Chemical Proteomic Characterization of a Covalent KRASG12C Inhibitor

Abstract

The KRASG12C protein product is an attractive, yet challenging, target for small molecule inhibition. One option for therapeutic intervention is to design small molecule ligands capable of binding to and inactivating KRASG12C via formation of a covalent bond to the sulfhydryl group of cysteine 12. In order to better understand the cellular off-target interactions of , a covalent KRASG12C inhibitor, we have completed a series of complementary chemical proteomics experiments in H358 cells. A new thiol reactive probe (TRP) was designed and used to construct a cellular target occupancy assay for KRASG12C. In addition, the thiol reactive probes allowed us to profile potential off-target interactions of with over 3200 cysteine residues. In order to complement the TRP data we designed , an alkyne containing version of, to serve as bait in competitive chemical proteomics experiments. Herein, we describe and compare data from both the TRP and the click chemistry probe pull down experiments.

Covalent Small Molecules as Enabling Platforms for Drug Discovery

“…Competition experiments with larger reactive molecules can then be used to assess engagement and selectivity across a vast number of reactive proteins, down to a residue‐specific level . Recently, this approach has been used to profile the covalent KRASG12C inhibitor ARS‐1620 (Figure B) . Studies by Chalmers et al .…”

“…Recently, this approach has been used to profile the covalent KRASG12C inhibitor ARS‐1620 (Figure B) . Studies by Chalmers et al . and Liu et al .…”

“… A) General ABP structure. B) Selected examples of reactivity‐driven ABPs targeting cysteine residues, including those used to profile covalent inhibitors such as the covalent KRAS inhibitor ARS‐1620 . C) Selected examples of emerging reactivity‐driven ABPs targeting novel reactive residues such as lysine, methionine, oxidised sulfur, alcoholic side‐chains, and electrophilic PTMs …”

“…A) Structural summary of the KiNativ ATP probe for enriching kinases from cell lysates, fluorophosphonate (FP)‐based probes for enriching serine hydrolase enzymes, and XO44 for enriching kinases in live cells . B) Selected examples of published ABPs derived from clinical candidates and approved drugs ABX‐1431, ARS1620, ibrutinib, afatinib, and BIA 10‐2474 …”

“…A competition experiment between the parent inhibitor and the probe is typically used to deconvolute and identify true targets of the parent inhibitor. These techniques were also employed in the ARS‐1620 and ABX‐1431 studies to provide an orthogonal readout to the broad‐spectrum ABPs. The ARS‐1620 study showed good target overlap between the promiscuous ABP experiment and the derivatised probe (ARS1620_ABP, Figure B), with the derivatised probe identifying some lower abundance targets .…”