Twentieth-Fourth Annual Discovery on Target Meeting

Cambridge Healthtech Institute held its annual Discovery on Target (DoT) meeting in Boston last week. Attendance may have set a record, with over 1000 participants. There were sessions on antibodies, conjugates, and radioligands, but I’ll just touch on some of the small molecule themes most relevant to this blog.

 

Covalent approaches

As with last year, covalent approaches to drug discovery were well-represented. Dhirendra Simanshu (Frederick National Laboratory) described several drugs birthed in the NCI RAS Initiative, including ones targeting the G12C mutant of KRAS and C242 on the RAS-binding domain of PI3Kα. He also described how various KRAS mutants cause resistance to drugs, finding that most of them affect the K_I term (binding) more than the k_inact term (bonding). Continuing on the KRAS theme, Jack Sloan discussed efforts at BMS to discover inhibitors targeting both the on- and off-states of KRAS G12C.

 

Kasia Handing (Tango Therapeutics) presented both covalent and non-covalent approaches against VRK1, a synthetic lethal target for glioblastoma. A biochemical screen yielded covalent inhibitors that were characterized crystallographically, but they turned out to be too non-selective to progress. In the meantime, an in-cell DNA-encoded library (DEL) screen led to more advanceable molecules.

 

DEL was indeed a major topic of the conference, and here too covalent approaches are being explored. Elizabeth D’ambrosio described how GSK is identifying covalent binders from DEL screens, a process which includes testing for time-dependence as well as aggressive washing steps under denaturing conditions to quench unreacted molecules and wash away any sticky, non-covalently bound compounds. And Xiaojie Bruce Lu (Chinese Academy of Sciences) described applying DEL to a variety of targets, including some covalently targeting non-cysteine residues.

 

One of the advantages of DEL is being able to screen large libraries, and increasing throughput is also of interest to Harvard’s Steve Gygi, who is screening covalent fragments against cells and cell lysates. In 2024 we described how he was using tandem mass tag (TMT) multiplexing to analyze 18 samples in a single mass-spectrometry run, and he has now extended this to 35 samples. What’s more, Steve is combining 16 different fragments in each sample and introducing clever deconvolution approaches to further accelerate the process.

 

Not everyone is targeting cysteine residues. Mark Tye described the discovery of Revolution Medicines’ zoldonrasib, which uses an aziridine warhead to target the G12D mutant of KRAS. And Balyn Zaro (University of California San Francisco) described using ninhydrin derivatives to target arginine. Most organic chemists are familiar with ninhydrin’s reaction with primary amines such as the N-termini of peptides, but Balyn has been able to optimize selectivity for arginine and has used the resulting chemical probes to identify more than 7000 arginine residues in cells, about 5% of which are hyperreactive. Interestingly, some cell lines seem to have a greater proportion of reactive arginine residues than others.

 

Direct-to-Biology (D2B) approaches

Another recurring theme was direct-to-biology, in which crude reaction mixtures are screened directly in various assays without purification. Indeed, Nicholas Bland of Domainex said that after looking at the agenda of the meeting he considered skipping the introductory slides – and this was on the first official day of the conference. Nicholas described using D2B for optimizing PROTACs as well as for improving fragment hits against the GPCR A_2A receptor.

 

Jack Sadowsky, who also presented at DDC this past spring, described how he and his colleagues at Kimia are using D2B in combination with machine learning to develop selective inhibitors of kinases while also improving their in vitro ADME properties. This effort entailed making and testing tens of thousands of compounds from a virtual space of some 80 million molecules.

 

But even small-scale D2B can be valuable. Trevor Sherwood described how he and his colleagues at BMS used D2B to make and test 663 molecules to improve the potency of an EP4 agonist identified from a high-throughput screen. This involved multiple reaction types, including photoredox chemistry, for which they built a custom-designed reactor to evenly heat and illuminate the microtiter plates. The D2B work was done in parallel with medicinal chemistry, but interestingly the development candidate BMS-986526 came directly from the D2B work and has entered a phase 1/2a trial for rheumatoid arthritis.

 

There were other talks and posters on D2B that I missed, but I did attend two breakfast roundtables on the topic that were quite lively despite happening at 7:30 – a testament to the interest in the topic.

 

“Traditional” fragment-based drug discovery

At a plenary panel moderated by Drug Hunter’s Dennis Hu on drugging difficult targets, Biogen’s Klaus Urbahns asked the other panelists if they were still doing fragment-based lead discovery. John Tallarico of Novartis and Ian Storer of AstraZeneca immediately said yes. And in a separate presentation, Kris Borzilleri described how she and her Pfizer colleagues used fluorine-detected NMR and fragment optimization to identify ligands for an orphan GPCR.

 

Debanu Das described using high-throughput crystallography screening at XPose Therapeutics, now part of Mid-Atlantic BioTherapeutics, to identify inhibitors of DNA damage response pathway proteins. Although the initial hits had no detectable activity, SAR by catalog was able to get to nanomolar inhibitors of the protein APE1, which is overexpressed in ovarian cancer, in a matter of weeks.

 

But perhaps the most robust validation of FBDD was provided by Steve Fesik of Vanderbilt, who described a decades-long effort to drug the MYC pathway, which is probably dysregulated in almost all cancers. We described some of his published work on WDR5 here, but he is now making progress against MYC itself. This has not been easy: MYC is intrinsically disordered, and SAR-by-NMR screens against both the transactivation domain and the DNA-binding domain failed. However, MYC forms a structured leucine-zipper heterodimer with the protein MAX, and screening this complex yielded hits at the DNA binding interface. (Earlier this year we described work from Novartis on a different leucine zipper.) The hits have been optimized to low nanomolar binders that disrupt DNA binding, unlike a slide full of other (alleged) MYC inhibitors, including high-profile commercially available compounds such as MYCi361 and MYCi975.

 

I’ll close – hopefully before you stop reading. If you attended, what talks or posters stood out? And if you wish you had attended, mark your calendar for Oct. 18-21 next year, when DoT returns to Boston in time for peak leaf-peeping season.

Twenty-First Annual Fragment-Based Drug Discovery Meeting

Last week some 875 people attended the CHI Drug Discovery Chemistry (DDC) meeting in San Diego. I can’t do justice to the 40 or so presentations I attended over four days but can highlight some of the main themes.

 

Reversible fragments

Membrane targets such as G protein-coupled receptors (GPCRs) pose a challenge for biophysical methods, but three talks presented progress. Matthew Eddy (University of Florida Gainesville) described high-resolution magic angle spinning (HRMAS) NMR, which entails spinning isolated cellular membranes containing GPCRs at high speed (4 kHz!), which miraculously yields sharp NMR signals for bound ligands. Matthew demonstrated applications with the human adenosine A_2A receptor and weak (mM) ligands. He noted that the technique can work with native, poorly expressed proteins, though data collection times can be upwards of 30 minutes.

 

Kris Borzilleri described using ¹⁹F NMR to find ligands against an orphan GPCR at Pfizer. The 2287 fragments screened yielded 87 hits, of which 38 confirmed by SPR. SAR studies eventually yielded low micromolar ligands, but these were difficult to advance in the absence of structure (see here for a more successful example from Merck).

 

Vanessa Porkolab (Eurofins Cerep) described using the Nanotemper Spectral Shift technology to screen 826 fragments against the adenosine A_2A receptor at 300 µM, with a 9.2% hit rate. Many of these ligands stabilized the GPCR in a thermal shift assay and seven were even active (as antagonists) in a cellular assay.

 

Turning to soluble proteins, Paola Di Lello presented a case study from Genentech and Vernalis applying ligand-observed NMR to the protein phosphatase PTPN22. Subsequent protein-observed NMR revealed that most of the 16 validated hits bound to two pockets some distance from the active site. The fragments were optimized to mid-micromolar affinity but showed no functional activity.

 

And Charlotte Hodson presented the eIF4E story from Astex. As we discussed last year, this yielded a low nanomolar ligand that did not have the desired cellular effects. Charlotte noted that subsequent genetic experiments were consistent with the limited efficacy. Still, the target was sufficiently interesting that a chemical probe would have been pursued even knowing it would be high-risk.

 

Covalent ligands

Covalent approaches made appearances throughout the conference. Keriann Backus (UCLA) described chemoproteomic approaches to find cysteine-targeting ligands; she noted that gain of cysteine residues (such as G12C in KRAS) are the most common missense variants in cancer. Keriann also warned how covalent compounds can cause potentially misleading effects in cells, as she described in Nat. Chem. Biol. last year.

 

In 2021 we wrote about the SpotXplorer fragment library from György Keserű (Hungarian Research Centre for Natural Sciences). György has now prepared a PhotoXplorer library, which uses diazirine tags for photochemical screening, which we described here. The new library has produced high hit rates across a variety of targets. György also described a new sulfozone-based photoprobe that is easier to prepare than diazirines.

 

Kelly Craft recounted a DNA-encoded library (DEL) screen at AbbVie against the target BCL2A1, also known as BFL1. This produced an aldehyde-containing low micromolar binder that formed an imine with buried lysine 102. Uncomfortable progressing an aldehyde, the researchers sought to covalently engage cysteine 55, the same cysteine targeted by AstraZeneca, as we wrote about here. The progression included at least one dual-warhead molecule which was crystallographically confirmed to bind both the lysine and cysteine. The effort ultimately yielded cysteine-selective leads.

 

Earlier this year I described the dDRTC method we developed at Frontier Medicines for determining k_inact/K_I, and Svetlana Kholodar presented a nice overview of its scope and utility. My colleague Johannes Hermann spoke in more detail about our covalent technologies, particularly those using AI.

 

Chemical space and the exploration thereof

Brian Shoichet (UCSF) gave an entertaining and wide-ranging account of “directed and random walks in chemical space.” Brian has consistently been on the bleeding edge of high-throughput in silico screens, from 67,000 compounds in 2009 to 138 million molecules in 2019 to 4 billion molecules today. When docking artifacts are avoided (as we discussed here), bigger libraries consistently produce more potent hits for more targets – an observation strikingly consistent with Alex Shaginian’s in 2023 as HitGen expanded their DEL libraries from billions to more than a trillion molecules. Brian is developing methods to computationally screen the >4 trillion make-on-demand molecules now available from companies such as Enamine.

 

Direct-to-biology (DTB) approaches, which rely on microscale chemical reactions screened without purification, have become increasingly popular methods for exploring chemical space. Jack Sadowsky correctly stated that Carmot was the first company formed around this approach; we previously wrote about the role Chemotype Evolution played in the discovery of sotorasib. Jack described how Kimia, which spun out of Carmot, has continued to advance the technology, applying it to find inhibitors selective for single members of closely related kinase families.

 

Allan Jordan described how Sygnature Discovery is applying DTB in a variety of assays including microsome stability and crystallography. (We wrote about crude reaction screening by crystallography earlier this year.) Expanding beyond DTB, Allan called their platform direct-to-discovery, and discussed how it led to a preclinical candidate with STORM Therapeutics in just 18 months.

 

WuXi Apptec is also using DTB. Peichuan Zhang described starting with ligands derived from fragment and DEL screens against the E3 ligase GID4 to make PROTACs to degrade BRD4; DTB was used to explore a wide range of different linkers. And Daniel Blair (St. Jude) described using DTB and affinity selection-mass spectrometry (AS-MS) to find new molecular glues for the oncology target LCK.

 

Computers, DEL, and DTB are not the only way to explore chemical space. Last year we covered Tom Kodadek’s bead-based screening approach at University of Florida Scripps, and Tom presented two talks on the topic, one using macrocycles to find binders to difficult targets such as PTP1B and one using small molecules to find molecular glues.

 

Speaking of PROTACs and glues, plenary keynote speaker Alessio Ciulli (University of Dundee) discussed the “evolution and future of targeted protein degradation.” Alessio noted that there are >25 PROTAC degraders and >10 glues in the clinic, though these collectively target only a small number of E3 ligands, so there is plenty of opportunity for the area to expand.

 

For many of us in industry, drugs represent the most privileged points in chemical space, and these often look quite different than we assume, as Dean Brown (Jnana) noted in his recent analysis of 104 oral small molecule drugs approved by the FDA from 2020 to 2024 (which we mentioned here). Some drugs contain eye-raising moieties such as acetylenes, styrenes, N-O bonds, and nitro groups. Indeed, it is worth remembering that venetoclax, arguably the most successful fragment-derived drug, sports a nitro group.

 

But before getting too complacent, Jonathan Baell (Manas) warned about frequent hitters in libraries of FDA-approved drugs. He notes in Eur. J. Med. Chem. earlier this year that many commercial libraries are actually enriched for molecules that cause spurious biological activity. Jonathan calls on library vendors to remove particularly egregious compounds, though I’d settle for world peace.

 

I’ll close on that pleasant thought, but please feel free to comment. I hope to see you in San Diego next year April 19-22 for the twenty-second iteration of DDC.

Fragment events in 2026

Lots of interesting events coming up this year - hope to see you at one!

February 17-19:  The Twelfth NovAliX Conference will be held for the first time in San Diego! You can read my impressions of the 2018 Boston event here, the 2017 Strasbourg event here, and Teddy's impressions of the 2013 event here, here, and here. 

 

April 13-16: CHI’s Fragment-Based Drug Discovery turns 21, old enough to legally drink in the US! The longest-running annual fragment event returns as usual to San Diego. This is part of the larger Drug Discovery Chemistry meeting. You can read impressions of the 2025 meeting, the 2024 meeting, the 2023 meeting, the 2022 meeting, the 2021 virtual meeting, the 2020 virtual meeting, the 2019 meeting, the 2018 meeting, the 2017 meeting, the 2016 meeting; the 2015 meeting here, here, and here; the 2014 meeting here and here; the 2013 meeting here and here; the 2012 meeting; the 2011 meeting; and the 2010 meeting. 

May 10-12: Industrial Biostructures of America will be held in Cambridge, MA. The meeting covers all aspects of structural biology including FBLD, membrane proteins, allostery, cryo-EM, machine learning, and more. 

September 14-16: Fragments X, RSC-BMCS Tenth Fragment-based Drug Discovery Meeting, will be held in Cambridge, UK.  You can read my impressions of the 2024 meeting, the 2013 meeting, and the 2009 meeting.

September 28 to October 1: CHI’s Twenty-Fourth Annual Discovery on Target will be held as always in Boston. As the name implies this event is more target-focused than chemistry-focused, but there are always plenty of FBDD-related talks. You can read my impressions of the 2025 meeting, the 2024 meeting, the 2023 meeting, the 2022 meeting, the 2021 meeting, the 2020 virtual meeting, the 2019 meeting, and the 2018 meeting.

 

November 10-12: CHI holds its third Drug Discovery Chemistry Europe in beautiful Barcelona. This will likely include tracks on lead generation, protein-protein interactions, degraders and glues, and machine learning, with multiple fragment talks throughout. 

Know of anything else? Please leave a comment or drop me a note.

Twentieth-Third Annual Discovery on Target Meeting

The CHI Discovery on Target (DoT) meeting was held last week in Boston. More than 850 people from 24 countries attended, 75% from industry. As usual I’ll just touch on some broad themes.

 

Covalent approaches

Covalent approaches were prominent throughout the conference. One of the very first talks was by Stefan Harry (Harvard/MGH), who described screening 416 cancer cell lines with three reactive “scout probes,” identifying some 6000 cysteine residues that could be covalently liganded. There are some interesting cell and context-dependent differences, and all the data are publicly available and easily searchable through a free online portal called DrugMap. He is now profiling a library of dual-electrophile-containing compounds to identify molecular glues.

 

Knowing which cysteines can be targeted is the first step for covalent drug discovery, and Sherry Ke Li described how she and colleagues at Genentech go about finding ligands. They’ve experimentally determined the reactivity of more than 6400 compounds against free cysteine and used this to train a machine-learning model to predict chemical (as opposed to specific) reactivity. Mass spectrometry (MS) using isolated proteins is the workhorse screening approach, but Sherry also described using variable temperature surface plasmon resonance (SPR) to dissect the individual components of k_inact/K_I.

 

AstraZeneca has also been doing considerable covalent screening, and Hua Xu briefly described the BFL1 story we wrote about here. In addition to pure proteins, they are now also starting to screen their covalent library in cells. Hua also presented earlier work from Pfizer on the discovery of the covalent kinase inhibitor ritlecitinib, which started with a noncovalent binder. Proteomic studies revealed that in addition to the intended target JAK3, it hits other TEC-family kinases too.

 

Adding a covalent warhead to a reversible binder is also the approach taken by MOMA Therapeutics in the discovery of their clinical WRN inhibitor MOMA-341, as presented by Momar Toure. They ended up targeting the same cysteine as Vividion (see here), though the binding mode is somewhat different. MOMA is also pursuing covalent fragment screening using intact protein MS, and Brian Sosa-Alvarado described how they were able to identify nanomolar inhibitors of RAD54L within six months of starting the program, aided by DNA-encoded libraries (DEL).

 

Not everyone is pursuing cysteine: Ken Hsu described how he and his team at University of Texas Austin are using sulfur-triazole exchange chemistry (SuTEx) to target tyrosine residues across the proteome. He noted that although cysteine could react with this warhead, the resulting thiosulfonate would be unstable. This is true in general, but I wonder if, just like the reversible cyanoacrylamide warheads we wrote about more than a decade ago, they could be stabilized within folded proteins.

 

Noncovalent approaches

Covalency is not the only game in town, as exemplified in a talk by Emma Rivers on “integrated hit discovery” at AstraZeneca. I was tickled that she grouped FBLD with HTS as “traditional” approaches, onto which they’ve added DEL and peptide libraries. Importantly, they’re focused on generating and capturing as much high-quality data as possible to enable machine learning – a topic we’ll touch on more below.

 

Nor are proteins the only target; there was a whole track on RNA- and DNA-targeting small molecules, where Benjamin Brigham described the plate-based equilibrium dialysis-based approach taken at Atavistik to screen metabolites and metabolite-like molecules. This led to two fragment-sized hits against RNA encoding SERPINA1, and although the affinities are modest, they do inhibit translation in a cell-free system.

 

At FBLD 2018 Astex presented the first cryo-EM structure of a fragment-protein complex, noting that throughput was an issue. The company has leaned into that challenge and now has three microscopes, including a top-of-the-line Krios, with another on the way. Miguel Zamora-Porras described how they have now solved hundreds of structures. Their Krios can collect data on two compounds per day, and the full cycle time from protein-ligand preparation to structure is about a week. Miguel described how structures of ligands bound to the ion channel protein TRPML1 helped reveal why some were agonists and others antagonists.

 

Data and its discontents

On the subject of structures, Steve Burley (Rutgers) gave an eloquent history and defense of the “RCSB Protein Data Bank: an open access research resource that benefits all humanity.” From its humble beginnings with just seven structures in 1971, the PDB now contains more than 240,000. And these are not just of scientific interest: all 88 of the new molecular entities the FDA approved for oncology between 2010 and 2023 had PDB structures that informed the biology or druggability, and 75% of the efforts involved structure-based design. Steve also mentioned that the question of where and how to store large-scale crystallographic data will be discussed in a meeting sometime in the spring of next year. Finally, Steve is hoping to retire from his position as Director of the RCSB PDB, so if you’re looking to make an impact, please apply.

 

The dramatic advances in protein structure prediction exemplified by AlphaFold would not have been possible without the PDB, but unfortunately the same high-quality information on protein-ligand binding modes and affinities is not available, as noted by Woody Sherman of Psivant. To illustrate the importance of training data, Woody asked ChatGPT to produce a picture of an analog clock set to 6:32. The result? A clock with three hands, one at 10, one at 2, and one at 6, because most images of clocks are set to 10:10.

 

Woody asked whether machine-learning-based docking can extrapolate or just interpolate. Although impressive results have been reported for some protein-ligand complexes, it turns out that there are often similar ligands in the training data. For truly novel ligands, the predictions tend to fall flat. Similarly, allosteric ligands are often (mis)placed into an orthosteric site – just because the model has been trained that that’s where ligands should go. Indeed, although Psivant is heavily invested in computational approaches, Woody mentioned that they often use “wet” approaches for finding initial chemical matter.

 

On the subject of dubious data, Al Edwards (University of Toronto) noted that a third of all immunofluorescence images in the literature use antibodies that give signals in knockout cells. And as we wrote just last month, many reported small molecule “probes” are just as bad. Al is CEO of the Structural Genomics Consortium, whose ambitious Target 2035 aims to find a pharmacological probe for every target in the human genome. As a starter, they’re aiming for 2000 probes in the next five years. They’re using affinity-selected mass spectrometry (ASMS), screening pools of 500 compounds and 8 proteins at a time, and are getting micromolar hits against about 30% of targets. They’re accepting protein submissions, so if you’re looking for starting points against your favorite protein contact them.

 

I’ll end here, but please leave comments. And mark your calendar for Sep. 28 to Oct. 1 next year, when DoT returns to Boston.

Fragment events in 2025 and 2026

For better or for worse, 2025 is half-way over. There are still some good conferences coming up, and 2026 is also starting to take shape.

September 21-24: FBLD 2025 will be held in the original Cambridge (UK),  where it was supposed to be held in 2020. This will mark the ninth in an illustrious series of conferences organized by scientists for scientists. You can read impressions of FBLD 2024, FBLD 2018, FBLD 2016, FBLD 2014, FBLD 2012, FBLD 2010, and FBLD 2009. 

 

September 22-25: You'll need to make a tough choice: FBLD 2025 or CHI’s Twenty-Third Annual Discovery on Target in Boston. As the name implies this event is more target-focused than chemistry-focused, but there are always plenty of FBDD-related talks. You can read my impressions of the 2024 meeting, the 2023 meeting, the 2022 meeting, the 2021 meeting, the 2020 virtual meeting, the 2019 meeting, and the 2018 meeting.

 

November 11-13: CHI holds its second Drug Discovery Chemistry Europe in beautiful Barcelona. This will include tracks on lead generation, protein-protein interactions, degraders and glues, and machine learning, with multiple fragment talks throughout. 

2026

February 17-19:  The Twelfth NovAliX Conference will be held for the first time in San Diego! (Please note the date and location change.) You can read my impressions of the 2018 Boston event here, the 2017 Strasbourg event here, and Teddy's impressions of the 2013 event here, here, and here. 

 

April 13-16: CHI’s Fragment-Based Drug Discovery turns 21, old enough to legally drink in the US! The longest-running annual fragment event returns as always to San Diego. This is part of the larger Drug Discovery Chemistry meeting. You can read impressions of the 2025 meeting, the 2024 meeting, the 2023 meeting, the 2022 meeting, the 2021 virtual meeting, the 2020 virtual meeting, the 2019 meeting, the 2018 meeting, the 2017 meeting, the 2016 meeting; the 2015 meeting here, here, and here; the 2014 meeting here and here; the 2013 meeting here and here; the 2012 meeting; the 2011 meeting; and the 2010 meeting. 

September 14-16: RSC-BMCS Tenth Fragment-based Drug Discovery Meeting will be held in Cambridge, UK.  You can read my impressions of the 2024 meeting, the 2013 meeting, and the 2009 meeting.

 

Know of anything else? Please leave a comment or drop me a note.

Twentieth Annual Fragment-Based Drug Discovery Meeting

Last week’s CHI Drug Discovery Chemistry (DDC) meeting was held as usual in San Diego. More than 850 people attended, 96% in person, with 70% from industry and 28% from outside the US. I personally attended more than three dozen talks over the four days and will just touch on some broad themes.

 

Noncovalent approaches

Steve Fesik (Vanderbilt) gave two talks, the first of which was focused on “FBDD tips for success.” This opinionated and entertaining romp revealed lessons learned across several projects on difficult targets such as KRAS. Another holy grail oncology target is MYC, which is largely disordered. A two-dimensional NMR screen against the protein failed to yield any hits, but a screen of the MYC:MAX heterodimer provided hits which have been optimized to high nanomolar potency and are able to block DNA binding.

 

The second talk was focused on E3 ligases, a target class Steve has been pursuing for the past decade. Steve is particularly interested in E3 ligases such as CBL-C, TRAF4, and KLHL12 that are differentially expressed in certain tissues. In the case of KLHL12, which is not found in heart tissue, an NMR-based screen led to fragment hits that were ultimately optimized to mid-nanomolar binders and could be turned into bivalent degraders for Bcl-xL and β-catenin.

 

When asked about his second favorite fragment-finding method after protein-detected NMR, Steve mentioned SPR. The throughput for SPR has historically been modest, but John Quinn (Genentech) described the new Carterra Ultra, which is capable of screening 96 proteins simultaneously while retaining good sensitivity. John screened 3000 fragments at 500 µM against multiple proteins in just two weeks, which provided an immediate assessment of both protein ligandability and fragment selectivity. Interestingly, and in contrast to some other analyses, shapelier fragments had similar hit rates to flatter fragments.

 

Several talks focused on fragment-to-lead success stories, some of which we’ve covered on Practical Fragments, such as RIP2 kinase inhibitors that started from flat fragments and were evolved to more three-dimensional leads as described by Mark Elban (GSK). John Taylor discussed pan-RAS inhibitors discovered at Cancer Research Horizons, the subject of an upcoming post. Andrew Judd (AbbVie) described the discovery of ABBV-973, a potent STING agonist that could be useful for certain types of cancer. And Justyna Sikorska described the discovery of a non-covalent WRN inhibitor at Merck. This is a nice complement to Vividion’s covalent WRN inhibitor, which we wrote about here and which was presented by Shota Kikuchi. Interestingly, structural biology was not enabled until late in this project.

 

One of the earliest arguments for fragment linking was the concept of avidity, and this underlies the basis of a technology discussed by Tom Kodadek and Isuru Jayalath at University of Florida Scripps. The idea is to immobilize fragments onto TentaGel beads, each the size of a red blood cell. These can be screened against multivalent proteins using either simple plate-based assays or FACS, the idea being that even if an individual protein-ligand interaction is weak, a multimeric protein can interact with several ligands on a single bead for enhanced binding. The researchers validated the concept with streptavidin, and also used it to find millimolar binders to the proteasome subunit Rpn13.

 

Last year we wrote about using photoaffinity crosslinking with fully functionalized fragments (FFFs) to identify non-covalent ligands to thousands of proteins in cells, and this was the subject of several talks. Chris Parker (Scripps) has mapped more than 7000 binding sites and described the discovery of an inhibitor against the inflammatory target SLC15A4. Interestingly, the molecule binds what appears to be a disordered region, though Chris speculated that it adopts a more defined structure in cells.

 

Belharra has gone all in on using FFFs, and Jarrett Remsberg and Andrew Wang described the construction of a diverse >11,000-membered FFF library, 88% of which consists of enantiomers. This has been screened against 13 different oncology and immunology cell lines to identify enantioselective or chemoselective hits against >4000 proteins including STAT3, IRF3, and AR.

 

Covalent approaches

The FFF approach uses covalent bond formation to trap a noncovalent ligand, but of course covalent ligands are all the rage these days, as we noted just last week. Dan Nomura (UC Berkeley) described the identification of stereoselective covalent ligands against a disordered region of cMYC that seem to work by destabilizing the protein in cells. Similarly, covalent ligands against the largely disordered AR-V7 also seem to destabilize the protein. It will be interesting to explore the mechanism of these molecules to see whether the proteins are more ordered inside cells.

 

Jin Wang (Baylor College of Medicine) described a chemoproteomic approach called Fragment Probe Protein Enrichment (FraPPE) which entails linking covalent fragments to a desthiobiotin tag. Labeled proteins are then pulled down, proteolyzed, and analyzed by mass spectrometry. In contrast, competition methods such as those described last year pull down labeled peptides after proteolysis. The advantage of FraPPE is that it can capture multiple peptides from each pulled-down protein, leading to fewer false negatives.

 

Of course, not every application of covalent discovery involves chemoproteomics. Joe Patel, who co-organized FBLD 2016, described the Nexo Therapeutics platform. They’ve built from scratch a library of >12,000 fragments, a third of which contain stereocenters. Each member is rule-of-three compliant before adding the warhead, meaning that the final molecules can be larger, which as we noted earlier this month is probably a good idea. To date Nexo has successfully screened more than a dozen targets using intact protein mass spectrometry.

 

The Nexo library targets not only cysteines but other residues as well, and Maurizio Pellecchia (UC Riverside) described using sulfonyl fluorides and fluorosulfates to target histidine residues. He and his group screened a library of 600 fluorosulfate-containing fragments (MW 250-350 Da) against the oncology target MCL1 and found several that stabilized the protein towards thermal denaturation. Crystallography confirmed covalent bond formation.

 

Most covalent fragments are electrophilic so that they can react with nucleophilic protein residues, but as we noted in 2022 it is possible to do the reverse. Megan Matthews (University of Pennsylvania) described how she used chemoproteomics to discover the mechanism of action for hydralazine, a drug that has been used since 1949 to treat hypertension. This fragment-sized (MW 160 Da!) molecule irreversibly alkylates a histidine residue within the active site of the enzyme ADO, a target that has also been implicated in gliobastoma.

 

Plenary Keynotes

The approval of the covalent BTK inhibitor ibrutinib in 2013 arguably marks the start of the modern era of covalent drug discovery, and Chris Helal described Biogen’s efforts against this target using reversible inhibitors, irreversible inhibitors, and degraders. Chris traced the origin of their phase-2 BIIB091 to a collaboration with Sunesis that used Tethering, so perhaps we should include this molecule in our list of fragment-derived clinical compounds.

 

Phil Baran of Scripps, who last spoke at the conference in 2020, gave the secondary plenary keynote. After stating that “medicinal chemists are the backbone of society,” he then detailed multiple examples of how they’ve been doing things wrong. Fortunately, he provided useful chemistry solutions, with “useful” defined as reactions that are operationally simple, have wide scope, and require only readily available reagents. Rather than deploying tedious protecting group installations and deprotections, Phil uses radical chemistry to directly generate carbon-carbon bonds between or within complicated molecules. His goal is to make the chemistry so simple and practical as to be boring, and he illustrated the point by showing his teenage daughter successfully running a reaction.

 

I’ll end here, but please leave comments. And mark your calendar for April 13-16 next year, when DDC returns to San Diego.

Fragment events in 2025

After a bumper year for conferences in 2024, this year is also shaping up to be eventful. 

April 14-17: CHI’s Twentieth Annual Fragment-Based Drug Discovery, the longest-running annual fragment event, returns as always to San Diego. This is part of the larger Drug Discovery Chemistry meeting. You can read impressions of the 2024 meeting here, the 2023 meeting here, 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 here, here, 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. 

 

May 5-6: Returning after a five year hiatus, Industrial Biostructures of America will be held in Cambridge, MA and includes a session on FBLD. 

June 2-4:  The Eleventh NovAliX Conference returns to the stunning city of Strasbourg. You can read my impressions of the 2018 Boston event here, the 2017 Strasbourg event here, and Teddy's impressions of the 2013 event here, here, and here.

September 21-24: FBLD 2025 will be held in the original Cambridge (UK),  where it was supposed to be held in 2020. This will mark the ninth in an illustrious series of conferences organized by scientists for scientists. You can read impressions of FBLD 2024, FBLD 2018, FBLD 2016, FBLD 2014, FBLD 2012, FBLD 2010, and FBLD 2009. 

 

September 22-25: You'll need to make a tough choice: FBLD 2025 or CHI’s Twenty-Third Annual Discovery on Target. 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 2024 meeting here, the 2023 meeting here, the 2022 meeting here, the 2021 event here, the 2020 virtual event here, the 2019 event here, and the 2018 event here.

 

Finally, from November 11-13 CHI holds its second Drug Discovery Chemistry Europe in beautiful Barcelona. This will include likely tracks on lead generation, protein-protein interactions, degraders, machine learning, and probably several fragment talks. 

   

Know of anything else? Please leave a comment or drop me a note.

Discovery on Target 2024

Last week Boston hosted CHI's 22^nd Annual Discovery on Target. With dozens of talks spread across seven or eight concurrent tracks over three days, and an additional day of pre-conference symposia, I’ll just touch on a few themes.

 

Computational Approaches

Artificial intelligence and machine learning were well represented. Brandon White described an ML model built at Axiom to predict liver toxicity, responsible for a quarter of clinical trial failures. As we noted last week, good ML models require lots of data, and Axiom has tested 50,000 small molecules in primary human hepatocytes from multiple donors using assays including high-content imaging. Just input a chemical structure and the model will predict toxicity. When run against the FDA’s database of drug-induced liver injury, the model performed with 74% sensitivity and 97% specificity, and even gave good dose predictions.

 

Woody Sherman (Psivant) laid out a series of “grand challenges for computers in drug discovery.” This is the working title for a publication he is spearheading to focus attention on key problems. They fall into five categories: chemistry (including synthesis, stability, and covalency), structure predictions (including protein-ligand structures, dynamics, and cryptic pockets), energetics (including affinity, selectivity, and kinetics), ADME (including everything from solubility and aggregation to bioavailability), and pharmacology (including toxicity). A sixth category, human considerations (including intellectual property and interpreting experimental data), is also being considered.

 

The success of AlphaFold to predict protein structures shows what computers can achieve, but in that case the effort was enabled by massive amounts of high-quality public data in the Protein Data Bank. Few of these challenges can draw on anything approaching the PDB. Indeed, even parameters as seemingly simple as solubility can change dramatically depending on crystal form and subtle changes to pH.

 

Because these computational challenges are so daunting, collecting them into one forum may prove salutary. And other categories may be worth including, such as target discovery. Woody is looking for co-authors, so reach out to him if you’re interested.

 

Covalent approaches

Covalent approaches to drug discovery have gone mainstream, at least if this conference is any indication. But they are not without risk: Doug Johnson (Biogen) described research implicating the piperidine acrylamide pharmacophore in approved BTK inhibitors with inhibition of ALDH1A1 and possible liver injury.

 

Several talks focused on methodologies. Alexander Federation (Talus) described data-independent acquisition (DIA) mass spectrometry methods, which can be more comprehensive than the more commonly used data-dependent acquisition (DDA) methods in identifying peptides in chemoproteomic studies, which we first discussed here. Talus is focused specifically on transcription factors.

 

As we noted earlier this year, Steve Gygi (Harvard) has been at the forefront of increasing the throughput of mass spectrometry methods, and he described how to increase the number of samples that can be analyzed simultaneously from 18 to 35. He also described two approaches, GoDig and CysDig, to look for up to 200 pre-specified proteins in a sample, ensuring identification of even low-abundance targets.

 

Turning to specific targets, Wai Cheung Adrian Chan described work done at Harvard to find covalent inhibitors against deubiquitinating enzymes (DUBs), reporting that screens of a small library of 178 covalent fragments in cell lysates found hits against several dozen DUBs. (We previously wrote about non-covalent USP7 inhibitors.)

 

Brooke Brauer described the optimization of a covalent inhibitor of Bfl-1 at AstraZeneca, an interesting oncology target. AZ has published some nice papers on this project which I’ll write about soon.

 

Last week we mentioned work Michelle Arkin and collaborators had done on 14-3-3 proteins, and Lynn McGregor described work done at Novartis on the same system. A screen of 6000 covalent compounds identified hits that modified a specific cysteine in 14-3-3 more rapidly in the presence of a peptide derived from the estrogen receptor. Stabilizing this interaction could be useful for treating certain cancers.

 

Not everyone is focused on cysteine: Andrea Zuhl described work done at Hyku Biosciences, which as the name suggests is targeting histidine, tyrosine, and lysine. This has necessitated building a fragment library of more than 6000 compounds, more than 70% of which are stable in buffer. Andrea presented one example targeting the catalytic lysine residue of the oncogenic ALK fusion protein, though the selectivity against other kinases was not disclosed.

 

All of these examples focused on covalent molecules in which the warhead is maintained during optimization. But as we first wrote about here, fully functionalized fragments (FFFs) contain a photoreactive moiety that reacts covalently with nearby proteins but is subsequently discarded. Sherry Niessen described how Belharra has industrialized this process by creating a library of about 11,000 FFF probes. Because of the low efficiency of protein crosslinking (typically <5%), most of the library consists of enantiomeric pairs to facilitate hit identification. Also, the average molecular weight of the library is around 350 Da, and these super-sized fragments tend to perform better than the strictly rule-of-three compliant molecules.

 

Covalent success stories

At least two presentations covered covalent fragment-based drug candidates. Shota Kikuchi (Vividion) described the discovery of VVD-214/RO7589831, a WRN inhibitor we wrote about earlier this year. As I speculated at the time, the cyclopropyl group was introduced to lower the reactivity of the vinyl sulfone warhead. Interestingly though, even early molecules were quite selective for WRN. Like sotorasib, binding is largely driven by the k_inact term of k_inact/K_i, again demonstrating that high reactivity for the target does not necessarily mean high chemical reactivity.

 

Finally, in his plenary keynote Steve Fesik (Vanderbilt University) covered multiple success stories, including the discovery of the KRAS^G12C inhibitor BI 1823911, which we wrote about here. Boehringer Ingelheim has since published molecules that hit multiple KRAS mutants as well as KRAS degraders, and Steve noted that all of these contain the same “squirrely-looking” fragment identified from SAR by NMR, an illustration of the power of fragment-based methods to explore new regions of chemical space.

 

I’ll close there, but please add your thoughts. There are is still at least one good conference coming up this year, and 2025 is quickly approaching.

FBLD 2024

The FBLD meetings have always been calendar highlights. Starting in 2008, before Practical Fragments even existed, they have graced cities around the world in 2009, 2010, 2012, 2014, 2016, and 2018. The plan was for 2020 to be held in Cambridge, UK, but for obvious reasons that didn’t happen. Last week, Boston hosted a triumphant return of the event. With more than 30 talks and dozens of posters I’ll just touch on a few major themes.

 

Crystallography

High-throughput crystallography was prevalent, as befits its growing role in fragment finding. (If you haven’t yet voted in our methods poll on the right side of the page please do so!) Debanu Das (XPose Therapeutics) described how crystallographic screens of just a few hundred fragments identified hits against DNA-damage response proteins such as APE1; these have been advanced to high-nanomolar inhibitors with cell activity. And Andreas Pica described the ALPX platform that enabled screening >4000 hits from an HTS screen against PDEδ resulting in >500 structures.

 

The Diamond Light Source was a pioneer in developing high-throughput crystallography methods, and several speakers described continued progress. Blake Balcomb noted that since 2015 they have collected >240,000 datasets and identified >30,000 ligands. Of these, some 3750 have been deposited into the Protein Data Bank.

 

A crystallographic fragment hit is just the start, and Frank von Delft emphasized that “fragment progression is neither fast nor cheap.” His goal is to take a 100 µM binder to a 10 nM lead in less than a week for less than £1000. Toward this end he and his team are using rapid chemical synthesis and crude reaction screening along with various computational approaches and crowd-sourced science. The COVID Moonshot, which we wrote about here, is one model, and Diamond is trying to create a “Moonshot factory” to pursue other viral targets.

 

Computational Approaches

Computational methods are potentially the least expensive fragment-to-lead method, and these were well represented. One challenge is screening the massive chemical space represented by make-on-demand libraries, and Pat Walters (Relay) described how this can be done using Thompson Sampling, an active-learning method that traces its origins to 1933. Applied to lead discovery, the method involves breaking larger molecules into component fragments and iteratively searching for better binders. Pat showed that searching just 0.1% of a library of 335 million molecules consistently found 90% of the best hits.

 

Most computational methods rely on experimental data, and over the past 25 years Astex has generated >100 crystal structures on each of more than 40 targets, with >6600 bound fragments in total. Paul Mortenson described how these are being used to develop generative models, with chemists providing feedback on suggested molecules.

 

Artificial intelligence is the centerpiece of Isomorphic Labs, which has unfettered access to AlphaFold 3. Rebecca Paul described an example starting from a literature fragment in which the predicted affinities matched well with experiment – and the molecules were considerably more potent than those suggested by an experienced medicinal chemist.

 

Recognizing the need for experimental affinity data for fragments, Isomorphic worked with Arctoris to screen 5420 fragments against 65 kinases covering the diversity of the kinome. After carefully curating the data, including rescreening the actives at a different CRO, they found 485 fragments with an IC₅₀ of 300 µM or better. Interestingly, only about half of these fragments are known kinase binders.

 

Sandor Vajda (Boston University) suggested there may be limitations to machine learning models. He found that using AlphaFold 2 to find cryptic pockets was dependent on their representation in the PDB, with rare experimental states not being predicted. Sandor also proposed an interesting hypothesis that cryptic pockets created only by the movement of side chains are not very ligandable because the side chains move on such a rapid time scale that they effectively act as competitive inhibitors to ligands.

 

Success Stories

No FBLD meeting would be complete without success stories, and FBLD 2024 was no exception. Chaohong Sun noted that nearly 80% of the targets at AbbVie taken into fragment-based screening are novel. Of these, more than 80% yield actionable hits, though 44% are not pursued for a variety of reasons, including finding hits from other sources, hits at novel sites with no obvious function, and changes to the portfolio. Chaohong described a series of STING agonists that was taken forward to low nanomolar leads with in vivo activity.

 

Michelle Arkin (UCSF) described progress on creating molecular glues to link 14-3-3 proteins to the estrogen receptor, which we last wrote about here. Covalent binders to the 14-3-3 protein stabilize the interaction with ERα by more than 100-fold and show activity in cancer cell models.

 

Multiple talks focused on SARS-CoV-2 targets. Ashley Taylor (Vanderbilt) described fragment screens against the papain-like protease PL^Pro that led to both covalent and non-covalent inhibitors. James Fraser (UCSF) described how a massive crystallographic screen against the Nsp3 macrodomain Mac1 led to high nanomolar compounds, which we wrote about here. And Adam Renslo (UCSF) discussed the further optimization of Mac1 inhibitors to yield molecules that could protect mice from a fatal challenge of the virus.

 

A drawback of pursuing novel targets is that sometimes the biology proves uncooperative. Andrew Woodhead described a successful fragment screen at Astex against the oncology target elF4E that led to mid-nanomolar binders that could disrupt the protein-protein interaction with eIF4G in cells. Surprisingly, these molecules had no effect on cell viability, and a series of mutational and targeted-protein degradation experiments suggested that blocking a larger region of the protein-protein binding site might be necessary.

 

Drugs are the ultimate success stories, as David Rees reminded participants in “25 years of thinking small.” In addition to providing an overview of FBLD at Astex, David added up the sales of all seven FDA-approved fragment-derived drugs, which totals more than $3 billion. Harder to quantify—though infinitely more valuable—are the added years of life for patients with once-untreatable cancers. These numbers will only grow as the dozens of fragment-derived molecules in the clinic continue to advance.

 

I’ll close on that note. If you missed FBLD 2024, you’ll have another chance next year: FBLD 2025 is planned for Cambridge (UK) September 21-24 next year. Barring global pandemics.

Fragment-based Drug Discovery Down Under (FBDD-DU) 2024

The end of June brought me to Brisbane for the fifth FBDD-DU Conference, which was meeting for the first time outside Melbourne. This was also my first FBDD-DU conference since 2019, and it was nice to see a wide range of talks from around Australia and beyond. As always, I won’t attempt to be comprehensive, so if you attended, please feel free to add your observations.

 

Techniques

Experimental techniques received considerable attention. Félix Torres (NexMR) described using an inexpensive benchtop NMR that doesn’t require liquid helium. Fragments were screened using photochemically induced dynamic nuclear hyperpolarization (photo-CIDNP). The method is so rapid that it is limited more by sample handling than data collection, and the Torres team is speeding things up using flow technology. Right now photo-CIDNP is still very much DIY, but rumor has it that Bruker may soon launch a photochemical module for their benchtop instrument.

 

We’ve written about high-throughput crystallographic screening at the Diamond Light Source, and synchrotrons around the world are building similar platforms. Kate Smith described integrated systems at the Swiss Light Source which automate crystallization, fragment screening, data collection, and data processing. She also described increasing automation of fragment screening using the free-electron laser (FEL), which we wrote about here. Current throughput is around 40 compounds per day and requires large amounts of protein, but these are still early days.

 

Australia is building their own high-throughput crystallography platform, and various components were described by Roxanne Smith (University of Melbourne), Gautham Balaji (Monash Univesrity), and Yogesh Khandokar (ANSTO-Australian Synchrotron). Watch this space!

 

Speaking of Australia, Nyssa Drinkwater described Compounds Australia, a national repository of more than 2.5 million molecules, including several fragment collections. Members, who can be from outside Australia, can store their own libraries within the facility to ease collaborations with other groups, and they can also access public libraries of compounds, including unusual Antipodean natural product extracts. I was fortunate to be able to visit the facility at Griffith University and can attest that it is easily the equal of those in large pharma.

 

Turning to mass spectrometry, Sally-Ann Poulsen (Griffith University) described covalent library screening against PRMT5, a target we’ve written about here. Sally-Ann is also a pioneer of (conventionally non-covalent) native mass spectrometry, and she described applying this methodology to screen small molecules against RNA.

 

But the star of the conference was SPR, appearing in multiple talks. Long-time readers may recall an instrument made by SensiQ, with its gradient injection capability to accelerate data collection. This is now marketed by Sartorius, and Lauren Hartley-Tassell (Griffith University) described using it to screen a glycoprotein. The larger plumbing in the instrument is less prone to clogging, and Lauren said it can even accommodate screening of whole cells.

 

Anything to accelerate the (sometimes painful) process of advancing fragments is always welcome. As Jason Pun (Monash University) noted, eight of nine targets screened in Martin Scanlon’s group started with fragments having affinities worse than 100 µM. Off-rate screening, an SPR technique we wrote about here, can rapidly identify more potent molecules from crude reaction mixtures, but data processing can be tedious. Jason described new software tools to automate this process, and hopefully he will publish the methodology and code. (An aside: over coffee Yun Shi of Griffith University noted that off-rate screening, or ORS, should really be called off-rate constant screening, which would give the more amusing acronym ORCS.)

 

Targets

Turning to targets, Ben Davis (Vernalis) described a collaboration with Servier to advance oncology target USP7 inhibitors from a literature fragment to a preclinical candidate. Crude reaction mixture screening was used extensively, not just by SPR but even in microsome stability studies. Unfortunately the project ended when on-target toxicology effects emerged, which were perversely more severe in higher animal species than they were in mice.

 

Yun Shi described finding tiny heterocyclic fragments that react with the NAD⁺ cofactor of neurodegenerative target SARM1 in situ to generate a potent inhibitor, as we wrote about here. Yun is using ¹⁹F NMR to follow the base-exchange reaction to identify inhibitors to other glycohydrolases too.

 

Deaths due to E. coli are – somewhat surprisingly – more common than those caused by any other pathogen, and Christina Spry described her work at the National Australian University to discover inhibitors of the essential dephosphocoenzyme A kinase (GPCK) enzyme, which catalyzes the final step in the synthesis of Coenzyme A (CoA). Fragment screening by DSF and NMR identified a weak (K_D=380 µM) binder, and fragment growing has led to a low nanomolar inhibitor that is selective against the human form of the enzyme.

 

Continuing the E. coli theme, several talks discussed efforts against the challenging bacterial virulence target DsbA, a twenty-year campaign in Martin Scanlon’s group at Monash as noted by Yildiz Tasdan. The enzyme has a shallow, hydrophobic active site, but the discovery of fragments binding to a cryptic site and crude-reaction screening by ORS (ORCS?) and affinity-selected mass spectrometry (ASMS) has finally led to molecules with dissociation constants around 1 µM.

 

Finally, in his closing keynote address Alvin Hung, who recently founded NeuroVanda, described a wide range of fragment success stories, many of them covered on Practical Fragments, against targets including pantothenate synthetase, GSK3β, PKC-ι, and MNK1/2. Although structural enablement helped in many cases, Alvin was not rigid about the need for atomic-level details: in response to the question whether he would advance a fragment in the absence of structure, he answered simply, “of course.” Perhaps it's time to redo my poll on this subject.

 

I’ll wrap up here, but if you missed this or earlier events this year there are still a couple more conferences in Boston, and 2025 is already starting to take shape.

Nineteenth Annual Fragment-Based Drug Discovery Meeting

Last week the CHI Drug Discovery Chemistry (DDC) meeting was held in San Diego. This was the largest ever, with more than 900 participants, 95% of whom attended in person, up from 87% last year. I won’t attempt to cover all fourteen tracks but will just touch on some of the main themes.

 

Computational approaches

All four days of the conference featured dedicated sessions on machine learning and artificial intelligence, but since I was in other sessions I don’t know how relevant they were to FBLD. If you attended an interesting talk please let me know so I can watch it on-demand.

 

Among computational talks I did see, Antonina Nazarova (University of Southern California) provided an update on V-SYNTHES, which we first wrote about here. This synthon-based screening approach now covers 36 billion molecules and has been tested against eight different proteins, four of which yielded nanomolar hits when tested experimentally.

 

Computational methods have historically treated proteins as rigid, though many targets are anything but. Diane Joseph-McCarthy (Boston University) described an improvement to the pocket finding approach FTMap, called FTMove, to incorporate molecular dynamics by starting with an ensemble of different crystal structures. A further advance is E-FTMap, which expands the number of virtual probes from 16 to 119 to more finely assess ligandable sites.

 

Benjamin Walters (Genentech) described using protein dynamics to find cryptic pockets using ESP, or Experimental Structure Prediction. In this approach, experimental data from hydrogen-deuterium exchange (HDX) or chemical shift perturbations (CSPs) are used to constrain multiple parallel computational simulations, leading to better models than flexible docking, even for weak fragments.

 

Experimental approaches

Protein-detected NMR was the first practical fragment-finding method, and Steve Fesik (Vanderbilt) described using SAR by NMR to find fragments binding to the papain-like protease of SARS-CoV-2. These have been advanced to molecules with nanomolar affinity and activity in cell-based assays.

 

Andreas Lingel described the new fluorine-containing fragment library built at Novartis and how ¹⁹F NMR was used to generate inhibitors of IL-1β. We wrote about that success last year, noting that the initial fragment hit was “super-sized,” and Andreas confirmed that for trifluoromethyl-containing fragments the upper molecular weight limit was relaxed to 350 Da.

 

Sriram Tyagarajan (Merck) presented a crystallographic screen against the neurodegeneration target TTBK1 which yielded hits at 15 sites. Several potential allosteric sites were identified, but fragment growing and linking were not successful, leading them to a quick (3 month) no-go decision on the protein.

 

Virgil Woods (City University of New York) described using crystallographic screening to find hits against the challenging phosphatase PTP1B both under conventional cryogenic temperatures as well as at room temperature. As we noted about related work, there was a surprisingly poor overlap between the two sets of hits, and some fragments bound in a different manner at different temperatures.

 

Integrating FBDD and DNA-encoded libraries (DEL) for lead generation was the topic of Chaohong Sun’s talk. She noted that of some two dozen targets at AbbVie screened by both methods, 60% found hits from both, 10% found only fragment hits, and 5% found only DEL hits, with a quarter of the targets producing no hits. Hits from both approaches can be combined, as we noted here. Chaohong also noted that for both FBDD and DEL, high quality protein is essential for successful screens.

 

Covalent approaches

Covalent approaches to drug discovery are becoming ever more acceptable as more covalent drugs are approved. Understanding these in depth was the focus of Micah Niphakis (Lundbeck), who characterized 22 approved drugs containing 18 different warheads. The stability in buffer, liver microsomes, and hepatocytes varied dramatically, though more recently approved drugs tended to be more stable. Chemoproteomic studies revealed many off-targets in cells; for example, all the kinase inhibitors tested hit BTK to some extent even when this was not the primary target. The fact that the drugs are (mostly) safe and well-tolerated is a useful reminder that just because we can detect something doesn’t mean it is relevant.

 

Henry Blackwell described building a 12,000-member covalent fragment library at AstraZeneca. Due to the presence of a warhead, they relaxed rule of three parameters, with MW ranging from 250-400 Da and ClogP from 0-4. Henry also discussed the successful use of this library to identify covalent hits against the anticancer target BFL1 that were optimized to k_inact/K_I ~ 7000 M^-1s^-1. This accomplishment is all the more impressive given that screens using ASMS, DSF, ¹⁹F NMR, and SPR had all failed to yield validated hits.

 

We recently wrote about electrophilic MiniFrags, and György Keserű (Research Center for Natural Sciences, Hungary) described screening these against HDAC8 and the main protease from SARS-CoV-2. He also mentioned that the set is available for purchase from Enamine, so you can try it yourself against your favorite target.

 

As covalent modifiers become more common we will see new metrics for characterizing them, as illustrated by Benjamin Horning’s (Vividion) presentation, “Ligand Efficiency Metrics in Covalent Drug Discovery.” He described Ligand Reactivity Efficiency (LRE), defined as pTE₅₀(target, 1 hr) – pTE₅₀(glutathione, 1 hr), where TE is the target (or glutathione) engagement. LRE is analogous to LLE but focused on reactivity rather than lipophilicity. Despite my post last week, the metric could be useful, and I look forward to seeing what Dr. Saysno and friends will make of it.

 

Most covalent modifiers bind to a target and remain intact, but Nir London (Weizmann Institute) has developed Covalent Ligand Directed Release (CoLDR), in which a portion of the small molecule leaves; applications include release of fluorescent or chemiluminescent probes. Useable warheads include α-substituted methacrylamides and sulfamate acetamides.

 

Although more recent covalent drugs have targeted cysteine residues, there is growing interest in other amino acid side chains. Nir mentioned that thio-methacrylate esters can react with lysine residues, thought the kinetics are slow. And Carlo Ballatore (University of California San Diego) described hydroxy-naphthaldehyde fragments that bound reversibly to a lysine on the vascular target KRIT1.

 

Both plenary keynote speakers focused heavily on covalent chemistry. Dan Nomura (UC Berkeley) described using chemoproteomics approaches to find covalent molecules that could inhibit, degrade, or change the cellular localization of myriad proteins.

 

Finally, K. Barry Sharpless (Scripps), one of only five people to have been awarded the Nobel Prize twice, gave a rich description of sulfur (VI) fluoride exchange chemistry (SuFEx), which included drawing chemical structures on a flip chart. He presented the discovery of a fluorosulfate that is bactericidal against multiple resistant forms of Mycobacterium tuberculosis. Interestingly, the molecule works by modifying a catalytic serine residue which then cyclizes to form a β-lactam. His passion for chemistry is obvious, but he also has personal reasons for pursuing the second most deadly infectious disease: his brother died of tuberculosis before effective drugs were developed. And with the rise of extensively drug resistant TB, we’ll need new ones.

 

I’ll end on that note, but please leave comments. And mark your calendar for April 14-17 next year, when DDC returns to San Diego.