In Silico Evaluation of the Thr58-Associated Conserved Water with KRAS Switch-II Pocket Binders

The KRAS switch-II pocket (SII-P) has proven to be one of the most successful tools for targeting KRAS with small molecules to date. This has been demonstrated with several KRAS(G12C)-targeting covalent inhibitors, already resulting in two FDA-approved drugs. Several earlier-stage compounds have also been reported to engage KRAS SII-P with other position 12 mutants, including G12D, G12S, and G12R. A highly conserved water molecule exists in the KRAS SII-P, linking Thr58 of switch-II and Gly10 of β1 sheet. This conserved water is also present in the cocrystal structures of most of the disclosed small-molecule inhibitors but is only displaced by a handful of SII-P binders. Here, we evaluated the conserved water molecule energetics by the WaterMap for the SII-P binders with publicly disclosed structures and studied the water behavior in the presence of selected inhibitors by microsecond timescale molecular dynamics (MD) simulations using two water models (total simulation time of 120 μs). Our data revealed the high-energy nature of this hydration site when coexisting with an SII-P binder and that there is a preference for a single isolated hydration site in this location within the most advanced compounds. Furthermore, water displacement was only achieved with a few disclosed compounds and was suboptimal, as for instance a cyanomethyl group as a water displacer appears to introduce repulsion with the native conformation of Thr58. These results suggested that this conserved water should be considered more central when designing new inhibitors, especially in the design of noncovalent inhibitors targeting the SII-P.