Abstract Although KRASG12C-specific inhibitors have been introduced, no approved targeted therapies exist for other clinically significant KRAS mutants, including KRASG12D and KRASG12V. We discovered BBO-11818, a potent, selective, orally bioavailable noncovalent pan-KRAS inhibitor capable of targeting multiple KRAS mutants in both the inactive GDP-bound (OFF) and active GTP-bound (ON) states. BBO-11818 binds in the Switch-II/Helix 3 pocket, inducing conformational changes incompatible with effector binding, and demonstrates high-affinity binding to mutant KRAS with strong selectivity over NRAS and HRAS. BBO-11818 potently inhibited MAPK signaling and cellular viability specifically in KRAS-driven lines and produced tumor regressions in KRAS-mutant xenograft models. Combination studies with anti–PD-1, anti-EGFR antibodies, and a RAS:PI3Kα breaker compound showed enhanced efficacy. BBO-11818 has entered phase I clinical trials for patients with various KRAS mutations in colorectal, pancreatic, and lung cancers (NCT06917079). Significance: We discovered BBO-11818, a potent and selective noncovalent KRAS inhibitor with activity against multiple KRAS mutants in both the active (ON) and inactive (OFF) states. BBO-11818 addresses the need for KRAS inhibitors targeting clinically relevant mutants such as KRASG12D and KRASG12V, either as monotherapy or in combination.

Supplementary Figure S6 shows that BBO-8520 shows anti-tumor activity in NCI-H358 CDX sotorasib-resistant tumors

Supplementary Figure S4 summarizes cysteine profiling, RNA-seq and kinomescan studies demonstrating that BBO-8520 is selective for KRAS G12C and MAPK inhibition.

Supplementary Figure S6 shows densitometry analysis for pERK and pAKT in a western blot experiment in KRASG12D and KRASG12D/A59G cells treated with BBO-11818 or DMSO.

Supplementary Table S2 summarizes the potency of BBO-8520, sotorasib, adagrasib and RMC-6291 on pERK and viability in a panel of KRAS mutant, wild type and BRAF V600E mutant cell lines

Supplementary Figure S2 shows the electron density map for BBO-8520 bound to KRAS G12C in the GDP and GppNHp states

Supplementary Figure S8 shows mean body weight and survival upon rechallenge in a KRASG12D syngeneic model treated with the combination of BBO-11818 and anti–PD-1 antibody. In addition, it shows mean body weight and mean tumor volume in KRASG12V and KRASG12D CDX models treated with BBO-11818 and BBO-10203 or BBO-11818 and cetuximab.

Supplementary Figure S1 shows the Kinact/KI values for BBO-8520, sotorasib and adagrasib in biochemical and cell-based assays

Disulfide tethering is a site-directed method of drug discovery used to identify hits for challenging targets. We applied tethering to target oncogenic KRAS, a small GTPase once considered undruggable due to its high nucleotide affinity and a perceived absence of binding sites. We prepared a library of 2160 disulfide-containing fragments. We screened over 1000 compounds against a panel of 83 engineered cysteine mutants of KRAS G12D in the active conformation and screened the full library for a subset of 30 mutants. For select mutants and hits, we performed 2-mercaptoethanol competition assays (βME-50) to prioritize ligands. Ligandability analysis comparing hit rates across mutant residues enabled the identification of druggable hot spots. Our studies confirmed known binding sites, including the Switch-II / α-helix 3 pocket. In addition, we identified previously undescribed cryptic pockets and validated select hits using computational chemistry and NMR spectroscopy. These pockets represent promising opportunities for future drug discovery campaigns.

This file contains the supplementary materials and methods for the manuscript.

Supplementary Figure S3 shows the activity of BBO-8520, sotorasib, and adagrasib on Ba/F3 cells with KRAS G12C mutations with altered states of GTP hydrolysis

Supplementary Figure S5 shows pAKT inhibition by BBO-11818 over a time course experiment in a KRASG12V cell line.

Supplementary Table S1 summarizes the crystallography data for BBO-8520 bound to GMPPNP- and GDP-bound KRAS G12C

Supplementary Figure S5 shows that BBO-8520 demonstrates in vivo pERK inhibition, KRAS G12C target engagement and is well tolerated

Supplementary Material and Methods

Supplementary Table S2 shows individual EC50 data for the inhibition of ERK phosphorylation in a panel of KRAS-mutant cell lines treated with BBO-11818.

Abstract KRAS oncogenic mutations, including the common G12C and G12D variants, impair GTP hydrolysis and occur at high frequency in cancer. The unexplained variations in RAS mutant allele frequency and tissue distribution, and the question of whether mutant-specific preferences for effector binding contribute to their prevalence in cancer, remain unanswered. Our NMR solution structures reveal that the SW2-pocket (SIIP) is larger in KRASG12D than in KRASG12C. This difference arises from distinct conformations of the P-loop, the SW2 loop, and the N-terminal a2 helix that are coordinated by perturbations in SW1 region. Codon 12 in the P-loop and residue Q61 within SW2 are key contributors to these structural differences. KRASG12D shows multiple D12 rotamers, whereas KRASG12C adopts a single C12 rotamer that restricts access to the nucleotide gamma-phosphate (gP) which imposes constraints on SIIP-directed inhibitor design. The Q61 side chain also adopts mutant-specific packing: in KRASG12C the side chain is constrained by interaction with Y96 leading to a narrowed pocket compared to that in KRASG12D where it shifts away from the SIIP, increasing its distance from the gP. These distinctions suggest that the mutants alter the catalytic environment for GTP hydrolysis. Our analysis of protein dynamics by NMR supports unique SW2 packing observed in KRASG12D. We identify a potent pan-KRAS small-molecule inhibitor, BBO-11534, that binds both GTP- and GDP-loaded KRAS. 31P NMR analyses demonstrate that BBO-11534 shifts the conformational equilibrium of GTP-bound KRASWT and KRASG12D toward the signaling-incompetent state 1. In contrast, this shift is modest in GTP-KRASG12C, likely due to confinement of the restricted C12 rotamer and smaller SIIP, which limits inhibitor access to the gP. This distinct conformational network within the active site provides mechanistic rationale for these allele-specific responses. To assess the impact of these structural differences between KRASG12D vs KRASG12C, we examined PI3K p110α binding in cells using a Bioluminescence Resonance Energy Transfer (BRET) assay. These measurements show that KRASG12D binds p110α with the highest affinity, followed by KRASG12C and then KRASWT. Chemical shift perturbation NMR and molecular dynamics simulations recapitulate this binding hierarchy (G12D > G12C > WT). Likewise, immunoprecipitation of FLAG-tagged KRASG12D in HEK293T cells captures more PI3Kα than the KRASG12C or KRASWT proteins, further supporting this trend. Together, this study identifies unique structural features in oncogenic KRAS mutants that demonstrate how these features influence protein function and provide guidance for the design of allele-specific therapeutics. Citation Format: ALOK K. SHARMA, Megan Rigby, MARCO TONELLI, Nicole FER, Patrick Alexander, JUN PEI, YUE YANG, DANA Rabara, Erik K. Larsen, Brian P. Smith, MA ROGER, Vandana Kumari, Marcin Dyba, Felice Lightstone, BIN WANG, PEDRO J. BELTRAN, Eli Wallace, Andrew G. Stephen, Dwight V. Nissley, Frank McCormick, Anna E. Maciag. KRAS G12C and G12D mutants exhibit distinct conformational flexibility in the helix 3–switch 2 pocket that drives differential protein function [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RAS Oncogenesis and Therapeutics; 2026 Mar 5-8; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(5_Suppl_1):Abstract nr A047.

Supplementary Table S1 shows detailed crystallographic data collection and refinement statistics for the interaction of KRASG12D and BBO-11818 in its GDP or GppNHp-bound forms, or for the interaction of HRASG12D/Q95H and BBO-11818 in its GppNHp-bound form.

Supplementary Figure S1 shows the sensorgrams curves used to calculate the binding kinetics of Compound 2 to three KRAS variants in their GDP- or GppNHp-bound forms in a Surface Plasmon Resonance (SPR) assay.