Fragments vs the SARS-CoV-2 main protease – this time by NMR

Last week we highlighted NMR screens against RNA from SARS-CoV-2. As we noted then, much of the drug discovery action has focused on the virus’s main protease, called M^pro or 3CLp. These efforts have included two separate screens by crystallography and/or mass spectrometry. A new (open-access) paper in Angew. Chem. Int. Ed. by Benoit Deprez, Xavier Hanoulle, and collaborators at CNRS and University Lille describes an NMR screen against this same protein.

 

The main protease is 306 amino acid residues long and forms a 67.6 kDa dimer in solution. Proteins this large are challenging for protein-detected NMR, both because the number of potentially overlapping resonances increases and because of line broadening. Nonetheless, the researchers used several sophisticated NMR techniques to assign more than 60% of backbone resonances as well as quite a few main-chain and side-chain hydrogens to gain information on binding locations.

 

A library of 960 fragments was purchased from Life Chemicals and Maybridge. These were pooled in groups of five, with each fragment at 377 µM, and screened by Water-LOGSY and, for 427 fluorinated fragments, ¹⁹F line broadening and chemical shift perturbation. This exercise yielded 159 hits.

 

These hits were validated in a protein-detected ¹H, ¹⁵N TROSY-HSQC experiment, which confirmed 38 fragments, giving an overall hit rate of around 4%, comparable to that seen in the crystallographic fragment screen against M^pro. Also in common with the previous screen is the fact that most of the fragments (32) bind somewhere in the active site, while a few (5) bind at the dimerization interface. Fragment hits tended to be both larger and more lipophilic than those in the overall library.

 

Earlier this year we highlighted an NMR study of non-covalent fragment hits from the crystallographic fragment screen, which found that only two of them had measurable affinities, and both were weak (K_D = 1.6 mM at best). In contrast, one of the new fragments, F01, has a dissociation constant of 73 µM. With a molecular weight of 287 Da and 20 non-hydrogen atoms this is a somewhat portly fragment, but it does have a ligand efficiency of 0.3 kcal mol^-1 per heavy atom. It also shows functional activity with an IC₅₀ = 54 µM in a biochemical assay and EC₅₀ = 150 µM in an antiviral assay. The researchers further characterized their molecule crystallographically, which confirmed that it binds to the active site; it makes three hydrogen bond interactions and multiple hydrophobic contacts with the protein.

 

Although crystallography has been receiving increasing attention among fragment-screening techniques, this paper is a reminder than NMR remains highly relevant, even for larger proteins that crystallize easily. And at the end of the day, it’s not how you screen but what you find and what you do next. Hopefully folks will follow up on F01. While PF-07321332 is making rapid progress in the clinic against this enzyme and the COVID Moonshot effort is moving molecules into animal studies, HIV has taught us that we’ll need multiple small molecule drugs.