Novel K-Ras G12C Switch-II Covalent Binders Destabilize Ras and Accelerate Nucleotide Exchange

Abstract

The success of targeted covalent inhibitors in the global pharmaceutical industry has led to a resurgence of covalent drug discovery. However, covalent inhibitor design for flexible binding sites remains a difficult task due to lack of methodological development. Here, we compared covalent docking to empirical electrophile screening against the highly dynamic target K-RasG12C. While the overall hit rate of both methods was comparable, we were able to rapidly progress a docking hit to a potent irreversible covalent inhibitor that modifies the inactive, GDP-bound state of K-RasG12C. Hydrogen-deuterium exchange mass spectrometry was used to probe the protein dynamics of compound binding to the switch-II pocket and subsequent destabilization of the nucleotide-binding region. SOS-mediated nucleotide exchange assays showed that, contrary to prior switch-II pocket inhibitors, these compounds appear to accelerate nucleotide exchange. This study highlights the efficiency of covalent docking as a tool for the discovery of chemically novel hits against challenging targets.

DUckCov: a Dynamic Undocking‐Based Virtual Screening Protocol for Covalent Binders

“…[30] Known covalentl igandsb ind to ah ighly flexible allosteric pocket, which traps KRas G12C in the inactiveG DP-bound state (thereby confirming its druggability). [30] In Table 1, the variousH -bond interactions are displayed for 10 KRas G12C structures containing ac ovalently bound acrylamide ligand, as well as the W QB values obtained for each interaction on the reference ligand. [30] In Table 1, the variousH -bond interactions are displayed for 10 KRas G12C structures containing ac ovalently bound acrylamide ligand, as well as the W QB values obtained for each interaction on the reference ligand.…”

“…[29] KRas is a small Gprotein, which is rendered constitutively active by the G12C mutation, leadingt oa bnormal cell growth.The mutation has been shown to be implicated in 40 %o fK Ras-driven lung cancers. [30] Known covalentl igandsb ind to ah ighly flexible allosteric pocket, which traps KRas G12C in the inactiveG DP-bound state (thereby confirming its druggability). [31] Additionally,covalent ligands can specifically target the mutatedK Ras G12C ,s paring the wildtype protein and offering the opportunity for oncogene-specific inhibition.…”

“…[31] Additionally,covalent ligands can specifically target the mutatedK Ras G12C ,s paring the wildtype protein and offering the opportunity for oncogene-specific inhibition. [30] In Table 1, the variousH -bond interactions are displayed for 10 KRas G12C structures containing ac ovalently bound acrylamide ligand, as well as the W QB values obtained for each interaction on the reference ligand. For the remaining two of the 12 selected structures (PDB IDs 4M21 and 6ARK),n oH -bonds could be reliably identified.…”

Rapid covalent-probe discovery by electrophile fragment screening

“…NV3CP was produced following the procedure described in Hussey et al 72 . K-Ras G12C (1-169) was expressed and purified as described in Nnadi et al 18 .…”

“…Historically, the most widespread approach for the design of such inhibitors relied on the incorporation of an electrophile into an already optimized reversible recognition element 4,[9][10][11] , most notably in kinase inhibitors 4,[12][13][14][15][16] . More recently, large-scale covalent virtual screens have also emerged as a method for the discovery of covalent binders [17][18][19][20][21][22] . While successful, in silico docking still has its limitations: it is limited to targets for which a crystal structure (or a high-quality model) is available; and second, it cannot efficiently address protein flexibility.…”