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.