Discovery of BBO-8520, a First-In-Class Direct and Covalent Dual Inhibitor of GTP-Bound (ON) and GDP-Bound (OFF) KRASG12C

Abstract

Approved inhibitors of KRASG12C prevent oncogenic activation by sequestering the inactive, GDP-bound (OFF) form rather than directly binding and inhibiting the active, GTP-bound (ON) form. This approach provides no direct target coverage of the active protein. Expectedly, adaptive resistance to KRASG12C (OFF)-only inhibitors is observed in association with increased expression and activity of KRASG12C(ON). To provide optimal KRASG12C target coverage, we have developed BBO-8520, a first-in-class, direct dual inhibitor of KRASG12C(ON) and (OFF) forms. BBO-8520 binds in the Switch-II/Helix3 pocket, covalently modifies the target cysteine, and disables effector binding to KRASG12C(ON). BBO-8520 exhibits potent signaling inhibition in growth factor-activated states, in which current (OFF)-only inhibitors demonstrate little measurable activity. In vivo, BBO-8520 demonstrates rapid target engagement and inhibition of signaling, resulting in durable tumor regression in multiple models, including those resistant to KRASG12C(OFF)-only inhibitors. BBO-8520 is in phase 1 clinical trials in patients with KRASG12C non-small cell lung cancer. Significance: BBO-8520 is a first-in-class direct, small molecule covalent dual inhibitor that engages KRASG12C in the active (ON) and inactive (OFF) conformations. BBO-8520 represents a novel mechanism of action that allows for optimal target coverage and delays the emergence of adaptive resistance seen with (OFF)-only inhibitors in the clinic. See related commentary by Zhou and Westover, p. 455.

Figure 1.

Discovery of BBO-8520. A, The compound progression chart shows the development of BBO-8520. Noncovalent KRAS^G12D inhibitor, compound 1, showed binding activity (SPR) to GppNHp-bound KRAS^G12D, and disruption of KRAS^G12D/RAF1(RBD) interaction in PPI assay. Compound 1 was equipped with a covalent warhead to generate compound 2. Optimization of cell potency and ADME properties gave rise to compound 3. Further improvement of the GTP-bound KRAS^G12C activity by replacing the aminobenzothiazole of compound 3 with a cyano-amino benzothiophene at quinazoline 7-position gave rise to BBO-8520. BBO-8520 shows covalent labeling of C12 in the active, GppNHp-, or GTP-bound KRAS^G12C, as well as disruption of KRAS^G12C/RAF1(RBD) binding. B, The table summarizes the ability of compounds to modify C12 of GDP-, GppNHp-, and GTP-bound KRAS^G12C as measured by MALDI-TOF mass spectrometry; and their ability to disrupt GppNHp- or GTP-bound KRAS^G12C binding to RAF1(RBD) in PPI assay. C, k_inact/K_i measurements of sotorasib, adagrasib, and BBO-8520 in the GDP-bound (OFF) and GTP-bound (ON) conformations of KRAS^G12C.

Figure 2.

Binding mode of BBO-8520 to KRAS^G12C (ON) and (OFF) conformation. A, GDP-bound (OFF; top, PDB 8V3A) and GppNHp-bound (ON; bottom, PDB 8V39) forms in surface and ribbon representations. The protein is colored light orange (OFF) or light blue (ON), with Switch-I, Switch-II, and Helix 3 highlighted in pink, cyan, and orange, respectively. BBO-8520 (light green at the top, pink at the bottom), nucleotide, C12, and T35 are shown as sticks, and Mg2⁺ (green) and the coordinating waters (red) are shown as spheres. B and C, Enlarged view of the binding pocket in the GDP-bound form (PDB 8V3A), focusing on the regions around (B) Switch-II and Helix 3, and (C) C12 and GDP. The protein and BBO-8520 are colored light orange and light green, respectively. H-bonds are indicated by dashed lines. D and E, Enlarged view of the binding pocket in the GppNHp-bound form (PDB 8V39), focusing on the regions around (D) Switch-II and Helix 3, and (E) C12 and GppNHp. The protein and BBO-8520 are colored light blue and light pink, respectively. F and G, Overlay of the GDP-bound and GppNHp-bound structures, focusing on the regions around (F) Switch-II and Helix 3, and (G) C12 and the nucleotide.

Figure 3.

Mechanism of action and selectivity of BBO-8520. A, BBO-8520 [ligand (L)] binding to KRAS^G12C-GTP [protein (P)] shifts the state 1–state 2 equilibrium of protein to the inactive, state 1-like conformation (induced γ_1PL peak located most downfield) in the protein–ligand (PL) binary complex spectrum. Peak β_1PL represents L binding induced inactive conformation of β GTP. Chemical shifts corresponding to peaks α₁, β₁, and γ₁ belong to state 1 (inactive, effector binding–deficient) conformation, whereas α₂, β₂, and γ₂ to the state 2 (active, effector binding–enabled) conformation. RAF1 RBD loading is unable to induce γ₂ (active conformation) population (see P + L + RBD spectrum). Shown on top and bottom are the control spectra (in the presence of DMSO) of P + RBD and P, respectively. The γ_1PL peak emergence is only noted in the presence of an inhibitor. The control spectra of KRAS^G12C-GTP alone or in the presence of RBD do not show this peak. B, BBO-8520 inhibits SOS-mediated nucleotide exchange of GDP with BODIPY-GDP KRAS. Avi-KRAS mutants indicated and Avi-NRAS WT were loaded with BODIPY-GDP, then BBO-8520 was added in a 2-fold dilution series starting at 30 nmol/L. The assay was started by the addition of SOS1 (aa564-1048) and GDP, then analyzed after 4 and 24 hours of incubation. KRAS^G12C shows the highest inhibition of nucleotide exchange with BBO-8520. NRAS WT was used as a control. C, pERK inhibitory activity of BBO-8520, sotorasib, and adagrasib against a panel of mouse embryonic fibroblasts (MEF) driven by KRAS mutants, HRAS, NRAS, and BRAF^V600E. BBO-8520 demonstrates the best pERK inhibitory activity in KRAS^G12C-driven MEF cells, compared with MEFs driven by other KRAS mutants or WT KRAS. BBO-8520 shows no pERK inhibitory activity in MEFs driven by HRAS, NRAS, or BRAF^V600E.

Figure 4.

BBO-8520 potently inhibits KRAS^G12C signaling in tumor cells. A, Target engagement and ERK phosphorylation time course in the KRAS^G12C cancer cell lines MIA PaCa-2 and SW1473. BBO-8520 at 20 nmol/L displays rapid pERK inhibition at 30 minutes which is sustained for up to 24 hours and is compared with 20 or 100-nmol/L sotorasib and adagrasib, which take longer and show less inhibition of pERK. B, HTRF analysis of phosphorylated ERK demonstrates time- and dose-dependent inhibition in response to BBO-8520 in MIA PaCa-2 and SW1463 cells, which is sustained for up to 34 hours posttreatment. C to E, Potent effects of BBO-8520 on 2-hour pERK inhibition, pAKT inhibition, and 7-day 3D viability measurements in KRAS^G12C cell lines as compared with sotorasib, adagrasib, and RMC-6291. The activity of BBO-8520 in KRAS^G12C cell lines is compared against a cell line panel comprising KRAS wild type along with G12C/D/V and S, G13D, and BRAF^V600Emutants. IC₅₀ (nmol/L) values for each cell line are captured in Supplementary Table S2.

Figure 5.

BBO-8520 maintains potency in the active state of KRAS^G12C. A, RAS:RAF ELISA assay in MIA PaCa-2 cells shows rapid dissociation of KRAS^G12C (ON) with RAF1 by BBO-8520 compared with the (OFF)-only KRAS^G12C inhibitors sotorasib, adagrasib, or GDC-6036. B and C, NCI-H358 cells were serum starved and then treated with 100 ng/mL of EGF, compound, and assayed for pERK HTRF 20 minutes after compound addition (B) or treated with 100 ng/mL of HGF and compound and assayed for a 5-day viability assay (C). Average potency shifts are shown to the right demonstrating the fold changes of IC₅₀ following EGF or HGF stimulation compared with vehicle. D, HeLa cells were engineered to express a KRAS^G12C/A59G double mutant known for attenuated GTP hydrolysis and assayed for pERK inhibition following treatment with 0.3, 1, 3, and 10 µmol/L of BBO-8520, sotorasib, or adagrasib. BBO-8520 demonstrated a potent inhibition of the pERK signal compared with the (OFF)-only inhibitors, which showed no activity. E, A long-term clonogenic assay using IC₉₀ concentrations (2-hour pERK) of BBO- 8520, sotorasib, and adagrasib, shows that only BBO-8520 can drive complete growth suppression for up to 35 days in culture compared with sotorasib or adagrasib.

Figure 6.

BBO-8520 demonstrates dose- and time-dependent inhibition of pERK and strong efficacy in KRAS^G12C models. A, BBO-8520 shows dose-responsive inhibition of pERK at 6 hours following a dose of 3, 10, and 30 mg/kg in a MIA PaCa-2 Matrigel plug PD assay (*, P < 0.01; **, P < 0.0001). B, Suppression of pERK was observed up to 72 hours following treatment with 30 mg/kg of BBO-8520 in the MIA PaCa-2 Matrigel plug PD (*, P < 0.01; **, P < 0.0001). C, In the corresponding MIA PaCa-2 CDX model, BBO-8520 showed significant antitumor activity at 0.1, 0.3, 1, 3, and 10 mg/kg following 28 days of treatment (*, P < 0.0001). D, In the NCI-H358 CDX model, BBO-8520 demonstrated significant and robust efficacy at 0.3, 1, 3, and 10 mg/kg following 28 days of treatment (*, P < 0.0001). E, In the KCP NSCLC GEMM, BBO- 8520 demonstrated significant and robust efficacy at 10 mg/kg (*, P < 0.0001). F, In the CT26-KRAS^G12C-luciferase syngeneic liver tumor model, BBO-8520 extended the median survival as a monotherapy or in combination with anti-PD-1.

Figure 7.

BBO-8520 is more efficacious than sotorasib and shows activity in sotorasib-resistant tumors. A, Efficacy of BBO-8520 and sotorasib in the RET-amplified LUN055 PDX model. Both BBO-8520 and sotorasib demonstrate antitumor activity (*, P < 0.0001), with BBO-8520 showing significant tumor regressions (23%) vs. 71% TGI for sotorasib. B, MIA PaCa-2 xenografts were grown under the presence of 10 mg/kg of sotorasib until tumors became resistant under treatment (day 35). On day 35, a cohort of eight mice were switched from sotorasib (10 mg/kg) to 30 mg/kg of BBO-8520. These mice showed strong responses with tumor volume regression. C, Analysis of sotorasib-resistant tumors showing a high proportion of KRAS^G12C amplification by ddPCR. D, Focused view of mice continuing on 10-mg/kg sotorasib or switched to 30-mg/kg BBO-8520 starting on day 35 (200 mm³). All mice treated with BBO-8520 had a statistically significant (P < 0.01) reduction in tumor volume compared with those continued on sotorasib. This included three mice with complete tumor regressions.