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 one FDA-approved drug. Several earlier stage compounds have been also 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 the Thr58 of switch-II and Gly10 of beta1-sheet. This conserved water is also present in the co-crystal 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 reveals the high-energy nature of this hydration site when co-existing with a SII-P binder, and that there is a preference for a single isolated hydration site in this location within the most advanced compounds. Furthermore, the water displacement is suboptimal and only achieved with a few disclosed compounds. These results suggest that this conserved water should be considered more central when designing new inhibitors, especially in the design of non-covalent inhibitors targeting the SII-P.