Multivalent Small-Molecule Pan-RAS Inhibitors

Abstract

Design of small molecules that disrupt protein-protein interactions, including the interaction of RAS proteins and their effectors, may provide chemical probes and therapeutic agents. We describe here the synthesis and testing of potential small-molecule pan-RAS ligands, which were designed to interact with adjacent sites on the surface of oncogenic KRAS. One compound, termed 3144, was found to bind to RAS proteins using microscale thermophoresis, nuclear magnetic resonance spectroscopy, and isothermal titration calorimetry and to exhibit lethality in cells partially dependent on expression of RAS proteins. This compound was metabolically stable in liver microsomes and displayed anti-tumor activity in xenograft mouse cancer models. These findings suggest that pan-RAS inhibition may be an effective therapeutic strategy for some cancers and that structure-based design of small molecules targeting multiple adjacent sites to create multivalent inhibitors may be effective for some proteins.

Keywords: GTPase; Hras; Kras; Nras; Ras; cancer; chemical biology; drug design; multivalent; small molecule.

Figure 1. Design of inhibitor of RAS proteins

(A) KRAS^G12D (PDB: 4DSN) with the switch I region shown in green and the switch 2 region in purple. (B) Location of three sites on KRAS^G12D targeted (D38 site in yellow, A59 site in blue, Y32 site in red), with pose of docked 3144 ligand. (C) Structure, docking score, chemical formula, mass and molecular weight of the small molecule 3144. See also Figure S1, Table S1 and Supporting Information.

Figure 2. 3144 binds to KRAS^G12D

(A) ¹⁹F NMR spectrum of 3144 titrated into KRAS^G12D (GppNHp-bound) at the indicated concentrations. (B) ITC binding assay with 3144 and KRAS^G12D-GppNHp. (C) (left panel) MST assay of the three-site compound 3495 and the two-site compound from which it was derived, 3443, using KRAS^G12D-GppNHp, (middle panel) MST assay with 3144 and its differential selectivity towards GppNHp-bound vs. GDP-bound KRAS^G12D, and (right panel) MST assay with two different point mutants within the predicted docking site on KRAS^G12D-GppNHp and 3144. (D and E) Summary of K_D values obtained for binding of 3144 to the indicated proteins by MST. (F) Melting temperatures of the indicated proteins with either endogenous nucleotide or GTP (after nucleotide exchange); the shift in melting temperature indicates successful exchange of bound nucleotide. See also Figure S2 and Supporting Information.

Figure 3. 3144 has RAS-dependent lethality

(A) (left panel) Inhibition of RAS expression in DLD-1 cells transfected with siRNAs targeting HRAS, NRAS and KRAS (si-panRAS) or a non-targeting siRNA control. Total Ras expression was determined with a pan-RAS antibody, as well as actin abundance. The total RAS abundance, normalized to actin abundance, is indicated. (middle and right panels) The concentration-dependent effects of 3144 and MG132 on DLD1 cells transfected with either si-panRAS or siNT was measured using Cell Titer Glo. (B) The effect of 3144 (left panel) and vemurafenib (right panel) on viability and growth of mouse embryo fibroblasts (MEFs) was measured at the indicated concentrations using Alamar blue. All compound measurements were performed in duplicate. Black line = Kras^lox/lox, Hras^−/−, Nras^−/− RERTn^ert/ert MEFs; red line = BRAF^V600E-CAAX; Kras^−/−, Hras^−/−, Nras^−/− RERTn^ert/ert MEFs. (C) The effect of 3144 was tested at the indicated concentrations in cultured patient-derived T-ALL samples. PDTALL6, 9, 13 and 19 = wt NRAS, PDTALL22 = NRAS^G13V, PDTALL26 = NRAS^G13D. See also Figure S3 and Supporting Information.

Figure 4. 3144 inhibits RAS-dependent signaling in cells

(A) 250,000 cells/well of the indicated MEFs were seeded in six-well dishes in medium with 10% FBS, then the next day the medium was changed to serum-free medium and the cells incubated for 24 h. 3144 or vehicle (DMSO) control were added the cells in serum-free medium for the indicated times. The cells were stimulated for 15 minutes with 10 ng/mL human EGF, then washed with cold PBS and analyzed by western blotting. The effects on abundance of phospho-ERK, total ERK, phospho-AKT, and total AKT were measured. The normalized relative abundance of pERK and pAKT is indicated next to these bands. (B) The effects of 3144, U0126 (MEK inhibitor) or BEZ-235 (PI3K inhibitor) on abundance of pAKT and total AKT (top left panel) and pERK and total ERK (bottom left panel) was determined at the indicated concentrations in BJeLR engineered human tumor cells (with HRAS^V12). (right panel) A similar experiment was performed in HT-1080 cells and Kras MEFs: For HT-1080 cells, 1 million cells/well (6 well dishes) were seeded, and incubated overnight. The medium was replaced with serum free medium containing the indicated concentration of 3144. After 2 h, 10ng/mL EGF was added as indicated for 15 min. MEF cells were seeded at 700k cells/well in a 12 well plate, and incubated overnight. The following day, normal growth media was replaced with serum free media containing the indicated concentration of 3144. Cells were treated for 80 min, after which the medium was replaced with medium containing the indicated concentration of 3144 +/− EGF at 10 ng/mL as indicated. Cells were treated with EGF for 15 min. (C) (top panel) BJeLR cells were treated with 3144 or U0126 as indicated and the amount of c-RAF bound to RAS proteins was determined. The cells were then lysed and the lysate was incubated with CRAF RBD-agarose beads for 2 h before being washed 2× with PBS, denatured and subsequently detected by western blotting. (Bottom panel) Effect of 3144 on RAS-RALGDS pathway. BJeLR cells were seeded in 2% FBS in DMEM 18 h prior to treatment with 3144 and U0126 in 2% FBS in DMEM for 3 h. Cells were then lysed and the lysate was incubated with RalBP1 agarose beads for 2 h before being washed 2× with PBS, denatured and subsequently detected by western blotting. (D) BJeLR cells were seeded in 10% FBS in DMEM 18 hr prior to treatment with 3144 in 10% FBS in DMEM for 3 h, HRAS was immunoprecipitated and the indicated proteins examined for co-precipitation. The relative intensity of each band is indicated. See also Figure S4 and Supporting Information.

Figure 5. 3144 shows activity in a mutant RAS cell line xenograft and a patient-derived T-ALL xenograft

(A) Stability of 3144 in human liver microsomes: 3144 and 7-ethoxycoumarin were incubated for 120 min with mouse or human liver microsomes and the amount of compound remaining at each time point was quantified by LC-MS. (B) Pharmacokinetics of 3144: male C57BL6 adult mice were dosed orally or intravenously with 3144 in 10% NMP/90% PEG-400 at 30 mg/kg. Concentration in the plasma was measured over the course of 12 h. (C) Effect of 3144 in an MDA-MB-231 xenograft. Eight week old nude female mice were injected with 7 million MDA-MB-231 cells. After tumors reached an average size of 58 cubic millimeters they were treated with vehicle orally (10 doses), 3144 orally (180 mg/kg, 10 doses), or by a combination of intravenous and intraperitoneal injections (30 mg/kg, 4 IV doses, 6 IP doses) over two weeks. (D) Effect of 3144 on a patient-derived T-ALL sample PDTALL22 as a luciferase-expressing xenograft. Mice were randomized into two treatment groups of 5 mice with equal loads of luciferase. Mice were treated with 3144 (30 mg/kg) or vehicle intraperitoneal once daily on days: 0, 1, 4, 5, 7 and 8. (E) Representative images of mice from each treatment group at days, 0, 4 and 8 (final day) of the study in (D), and bar graph representing spleen weight in grams and percentage of human CD45+ cells in the spleen of vehicle and 3144-treated mice. (F) Hematoxylin and eosin immunohistochemistry of spleen sections of mice treated with 3144 compared to vehicle. (G) Pre-treatment analysis (biopsy, Bx) and post-treatment analysis (necropsy, Nx) of tissue samples taken from KP^f/fC mice by western blotting for phosphorylated ERK1/2, total ERK1/2, phosphorylated AKT (S473), and total AKT. Samples were taken prior to, or after five days of, treatment from each of three mice receiving either 3144 (30 mg/kg) or vehicle dosed once daily, intraperitoneally. (H) Analysis of phosphorylated ERK1/2 and total ERK1/2, detected by immunohistochemistry. Shown are images from representative sections of the biopsy and necropsy samples from 3144 and vehicle treated mice. See also Figure S5 and Supporting Information.