Crystallography first, then virtual screening: application to PKA

Fragment-based screening is often funnel-shaped: a virtual screen might identify dozens or hundreds of potential hits that are tested in various assays, eventually leading to a few chosen for crystallography. But a paper we highlighted back in 2016 argued that many assays miss genuine hits, and crystallography should be moved to the front of the line. A paper just published in J. Med. Chem. by Serghei Glinca and collaborators at CrystalsFirst, BioSolveIT, Enamine, and elsewhere provides a proof of concept.

 

The researchers started with a set of 19 crystal structures of fragments bound to Protein Kinase A (PKA) from a campaign we wrote about in 2020. Four diverse fragments were chosen for further study. Importantly, the affinities of these fragments had not been measured; selection was based on the diversity of chemical structures and binding modes.

 

Next, the crystal structures of the four fragments were used as starting points for four virtual screens using 208,293 Enamine REAL Space fragments (see here for more on these). These were docked using BioSolveIT’s FlexX algorithm, and 50 from each of the four screens were then computationally grown. Just over half a million of these elaborated molecules were then docked, and after clustering, triaging, and visual selection, 106 were chosen for synthesis, of which 93 were delivered and 75 were soluble at 200 mM in DMSO.

 

The soluble fragments were tested in a functional assay, and 30 of these showed inhibition. Most were weak (double digit micromolar or higher) but fragment EN093 (derived from Frag2) was a low micromolar inhibitor. All of the initial fragments were very weak inhibitors, with at best millimolar activity.

The 75 soluble compounds were also tested in a thermal shift assay (each at 2.5 mM), revealing 29 hits, of which 19 were also active in the functional assay. These included EN093. Interestingly, only one of the initial fragments (not Frag2) showed any activity in the thermal shift assay.

 

To assess how well the docking performed, 13 of the most active compounds were tested in co-crystallization experiments, yielding 6 high-quality bound structures. These confirmed the virtual screens, with the rmsd for EN093 being 0.74 Å.

 

Impressively, the whole study, including compound synthesis and crystallographic validation, took just 9 weeks.

 

This “Crystal Structure First” is conceptually similar to the V-SYNTHES approach we discussed earlier this year, with the difference being that while V-SYNTHES is entirely virtual, Crystal Structure First starts with an actual structure. As the researchers state, “using crystallographically validated fragments and bound ligands for template-based docking can be thought of as introducing a ‘magnet’ to help find the needle in an ever-growing haystack in a more targeted way.”

 

This is a nice case study, and intuitively it makes sense to start with an experimentally determined structure. Indeed, the increasing number of publicly available fragment structures should be a boon for this approach. That said, it is interesting that most of the molecules made and tested are quite weak, and only two have ligand efficiencies equal to or greater than 0.3 kcal/mol per heavy atom. As we suggested earlier this year, crystallography may find ligands that are just too weak to be useful. Perhaps adding a functional screen before computational elaboration could lead to even more and better binders.