From fragment linking to covalent RAD52 inhibitor

Targeting proteins involved in DNA repair is a promising approach against cancer. However, finding effective small molecule inhibitors can be difficult because the protein-DNA interface tends to be very polar, with few classically ligandable pockets. In a new J. Am. Chem. Soc. paper, Nicolas Bocquet, Olivier Querolle, Ulrich Lücking, and collaborators at FoRx Therapeutics, WuXi AppTec, Diamond Light Source, University of Bern, Sandexis, and Weizmann Institute of Science provide a proof of concept.

 

The researchers were interested in RAD52, which mediates several DNA repair functions. The N-terminus of the protein forms an oligomeric ring structure, and each subunit contains a solvent-exposed groove that binds to single-stranded DNA. Since DNA is negatively charged, the researchers conducted a crystallographic screen against a library of 320 carboxylic acid containing fragments. This identified three related molecules, of which compound 3 had the highest occupancy. Because the compounds bind at the DNA-binding interface, the researchers developed a fluorescence polarization (FP) DNA-displacement assay and found that the fragments disrupt DNA binding at high micromolar concentrations.

 

Interestingly, the crystal structure revealed two copies of compound 3 bound to each subunit of RAD52: each positioned where a single nucleotide would normally bind, with the carboxylic acids making similar contacts as the phosphates. In other words, the fragments mimic two consecutive bases of a single-stranded oligonucleotide. Naturally, this suggested a linking approach.

Linking two close analogs of compound 3 led to compound 5, with a satisfying two orders of magnitude improvement in activity in the FP assay, which the researchers suggest is largely due to lowering the entropic penalty. Linking directly off the thiazole moieties produced similar results, and a macrocycle had slightly better activity, though not so much better as to justify the synthetic hassle. A crystal structure of compound 5 revealed a small hydrophobic cleft, and this was exploited with a phenyl moiety in compound 11, a high nanomolar inhibitor in the FP assay.

 

A crystal structure of compound 11 bound to RAD52 confirmed that the compound binds as designed. The structure also revealed a cysteine residue (C64) in close proximity to the phenyl moiety. Adding an acrylamide warhead yielded a covalent inhibitor, and further optimization ultimately led to FORX-864, which has low nanomolar activity in the FP assay after 4 hours. The researchers appropriately measured the k_inact/K_I, which came in at a respectable 230,000 M^-1s^-1, better than some covalent clinical compounds. Removing the warhead led to a dramatic loss in potency (IC₅₀ = 27 µM in the FP assay).

 

In a cellular assay to measure single-stranded annealing (SSA), FORX-864 showed roughly 1 micromolar activity, while a compound lacking the warhead was inactive. FORX-864 was also inactive in a cell line in which RAD52 had been knocked out, suggesting on-target activity. The researchers profiled the reactivity of FORX-864 in an activity-based proteomic profiling (ABPP) assay and found that it was reasonably selective. Intravenous dosing in mice showed encouraging pharmacokinetic properties, though with two carboxylic acid moieties oral dosing might be challenging.

 

Overall this is a nice example of liganding a difficult target. Given that FORX-864 mimics a dinucleotide, it may bind to other DNA- or RNA-binding proteins. However, covalent binding is required for high potency, and it is unlikely these other proteins will have cysteine residues in the appropriate position, which should help with selectivity. FORX-864 certainly appears to be the best chemical probe for RAD52 to date.

Fragments in the clinic: VVD-214

Just over two years ago we highlighted a new clinical candidate targeting WRN, a covalent inhibitor then called VVD-133214. An open-access paper published near the end of last year in J. Med. Chem. from Shota Kikuchi, David Weinstein, and colleagues at Vividion describes in detail the optimization of the covalent fragment hit to the clinical compound. (Shota presented some of this work at the 2024 DDC meeting.) This paper is also an interesting contrast to non-covalent fragment-finding approaches against this target we wrote about earlier this month.

 

The 2024 post described the chemoproteomic screening that identified compound 1a, which covalently binds to C727 in WRN. An early observation was that some molecules were cooperative with ATP while others were competitive. Given the high concentration of ATP in cells, the researchers prioritized the former category, which led to compound 1f. (Note that while I’m showing only the k_inact/K_I values, the researchers used biochemical and cell-based assays to drive SAR).

 

The vinyl sulfone warhead is unusual amongst covalent clinical compounds, so the researchers sought to characterize it. The rate of reaction with glutathione for compound 1f is comparable to the approved drug osimertinib: reactive, though acceptable. To try to lower the reactivity and also prevent isomerization of the double bond, the researchers introduced a methyl group. Compound 2a not only showed increased stability but also improved activity against WRN and sub-micromolar activity in a cell-based assay. A crystal structure of a later molecule revealed interactions with a hydrophobic patch on the protein, explaining the improvement in potency. Importantly, the other enantiomer was much less potent.

 

Other rings were tried, unsuccessfully, to replace the pyrimidine and the phenyl moieties. However, changing the cyclopentyl ring to a tert-butyl moiety (compound 5d) further improved the potency to the point where the compound could be tested in vivo, where it proved to be active in a mouse xenograft study. Mass spectrometry experiments revealed prolonged occupancy of C727 out to 24 hours after compound dosing even though the compound itself had been cleared, consistent with a long half-life of the WRN protein. The researchers note that high target engagement (TE) at 24 hours was predictive of tumor growth inhibition, which streamlines optimization since it is easier to run a one-day TE experiment than a multi-week efficacy study.

 

Further optimization of ADME properties ultimately led to VVD-214, which was active in a mouse xenograft study and showed good oral bioavailability and pharmacokinetics in mouse, rat, dog, and monkey. This compound was also profiled in a chemoproteomic assay and found to be quite selective for the C727 of WRN.

 

There are several important lessons in this paper. First, the initial fragment is larger than prescribed by the rule of three, consistent with an analysis of covalent inhibitors last year. Second, much of the SAR was empirical; crystallography was not used until relatively late in the campaign. When a crystal structure was finally solved of VVD-214 bound to WRN it revealed no polar contacts between the ligand and the protein, only hydrophobic interactions, which is rare for fragments, let alone drugs. Perhaps because of the lack of polar interactions, it was impossible to measure the inhibition constant (K_I), and saturating the warhead to make it unreactive completely abolished activity. In other words, the binding is largely driven by reactivity, but specific reactivity for WRN rather than generic chemical reactivity.

 

In 2024 just two WRN inhibitors had entered the clinic, the other being a noncovalent molecule called HRO761. We quoted the Vividion team as saying that “this presents a rare opportunity to compare two small molecule oncology drugs targeting the same protein by different mechanisms.” Since then, HRO761 has been quietly discontinued, as has another noncovalent drug, IDE275. Meanwhile, development of VVD-214 is ongoing, and another covalent compound, MOMA-341, which we mentioned here, has also begun human testing. (To be fair, so has yet another non-covalent molecule NDI-219216. And the binding mechanism of a sixth WRN drug, EIK1005, is undisclosed.) While it’s still early in the match, covalent drugs seem to be punching above their weight. May the best drug(s) prevail - Practical Fragments is rooting for them all.

From noncovalent fragments to covalent WRN inhibitors

Last week we described the discovery and early optimization of noncovalent inhibitors against the the DNA helicase WRN, an interesting oncology target both for its conformational flexibility as well as a ‘synthetic lethal’ approach to cancer drugs. Today we continue the theme with a Bioorg. Med. Chem. Lett. paper from Geoffrey Smith and colleagues at CHARM Therapeutics.

 

At the time the project began, no specific WRN inhibitors had been reported, but a crystal structure of the ADP-bound form of the protein had been published. The computational tool Fpocket, which we wrote about here, was used to identify several ligandable pockets.

 

To find actual ligands, the researchers crystallographically screened a library of 860 "poised" fragments from Enamine and used PanDDA (see here) to identify binders, even those with low occupancies. Several ligands occupied pockets that had been identified by Fpocket. More interestingly, five fragments bound in a previously cryptic site that had not been predicted. This pocket was formed by rotation of two phenylalanine residues as well as peptide backbone movements, consistent with sites able to support high affinity ligands, as we discussed in 2024. Thus, the focus turned to ligands that bind here.

 

Because of its low occupancy in the crystal, the orientation of compound 3 was ambiguous, so the researchers turned to a machine-learning-based protein-ligand co-folding algorithm called DragonFold. This revealed that the fragment binds in close proximity to a cysteine residue, C727, known to be reactive. Scaffold hopping and addition of a covalent warhead led to compound 4d. While the initial compound 3 showed no activity in a WRN helicase assay, compound 4d showed micromolar activity. Moreover, a crystal structure revealed binding to C727. Further SAR led to molecules such as compound 9b, the most potent WRN inhibitor reported in the paper.

 

Compounds were also tested against the closely related helicase Bloom syndrome protein, or BLM, and most of them were active, though the SAR varied between WRN and BLM. The activity against BLM is odd given that the residue corresponding to C727 is a serine, but the researchers note that the molecules might bind to other cysteine residues in BLM. Although no chemical reactivity data are provided for the ligands, I suspect they are somewhat reactive.

 

Several important lessons can be drawn from this brief paper. The fact that an experimental screen was able to identify cryptic pockets missed in a computational screen justifies empirical approaches. The identification of these pockets is all the more impressive given that pre-formed crystals were sufficiently flexible to undergo significant conformational changes. But computational approaches did prove their utility in refining the binding mode of the ligand. And finally, this is another example of appending a covalent warhead onto a non-covalent ligand.

 

Next week we’ll conclude this WRN trilogy with a covalent-first example.

Review of 2025 reviews

Turning and turning in the widening gyre

The falcon cannot hear the falconer…

 

In our small, annual counterweight to Yeats’ “mere anarchy," Practical Fragments looks back on 2025.

 

This year marked the thousandth post on Practical Fragments, a milestone neither Teddy nor I imagined when the blog launched back in 2008. In terms of conferences, I wrote about CHI’s Drug Discovery Chemistry in San Diego here and Discovery on Target in Boston here.

 

For the past decade I’ve participated in annual J. Med. Chem. perspectives covering fragment-to-lead success stories, two of which published this year. The first, spearhead by Rhian Holvey at Astex, covers the year 2023, while Astex’s David Twigg took the lead on covering the year 2024. In addition to the tabular summaries for which these reviews are best known, both also include tables of “near misses,” none of which made the main tables because the starting points were (sometimes just slightly) too large. Four out of six of these heavyweights are covalent fragments, suggesting that the rule of three may need to be relaxed for these. The most recent paper also includes a table showcasing the eight approved FBLD-derived drugs.

 

Two more general publications are also of interest. In a brief (open-access) editorial in J. Med. Chem. Weijun Xu and Congbao Kang at A*STAR summarize fragment-finding methods and approved drugs and discuss future applications of FBLD in PROTACs and targeting RNA. And in Curr. Res. Pharm. Drug Discov., Geoffrey Wells, Exequiel Porta, and colleagues at University College London present a “graphical review” which covers library design, screening strategies, hit validation, fragment optimization, and a few case studies of approved drugs, along with current challenges.

 

In Drug Des. Devel. Ther., Bangjiang Fang and colleagues at Shanghai University of Traditional Chinese Medicine present an open-access bibliometrics analysis of 1301 fragment-based drug design papers published between 2015 and the end of 2024, which includes top ten lists of institutions, authors, and papers as well as keyword trends and analyses. Annual growth has averaged 1.4%, and the field is both global and collaborative, with 35% of publications involving more than one country.

 

Targets

Two reviews focus on oncology. The first, in Bioorg. Chem. by Milind Sindkhedkar and collaborators at Manipal College of Pharmaceutical Sciences and Lupin Ltd., briefly covers the history and practice of FBDD before providing short summaries of seven of the eight approved drugs to come from it. The second, published open-access in Chem. Rev. by Vanderbilt’s Steve Fesik, is a concise and highly readable introduction and account of the author’s groundbreaking work on BCL-2 family proteins, KRAS, and WDR5.

 

A much longer open-access review in Chem. Rev. by Paramjit Arora and colleagues at New York University covers protein-protein interactions (PPIs). Much of the focus is on larger molecules such as cyclic peptides, peptide mimetics, and other macrocycles, but there are summaries of fragment-based approaches against KRAS and 14-3-3 proteins.

 

The topic of 14-3-3 proteins is treated more fully in an open-access Acc. Chem. Res. paper by Michelle Arkin and colleagues at UCSF. While the focus of most efforts against PPIs is to find inhibitors, for 14-3-3 the goal is to find stabilizers, or molecular glues. The Arkin lab and others have been succeeding using various approaches, particularly disulfide tethering. We wrote about these efforts most recently in 2023, and the new review provides a nice update.

 

Fragment finding methods and libraries

Sahra St. John-Campbell and Gurdip Bhalay, both at The Institute of Cancer Research, published a massive open-access perspective on “target engagement assays in early drug discovery” in J. Med. Chem., covering a host of biochemical, biophysical, and cell-based assays. A table lists more than 50 different techniques, almost half of which are applicable to FBLD. Each row shows what characteristic(s) are measured as well as critical requirements for  protein, sample, and equipment. The paper is also beautifully illustrated with dozens of figures: one shows which techniques are most useful for different types of targets, and each method gets its own diagram.

 

A more focused open-access review is provided by Stefanie Freitag-Pohl and colleagues at Durham University in Biophys. Rev. After surveying various biophysical techniques, the researchers focus on spectral shift analysis, and in particular the Dianthus instrument from NanoTemper Instruments. This plate-based, high-throughput microfluidics-free instrument can detect changes in fluorescence caused by environment or temperature. Examples demonstrate affinity measurements across several orders of magnitude, up to double-digit millimolar, and a nice scheme shows use of the Dianthus in a fragment-screening workflow.

 

Moving to specific techniques, Jia Gao, Ke Ruan, and colleagues at University of Science and Technology of China Hefei provide an open-access survey of “the rise of NMR-integrated fragment-based drug discovery in China” in Mag. Res. Lett. After a brief overview of NMR approaches, they cover case studies from China, most of which are focused on fragment screening rather than optimization.

 

A less common biophysical method is native mass spectrometry (nMS), the subject of an open-access opinion in RSC Med. Chem. by Louise Sternicki and Sally-Ann Poulsen at Griffith University. This is a good survey of the approach; we highlighted a more fragment-focused review by the same authors last year.

 

The most common fragment-finding approach, X-ray crystallography, is covered in two open-access reviews. The first, in Acta Cryst. F by Sarah Bowman and collaborators at University of Buffalo and Brookhaven National Laboratory, focuses on critical early stages, from protein characterization to sample preparation and various crystallization approaches. The second, in Curr. Opin. Strut. Biol. by Martin Noble and colleagues at Newcastle University, starts by briefly reviewing crystallographic fragment screening before turning to fragment libraries. The paper includes a nice table summarizing publicly available libraries at major synchrotrons, with the text describing these in more detail.

 

The provider of one of these libraries, EU-OPENSCREEN, is the subject of an open-access review in SLAS Discov. by Robert Harmel and collaborators at EU-OPENSCREEN ERIC and Fraunhover ITMP. As we wrote last year, EU-OPENSCREEN is a broad consortium whose mission is to advance early drug discovery by providing access to technology and expertise. The new paper summarizes the four compound collections, including the European Fragment Screening Library (EFSL), and surveys progress to date. It also lays out ambitious plans, including expanding to >30 sites in nine countries.

 

Computational approaches and cryptic sites

Despite the hype about artificial intelligence in the broader world, AI in fragment-based drug discovery has been less common. In Curr. Opin. Struct. Biol., Woong-Hee Shin and colleagues at Korea University College of Medicine summarize applications to fragment growing, merging, and linking. The open-access paper includes a handy table of 13 programs, and includes GitHub links where available.

 

Cryptic binding sites, defined by Ehmke Pohl and collaborators at Durham University and Cambridge Crystallographic Data Centre “as binding pockets that exist in the ligand-bound state of a protein but not in its apo form,” are the focus of an open-access review in Bioinform. Adv. The researchers cover earlier computational approaches for finding these, especially molecular dynamics (MD) and machine learning (ML). They note that a key challenge for ML is the limited quantity and quality of experimental data: undiscovered cryptic sites would be misclassified as non-binding sites.

 

Yowen Dong, Ge-Fei Hao, and colleagues at Guizhou University review “computational methods for identifying cryptic pockets” in Drug Discov. Today. As with the previous review, these are divided between molecular dynamics and AI-based techniques, which are discussed individually and then compared. The researchers apply six approaches to the model bacterial protein TEM-1 β-lactamase and find that, for this highly studied single protein, the AI-based methods are much faster (seconds instead of days) and just as accurate, though MD-based methods provide more insight into formation mechanisms of cryptic pockets.

 

Covalent ligands

Allosteric sites are an important sub-class of cryptic pockets, and in J. Med. Chem. Jianing Li and colleagues at Purdue University discuss covalent allosteric inhibitors. After briefly discussing advantages of covalent molecules, they review examples targeting protein phosphatases, kinases, and GTPases, such as KRAS.

 

Of course, covalent molecules are not limited to allosteric sites. An open-access review in Bioorg. Med. Chem. Lett. by Walaa Bedewy, John Mulawka, and Marc Adler at Toronto Metropolitan University summarizes published covalent protein ligands, grouping them by target site:  active sites, residues adjacent to an active site, protein-protein interfaces, cofactor binding sites, and allosteric sites.

 

Chem. Rev. published two massive reviews on covalent ligands, each with more than 300 references. The first, by Tomonori Tamura, Masaharu Kawano, and Itaru Hamachi at Kyoto University, covers a wide range of topics, from covalent drugs, to peptide- and protein-based covalent inhibitors, to chemical biology labeling and target engagement strategies, to covalent bifunctional molecules such as PROTACs and radionucleotide-based molecules, and even covalent modification of DNA and RNA. The paper includes 68 figures, many reproduced from the original publications.

 

The second (open-access) Chem. Rev. paper, by Ku-Lung Hsu and colleagues at University of Texas at Austin, focuses on covalent ligands targeting protein residues other than cysteine, particularly lysine and tyrosine; we highlighted some of Hsu’s work recently. The paper also discusses naturally occurring molecules that bind to lysine, such as pyridoxal phosphate and aldose sugars.

 

Methods for finding covalent ligands are the focus on an open-access review in JACS Au by Mengke You, Hong Liu, and Chunpu Li at Shanghai Institute of Materia Medica. Specifically, they review disulfide tethering, activity-based protein profiling (ABPP), covalent DEL, phage and mRNA display, and sulfur(IV) fluoride exchange (SuFEx), with examples for each.

 

The last paper on this topic, in J. Med. Chem., offers a brief but important overview of all covalent FDA-approved small molecule drugs through 2023. Samuel Dalton and collaborators at Isomorphic Laboratories and Merck counted 128 covalent drugs, about 7% of all small molecule drugs. More than half are antibiotics, and more than 85% target serine or cysteine. Only 10% are reversible, but this number is rapidly increasing, with 11 of the 13 reversible covalent drugs approved since 2010. Importantly, the names, chemical structures, indication, target and target residue, warhead, and key references for all the drugs are provided in the supporting information.

 

Miscellaneous

Deconstruction of ligands to smaller fragments that are then “reconstructed” into new leads is a venerable approach in FBLD and the subject of an open-access perspective in J. Med. Chem. by J. Henry Blackwell, Iacovos Michaelidies, and Floriane Gibault at AstraZeneca. Multiple examples dating as far back as the late 1990s are provided, along with appropriate caveats about potential changes in fragment binding modes and protein conformations.

 

Finally, an open-access perspective in J. Med. Chem. by Dean Brown (Jnana Therapeutics) examines the 104 oral drugs approved from 2020 through 2024, including structures, dosing, pharmacokinetics, and safety. Roughly a third of these drugs are dosed more than once per day, and almost a quarter have a black box warning, while 42% have at least one contraindication. Dean warns that “overly prescriptive [development candidate] criteria may inadvertently stifle the development of innovative drugs,” and that it is difficult but important “to be the champion for a compound that others perceive as ‘un-drug like.’” The growing success of covalent drugs illustrates that some organizations are taking this to heart.

 

And that’s it for 2025. Thanks for reading and special thanks for commenting. And in 2026, may the best of us be filled with passionate intensity.

Metal-binding fragments vs glutaminyl cyclases

Metal-binding fragments have a long history in FBLD; the first mention on Practical Fragments was back in 2010. The idea is to use the strong interaction between a fragment and a protein-bound metal as an affinity anchor for further optimization. The latest example, by Jie-Young Song, Soosung Kang and collaborators at Korea Institute of Radiological & Medical Sciences, Ewha Womans University, and elsewhere was published in ACS Med. Chem. Lett.

 

Glutaminyl cyclases such as glutaminyl-peptide cyclotransferase (QC) and glutaminyl-peptide cyclotransferase-like protein (isoQC) convert N-terminal glutamine or glutamate residues on proteins to pyroglutamates. This modification tends to stabilize proteins, and it has been implicated in several diseases. In particular, modification of CD47 by isoQC seems to be important for the ability of cancer cells to evade the immune system.

 

QC and isoQC are closely related enzymes with a zinc-containing active site. Capitalizing on this, the researchers tested a library of 36 potential metal-binding fragments in a functional assay against QC. Most of the compounds tested were inactive, though 11 had IC₅₀ values less than 0.8 mM. A few of these, including compound ab, were used to generate a second library of just half a dozen larger fragments, and compound 9 turned out to quite potent.

 

The researchers recognized that compound 9 has two potential zinc-binding moieties, and docking suggested the newly added amino-thiadiazole was likely responsible for the increased activity. Structure-based design ultimately led to compound 22b, with low nanomolar activity against QC and isoQC. The molecule did not seem to be generally cytotoxic, but it did increase phagocytosis of cancer cells in vitro, consistent with an effect on the “don’t eat me” function of CD47.

 

Unfortunately, no information is provided on the selectivity of compound 22b against other zinc-dependent enzymes. Moreover, unlike an earlier example of starting with metallophilic fragments, no ADME data are provided. But whether or not this particular series advances, it is nice to see metallophilic fragments being explored.

Sixteenth Annual Fragment-Based Drug Discovery Meeting

Last week Cambridge Healthtech Institute held its Sixteenth Drug Discovery Chemistry (DDC) meeting virtually for the second year in a row. The eight tracks, four in parallel, spanned three days. There were occasional kinks, and the 6:30 morning start time was painful for those of us on the Western edge of the US, but for the most part things went smoothly. Attendance was up 25% over last year, to 750 participants. Attendees from outside the US comprised 40%, similar to last year and up from 2019, while industry representation remained steady at 70%. As always I’ll just touch on a few broad themes – please feel free to add your impressions.

 

Methods

These meetings are always great venues to learn about new techniques, and one that stood out is “mass photometry” (MP). This label-free method allows visualization of single proteins in solution. The technology, which builds on interference reflection microscopy and interferometric scattering microscopy, rapidly provides information on the molecular weight of proteins to an accuracy of about 2 kDa. Stefan Geschwindner (AstraZeneca) used the method to follow a change in the monomer-dimer equilibrium of the protein PAD4 in response to ligand-binding. He noted that the instrument, sold by Refyn, provides measurements in about 10 seconds for proteins from 35 kDa to 4 MDa in size.

 

On the opposite end of reductionism, Bryan Roth (University of North Carolina, Chapel Hill) discussed customized high-throughput cell-based screens for 320 of the 358 GPCRs in the human genome. We’ve previously described how these assays have been used to screen molecules prioritized from massive computational screens. If you’d like to profile your own molecules, you can submit compounds here for free with no IP-entanglements.

 

Cryo-EM was the subject of our April 1 post last year, but as we discussed here the technique is no joke. Pamela Williams discussed how Astex has formed a consortium with several other companies to advance the technology. She noted that of the 270 membrane protein structures released last year with resolutions better than 3.5 Å, 167 came from cryo-EM while only 103 came from crystallography. Unlike in crystallography, resolution can vary over the protein; local resolution around the region of interest may be better or worse than global resolution. And there is still much to be done to truly industrialize the approach, particularly in sample preparation (specifically making the grids).

 

Speaking of crystallography, James Fraser (University of California, San Francisco) discussed a high-throughput crystallographic screen against the SARS-CoV-2 macrodomain protein, which we covered earlier this year. A fragment-linking program called “Fragmentstein” was used to generate molecules with low micromolar affinity.

 

At the last DDC meeting Frank von Delft (Diamond Light Source) described a high-throughput crystallographic screen of the SARS-CoV-2 M^Pro protein, and this year he provided an update on how the open-source COVID Moonshot effort has advanced hits to non-covalent 30 nanomolar binders with 100 nanomolar antiviral activity in cells. Current efforts are focused on improving metabolic stability and oral bioavailability, but Frank lamented the “astonishing inertia” on the part of funding agencies to support the effort. Although the spectacular success of vaccines should contain the outbreak, there are still occasional breakthrough infections, and not everyone can be vaccinated or mount a successful immune response. Small molecule drugs will be useful, and it would be nice if more people were working on them.

 

Success Stories

I’ll never turn down a crystal structure, but it is important to remember that fragments can be advanced even in the absence of structural information. As a case in point, Paolo di Fruscia (AstraZeneca) described the discovery of allosteric binders of MEK1; we wrote about these earlier this year.

 

On the other hand, Justin Dietrich described AbbVie’s discovery of TNFα inhibitors – an effort he said would not have been possible without structural information. As we discussed here, the team struggled with the lipophilic nature of some of their initial molecules; Justin noted that “if a protein wants grease you have to find a way to get it grease.” The physicochemical properties can then be improved during optimization.

 

Rod Hubbard (Vernalis) described the discovery of LpxC inhibitors. He emphasized the importance of using biophysical methods to carefully characterize and optimize the target protein, and noted that NMR can identify fragments that crystallography might miss. Also, as we noted when we wrote about this campaign last December, fragments can provide multiple starting points, and it can be useful to optimize fragments before embarking on a fragment-to-lead effort.

 

Jus Singh (Ankaa) provided a brief history of targeted covalent drugs. Some folks are still nervous about the potential for covalent molecules to cause idiosyncratic toxicity; indeed, this was one of the reasons Frank von Delft gave for pursuing non-covalent M^Pro inhibitors. However, Jus noted that five out of seven approved covalent kinase inhibitors are not associated with liver toxicity, while some non-covalent kinases inhibitors are. Jus also noted that all three FDA-approved BTK inhibitors are covalent, despite longstanding efforts to develop non-covalent inhibitors.

 

Finally, Micah Steffek (Genentech) described the discovery of LC3 binders, the first step in making an autophagy-based version of PROTACs. An NMR screen yielded hundreds of hits, but despite obtaining multiple crystal structures the team struggled to obtain molecules with better than mM potency. However, combining fragment information with results from a DEL-screen, akin to the story we described here, led to submicromolar inhibitors. Intriguingly, these covalently modified a lysine residue.

 

Odd and ends

Micah’s talk led to some discussion in a speaker panel as to the meaning of hit rates. Do you consider the primary hit rate or the confirmed hit rate? And what level of confirmation is required? As Rod Hubbard noted, different techniques may give different answers for good reasons, and you don’t want to throw away potential hits on the basis of the worst performing or least sensitive technique.

 

Mads Clausen (Danish Technical University) described his shapely library of fluorinated fragments (see here). Hit rates seem to be similar to flatter compounds, though with only four targets screened it is perhaps too early to draw conclusions.

 

But perhaps there is a limit to shapeliness: Justin Dietrich could not recall seeing any fully unsaturated Fsp³ = 1) fragments coming up as hits, despite being present in the AbbVie libraries. Andreas Lingel said he had occasionally seen these at Novartis, but they were never pursued. Similarly, no one could recall advancing any acyclic fragments. A quick glance at the 131 fragment-to-lead success stories captured in five annual reviews revealed that all of the fragments had at least one ring and one sp²-hybridized carbon.

 

And with that I’ll close. The Seventeenth installment of DDC is scheduled for April 18-21 of next year in San Diego, and the more biology-focused Discovery on Target is scheduled to take place both online and in-person in Boston from September 27-30. Hope to see you there!

A general fragment-based approach to… targeting RNA?

This is taken from the title of a recent open-access paper by Matthew Disney and collaborators at Scripps Research Institute Jupiter and Florida Atlantic University in Proc. Nat. Acad. Sci. USA. RNA has long been a target of FBLD: Practical Fragments first blogged about it in 2009, and a 2002 paper reported using fragment linking to obtain a low micromolar binder. So how general is the new approach?

 

The researchers describe chemical cross-linking and isolation by pull-down fragment mapping (Chem-CLIP-Frag-Map). This involves using photoaffinity probes that can crosslink to biomolecules such as RNA. The probes also have an alkyne tag that can be used to isolate bound molecules using click chemistry. We’ve written previously about such “fully functionalized fragments” (FFFs).

 

Earlier work had resulted in the identification of compound 1, which binds to a specific site on pre-miR-21, the precursor to a non-coding microRNA linked to cancer. An FFF version of compound 1 was shown to crosslink to pre-miR-21 after irradiation with UV light, and the site of modification could be mapped using a reverse-transcriptase-mediated primer extension, which stalled at the modified bases.

 

Next, the researchers screened 460 FFFs at 100 µM and found 21 that crosslinked to pre-miR-21. They were ultimately looking to link fragments with compound 1, and thus competition studies were used to eliminate fragments that bound at the same site. This left three fragments, and primer extension studies confirmed that these bound near but not at the binding site of compound 1.

 

Next, the researchers attached these three fragments to compound 1, with or without various linkers. Some of the resulting molecules had improved affinity, and compound 9 showed the tightest binding according to microscale thermophoresis (MST). Mutational and competition studies confirmed that the molecule binds to the expected site. Importantly, compound 9 not only bound to pre-miR-21, it also blocked processing by the enzyme Dicer. Moreover, it showed activity in cell models consistent with inhibition of pre-miR-21.

 

This is a nice paper, but there are several limitations. First, compound 9 is still a fairly modest binder with lackluster ligand efficiency. Indeed, while potency can be overrated, I would love to see a fully synthetic low nanomolar RNA binder. Second, while the approach may be general, it is not necessarily easy, and it requires specialized fragments. And as we noted last year, there is no relation between crosslinking efficiency and affinity. I wish the researchers had tried linking some of the non-selected fragments to see whether these were false negatives. Indeed, given the complexity of the approach, I wonder if the researchers would have been better off simply making and testing an anchor library around compound 1, in a similar fashion as described here.

 

But whether or not Chem-CLIP-Frag-Map turns out to be the solution to targeting RNA, I wholeheartedly agree with the conclusion: “It may be time to describe biomolecules that are perceived to be challenging small molecule targets as ‘not yet drugged’ rather than ‘undruggable.’”

Eighteenth Annual Discovery on Target Meeting

Last week CHI held Discovery on Target – virtually of course. There were 20 tracks over three days and more than 650 attendees, down from 1100+ last year. Because the event was more fragmented (pun intended) than the recent DDC, which had at most four parallel tracks, the Q&As and discussions seemed smaller, though that could have just been the ones I attended. On the other hand, one of the huge advantages of the format is being able to watch concurrently scheduled talks later. More thoughts on virtual conferences are here, and if you have not already done so please take our poll on the right side of the page.

 

This conference has always been more biology-focused than DDC, with tracks on antibodies, immunology, NASH, gene therapy, disease modeling, and fibrosis, among others. But there were also plenty of talks on targets and methodology, which is where I’ll focus most of this post. Please add your highlights and thoughts in the comments.

 

Julien Orts (ETH Zurich) presented an update on his NMR² method, which uses information from intermolecular NOEs to computationally determine protein-ligand structures without requiring full NMR assignment of the protein. We wrote about this technique in 2017 and at the time we questioned how applicable it would be to fragments due to low affinities, multiple binding modes, and fewer contacts. As it turns out, very: Julien described successes with proteins including HDM2, DsbA, bromodomains, and Pin1. Even with as few as 10-12 intermolecular NOEs he has been able to get good agreement with crystal structures. Currently he is applying this approach to SARS-CoV-2 proteins as part of the COVID-19-NMR project.

 

William Pomerantz (University of Minnesota Twin Cities) also presented NMR techniques. He is particularly known for his protein-observed ¹⁹F (PrOF) NMR screening, in which fluorinated tyrosine and tryptophan residues are introduced into proteins. Ligand binding changes the chemical shifts of the fluorine atoms, and by varying the concentration of the fragment, accurate dissociation constants can be determined. In early work, a screen of 930 fragments in pools of 5 against BRD4 took 11 hours (and another 11 hours for deconvolution) and provided multiple hits. We’ve covered some of his more recent work using shapely fragments here, and in unpublished work he has been screening the dual-domain construct of BRD4 and finding fragments that are ten-fold selective for one over the other bromodomain. He is further improving throughput by screening two proteins simultaneously.

 

Rounding out NMR, Andrew Petros (AbbVie) presented a beautiful fragment-to-lead success story on TNFα, a trimeric cytokine that has been the subject of numerous (often unsuccessful) lead-discovery efforts. A 2-dimensional NMR screen of 18,000 fragments gave just 11 hits. Crystallography of one showed two copies binding in close proximity, and linking these ultimately led to a low nanomolar binder. The series showed high clearance and no oral bioavailability, so they performed additional screens to identify different fragments that were ultimately advanced to potent compounds with animal efficacy. I look forward to reading the paper when it is published.

 

Finding fragments is important, but so is avoiding artifacts, the subject of a talk by Samantha Allen (Janssen). Around 2% of screening compounds can form small molecule aggregates that can interfere with assays, and if these aren’t weeded out they can quickly overwhelm an assay. Samantha described the use of resonance waveguide grating (RWG) technology, as used in the Corning Epic BT. This label-free technology is similar to SPR, but RWG can be run in 384 or 1536 well plates. Samantha showed that RWG compares favorably to dynamic light scattering for detecting aggregates. It is also 4-5 times faster and less prone to false-positives.

 

Covalent fragments were a theme of last month’s DDC, and they were prominent here as well. Four years ago we highlighted work out of Ben Cravatt’s lab doing covalent fragment screening in cells, but this was a rather time-consuming process. Steve Gygi (Harvard) has streamlined activity-based protein profiling and was able to screen 288 fragments in just 7 days and identify more than 1500 modified cysteine residues.

 

Dan Nomura (UC Berkeley) continued the theme with a wide-ranging presentation using chemoproteomics to discover covalent ligands for a variety of targets, including new E3 ligases, which can be used for developing targeted protein degraders. (Shameless plug/disclosure: Dan Nomura is a founder of my company, Frontier Medicines, and we are actively hiring across multiple positions and levels.)

 

Targeted protein degraders such as PROTACs were the subject of one track at last year’s DoT meeting, and this year two sequential tracks were devoted to the topic. As I suggested in 2018, fragments could be ideal starting points given that high affinity is not always necessary. This year, Stewart Fisher confirmed that he and his colleagues at C4 Therapeutics often “detune” chemical matter, lowering the binding affinity to get efficient degraders. That doing so can improve physicochemical properties is a nice bonus.

 

Finally, although not directly fragment-related, William Kaelin (Dana Farber Cancer Institute) gave an inspiring talk on the discovery and development of MK-6482, an allosteric HIF2α inhibitor in late-stage clinical trials for cancer linked to Von Hippel-Lindau disease; data released just last week shows durable responses in patients with kidney cancer. The science itself was lovely, but he reminded us of the ultimate stakes: “It’s not about what journal your paper is published in or whether you can fool reviewer 3, it’s about whether you publish things that are true and robust and can be built upon by others.”

 

Words to live by.

From noncovalent to covalent fragment for NSD1

As evident at the CHI Drug Discovery Chemistry meeting a couple weeks ago, covalent fragment-based lead discovery is becoming increasingly popular. Normally this entails screening a library of electrophile-containing fragments. However, it is also possible to start with a noncovalent fragment and add the “warhead” later. This is the approach taken by Jolanta Grembecka, Tomasz Cierpicki, and collaborators at University of Michigan, Memorial Sloan Kettering Cancer Center, and Columbia University in a paper just published in Nat. Chem. Biol.

 

The researchers were interested in NSD1, one of three related histone methyltransferases whose potential role in cancer is uncertain – in part due to the lack of good chemical probes. They started with a two-dimensional (¹H-¹⁵N HSQC) NMR screen of 1600 fragments in pools of 20, with each fragment present at 250 µM. BT1 was one of the hits, and synthesis of several analogs led to BT2, which showed low micromolar affinity as assessed by ITC as well as an IC₅₀ of 66 µM in a functional assay. Crystallography was unsuccessful, but NMR experiments suggested considerable changes in an autoinhibitory loop that blocks the substrate-binding region of the so-called SET domain.

For whatever reason, the researchers tested thiocyanate analog BT3 and found that this binds covalently as a disulfide to a cysteine in the autoinhibitory loop. They were able to get a crystal structure of BT3 bound to the protein, which revealed significant rearrangements that allow BT3 to bind deep in the SET domain, where it makes multiple polar and hydrophobic contacts. Prudently they chose to replace the unstable thiocyanate warhead, and while acrylamide derivatives were inactive, aziridine BT5 modified the protein as assessed by mass spectrometry.

 

BT5 inhibited NSD1 with an IC₅₀ of 5.8 µM after a four hour incubation and was somewhat selective against the related proteins NSD2 and NSD3 as assessed both by mass spectrometry and activity assays. It also showed good selectivity at 50 µM against 20 other epigenetic enzymes and 291 kinases. Interestingly though, at this concentration BTK was inhibited by 41% and EGFR was inhibited by 49%; both these kinases are targeted by approved covalent drugs.

 

Next, the researchers conducted multiple cell assays. A cellular thermal shift assay (CETSA) revealed that BT5 stabilized NSD1 but not NSD2 or NSD3. Growth of an NSD1-dependent cancer cell line was inhibited with GC₅₀ = 1.3 µM after 3 days, NSD1-mediated histone methylation was suppressed, and several target genes showed reduced expression. BT5 also inhibited growth of non-NSD1-dependent cell lines, though at 6-8 higher concentrations, and did not alter methylation or target gene expression. Finally, the compound impaired colony formation of cells from a primary patient-derived sample with an NUP98-NSD1 translocation.

 

This is a nice, carefully conducted study. Refreshingly, the researchers do not attempt to oversell their results, and acknowledge that “further optimization is needed to develop NSD1 chemical probes.” But they’re off to a good start, and it will be fun to see what they – and others – will come up with.

Fifteenth Annual Fragment-Based Drug Discovery Meeting

What a strange year for conferences! Everything fragment-related in the first half of the year was canceled or rescheduled. And when it turned out that postponing Drug Discovery Chemistry (DDC) from April to August was overly optimistic, Cambridge Healthtech Institute went virtual for the first time, with the meeting running last week from August 25-28. Although I was skeptical, in the end the meeting was quite successful, with more than 600 attendees – roughly three-quarters last year’s in-person attendance. 

 

To accommodate multiple time zones each of the parallel tracks was shorter. Days typically began around 10:00 AM EDT, which was 7:00 for me in San Francisco but midnight for folks in Melbourne. Despite this inconvenience, 40% of participants this year came from outside the US, up from 30% last year.

Most of the talks were pre-recorded, which meant that speakers could answer questions in real-time in a chat box. More importantly, the talks are available to attendees for a year, which means you can watch relevant talks from other tracks, thus relieving the FOMO inevitably experienced in meetings of this sort. It also means you can replay particularly relevant talks to your colleagues, though I wonder if this also makes speakers wary about disclosing the freshest information. The on-demand access to posters is especially useful, as it is too easy to overlook these in the usual melee. The organizers also did a nice job of trying to foster the feel of an in-person meeting, with multiple live Q&A panels, breakout sessions, and other interactive events.

There are drawbacks, chief among them the lack of spontaneous and serendipitous meetings that are one of the main benefits of in-person conferences. And of course there were technical snafus. Most talk slots were only 20 minutes, but some of the pre-recorded talks went as long as 28 minutes, forcing attendees to choose between missing part of one talk or another (or a live Q&A). Although you can fast-forward, it would be nice if you could also speed up playback speeds. And on at least one occasion the wrong talk was played, though I was able to go back and watch the correct one later.

But enough about process, what about the event itself? With dozens of talks over four days I can’t be exhaustive, so please add your thoughts to the comments.

Faster, cheaper, better.

This describes one consistent theme. Frank von Delft (Diamond Light Source) gave an update on their high-throughput crystallographic screening, which has led to > 3000 fragment hits on > 150 targets since 2016. We’ve written previously about their efforts against COVID-19, which have led to three separate sub-micromolar lead series against the viral protease M^Pro. This rapid progress has been enabled by crowdsourcing across more than 30 separate groups, but Frank is also moving toward crystallographic screening of crude reaction mixtures, similar to the REFiL approach described by Beatrice Chiew (Monash University, see here for a longer description). Frank hopes to make good compounds a commodity, with a 5-year vision of achieving “biologically relevant potency routinely, cheaply (£10k) and quickly (weeks).” It’s an audacious goal, and we’ll check back in 2025 to see how far the field has come.

Key to the success of these types of approaches is what plenary speaker Phil Baran (Scripps) calls “boring chemistry” that consistently works in multiple contexts. To Phil, inventing chemistry that becomes boring is a great compliment, and he showed examples of running interesting transformations in tea, beer, and wine. As the name of this blog suggests, I have a soft spot for this sort of thing, and wholeheartedly agree with his statement that “you can’t give a Nature paper to a cancer patient.”

Covalent fragments (or not)

Covalent drug discovery was also a major theme, with John McCarter (Amgen) and Matt Marx (Mirati) describing discovery of two covalent clinical compounds against an oncogenic mutant form of KRAS (see here). Dom Esposito (Frederick National Laboratory) is also pursuing KRAS using covalent Tethering. They have prepared 91 cysteine mutants and screened 18 of them against nearly 1200 disulfide-containing fragments, yielding a plethora of hits.

Alexander Statsyuk (University of Houston), Maurizio Pellecchia (UC Riverside), and Nir London (Weizmann Institute) all also discussed covalent lead discovery. Maurizio has been targeting lysine residues using sulfonyl fluorides and fluorosulfates; for the latter warhead he has been able to show reasonable pharmacokinetics in rodents. We’ve previously discussed some of Nir’s work, but here he described a nice case study against the challenging anti-cancer target Pin1 which led to potent and surprisingly selective chloroacetamides with activity in mice.

Interestingly, while chloroacetamides were the main class of M^Pro fragment hits, the three most advanced lead series Frank mentioned are all non-covalent. There were plenty of other nice non-covalent fragment-based success stories too, including potent selective inhibitors of the lipid kinase Vps34 (Jenny Viklund, Sprint Biosciences) and selective inhibitors of one kringle domain of apolipoprotein(a) (Jenny Sandmark, AstraZeneca).

Methods

As always, there were many talks on methods, especially during the Fifth Annual Biophysics Friday track. John Quinn (Genentech) provided a state-of-the-art update on SPR, and mentioned that they are able to screen 3000 fragments in a day or two using the Biacore 8K. This allows them to assess ligandability for new targets, and the ligandability score is predictive for how well the target performs in HTS or DEL screening.

Amit Gupta described NanoTemper’s new Dianthus instrument, which relies on the temperature-related intensity change (TRIC) of a fluorophore bound to a protein. This is similar to their MST approach though it appears to be higher-throughput, and a paper benchmarking these techniques against DSF and SPR should be coming out later this year.

Also on the subject of thermal shifts, Justin Hall (Pfizer) gave a provocative presentation on using these to determine ligand affinities. He noticed a correlation in his own research, but the prevailing wisdom held that irreversible thermal denaturation (as seen for most proteins) would not provide thermodynamic parameters. Nonetheless, perhaps because proteins are fundamentally similar (consisting as they do of chains of 20-odd amino acid residues), the temperature-dependent Arrhenius functions and activation energies of unfolding are also similar, and thus for heating rates of 4°C/minute and 100 µM ligand one can extract dissociation constants. However, he did mention that this approach is restricted to reasonably tight ligands (K_D < 20 µM). Also, if a ligand binds to the unfolded state of the protein, or to multiple sites, all bets are off.

In the interest of time I’ll stop here, but if you’d like to experience a virtual conference yourself, there will be a number of good FBLD talks at Discovery on Target next month, and I hope to “see” you there. But I especially hope that in-person conferences will resume next year once our industry – and competent governments – get COVID-19 under control.

Second harmonic generation (SHG) vs KRAS

Practical Fragments is currently running a poll on fragment-finding methods used by readers – please vote on the right-hand side. One biophysical method that perhaps we should have included is second harmonic generation (SHG). A recent paper in Proc. Nat. Acad. Sci. USA by Josh Salafsky, Frank McCormick, and collaborators at Biodesy, University of California San Francisco, and elsewhere describes the technique and its application to find fragments that bind to the oncogenic protein KRAS.

In SHG, two photons of the same energy are absorbed by a material which then emits a single photon with twice the energy. In the commercial instrument developed by Biodesy, a powerful 800 nm laser irradiates a dye, and the 400 nm photon it emits is detected. The intensity of the signal is exquisitely sensitive to the precise orientation of the dye. If a protein is labeled with an SHG-active dye and then immobilized on a glass surface, even subtle changes in conformation will be detected.

The researchers chose the G12D mutant form of KRAS, which is one of the most common variants and is associated with particularly aggressive tumors. They labeled the protein with a lysine-reactive SHG dye under conditions in which each protein would, on average, have one covalently-bound dye molecule (though some would have none and others would have more than one). Proteolysis and mass-spectrometry analysis revealed that the dye molecule labeled three different lysine residues, which the researchers viewed as a feature since a ligand causing a conformational change to any of the lysine residues would generate a signal. The researchers also demonstrated that the dye modification did not interfere with the ability of KRAS to bind to the RAS-binding domain of RAF.

Labeled KRAS was then immobilized and tested against several proteins known to bind it, including antibodies and the nucleotide exchange factor SOS. These produced SHG signals, presumably by causing conformational changes to KRAS, while non-binders such as tubulin did not.

Having established that the assay could detect binders, the researchers screened 2710 fragments at 250 and 500 µM, and obtained a whopping 490 hits. These were then triaged by screening at lower concentrations and performing dose-titrations, and 60 were then characterized by SPR.

Fragment 18, 4-(cyclopent-2-en-1-yl)phenol, showed binding by both SHG and SPR, and was further studied by 2-dimensional NMR (¹H-¹⁵N HSQC). This technique allowed measurement of the weak 3.3 mM dissociation constant. More importantly, it allowed the researchers to establish the binding location as being near the so-called “switch 2” region where SOS normally binds. This is the same region where a previous NMR screen had identified the slightly more potent fragment DCAI. The current paper confirmed that finding, though the researchers found evidence that DCAI may bind to other sites too. Docking studies using SILCS suggested that fragment 18 likely binds in a similar orientation as DCAI. Not surprising given the low affinity, the new fragment did not show functional activity in a biochemical screen.

SHG is an interesting approach, and the ability to rapidly assess protein conformational changes distinguishes it from other biophysical techniques. Site-specific labeling would produce more informative data on which regions of a protein move. However, I wonder if SHG is perhaps too sensitive, as evidenced by the large number of hits. Indeed, the researchers demonstrated that the promiscuous lipophilic amine mepazine also generated a strong SHG signal with KRAS. It would be interesting to do a head-to-head comparison with other similarly rapid techniques such as DSF or MST. Have you tried using SHG, and if so, how did it perform for you?

Fragment Based Lead Discovery San Diego 2011

Zenobia Therapeutics hosted a one-day FBLD meeting in San Diego last Friday. It was a very full day of seven talks and plenty of informal discussion, with both lunch and happy-hour on a rooftop terrace overlooking the ocean. I’ll try to give some flavor of the event below – those of you who were there, please chime in with your own impressions.

In the first talk of the day, Michael Recht of Xerox’s PARC discussed the construction and use of array-based nanocalorimetry devices, which analyze samples as small as a couple hundred nanoliters. Although conceptually related to isothermal titration calorimetry, the scale of nanocalorimetry makes direct measurement of binding enthalpy difficult. However, the devices are well-suited to measuring the heat output from enzymatic reactions, thus allowing detailed study of enzyme kinetics and inhibitor characterization (see here for a full description and here for a review, both open-access).

Maurizio Pellecchia of the Sanford-Burnham Medical Research Institute gave a wide-ranging talk with the common theme of NMR as an enabling method. In particular he noted that, due to its low protein requirements, ILOE (which we’ve written about previously) could be considered the “homeopathy of NMR;” a recent paper in J. Med. Chem. describes its application to generate hits against Bcl-family proteins. He used a different NMR technique to discover a potent and selective JNK inhibitor that binds in both the ATP and substrate binding sites. On a completely different topic, Maurizio noted that roughly one-third of drug targets contain metals, prompting the construction of a fragment-library designed with metal-chelation in mind, a strategy we’ve written about previously (see also here). Many of these techniques and libraries have now been licensed into a new company, AnCoreX Therapeutics, founded by Maurizio.

Schrödinger was a major sponsor of the meeting, and company scientist Kathryn Loving gave a good overview of the potential of their suite of software tools for fragment-based lead discovery. After lunch I presented a PDK1 story (see here) as well as applications of Carmot’s Chemotype Evolution, and Michelle Arkin from UCSF discussed some of the capabilities of the university’s Small Molecule Discovery Center, with special focus on protein activators and protein-protein disruptors.

Tony Giannetti of Genentech gave a nice presentation on caspase-6 inhibitors and also discussed the power of SPR, a topic on which he’s written extensively. He noted that as long as one is careful about conducting the experiment, the number of promiscuous or non-specific binders found by SPR tends to be less than the number of true hits – he described the former as “a managed headache.”

Tony is in the enviable position of having screened numerous targets using the same platform, allowing him to do some very interesting data mining. One phenomenon that we’ve previously commented on is the fact that, across multiple companies, only about a third of fragments hit in one or more screens, and Roche is no exception (36% across 13 screens). With the merger of Genentech and Roche now complete, Tony has been able to compare the fragment libraries assembled separately at each institution. Interestingly, although they contain < 5% common fragments and < 10% similar fragments, the libraries are, by broad measurements such as size or lipophilicity, statistically indistinguishable. However, the hits from the Roche collection seem to have higher ligand efficiencies. Also, the most potent binders tend to have 13-17 atoms, with the most ligand-efficient molecules generally having a molecular weight < 200. Finally, there has been a lot of recent discussion on making fragments less flat (aromatic) and more three-dimensional (often aliphatic). However, as I predicted 2 years ago, one needs to be cautious: it turns out that hits were actually enriched for flatter fragments, although Tony did mention that most of the screens were against kinases, which may be particularly “flatophilic”.

Vicki Nienaber, founder and president of Zenobia, closed the conference by discussing the potential for fragment-based approaches to tackle diseases of the central nervous system (CNS). The rationale is that, since CNS drugs need to be small to cross the blood-brain-barrier, it pays to start with fragments. Indeed, Zenobia’s fragment library has a molecular weight averaging just 150-175, with an upper limit of 225, which should allow them to avoid molecular obesity. Vicki described the discovery of nanomolar leads against LRRK2, a Parkinson’s disease target, and against PDE10, a Huntington’s disease target.

As far as I know this was the last face-to-face fragment event this year, but there are still several webinars this month and next, and it’s not too soon to start planning for 2012; please see here for upcoming events.