Covalent fragments meet high-throughput synthesis and crystallography

Two hot areas of FBLD include covalent fragments (see here for example) and high-throughput crystallography (see here and here). Two new papers add to the excitement but also reveal some of the challenges.

 

The first, published open access in J. Med. Chem. by Charles Eyermann (University of Cape Town), Christopher Schofield (Ineos Oxford Institute of Antimicrobial Research), Christopher Dowson (University of Warwick) and collaborators at Diamond Light Source focuses on an antibacterial target, the penicillin binding protein PaPBP3 from Pseudomonas aeruginosa.

 

Some members of the group had previously screened a library of 40 compounds against this protein and identified a single hit that bound covalently. Inspired by this result, they assembled a library of 262 commercial covalent fragments, 152 of which contained boron, an element known to react with serine and threonine hydrolases. These were screened crystallographically at 250 mM, resulting in 34 boron-containing hits “with various levels of electron density observed at the active site.” Many of these seem to have given ambiguous density, and only a handful of fragment structures are reported.

 

Next, the researchers attempted fragment growing; the team ultimately obtained 10 structures, drawing from the original fragments and the elaborated molecules. Interestingly, these showed three different binding modes: a monocovalent complex with the active-site serine, a dicovalent complex with the active-site serine and a neighboring serine, and a tricovalent complex with these two serine residues and a nearby lysine side chain.

 

Despite having up to three covalent bonds to the enzyme, even the elaborated molecules showed at best double-digit micromolar inhibition of PaPBP3, and none showed antimicrobial activity. The bond between boron and serine or lysine is reversible, so while disappointing, this weak activity is not entirely surprising. The result is reminiscent of efforts against the SARS-CoV-2 main protease, M^pro or 3CLpro, where many of the crystallographic hits turned out to be very weak binders.

 

The second paper, published open access in Angew. Chem. Int. Ed. by Alexander Dömling and collaborators at University of Groningen and the Paul Scherrer Institute, also looked at 3CLpro. Here though, rather than starting with commercial fragment libraries the researchers used high-throughput synthesis to make their own.

 

The Dömling lab has had a long-standing interest in multi-component reactions in which several reagents are combined to generate products. The researchers explored the Passerini reaction (which uses an aldehyde or ketone, a carboxylic acid, and an isocyanide) and the Ugi reaction (which uses all of these as well as an amine). For the carboxylic acid, they chose an electrophile such as acrylic acid. They initially worked out the conditions at 0.5 mmol scale in 96-well plates and then purified the products using chromatography or precipitation. The majority of wells yielded product in good yields and purity.

 

Next, the researchers turned to high-throughput methods, performing the reactions in 384-well plates using acoustic liquid handling. This is similar to the approach I wrote about here that was used to discover covalent KRAS inhibitors, ultimately leading to the approved drug sotorasib.

 

The researchers then soaked 181 of their compounds against the SARS-CoV-2 protein 3CLpro, with each compound at 10 mM. Unlike the crude reaction screening described here, the researchers used pure compounds. This effort resulted in five hits, though one of them turned out to bind noncovalently. Three of the molecules had low micromolar activity.

 

In the end, while both of the papers do report the discovery of covalent modifiers, the functional activities in the first paper are modest, and the active warheads in the second paper (chloroacetamides and acrylates) are likely too reactive to be advanceable. A nice feature of crystallography is that it can provide structural information for extremely weak hits. But as we’ve asked previously, how weak is too weak? Getting more crystal structures more rapidly is one thing, but figuring out which ones are useful requires even more skill, creativity, and luck.