Revealing the mechanism of action of a first-in-class covalent inhibitor of $\mathrm{KRASG12C}$ $\mathrm{(ON)}$ and other functional properties of oncogenic $\text{KRAS}$ by 31P $\mathrm{NMR}$

Sharma, Alok K.; Pei, Jun; Yang, Yue; Dyba, Marcin; Smith, Brian; Rabara, Dana; Larsen, Erik K.; Lightstone, Felice C.; Esposito, Dominic; Stephen, Andrew G.; Wang, Bin; Beltran, Pedro J.; Wallace, Eli; Nissley, Dwight V.; McCormick, Frank; Maciag, Anna E.

doi:10.1016/j.jbc.2024.105650

Revealing the mechanism of action of a first-in-class covalent inhibitor of $$\mathrm{KRASG12C}$$ $$\mathrm{(ON)}$$ and other functional properties of oncogenic $$\text{KRAS}$$ by ³¹P $$\mathrm{NMR}$$

Journal Article · Tue Jan 16 00:00:00 EST 2024 · Journal of Biological Chemistry

DOI:https://doi.org/10.1016/j.jbc.2024.105650· OSTI ID:2345116

^[1]; Pei, Jun ^[2]; Yang, Yue ^[2]; ^[1]; Smith, Brian ^[1]; ^[1]; ^[1]; Lightstone, Felice C. ^[2]; Esposito, Dominic ^[1]; Stephen, Andrew G. ^[1]; Wang, Bin ^[3]; Beltran, Pedro J. ^[3]; Wallace, Eli ^[3]; ^[1]; McCormick, Frank ^[4]; Maciag, Anna E. ^[1]

Frederick National Laboratory for Cancer Research, Frederick, MD (United States)
Lawrence Livermore National Laboratory (LLNL), Livermore, CA (United States). Physical and Life Sciences (PLS) Directorate
BridgeBio Pharma, Inc., Palo Alto, CA (United States)
Frederick National Laboratory for Cancer Research, Frederick, MD (United States); BridgeBio Pharma, Inc., Palo Alto, CA (United States); University of California, San Francisco, CA (United States)

Individual oncogenic KRAS mutants confer distinct differences in biochemical properties and signaling for reasons that are not well understood. KRAS activity is closely coupled to protein dynamics and is regulated through two interconverting conformations: state 1 (inactive, effector binding deficient) and state 2 (active, effector binding enabled). Here, we use ³¹P NMR to delineate the differences in state 1 and state 2 populations present in WT and common KRAS oncogenic mutants (G12C, G12D, G12V, G13D, and Q61L) bound to its natural substrate GTP or a commonly used nonhydrolyzable analog GppNHp (guanosine-5'-[(β,γ)-imido] triphosphate). Our results show that GppNHp-bound proteins exhibit significant state 1 population, whereas GTP-bound KRAS is primarily (90% or more) in state 2 conformation. This observation suggests that the predominance of state 1 shown here and in other studies is related to GppNHp and is most likely nonexistent in cells. We characterize the impact of this differential conformational equilibrium of oncogenic KRAS on RAF1 kinase effector RAS-binding domain and intrinsic hydrolysis. Through a KRAS G12C drug discovery, we have identified a novel small-molecule inhibitor, BBO-8956, which is effective against both GDP- and GTP-bound KRAS G12C. We show that binding of this inhibitor significantly perturbs state 1–state 2 equilibrium and induces an inactive state 1 conformation in GTP-bound KRAS G12C. In the presence of BBO-8956, RAF1–RAS-binding domain is unable to induce a signaling competent state 2 conformation within the ternary complex, demonstrating the mechanism of action for this novel and active-conformation inhibitor.

View Accepted Manuscript (DOE)

Research Organization:: Lawrence Livermore National Laboratory (LLNL), Livermore, CA (United States)

Sponsoring Organization:: BridgeBio Pharma; National Institutes of Health (NIH); USDOE National Nuclear Security Administration (NNSA)

Grant/Contract Number:: AC52-07NA27344

OSTI ID:: 2345116

Report Number(s):: LLNL--JRNL-853388; 1080606

Journal Information:: Journal of Biological Chemistry, Journal Name: Journal of Biological Chemistry Journal Issue: 2 Vol. 300; ISSN 0021-9258

Publisher:: American Society for Biochemistry and Molecular BiologyCopyright Statement

Country of Publication:: United States

Language:: English

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31P NMR
59 BASIC BIOLOGICAL SCIENCES
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state 1 (inactive) conformation
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Revealing the mechanism of action of a first-in-class covalent inhibitor of $$\mathrm{KRASG12C}$$ $$\mathrm{(ON)}$$ and other functional properties of oncogenic $$\text{KRAS}$$ by 31P $$\mathrm{NMR}$$

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References (55)

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Related Subjects

Revealing the mechanism of action of a first-in-class covalent inhibitor of $$\mathrm{KRASG12C}$$ $$\mathrm{(ON)}$$ and other functional properties of oncogenic $$\text{KRAS}$$ by ³¹P $$\mathrm{NMR}$$