Discovery of RMC-5552, a Selective Bi-Steric Inhibitor of mTORC1, for the Treatment of mTORC1-Activated Tumors

Abstract

Hyperactivation of mTOR kinase by mutations in the PI3K/mTOR pathway or by crosstalk with other mutant cancer drivers, such as RAS, is a feature of many tumors. Multiple allosteric inhibitors of mTORC1 and orthosteric dual inhibitors of mTORC1 and mTORC2 have been developed as anticancer drugs, but their clinical utility has been limited. To address these limitations, we have developed a novel class of "bi-steric inhibitors" that interact with both the orthosteric and the allosteric binding sites in order to deepen the inhibition of mTORC1 while also preserving selectivity for mTORC1 over mTORC2. In this report, we describe the discovery and preclinical profile of the development candidate RMC-5552 and the in vivo preclinical tool compound RMC-6272. We also present evidence that selective inhibition of mTORC1 in combination with covalent inhibition of KRAS^G12C shows increased antitumor activity in a preclinical model of KRAS^G12C mutant NSCLC that exhibits resistance to KRAS^G12C inhibitor monotherapy.

Figure 1

Rapamycin, FK-506, Rapalink-1, and representative mTOR inhibitors.

Scheme 1. Synthesis of C40-Ether Triazole-Linked Bi-Steric Inhibitors

Reagents and conditions: (i) hex-5-yn-1-yl trifluoromethanesulfonate, 2,6-di-tert-butyl-4-methylpyridine, DCM, from 0 °C to rt, 30% yield; (ii) RN₃, Cu(MeCN)₄PF₆, TBTA, DMSO, rt, 20–62% yield; (iii) RN₃, CuSO₄, sodium ascorbate, MeOH, rt, 18–37% yield.

Scheme 2. Synthesis of C40-Carbamate-Linked Bi-Steric Inhibitors

Reagents and conditions: (i) R = C=O (rapamycin); p-nitrophenyl chloroformate, py, DCM, −78 °C, 59% yield. (ii) R = (R)-CH(OH); p-nitrophenyl chloroformate, py, 4 Å molecular sieves, DCM, from −15 °C to −10 °C, 63% yield or R = (R)–CH(OMe), from −10 °C to rt, 85% yield. (iii) R₁NH₂, DIPEA, DMA, rt, 36–63% yield.

Scheme 3. Synthesis of Ring-Opened Seco-Analogs, and Structure of Secorapamycin

Reagents and conditions: (i) NH₄OAc, DMA, 40 °C, 28% yield.

Figure 2

Structural representation of binding sites for FKBP12-rapamycin and active-site inhibitors in mTORC1 and mTORC2. Rapamycin has reduced affinity for mTORC2 due to partial occlusion of the FKBP12-rapamycin binding (FRB) domain, while active-site inhibitors have similar affinity for both complexes. Reprinted by permission from Springer Nature ref (58). Copyright 2021.

Figure 3

Bi-steric inhibitor 16 undergoes ring opening of the rapamycin macrocycle in vivo to form ring-opened 32. (A) Mouse PK: male Balb/c mice (n = 3), IP = 3 mg/kg. (B) Rat PK: male Sprague–Dawley rats (n = 3), IV bolus = 1 mg/kg; vehicle = transcutol/solutol HS15/H₂O 5%/5%/90% (v,w,v).

Figure 4

Rapamycin analogs 11 (PDB 8ER6) and 12 (PDB 8ER7) bind at FKBP12’s canonical rapamycin binding site. (A) Hydroxy analog 11 (darker orange) binds in the FKBP12 (red) pocket partially defined by residue F47. C32 hydroxyl residue donates an intramolecular hydrogen bond with the adjacent ester oxygen (dashed line). (B) Methyl ether 12 (lighter orange) is not able to form this intramolecular hydrogen bond, and the methyl group is oriented toward FKBP12 residue F47. As a result, F47 must shift upward to accommodate this additional steric bulk.

Figure 5

(A) Local resolution map of the mTORC1-RMC-5552-FKBP12 cryo-EM structure (PDB 8ERA). Higher resolution core of the map allows for confident modeling of side chains and both the rapalog-like and the ATP-competitive moieties of RMC-5552. N-HEAT domain of mTOR (right) and WD40 domain of Raptor (left) are more disordered than other regions of the map and consequently approach 7 Å. (B) Cryo-EM map of mTORC1-FKBP12-RMC-5552 38 complex. mTORC1-FKBP12-RMC-5552 38 complex cryo-EM map is shown colored by protomer. Blue is mTOR, green is Raptor, purple is mLST8, and red is FKBP12. (C) Cryo-EM map reveals density for rapalog moiety of RMC-5552 38 binding at its expected location between FKBP12 and the FRB domain at threshold 0.0033. (D) Cryo-EM map reveals density corresponding to the ATP-competitive moiety of RMC-5552 38 (orange) binding mTOR (blue) at its orthosteric site at threshold 0.024. Multiple hydrogen bonds are formed between mTOR and ligand, including with mTOR residues K2187, E2190, G2238, and V2240 (black dashed lines). 4-Aminopyrazolo[3,4-d]pyrimidine core of RMC-5552 π stacks with the aromatic side chain of mTOR residue W2239 (purple dashed line). Density (gray transparent surface) does not extend beyond the beginning of the linker region.

Figure 6

Tumor PD after a single dose of each inhibitor in the MCF-7 CDX model: (A) RMC-5552 38 and RMC-6272 40; (B) sapanisertib 5 and everolimus 4.

Figure 7

Mean tumor volume over time in MCF-7 CDX model for (A) RMC-5552 38 and (B) RMC-6272 40. Data were analyzed by two-way repeated measures ANOVA; ** p < 0.01 and *** p < 0.001, as compared to control at end of study. Mean percentage body weight change for (C) RMC-5552 38 and (D) RMC-6272 40.

Figure 8

(A) Mean tumor volume over time, (B) mean percentage body weight change, and (C) caspase-3 cleavage staining 24 h post a single dose for RMC-6272 40, sotorasib, and the combination in the NCI-H2122 CDX model.