Chemical remodeling of a cellular chaperone to target the active state of mutant KRAS

Abstract

The discovery of small-molecule inhibitors requires suitable binding pockets on protein surfaces. Proteins that lack this feature are considered undruggable and require innovative strategies for therapeutic targeting. KRAS is the most frequently activated oncogene in cancer, and the active state of mutant KRAS is such a recalcitrant target. We designed a natural product-inspired small molecule that remodels the surface of cyclophilin A (CYPA) to create a neomorphic interface with high affinity and selectivity for the active state of KRAS^G12C (in which glycine-12 is mutated to cysteine). The resulting CYPA:drug:KRAS^G12C tricomplex inactivated oncogenic signaling and led to tumor regressions in multiple human cancer models. This inhibitory strategy can be used to target additional KRAS mutants and other undruggable cancer drivers. Tricomplex inhibitors that selectively target active KRAS^G12C or multiple RAS mutants are in clinical trials now (NCT05462717 and NCT05379985).

Fig. 1.. Development of a tri-complex inhibitor targeting the active state of KRAS^G12C.

(A) Tertiary structure of the primitive CYPA:Compound-1:KRAS^G12C-GMPPNP complex at 1.40 Å resolution. (B) The chemical structure of Compound-1 and the macrocyclized scaffold for subsequent compounds, with the sanglifehrin-derived CYPA-binding moiety shown in blue. R denotes divergent chemical moieties comprising non-covalent (black) and covalent (pink) derivatives. (C) The interaction between the indicated KRAS proteins (50 nM) and CYPA (20 μM) in the presence of increasing compound concentrations was determined by TR-FRET (mean ± SEM, n=3, N=2). (D) The rate (k_obs) of covalent modification of KRAS^G12C in the presence of CYPA (25 μM) and the indicated compounds (mean ± SEM, N=3). (E) Schematic of tri-complex formation by RMC-4998 along with rate constants for each reaction. (F) Mapped pairwise atomic distances between structures of apo-CYPA (PDB: 3K0N) and RMC-4998-bound CYPA showing the structural rearrangements (white to green gradient) that gave rise to the high-affinity neomorphic binding interface for KRAS^G12C. Interactions between RMC-4998, CYPA, and the Switch I (G) or Switch II (H) regions of KRAS^G12C in the crystal structure of the mature tri-complex (1.53 Å). (I) The interaction between KRAS^G12C (2 μM), RMC-4998 (10 μM), and CYPA (2 μM) was determined by native PAGE. (J) As in J, but KRAS^G12C was loaded with non-hydrolysable GTPɣS (active state) or GDP (inactive state). A representative of at least two independent experiments is shown in (I) and (J). (K) HEK293 cells co-expressing small bit luciferase-tagged RAS variants and large-bit luciferase tagged CYPA were treated with RMC-4998 (100 nM) for 2h followed by determination of luciferase activity (mean ± SEM, N=4).

Fig. 2.. Structural constraints for tri-complex formation and active state selective KRAS inhibition.

(A) Superimposition of the structures of the CYPA:RMC-4998: KRAS^G12C tri-complex and the KRAS:CRAF^RBD/CRD complex (PDB: 6XI7) (31). (B) The effect of RMC-4998 on the interaction between the indicated KRAS proteins (12.5 nM) and BRAF^RBD (50 nM) in either the presence or the absence of CYPA was determined by TR-FRET (mean ± SD, N=4). HEK293 cells co-expressing small bit luciferase-tagged KRAS^G12C and large bit luciferase-tagged CYPA (C) or full-length CRAF (D) were treated with RMC-4998 (100 nM) followed by determination of reconstituted luciferase activity in live cells (mean ± SEM, N=3). (E) The indicated parental, CYPA-null or CYPA rescued H358 cells were treated as shown and their extracts were analyzed by RBD-pulldown and immunoblotting to determine the effect on KRAS activation. A representative of two independent experiments is shown. (F) KRAS mutant cells were infected with a dox-inducible saturation mutagenesis library based on a KRAS^G12C backbone and treated with either DMSO or RMC-4885 (100 nM) for two weeks. Shown is the log(fold-change) (logFC) in abundance relative to T0 (mean ± 95%CI, N=3) for variants meeting the threshold for statistical significance (see Methods). (G) The effect of the indicated CYPA variants on tricomplex formation in live cells was determined as in C (mean ± SEM, N=3).

Fig. 3.. Cellular effects of active state selective KRAS^G12C inhibition.

(A) Extracts from the indicated KRAS^G12C mutant cell lines were treated with RMC-4998 for 2 h before lysis and immunoblotting. The levels of active KRAS (KRAS-GTP) were determined by pull-down with the RAS-binding domain (RBD) of CRAF. A representative of at least two independent experiments for each cell line is shown. (B) The kinetics of target inhibition in live cells treated with an active state (RMC-4998, 100 nM) or an inactive state (sotorasib, 10 μM and adagrasib, 1 μM) selective inhibitor were determined as in Fig. 2C (mean ± SEM, N=3) (C) The indicated cell lines were treated as shown in the presence or absence of growth factor (GF) stimulation to determine the effect on ERK phosphorylation (pERK/total, 4 h) or cell proliferation (120 h) (mean ± SEM, N=3-4). EGF: epidermal growth factor, HGF: hepatocyte growth factor.

Fig. 4.. Potent and selective suppression of KRAS^G12C-driven tumor growth by tri-complex inhibitors.

(A) The indicated cells were treated with increasing concentrations of RMC-6291, RMC-4998, or adagrasib for 120h and the effect on cell viability was determined using the 3D CellTiter-Glo assay. Each point represents an individual cell line (n = 17 for G12C, n=11 for non-G12C) and the gray line indicates the median IC₅₀ for each group of cell lines. (B) Mice bearing H358 CDX tumors were treated with RMC-6291 at the indicated dose, administered orally once daily, and the tumor volume was assessed for 28 days. ***adj.p<0.001 for RMC-6291 (all dose groups) vs control, using repeated measures 2-way ANOVA (n=6/group for control, n=8/group for RMC-6291); adjusted based on multiple comparison via Dunnett’s test on the final tumor measurement. (C) The unbound plasma concentration of RMC-6921 and the expression of DUSP6 mRNA in H358 tumors following administration of a single oral dose of RMC-6291 at the indicated doses (mean ± SEM, n=3). (D, E) The indicated NSCLC (D) or CRC (E) xenograft models were treated with RMC-6291(200 mg/kg administered orally once daily) to determine the effect on mean tumor growth or regression after 28±2 days (% change from baseline, mean ± SEM, n as indicated in table S3). The dashed line indicates 10% reduction in tumor volume from baseline.