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

Abstract

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.

Figure 1

Structure of the KRAS G-domain and location of the Thr58-associated conserved water. (A) KRAS structure with selected highlighted areas (PDB ID: 5F2E). SII-P (green transparent surface) is enclosed by switch-II (blue cartoon) and reachable from position 12 (yellow transparent surface) located in the P-loop (yellow cartoon). The conserved water (red sphere) resides in SII-P next to Thr58, Gly10, and Tyr96 (shown in sticks). This water appears in numerous KRAS crystal structures, as highlighted here with examples of electron density maps of (B) KRAS Q61H bound to GTP (PDB ID: 6MNX), (C) KRAS WT bound to GNP (PDB ID: 6VC8), and (D) KRAS bound to GNP in complex with effector protein RAF1 (RBD) (PDB ID: 6VJJ). Electron density (blue transparent surface) displayed at 2Fo – Fc σ = 1.5.

Figure 2

KRAS(G12C) inhibitors with and without the Thr58-associated conserved water. (A) Compound 6 of the first described series of G12C inhibitors by Ostrem et al. has adjacent hydration sites (ΔG = +1.93 and +5.20 kcal/mol) next to the conserved water (ΔG = +4.78 kcal/mol). (B) ARS-853, the first cell active G12C inhibitor, appears with an isolated Thr58-associated hydration site (ΔG = +5.09 kcal/mol). (C) ARS-1620, the first in vivo active G12C inhibitor, displays a high-energy conserved hydration site (ΔG = +10.93 kcal/mol). (D) Sotorasib, the first FDA-approved G12C inhibitor, exists with high-energy conserved water (ΔG = +10.82 kcal/mol). (E) Bayer compound 13 displaces the conserved water and forms a H-bond with Gly10. However, it is suboptimal to the native conformation of Thr58 (occupancy of 0.5 in the shown configuration; see also Figure S5). (F) Adagrasib, the second FDA-approved G12C inhibitor, displaces the conserved water by a cyanomethyl group, simultaneously promoting a non-native flipped configuration of Thr58. The conserved water in (A)–(D) is also found in the crystal structures (see Table 1).

Figure 3

KRAS(G12C) inhibitors that have displaced the Thr58-associated conserved water. The conserved water location is highlighted with a blue circle. PDB IDs for the cocrystal structures are given in parentheses. Three-dimensional (3D) visualizations of their WaterMap results are provided in Figures 2E–F and S7.

Figure 4

KRAS(G12D) inhibitors exist with Thr58-associated conserved water. (A) MRTX1133 displays an isolated high-energy conserved hydration site (ΔG = +9.24 kcal/mol). (B) Compound 15, which exhibits lower affinity compared to MRTX1133, displays an additional adjacent high-energy hydration site (ΔG = +9.22 kcal/mol) next to the Thr58-associated conserved water (ΔG = +5.34 kcal/mol). (C) Based on the crystal structure, 8-ethynyl of MRTX1133 interacts with the conserved water (PDB ID: 7RPZ).(D) Adjacent water is also found in the cocrystal structure of compound 15 (PDB ID: 7RT1).

Figure 5

KRAS(G12S) inhibitors interact with the Thr58-associated conserved water in their covalently bound state. (A) G12Si-1 displays a single isolated conserved water (ΔG = +5.41 kcal/mol). (B) Interactions of the conserved water when in complex with G12Si-1 in the energy-minimized crystal structure. H-bonds are displayed with yellow dashed lines, and halogen bonds are displayed with magenta dashed lines. Also, the other H-bonds of the hydroxyl group of G12Si-1 (to Gly10 and Tyr96) are shown. (C) With chain A of G12Si-5, a higher energy (ΔG = +7.42 kcal/mol) is estimated for the conserved hydration site. (D) With chain B of G12Si-5, comparable energy to G12Si-1 for the conserved water is observed (ΔG = +5.79 kcal/mol). (See Figure S12 for the conformation differences of Lys16 in C and D.) The conserved water is also found in the crystal structures (see Table 3).

Figure 6

KRAS(G12R) inhibitor compound 4 displaces the Thr58-associated conserved water. (A) In chain A, an adjacent high-energy hydration (ΔG = +10.23 kcal/mol) next to the displaced conserved site is observed. (B) In chain B, a flipped Thr58 configuration is observed (probability of 0.71) and no adjacent hydration sites to the displaced conserved water are predicted by WaterMap. However, between the switch-II loop and Arg68 side chain, a unique high-energy hydration site is observed (ΔG = +5.33 kcal/mol). (C) Superimposition of all published cyanomethyl displacers reveals that G12C inhibitors adagrasib (red structure) and compound 12a (gray) are better-positioned and capable of introducing H-bonding interaction with Gly10 (yellow dashed lines), while cyanomethyl of G12R inhibitor compound 4 resides further away (gold and green). With chain A of 8CX5, which displays the longest cyanomethyl distance, Thr58 is not flipped.

Figure 7

Selected KRAS SII-P binders for the microsecond timescale molecular dynamics simulations. KRAS(G12C) targeting inhibitors: sotorasib, JDQ443 and AZD4625; KRAS(G12D) targeting inhibitors: MRTX1133 and compound 5B; KRAS(G12S) targeting inhibitor: G12Si-1. PDB IDs for the cocrystal structures are given in parentheses.

Figure 8

Selected protein–ligand interactions of the KRAS SII-P binders in the microsecond timescale molecular dynamics simulations. Representative snapshots of the MD simulations (A–F) highlighting the binding modes of the compounds and the locations of the discussed interactions. Frequencies of the selected interactions: (G) Asp69, (H) Gly60, (I) Gly10, and (J) Lys16. Data in (G)–(J) consist of 10 μs for each system, which was analyzed every nanosecond (=10,000 data points for each system). The more complete ligand-specific interaction frequencies for individual ligands are provided in Figures S17 and S18.

Figure 9

Water behavior on the conserved water location in microsecond timescale MD simulations. (A) Observed H-bond frequencies between Thr58 O and water in simulated systems. (B) Observed H-bond frequencies between Gly10 NH and water in simulated systems. (C) Minimum distance of a water molecule to the oxygen (O) atom of Thr58. (D) Minimum distance of a water molecule to the nitrogen (N) of Gly10. (E) Minimum distance of a water molecule to the side chain atoms of Val9. In (C)–(E), the line displays the average, and the shaded area represents SD.