The Pan-RAF-MEK Nondegrading Molecular Glue NST-628 Is a Potent and Brain-Penetrant Inhibitor of the RAS-MAPK Pathway with Activity across Diverse RAS- and RAF-Driven Cancers

Abstract

Alterations in the RAS-MAPK signaling cascade are common across multiple solid tumor types and are a driver for many cancers. NST-628 is a potent pan-RAF-MEK molecular glue that prevents the phosphorylation and activation of MEK by RAF, overcoming the limitations of traditional RAS-MAPK inhibitors and leading to deep durable inhibition of the pathway. Cellular, biochemical, and structural analyses of RAF-MEK complexes show that NST-628 engages all isoforms of RAF and prevents the formation of BRAF-CRAF heterodimers, a differentiated mechanism from all current RAF inhibitors. With a potent and durable inhibition of the RAF-MEK signaling complex as well as high intrinsic permeability into the brain, NST-628 demonstrates broad efficacy in cellular and patient-derived tumor models harboring diverse MAPK pathway alterations, including orthotopic intracranial models. Given its functional and pharmacokinetic mechanisms that are differentiated from previous therapies, NST-628 is positioned to make an impact clinically in areas of unmet patient need. Significance: This study introduces NST-628, a molecular glue having differentiated mechanism and drug-like properties. NST-628 treatment leads to broad efficacy with high tolerability and central nervous system activity across multiple RAS- and RAF-driven tumor models. NST-628 has the potential to provide transformative clinical benefits as both monotherapy and vertical combination anchor.

Figure 1.

NST-628 is a pan-RAF–MEK nondegrading molecular glue. A, Chemical structure of NST-628. B, MEK1 immunoprecipitation in HCT116 cell treated with indicated concentrations of NST-628 (4-100 nmol/L) for 2 hours, and blot analysis was performed for ARAF, BRAF, CRAF, phospho-MEK, MEK1, phospho-ERK, and vinculin as a loading control. C, MEK1–RAF complex formation monitored by AlphaLISA protein–protein interaction assays after treatment with various concentrations of NST-628 for 30 minutes at RT. D, Table of binding constants from SPR-based ternary complex assays where MEK1 is titrated with immobilized GST-BRAF or GST-CRAF in the presence and absence of 3 μmol/L NST-628.

Figure 2.

NST-628 engages pan-RAF–MEK complexes with active and inactive RAF conformations. A, Overview of MEK1–RAF heterodimers in the crystal structures of MEK1–ARAF (2.42 Å resolution), MEK1–BRAF (2.07 Å resolution), and MEK1–CRAF (2.59 Å resolution) with NST-628 (shown as spheres) and active RAF. Insets show electron density for NST-628 (blue mesh) in the interfacial allosteric site with key interactions highlighted by black dashes and waters represented as red spheres. B, Overlay of the distinct RAF conformations observed in crystal structures. C, Table of binding constants from SPR-based ternary complex assays titrating MEK1 with immobilized full-length WT BRAF or full-length S365A BRAF in the presence or absence of 3 μmol/L NST-628. D, Overview of cryo-EM structure of MEK1–CRAF–14-3-3 with NST-628 (4.36 Å resolution). Insets show electron density for NST-628 and the interfacial allosteric pocket.

Figure 3.

NST-628 inhibits BRAF class II/III mutants and does not induce the formation of RAF heterodimers. A, Table of binding constants from SPR-based ternary complex assays titrating MEK1 with immobilized GST-BRAF G466A or GST-BRAF G469A in the presence and absence of 3 μmol/L NST-628. B, BRAF immunoprecipitation in the NCI-H1666 cell line treated with 100 nmol/L of the indicated inhibitors for 2 hours, and blot analysis was performed for ARAF, BRAF, CRAF, MEK1, phospho-ERK, and vinculin as a loading control. C, BRAF class II/III mutant cell line panel (OV90, NCI-H1666, NCI-H1755, NCI-H2405, WM1963, WM3629, WM3670, WM3912, and WM3928) was treated for 72 hours with a dose titration of NST-628, avutometinib, naporafenib, exarafenib, or plixorafenib, and viability was measured by CellTiter-Glo.

Figure 4.

NST-628 inhibits the growth of RAS- and RAF-driven cancers. A, OMNI cell line panel was treated with a dose-response of NST-628 for between 3 and 7 days, and viability was measured by CellTiter-Glo. Response rates in each mutational background were calculated for models with a GI₅₀ of ≤100 nmol/L. B, HCT116 (C) IPC-298, (D) SK-MEL-2, and (E) MeWo cell lines were treated with 4, 20, or 100 nmol/L NST-628 for 48 hours and were stained with Annexin V and DAPI, and live, early apoptosis, late apoptosis, and necrotic cells were analyzed by flow cytometry. F, HCT116 (G), IPC-298 (H), SK-MEL-2, and (I) MeWo cells were treated with a dose response of NST-628, trametinib, avutometinib, cobimetinib, belvarafenib, or tovorafenib for 72 hours, and viability was assessed by CellTiter-Glo.

Figure 5.

NST-628 displays potent antitumor activity in KRAS- and NRAS-mutant models. HCT116 tumors treated with a single dose of 3 mg/kg qd, 5 mg/kg qd, or 1.5 mg/kg b.i.d. NST-628 and assessed for (A) phospho-ERK or (B) phospho-MEK by immunoblot 4, 8, or 24 hours after treatment. C, Body weights of HCT116 tumor-bearing mice treated with 0.3 or 1 mg/kg qd trametinib, 0.3 or 1 mg/kg qd avutometinib, or 3 or 5 mg/kg qd NST-628. D, Day 10 tumor volume of HCT116 tumors treated with 0.3 mg/kg qd trametinib, 0.3 mg/kg qd avutometinib, 3 mg/kg qd, 5 mg/kg qd, or 1.5 mg/kg b.i.d. NST-628; tumors are normalized to D0 starting volume. IPC-298 tumors treated with 0.5 mg/kg b.i.d., 1.5 mg/kg b.i.d., or 5 mg/kg qd NST-628, cobimetinib (5 mg/kg qd), belvarafenib (15 mg/kg qd), or a combination of cobimetinib (5 mg/kg daily) and belvarafenib (15 mg/kg) and immunoblotted for (E) phospho-ERK or (F) phospho-MEK 4 hours after treatment. G, IPC-298 tumor volume and (H) body weights of tumor-bearing mice treated as in E and F. Tumor volume and body weights are normalized to D0. I, CTG-0723, CTG-1684, CTG-1351, CTG-0308, CTG-3063, CTG-0889, CTG-1086, CTG-1375, CTG-0941, CTG-1612, CTG-0302, CTG-0881, CTG-1471, CTG-1358, CTG-0381, CTG-0291, CTG-1441, CTG-2841, CTG-0964, CTG-3059, CTG-0314, CTG-1068, and CTG-1501 (left–right) PDX tumors treated with 3 mg/kg qd NST-628. Data are represented as day 13/14 or maximum response tumor volume, normalized to D0.

Figure 6.

NST-628 is a fully brain-penetrant inhibitor. A,In vivo half-life, rat brain Kp, Kp, uu, and mouse Kp values for NST-628, trametinib, and avutometinib. B, Plasma concentration compared with phospho-ERK levels 4 hours after dose in normal mouse brains of mice treated with 1 mg/kg trametinib, 1 mg/kg avutometinib, 5 mg/kg cobimetinib, or 0.3, 1, or 3 mg/kg NST-628. C, Bioluminescent imaging of SK-MEL-2-luc intracranial tumors treated with vehicle or 1.5 mg/kg b.i.d. NST-628 at D0, D7, and D18 of the study. D, Intracranial tumor volume measured by bioluminescent imaging of SK-MEL-2-luc tumors treated with 0.3 mg/kg qd trametinib, 0.3 mg/kg qd avutometinib, 3 mg/kg qd or 1.5 mg/kg b.i.d. NST-628. E, Tumor volume as measured by bioluminescent imaging of intracranial MeWo-luc tumors treated with 25 mg/kg qd tovorafenib or 0.3, 1, 3 mg/kg qd NST-628. F, DUSP6 transcript levels in intracranial MeWo-luc tumors treated with 25 mg/kg tovorafenib or 3 mg/kg NST-628 at 4, 8, and 24 hours after single dose of inhibitor.

Figure 7.

NST-628 enhances the efficacy of KRAS^G12C inhibition. A, Tumor volume of NCI-H23 tumors and (B) body weights of NCI-H23 tumor-bearing mice treated with 2 mg/kg NST-628, 100 mg/kg qd sotorasib, or a combination of NST-628 and sotorasib. C, Phospho-ERK levels in NCI-H23 endpoint tumors from A and B collected 4 hours after last dose.