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.

A library of covalent fragments vs a library of kinases

Protein kinases have proven to be a fruitful class of targets, as evidenced by more than 80 FDA-approved drugs, five of which came from fragments. Because all protein kinases bind ATP, selectively inhibiting just one of the more than 500 family members can be challenging. This is a bit easier for the 215 protein kinases that contain a cysteine within the ATP-binding pocket capable of reacting with covalent ligands. In a recent (open access) Angew. Chem. Int. Ed. paper, Matthias Gehringer, Stefan Knapp, and collaborators at Johann Wolfgang Goethe-University and Eberhard Karls University Tübingen provide such starting points for dozens of kinases.

 

The researchers built a small library of 47 fragments consisting of six classic hinge-binding moieties such as pyrazole and azaindole coupled through nine aryl linkers at varying positions to an electrophilic acrylamide warhead. Although most of the compounds are rule-of-three compliant, the researchers note they “reside at the upper end of fragments space,” similar to what we discussed last week. Chemical reactivity towards the abundant cellular thiol glutathione was tested and found to be lower than the approved drug afatinib, meaning the fragments might be good starting points for optimization.

 

Each member of the fragment library was screened against 47 different protein kinases chosen to present cysteine residues at a variety of positions around the ATP binding site. Two types of screens were conducted: intact protein mass spectrometry to assess covalent binding and differential scanning fluorimetry (DSF) to assess protein stabilization. Screens were run at fairly high concentrations, 50 µM protein and 100 µM fragment.

 

The results, plotted as a two-dimensional figure with kinases on one axis and compounds on the other, provide a wealth of information. Some compounds hit multiple kinases while others hit few or none. Similarly, some kinases are hit by multiple compounds while others are recalcitrant.

 

A couple more general observations emerged. First, there was little if any correlation between the inherent reactivity of a given fragment (as assessed by reactivity with glutathione) and the number of kinases hit, suggesting that covalent modification was driven by specific interactions rather than nonspecific reactivity. Second, there was also no clear correlation between the ability of a fragment to stabilize a given kinase and the ability of the same fragment to covalently bind to that kinase. This latter observation isn’t surprising, since one could imagine a fragment binding noncovalently to a kinase and stabilizing it without forming a covalent bond.

 

Most proteins contain multiple cysteine residues, and the researchers confirmed that the fragments were covalently modifying the cysteines in the ATP-binding pocket using mutagenesis, trypsin digestion, or, for MAP2K6, RIOK2, MELK, and ULK1, crystallography. The crystal structures were particularly informative in showing hydrogen bond interactions between the covalently-bound fragments and the hinge region.

 

As we’ve noted, the best metric for characterizing irreversible covalent inhibitors is k_inact/K_I, and the researchers determined these for covalent inhibitors of PLK1, PLK3, RIOK2, CHEK2, and CSNK1G2. The values ranged from 2 to 8 M^-1s^-1, comparable to other early covalent fragments.

 

This is a lovely, systematic paper that is in some ways an irreversible complement to a study we wrote about in 2013 focused on reversible covalent kinase inhibitors. The fact that hit rates are relatively high likely reflects the fact that all the fragments contain privileged hinge-binding pharmacophores.

 

Perhaps most importantly, all the data are available in the supporting information. If you’re interested in pursuing any of these 47 kinases, you may find good starting points here.

Do covalent fragments need to be larger?

A few months ago we highlighted work out of AstraZeneca detailing how to build a covalent fragment library. One of the design features was including larger molecules beyond the traditional rule of three (Ro3) criteria. A new open-access paper in J. Med. Chem. by György Keserű and collaborators at the HUN-REN- Research Centre for Natural Sciences and the Weizmann Institute of Science explores “size-dependent target engagement of covalent probes.”

 

The paper starts with a theoretical discussion of covalent inhibitors, focusing on the classic two-step mechanism in which binding of a ligand to a protein is followed by covalent bond formation. These steps are characterized by the inhibition constant (K_I) and the inactivation rate constant (k_inact). The most appropriate way to assess an irreversible covalent inhibitor is by the ratio k_inact/K_I, as we discussed last year.

 

A two-step mechanism is not the only possibility: the researchers also consider a three-step model in which binding of the ligand is followed by a second step, deprotonation of the amino acid nucleophile, before the final bond-forming step.

 

Fragments typically have lower affinities than lead-size or drug-size molecules, and thus k_inact will usually need to be higher for smaller molecules in order to see significant protein labeling. Simulations in which K_I is held constant show that at the high micromolar affinities often seen for fragments, protein modification requires either long incubation times or high reactivities. In addition to these simulations, the researchers also reanalyze publications we’ve previously covered such as this and this to argue that “reactivity contributes to labeling when the effects of other factors cancel out.”

 

Next, the researchers examine the kinase BTK and the oncology target KRAS, both of which have been successfully drugged with covalent molecules, ibrutinib and adagrasib, respectively. They trimmed back these molecules to smaller lead-like and fragment-like molecules and found that while some lead-sized molecules could still label the proteins, this was not the case for the fragment-sized molecules. From this they conclude that “fragment-sized covalent agents do not offer smooth optimization and are not ideal starting points.”

 

Two examples do not a trend make, but the researchers point to other examples in the literature. In 2020 we noted the larger size of covalent KRAS hits, and Vividion’s WRN inhibitor also started from a molecule with a molecular weight of 337 Da, while GSK’s starting point weighs in at 312 Da. The AstraZeneca library we mentioned at the start of this post yielded a hit against BFL1 that also just missed the Ro3 cutoff, coming in at 302 Da.

 

That said, there are counterexamples. Just last month we highlighted a covalent fragment hit that fits comfortably within the rule of three. Fragment-sized covalent hits can be found, but don’t expect them to be common. The alternative approach, screening lead-like compounds, will also likely require screening more compounds due to lower coverage of chemical space. Either way, libraries containing more molecules are likely to be beneficial for finding covalent starting points.

Fragments meet crypto!

Two years ago today, Practical Fragment$ launched a line of non-fungible tokens. Unfortunately, the NFT craze didn't last much longer than that for sea shanties. But another virtual confection, cryptocurrency, appears to be less ephemeral, and today SkyFragNet has announced the launch of FragCoin.

 

One of the problems with crypto is that it uses a huge amount of (arguably) wasted energy. What if the effort to mine crypto could be put to practical use?

 

As we noted six years ago today, SkyFragNet has automated the entire drug discovery pipeline. It is also building a powerful generative AI positronic brain, to be trained using in-house experimental data. But even though the prospective computational docking methods are best in class, they still need to be improved, and for this SkyFragNet is turning to everyone with a computer.

 

To mine new FragCoins, miners will dock fragments against various targets, and these results will be compared with ground truths at SkyFragNet: each successful docking event will initially result in one FragCoin. Think of it as Folding@home but for profit as well as fun.

 

As FragCoins are mined, the docking software will be continually improved. Over time the number of successful docking events required for a FragCoin will increase. 

 

Just like bitcoin, the number of FragCoins will be strictly limited, in this case to 977,468,314, the number of fragments in GDB-13. So don't delay: start docking today!