25 April 2022

Seventeenth Annual Fragment-Based Drug Discovery Meeting

Last week the CHI Drug Discovery Chemistry (DDC) meeting returned triumphantly to San Diego. This was the best conference I’ve attended in years, which reflects not just the quality of the meeting itself but the fact that three-dimensional events are vastly superior to their 2D counterparts.
About 75% of the more than 700 attendees were physically present, though having a virtual option turned out to be wise; at least three of the speakers had COVID but were still able to present remotely. Although the FBDD track lasted just a day and a half, fragments were well-represented across the four days and ten tracks. I won’t attempt to summarize the more than 40 talks I attended but will just cover some broad themes.
Computational Methods
Seva Katritch (USC) described the V-SYNTHES approach we highlighted in January. This modular method enables computational fragment growing, in effect facilitating a search of 11 billion molecules from just 600,000 scaffolds. The method as described makes heavy use of Enamine’s make-on-demand molecules, and I think everyone in the audience was excited to hear that the company has started making and shipping compounds from Kyiv again.
In the comments to the blog post on V-SYNTHESES someone mentioned BioSolveIT, and Paul Beroza (Genentech) described using their software for a similar approach. One of the targets they investigated, ROCK1, was also investigated with V-SYNTHES, and both techniques yielded unique nanomolar inhibitors.
Jan Wollenhaupt (Helmholtz Zentrum Berlin) also mentioned BioSolveIT in the context of fragment growing by catalog. Fragments identified crystallographically from their F2X libraries (see here) were grown to low micromolar endothiapepsin binders. Interestingly an unbiased docking screen did not find these molecules, illustrating the utility of stepwise computational approaches.
DOTS is another approach to computational growing and docking enabled by rapid synthesis we’ve previously written about, and Xavier Morelli (CNRS) gave an update, including the fact that they plan to launch a webserver soon.
Physical Methods
Tim Kaminski (InSingulo) described an intriguing method for screening liposome-bound proteins such as GPCRs. Dyes incorporated into the liposome are visualized using single molecule microscopy, and the liposomes can be observed in real time binding to immobilized targets in 384-well plates. Tim mentioned that the instrument should be available for purchase next year.
A new take on an old method was described by Félix Torres (ETH), who discussed using photochemically induced dynamic nuclear polarization (photo-CIDNP) to increase the sensitivity of NMR, thereby reducing experimental times by a factor of 100. The method requires specialized fragments and a customized NMR, but they can currently screen 1500 fragments per day, and the approach could be particularly valuable for screening hard-to-express proteins.
Sticking with the theme of photochemistry, Rod Hubbard (Vernalis by way of Hitgen) discussed a DNA-encoded library of more than 130,000 fragment-linker combinations each containing a photoaffinity tag. Screening this against PAK4 yielded 425 hits, and of the 30 chosen for validation more than 90% confirmed by NMR or crystallography. As we noted in 2020, combining DEL and FBLD provides new opportunities for exploring chemical space.
It’s been a few years since we discussed weak affinity chromatography, and Kirill Popov (WAC) provided an update. They’ve applied the approach to more than 50 targets and have obtained hit rates up to 20%. An example against SMARCA4 yielded hits that were subsequently found to bind at two sites, one of which had not previously been described.
Covalent fragments continue to increase in popularity. FragNet alum Lena Muenzker (BI) described an intact-protein mass spectrometry screen of the E3 ligase SIAH1 against 1260 acrylamides, resulting in 214 hits. Crystallography has been successful, and they are planning to use these to generate covalent PROTACs.
We’ve previously written about screening covalent fragments in cells, and Benjamin Horning (Vividion) and Madeline Kavanagh (Scripps) described a nice chemoproteomics case study in which an alkynamide-containing fragment was identified that binds to cysteine 817 in the kinase JAK1. Optimization led to a low nanomolar binder that inhibits JAK1 signaling.
Cysteine is not the only amino acid amenable to covalent modification. Plenary keynote speaker Laura Kiessling (MIT) described squarate derivatives as tunable “Goldilocks” warheads for lysine, with the right balance of reactivity and stability.
Success Stories
Cases studies were abundant, including some new disclosures that I’ll hold off describing until they publish. Of course, drugs are the ultimate success stories, and several of these were presented. Svitlana Kulyk recounted the discovery of MRTX1719, Mirati’s MTA-cooperative PRMT5 inhibitor, including some interesting tangents not discussed in the publication.
Steve Fesik (Vanderbilt) gave two presentations on near-clinical compounds, one targeting MCL1 and the other WDR5. In both cases weak fragments were advanced to picomolar binders within one to two years, but it has taken much longer to optimize other properties of the molecules.
Indeed, this turned out to be something of a theme. Valerio Berdini (Astex) discussed the discovery of erdafitinib, the third approved FBLD-derived drug. The program started in 2006, and it took just nine months to go from the fragment hit to late lead optimization. But the compound didn’t enter the clinic until 2012, and it took until 2019 to be approved.
Similarly, Wolfgang Jahnke (Novartis) described the story of the sixth approved fragment-based drug. The fragment screen against ABL was conducted in 2006, but the project went through two near-death experiences. Asciminib finally entered the clinic in 2014, and it was approved last year.
But timelines are not destined to be long. We’ve previously written about vemurafenib, the first FBLD-derived drug, which took just six years from project initiation to approval. Ryan Wurz (Amgen) gave a retrospective on sotorasib, the fifth approved FBLD-derived drug. Amgen started the program in August 2012, sotorasib was first synthesized in early 2017, first dosed in humans in 2018, and approved in May of last year. Fast doesn’t mean easy: it took 110 co-crystal structures, and I counted more than 100 names on the acknowledgement slide. But success against KRAS is a welcome reminder that sometimes we really can accomplish the impossible when we work together.
This is a good point on which to close. Assuming SARS-CoV-2 doesn’t intervene, DDC is scheduled to return to San Diego April 10-13 next year. I hope to see you there!

18 April 2022

Fragments win in a virtual screen against Notum

Wnt proteins are implicated in a variety of diseases, from Alzheimer’s to colorectal cancer. The enzyme Notum shuts down signaling by removing a palmitoyl group from Wnt. Last year Practical Fragments highlighted several series of Notum inhibitors identified from biochemical and crystallographic fragment screens. The researchers behind those efforts, including Paul Fish and Fredrik Svensson (University College London), have now published a successful virtual screen against the enzyme in J. Med. Chem.
Starting with 1.5 million compounds available from ChemDiv, the researchers chose 534,804 based on a variety of computational filters including molecular weight (200-500 Da), number of hydrogen bond donors (<=2) and ClogD (-4 to 5). A virtual screen of these (using Glide) produced 1330 high-scoring hits, of which 1088 were chosen for purchase. Of these, 952 were available, a much higher percentage than the ZINC15-reliant paper we wrote about earlier this year.
All 952 compounds were tested in a biochemical assay, and the 44 that gave >50% inhibition at 1 µM were then tested in dose-response format. This yielded 31 compounds with IC50 values < 500 nM. These could be subdivided into four structurally related clusters and eight singletons. Further triaging removed compounds likely to cause assay interference as well as those similar to known Notum inhibitors. This left two clusters and two singletons.

Compound 1f was the most potent member of a series of 9 related (and possibly covalent) inhibitors. Although these strongly inhibited the enzyme in the biochemical assay, they were essentially inactive in a cell-based assay. They were also highly insoluble and showed low cell permeability, and were thus dropped.
Compound 2a was one of two related molecules that were also quite potent when initially tested. Unfortunately, when the molecules were resynthesized they turned out to be significantly weaker and were also not very soluble, so this series was also halted.
The singleton compound 3 turned out to be a covalent inhibitor; the catalytic serine formed an ester with the molecule. The mechanism is more fully described in this open-access J. Med. Chem. paper.
That leaves the second singleton. Compound 4d was not just active in the biochemical assay, it also showed sub-micromolar cell activity. SAR, guided by crystallography, ultimately led to low nanomolar inhibitors. The pKa of compound 4d was measured to be 7.9, which is less acidic than many previously reported Notum inhibitors and thus more likely to be cell permeable. This turned out to be the case experimentally, and the compound was also stable in mouse liver microsomes. Pharmacokinetics in mice were promising for several compounds, but unfortunately brain penetration – which the researchers were hoping for – was negligible. (This could be an advantage for peripheral diseases.)
This is a nice example of lead discovery in academia. Like last week’s post, it also illustrates that fragments themselves can be quite potent. Indeed, although the researchers were looking for molecules up to 500 Da in their virtual screen, all of the best hits were fragment-sized. Another illustration that small is beautiful.

11 April 2022

Nucleophilic fragments vs SARM1: in situ inhibitor assembly

Recently Practical Fragments wrote about nucleophilic fragments that could react with proteins or cofactors. Previously we’ve also written about in situ chemistry, in which a protein catalyzes the formation of an inhibitor. An interesting marriage of these concepts has just been published (open access) in Mol. Cell by Robert Hughes (Disarm Therapeutics), Thomas Ve (Griffith University) and a group of international collaborators.
The researchers were interested in the protein SARM1, which is implicated in the axon degeneration associated with several neurodegenerative disorders. Last year the researchers published a Cell Rep. paper (also open access) in which a biochemical screen of roughly 200,000 molecules led to the discovery of isoquinoline as a 10 µM inhibitor of SARM1. Optimization led to 5-iodoisoqinoline, dubbed DSRM-3716, a 75 nM fragment-sized inhibitor. The paper goes on to demonstrate that the molecule not only prevents axonal degeneration but can even promote recovery of injured axons. The new paper explores the mechanism of action.
SARM1 is an NADase: it cleaves the critical cofactor nicotinamide adenine dinucleotide (NAD+). While using NMR to study the mechanism of inhibition, the researchers found that DSRM-3716 reacts with NAD+ to form the new compound shown. In this sense, DSRM-3716 acts as a prodrug, somewhat analogous to sulfanilamide antibiotics which act as PABA mimics to block folate biosynthesis.

What’s behind the inhibition of SARM1? A series of crystallographic and cryo-EM studies of SARM1 reveal that the protein can self-associate into multimers which are either inactive or active depending on the relative orientations of the individual proteins. NAD+ normally binds at the interface between two SARM1 proteins. The compound made from NAD+ and DSRM-3716 binds here as well, blocking further activity. The crystal structures also revealed a clear halogen bond (see here) with the iodine in DSRM-3716, explaining the increased activity over isoquinoline itself.
Unlike the nucleophilic fragments we wrote about last month, isoquinoline probably won’t raise too many eyebrows among medicinal chemists, as the moiety is found in a handful of approved drugs. The researchers also demonstrated that DSRM-3716 itself is selective for SARM1 in a panel of other enzymes that use NAD+.
This is a lovely case of high-throughput screening in which the hit turns out to be a fragment. Indeed, the highly charged compound that actually inhibits SARM1 would not be cell-permeable, but that's just fine since it is formed inside cells. It is worth noting that nearly 1000 approved drugs could be classified as fragments in terms of molecular weight. In the case of CNS drugs, small is beautiful, and it will be fun to watch how far DSRM-3716 derivatives will be able to advance.

01 April 2022

Fragments in space!

Practical Fragments has discussed fragments on Mars and Venus, but those planets are just two small specks in a vast universe. Always thinking big, the luminaries at DREADCO (who previously brought us fragment screening in cells using cryo-EM) have set their sights on deep space. Their theoretical proposal has just been published in the Journal of Extraterrestrial and Space Technologies.
One of the big unknowns in molecular recognition is precisely how small molecule ligands approach proteins. To find out, the researchers propose creating a library of fragments, each of which is attached to a very tiny mirror. Proteins of interest would also have tiny mirrors affixed to them. Laser interferometry would be used to study the interactions of proteins and ligands in extremely dilute solutions.
One potential problem with this approach is gravity, which is hard to escape on Earth, so the researchers propose running their experiment at a Lagrange point. They had hoped to catch a ride on the James Webb Space Telescope, but the mirror fabrication has taken longer than expected.
Even for a secretive multinational megacorporation like DREADCO this will be an expensive endeavor, so they’ll probably have to wait until they’ve eradicated human disease before launching this project. In the meantime, they’re taking suggestions for protein targets – feel free to leave yours in the comments!