Covalent fragment linking on the SARS-CoV-2 Main Protease

Fragment linking is a compelling approach to advancing fragments, but, as our poll from several years ago showed, it often works better in theory than practice. Also, most examples of fragment linking involve non-covalent molecules. In a new open-access ChemMedChem paper, György Keserű and collaborators at HUN-REN Research Centre for Natural Sciences, University of Ljubljana, and Diamond Light Source apply linking to a covalent fragment.

 

The main protease (3CL^pro or MPro) of SARS-CoV-2 was the first target pursued in the fight against COVID-19. A crystallographic fragment screen was conducted just weeks into the pandemic, yielding dozens of hits. Some of these, such as compound 1, formed covalent bonds to the active-site cysteine. Compound 2 bound noncovalently nearby. In the new paper the researchers used the program DeLinker, a “graph-based deep generative method,” to try to link them.

 

Just three linked molecules were made and tested, each with a different amide connectivity. While two were quite weak, compound 5 showed low micromolar activity. This is especially impressive given that both compounds 1 and 2  themselves were inactive. SAR studies revealed that the nitrile warhead is essential for activity, and a crystal structure of compound 5 bound to 3CL^pro confirmed covalent binding. Surprisingly though, the structure showed that the moiety derived from compound 2 binds in a “flipped” position relative to the non-covalent complex. Further fragment-growing led to compound 28, the most potent compound described in the paper.

 

The researchers also tried warhead swapping, replacing the reversible nitrile warhead with a chloride for irreversible aromatic nucleophilic substitution. Although some of these molecules showed activity, the best was about 30-fold less potent. This observation makes sense, as the cysteine sulfur atom must take different trajectories to react with the different warheads.

 

As the researchers point out, more potent inhibitors of 3CL^pro have been reported; the approved drug nirmatrelvir targets this enzyme. However, the paper is still a rare example of fragment linking starting from a covalent fragment; see here for fragment merging on a different target.

 

Personally what I find most interesting is the fact that it took just three molecules to go from inactive fragments to a low micromolar inhibitor. But given that this  molecule binds in a somewhat different manner than predicted, perhaps a high-throughput approach, such as direct-to-biology, would generate even more potent molecules.