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. 2024 Apr 8;629(8013):919–926. doi: 10.1038/s41586-024-07205-6

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 2 — a,b,c,d, Superimposition of the CYPA:RMC-7977:KRAS wild-type (WT) tri-complex structure (PDB: 8TBF) with KRAS:CRAF RBD-CRD complex (a, PDB: 6XI7), HRAS:PIK3 CD (b, PDB: 1HE8), HRAS:RALGDS (c, PDB: 1LFD), or KRAS:SOS1 REM-CDC25 (d, PDB: 1XD2). Note steric clashes caused by CYPA occupying the Switch I and II motif of KRAS. e,f, Steady state (e) and kinetic sensorgram (f) of RMC-7977 and CYPA binding measured by SPR response units (RU). g,h, Steady state (g) and kinetic sensorgram (h) of RMC-7977 binding to KRAS^G12C measured by SPR. Measurements taken in the presence or absence of CYPA, and GMPPNP or GDP nucleotides. e,g, Datapoints represent mean ±s.d. of 3 biological replicates. i, RAS interacts with CYPA and RMC-7977 through the conserved effector lobe. Isoform divergent residues are distal from the site of interaction, allowing pan-isoform inhibition. j, Alignment of wild-type HRAS, wild-type NRAS, and KRAS^G12X (A, C, D, R, S, V) mutant tri-complexes shows functionally identical binding modes to the CYPA:RMC-7977 binary complex. 12th position mutant sidechains shown as sticks (PDB: 8TBF, 8TBG, 8TBH, 8TBI, 8TBJ, 8TBK, 8TBL, 8TBM, 8TBN). k, Tri-complex formation assay (KRAS^G12C:RMC-7977:CYPA binding in U2OS cells) in the presence of 100 nM RMC-7977 and the indicated concentration of adagrasib, normalized to the expected min. (100 nM RMC-7977, no adagrasib) and max. (0 nM RMC-7977, no adagrasib) BRET signal. Cells were treated with inhibitor for 4 h. Datapoints represent mean ±s.d. of 6 biological replicates. l, Time resolved-FRET between recombinant KRAS^G12V and CYPA in the presence of RMC-7977 (50 nM of each) treated with the indicated concentrations of recombinant BRAF RBD, normalized as % of DMSO (EC₅₀ = 357 nM, 95% CI = 270-505 nM). n = 3 biological replicates, plotted as mean ±s.d. normalized to control.

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