegenerative diseases such as Alzheimer’s. Because of the chronic nature of these conditions, selectivity was all the more important, as was brain penetration. Screens of the Astex fragment library, along with a set of known zinc-binding fragments, yielded 35 crystallographically validated hits. These included compound 3, which forms bidentate interactions with the catalytic zinc through the amine and carbonyl moieties. 
An additional fragment (compound 4, not shown) bound in a pocket at the “foot” of the catalytic site, which is normally partially blocked by a side chain residue. Yet another fragment bound near the entrance tunnel. Growing compound 3 in both directions led to compound 7, the first molecule of the series with measurable activity in a fluorescence assay. Rigidification and further growing led to compound 9, with low micromolar activity.
Interestingly, merging compound 9 directly with foot-pocket binder compound 4 did not improve activity, but adding a chlorine atom to fill a small subpocket increased affinity two-fold (compound 13). Finally, optimization of the moiety at the entrance tunnel (the isoindoline of compound 13) yielded compound 17, with high nanomolar potency.
Compound 17 caused increased acetylation of H4K12 in cells. More importantly, it showed good brain exposure when orally dosed in mice, and H4K12 acetylation was observed in mouse brain tissue. The compound hits HDAC1 and HDAC8 with similar potency as HDAC2, and these could be contributing to the biological effect. Nonetheless, while potency and selectivity still need to be improved, compound 17 is an attractive lead for further optimization. 
In a comment to the Notum work we highlighted back in May, Daniel Beck wondered whether “fragments are especially good starting points for CNS target campaigns.” This paper suggests the answer is yes.

21 November 2022

Fragments in the clinic: 2022 edition

In the US we're about to celebrate Thanksgiving. One of the things I'm thankful for is the discovery of new medicines, so this seems like an appropriate time to update our tally of fragment-derived drugs.
As always, this table includes compounds whether or not they are still in development (indeed, some of the companies no longer even exist). Because of this, the Phase 1 section contains a higher proportion of compounds that are no longer progressing. The full list contains 58 molecules, up from 52 last year, with more than 40% approved or in active trials: not bad given that only about 10% of drugs that make it into the clinic are ultimately approved.
Drugs reported as still active in clinicaltrials.gov, company websites, or other sources are in bold, and the 36 that have been discussed on Practical Fragments are hyperlinked to the most relevant post. The list is almost certainly incomplete, particularly for Phase 1 compounds. If you know of others please leave a comment.


PexidartinibPlexxikonCSF1R, KIT
Amgen KRASG12C
VenetoclaxAbbVie/GenentechSelective BCL-2
Phase 3

Navitoclax (ABT-263)AbbottBCL-2/BCLxL
Pelabresib (CP-0610)
Phase 2

AT9283 AstexAurora, JAK2
IndeglitazarPlexxikonpan-PPAR agonist
MAK683NovartisPRC2 EED
Cullinan Oncology / Wistar
Phase 1

ABBV-744AbbottBD2-selective BET
ABT-518AbbottMMP-2 & 9
AT13148AstexAKT, p70S6K, ROCK
AZD5099AstraZenecaBacterial topoisomerase II
BI 1823911Boehringer IngelheimKRASG12C
BI 691751Boehringer IngelheimLTA4H
CFTX-1554Confo TherapeuticsAT2 receptor
HTL0014242Sosei HeptaresmGlu5 NAM
HTL0018318Sosei HeptaresM1-receptor partial agonist
HTL9936Sosei HeptaresM1-receptor partial agonist
NavoximodNew Link/GenentechIDO1

14 November 2022

The agony and ecstasy of thiazoles

Earlier this year we highlighted an analysis of rings found in drugs. Thiazoles are tied for thirteenth place, occurring in at least 30 drugs. (A substructure search in DrugBank pulls up 49.) They pack a lot of diversity into just 5 heavy atoms, with a nitrogen atom capable of acting as a hydrogen bond acceptor as well as a sulfur atom. But they can also be tricksy: an analysis several years ago found that 2-aminothiazoles are over-represented as hits in fragment screens but are often not advanceable. A new open-access paper in ACS Med. Chem. Lett. by Rok Frlan and collaborators at the University of Ljubljana confirms and broadens these conclusions.
The researchers assembled a library of 44 fragment-sized 1,3-thiazoles and five 1,3,4-thiadizaozles. These were then screened at 0.5 or 0.625 mM against four unrelated enzymes in biochemical assays. Two of the enzymes contain catalytic cysteine residues, and these had high hit rats: 14 hits for the SARS-CoV-2 3CLpro enzyme and 26 for the E. coli MurA enzyme. In contrast, MetAP1a had only 3 hits, while DdlB had none. Are any of these hits real?
None of the compounds had been classified as PAINS, and aggregation was deemed unlikely for all but one compound based on chemical searches and the presence of detergent in the assays for MurA, DdlB, and 3CLpro. One compound also seemed to interfere with the fluorescent assays and was ruled a false positive. So far, so good.
However, 8 of the compounds turned out to be unstable in aqueous buffer. Moreover, four compounds turned out to be redox active in at least one of three different assays. Redox cycling can generate reactive oxygen species, which inhibit cysteine-dependent proteins nonspecifically.
Next the researchers tested to see whether their fragments reacted with a small test thiol, 5-mercapto-2-nitrobenzoic acid. Shockingly, 19 of them did, and most of these inhibited at least one of the enzymes. Many of these contain potential leaving groups such as halogen atoms, but some didn’t, leaving the nature of the reaction unclear. Still, the results suggest that the fragments are more thiol-specific than protein-specific, and so another potential source of false leads.
When the researchers retested the ability of the fragments to inhibit the enzymes in the presence of the reducing agent DTT, only one of the CLpro hits reproduced – and that was the compound that showed fluorescence interference. The results were not quite so bad for MurA, though many hits fell out.
Finally, the researchers tried to correlate reactivity with quantum-mechanical calculations using several different methods. Unfortunately, as they note, “no meaningful relationships were observed.” Laudably, data for all the compounds are provided, so interested readers are free to try their own analyses.
In the end it is not clear whether any of the hits will be useful, but the high correlation between pathological mechanisms and activity does not make one optimistic. As the first paragraph above makes clear, this does not mean that thiazoles should be avoided. Indeed, the researchers explicitly state that “we do not want to establish a general knockout criterion to exclude thiazole or thiadizaole screening hits from further development, but it is essential to evaluate their reactivity if they prove to be hits.” This is where orthogonal biophysical methods, such as crystallography, can distinguish true hits from artifacts.

07 November 2022

Fragments vs IL17A: merging and linking

One of the talks at the Discovery on Target meeting last month described the discovery of small molecule inhibitors of IL17A, a pro-inflammatory cytokine. Antibody-based drugs against this protein are useful for psoriasis and other diseases, but they require regular injections. Also, because the antibodies stick around for a long time and dampen the immune response, they could leave patients less able to combat an infection. An orally available small molecule could solve both these problems, but blocking protein-protein interactions is generally difficult. Early progress towards this goal has been published (open access) in Sci. Reports by Eric Goedken and collaborators at AbbVie.
The researchers started by 13C-labeling the methyl groups of isoleucine, leucine, valine, and methionine. They then performed a 2D NMR screen ([1H, 13C]-HSQC) of ~4000 fragments in pools of 12, with each fragment at 1 mM. This yielded multiple hits, including compound 4. Interestingly, the chemical shift perturbations (CSPs) caused by this fragment were distinct from those caused by known previously disclosed binders, suggesting a different binding site. In particular, one of these CSPs could be traced to a methionine residue. The full NMR assignment of the protein was not conducted, but modeling and mutagenesis narrowed the possibility down to a methionine near the C-terminus.
Surface plasmon resonance (SPR) experiments revealed that compound 4 had very low affinity, but two rounds of optimization led to compounds 5 and 6. At this point the researchers were able to solve the crystal structures of these molecules bound to IL17A. The protein exists as a homodimer, and the molecules bind symmetrically, with two copies per homodimer. Moreover, the two copies bind close to one another.

In addition to these fragments, the researchers had also identified compound 7, and a crystal structure revealed that this binds in a similar fashion. Merging compound 7 with compound 6 and linking two copies of the resulting monomer led ultimately to dimeric compound 10, with nanomolar affinity by both SPR and isothermal titration calorimetry (ITC). This molecule inhibited the binding of IL17A with its receptor in a biochemical assay and also showed low micromolar activity in a cellular assay.
This is a nice paper that bears some similarity to previous work we highlighted from AbbVie on a different cytokine, TNFα. There too fragment linking was used initially. As in the present case, that effort led to a drop in ligand efficiency and a significant increase in the size of the molecules, resulting in suboptimal pharmaceutical properties. Identifying drug-like small molecules has been an ongoing challenge for IL17A; peptide-based inhibitors and macrocycles have been found that bind to other sites on the protein, but many of these are also well beyond rule-of-five space. As the researchers conclude, “we look forward to seeing which of these sites prove to be the most amenable to producing optimized drug candidates.”

31 October 2022

From noncovalent fragment to covalent KRASG12C inhibitor

Last week we highlighted Steve Fesik’s presentation at the Discovery on Target meeting in which he discussed the discovery of a covalent inhibitor of the oncology target KRASG12C. The paper describing this work, by Joachim Bröker, Alex Waterson, and collaborators at Boehringer Ingelheim and Vanderbilt University, has just appeared (open access) in J. Med. Chem.
A decade ago we described how the Fesik lab reported finding millimolar fragments that bind to the so-called switch I/II pocket on KRAS. In collaboration with researchers at Boehringer Ingelheim, these were optimized to sub-micromolar ligands that block nucleotide exchange (see here). However, these molecules hit all RAS isoforms and show only modest cell activity. In contrast, the approved drug sotorasib binds in a different pocket, called switch II, and forms a covalent bond with an oncogenic cysteine mutation, G12C.
To find molecules that would bind in the switch II pocket, the researchers first needed to block the switch I/II pocket, which seems to be a hot spot for fragment binding. They did so by introducing a cysteine mutation near the pocket and linking this via a disulfide to a small fragment. All this was done in the context of KRASG12V, a mutant that is more common in cancer than KRASG12C. The modified protein was then screened using two-dimensional protein-observed (HSQC) NMR against 13,000 fragments. This process identified 20 fragments that bind outside of the switch I/II pocket, including compound 1, which bound to the modified protein with mid-micromolar affinity.
A combination of SAR-by-catalog and synthesis suggested the importance of both the amino group and the nitrile, and these observations were confirmed by a crystal structure of compound 1 bound to the protein deep in the switch II pocket, as predicted from the NMR data. The crystal structure also revealed a vector to grow the molecule, leading to compound 12. This molecule had sufficiently high affinity to bind to KRASG12V without the introduction of the switch I/II blocking fragment. Further growing to compound 19 and addition of a phenyl group (compound 20b) led to low micromolar binders. Installation of an acrylamide warhead and further decoration led to BI-0474, which rapidly reacted with the mutant cysteine in KRASG12C. Interestingly, the initial fragment is carried through unchanged.
In addition to potent biochemical activity, BI-0474 showed low nanomolar cell activity. The “bioavailability was not yet optimized,” but intraperitoneal administration led to anti-turmor activity in mouse xenograft models. The paper also notes that “a more advanced orally available analogue from this series has recently entered phase I clinical trials.” As we noted earlier this year, this is BI 1823911.
It is worth contrasting this work with the discovery of sotorasib, which we discussed in 2020. Sotorasib traces its origins to covalent fragment screens, and an electrophile was maintained throughout the optimization process. In contrast, the new paper starts with a non-covalent fragment that was optimized before an electrophilic warhead was introduced. This is probably more typical of how covalent drugs are discovered, as exemplified last year for the BTK inhibitor TAK-020. However, it is not necessarily easy; Steve mentioned in his presentation earlier this month that achieving the optimal configuration of the warhead took some effort.
KRAS has become a poster child for the power of fragment-based approaches to deliver drugs against previously intractable targets. The fact that the new molecules have good non-covalent affinity broadens the range of ligandable oncogenic mutants beyond KRASG12C. Indeed, the researchers end by noting that their approach has led to “molecules that are highly attractive for further use in the discovery of inhibitors against other KRAS mutants.”
Let’s hope they – and others – succeed.