A method for the second-site screening of K-Ras in the presence of a covalently attached first-site ligand

Abstract

K-Ras is a well-validated cancer target but is considered to be "undruggable" due to the lack of suitable binding pockets. We previously discovered small molecules that bind weakly to K-Ras but wanted to improve their binding affinities by identifying ligands that bind near our initial hits that we could link together. Here we describe an approach for identifying second site ligands that uses a cysteine residue to covalently attach a compound for tight binding to the first site pocket followed by a fragment screen for binding to a second site. This approach could be very useful for targeting Ras and other challenging drug targets.

Fig. 1

Six residues around the primary binding pocket were mutated to cysteine. A previously identified K-Ras inhibitor (1) is depicted as a stick figure to indicate the location of the primary binding pocket

Fig. 2

Crystal structures of various K-Ras mutants covalently attached to compound 2 (cyan). a) In mutant T74C, the covalently attached compound 2 is pointing towards the solvent. b) In the mutant S39C, the linked compound is sitting inside of the primary pocket, but the NH group of the indole is pointing in the opposite direction compared to compound 1 (green). c) The modified Q70C mutant crystallized as a dimer with the indole portion of the compound occupying the pocket of a neighboring molecule. (PDB code 4PZY) None of these modifications blocks the probe compound 1 from binding to the first-site

Fig. 3

Ribbon and molecular surface representations of the crystal structures of GDP-bound K-Ras S39C that was reacted with thiol-reactive compounds (cyan) a) 3, (PDB 4PZZ) b) 4, (PDB 4Q01) c) 5, (PDB 4Q02) and d) 6. (PDB 4Q03) All these compounds completely block the pocket and prevent the probe compound from interacting with the protein. Among them, compound 3 perfectly overlays with the probe compound 1 (green)

Fig. 4

¹H/¹⁵N HMQC spectra of the uniformly 15N-labeled K-Ras S39C mutant covalently modified by compound 3 recorded in the absence (blue) and presence (red) of a fragment hit