A Top-Down Proteomic Assay to Evaluate KRAS4B-Compound Engagement

Abstract

Development of new targeted inhibitors for oncogenic KRAS mutants may benefit from insight into how a given mutation influences the accessibility of protein residues and how compounds interact with mutant or wild-type KRAS proteins. Targeted proteomic analysis, a key validation step in the KRAS inhibitor development process, typically involves both intact mass- and peptide-based methods to confirm compound localization or quantify binding. However, these methods may not always provide a clear picture of the compound binding affinity for KRAS, how specific the compound is to the target KRAS residue, and how experimental conditions may impact these factors. To address this, we have developed a novel top-down proteomic assay to evaluate in vitro KRAS4B-compound engagement while assessing relative quantitation in parallel. We present two applications to demonstrate the capabilities of our assay: maleimide-biotin labeling of a KRAS4B^G12D cysteine mutant panel and treatment of three KRAS4B proteins (WT, G12C, and G13C) with small molecule compounds. Our results show the time- or concentration-dependence of KRAS4B-compound engagement in context of the intact protein molecule while directly mapping the compound binding site.

Figure 1

Top-down characterization of compound engagement to the active state of KRAS4B. MS1 region containing the 21+ charge state following incubation with (a) 917101, (b) 917969, and (c) 917178 at 500 μM for 1 h. Compound engagement for rKRAS4B^C118S (top); rKRAS4B^G12C/C118S (middle); and rKRAS4B^G13C/C118S (bottom). Peaks corresponding to each compound addition are labeled (cyan: 1 compound addition; orange: 2 compound additions; and green: 3 compound additions).

Figure 2

Top-down characterization of compound engagement to the inactive state of KRAS4B. MS1 region containing the 21+ charge state following incubation with (a) 917101, (b) 917969, and (c) 917178 at 500 μM for 1 h. Compound engagement for rKRAS4B^C118S (top); rKRAS4B^G12C/C118S (middle); and rKRAS4B^G13C/C118S (bottom). Peaks corresponding to each compound addition are labeled (cyan: 1 compound addition; orange: 2 compound additions; and green: 3 compound additions).

Figure 3

Relative quantitation of compound engagement by intact mass. Fractional abundances of all proteoforms were plotted as a function of compound concentration for 917101 (a,b), 917969 (c,d), and 917178 (e,f) with rKRAS4B in the active state (upper row) and inactive state (lower row). rKRAS4B^C118S proteins are in purple, rKRAS4B^G12C/C118S proteins are in blue, and rKRAS4BG^G13C/C118S proteins are in black.

Figure 4

Top-down characterization of FDA-approved compounds. MS1 region containing the 21+ charge state following incubation with Sotorasib (AMG510; a-b) or Adagrasib (MRTX849; c-d) at 500 μM for 1 h. Complete engagement was observed with rKRAS4B in the inactive GDPβS state with targeted MS2 fragmentation confirming the localization of each compound to C12 (a and c). Compound engagement was also observed with rKRAS4B in the active, GppNHp state to C12 as confirmed with targeted MS2 fragmentation (b and d).