Journal: PLoS ONE

Article Title: IODVA1, a guanidinobenzimidazole derivative, targets Rac activity and Ras-driven cancer models

doi: 10.1371/journal.pone.0229801

Figure Lengend Snippet: (A) IODVA1 inhibits EGF-induced lamellipodia formation in MDA-MB-231 cells. MDA-MB-231 cells were plated on fibronectin-coated coverslips, serum starved for 4 h, incubated with the indicated concentrations of IODVA1 for 1 h, then EGF (50 ng/mL) stimulated, fixed and stained with Phalloidin Alexa Fluor 594 (F-Actin, pseudocolored red) and DAPI (nuclei, pseudocolored blue). Representative images show lamellipodia formation and enrichment of actin staining at the leading edge (white closed arrowheads) at 0 and 0.3 μM concentrations and lack of lamellipodia with rounded cell morphology at 1 and 3 μM. Note equally distributed phalloidin staining with presence of stress fibers in the cell body of the 1 and 3 μM treated-cells (white open arrowheads) indicative of stationary cells. Scale bar = 10 μm. Results are representative of three independent experiments. (B) IODVA1 inhibits PDGF-induced CDR formation in 3T3 fibroblasts. NIH-3T3 cells were plated on fibronectin-coated coverslips, serum starved for 4 h, incubated with the indicated concentrations of IODVA1 for 1 h, then PDGF (50 ng/mL) stimulated, fixed and stained with Phalloidin Alexa Fluor 594 (F-Actin, pseudocolored magenta) and DAPI (nuclei, pseudocolored blue). Closed white arrowheads indicate circular dorsal ruffles, arrows indicate lack of elongated morphology typical in stimulated fibroblasts. The percentage of cells with CDRs was counted as the number of cells with CDRs normalized to the total number of cells in the field. Around 150 cells were counted per condition per experiment. Cells with multiple CDRs were counted only once. Results are representative of three independent experiments. Scale bar = 10 μm. (C) MDA-MB-231 cells were incubated with IODVA1 (IO1) at the indicated concentrations for 1 h, lysed, and incubated with GST-PAK-GBD (binds active Rac and Cdc42) and GST-Rhotekin RBD (binds active RhoA). The protein complexes were resolved on SDS-PAGE and immunoblotted with pan-Rac, Cdc42 or RhoA antibodies. Levels of active Rac (RacGTP, % of control), active Cdc42 (Cdc42GTP, % of control) and active RhoA (RhoAGTP, % of control) were quantified using ImageJ and ImageLab and show combined data as mean ± s.e.m. from at least 3 independent experiments. (D) Left panel—MDA-MB-231 cells were incubated with IODVA1 (IO1, 1 μM) for 30 min, lysed, and immunoblotted for pPAK1(T423)/ pPAK2(T402). Lysates were loaded in two replicates. Results shown are mean ± s.e.m. of two independent experiments. Right panel–MDA-MB-231 cells were incubated with IODVA1 (IO1, 0.3 and 1 μM) for 30 min or 3 hours, lysed and immunoblotted for pPAK4(S474)/ pPAK5(S602)/ PAK6(S560). Results shown are mean ± s.e.m. of two independent experiments. n.s.–not significant, *—p < 0.05, **—p < 0.01, ****—p < 0.0001.

Article Snippet: The following antibodies were used–GAPDH (GeneTex GTX627408, 1:5000), b-Actin (CST 4970S, 1:10,000), pERK1/2 T202/Y204 (CST 4370, 1:2000), total ERK1/2 (CST 4696, 1:2000), pAKT S473 (CST 9271, 1:1000), pPAK1 T423/ pPAK2 T402 (CST 2601S, 1:1000), PAK1/2/3 (CST 2604, 1:2000), PAK1 (CST 2602, 1:1000), pPAK4 S474/pPAK5 S602/ pPAK6 S560 (CST 3241, 1:1000), PAK4 (CST 62690, 1:1000), anti-mouse Eu (Molecular Devices R8205), anti-rabbit Eu (Molecular Devices R8204), anti-mouse-HRP (CST 7076, 1:5000), and anti-rabbit-HRP (CST 7074, 1:5000), Ki67 (Abcam, IHC 1:100), cleaved caspase 3 (Asp175, CST 9661, IHC 1:100), goat anti-rabbit Alexa Fluor 568 (Abcam, IF 1:500).

Techniques: Incubation, Staining, SDS Page, Control

Journal: Cancer Discovery

Article Title: 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

doi: 10.1158/2159-8290.CD-24-0139

Figure Lengend Snippet: 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.

Article Snippet: Immunoprecipitation samples and input samples were resolved on 4% to 12% Bis-Tris Gels, and Western blotting was performed using antibodies against ARAF (Abcam,# ab200653), BRAF (Santa Cruz Biotechnology, #sc-5284), CRAF (BD #610152), MEK1 (Cell Signaling Technology, #2352), phospho-MEK (S217/221; Cell Signaling Technology, #9154), phospho-ERK (T402/Y404; Cell Signaling Technology, #4370), phospho-p90 RSK (Abcam,# ab32413), and vinculin (Millipore Sigma, #V9131).

Techniques: Immunoprecipitation, Control, Binding Assay

Journal: Cancer Discovery

Article Title: 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

doi: 10.1158/2159-8290.CD-24-0139

Figure Lengend Snippet: 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.

Article Snippet: Immunoprecipitation samples and input samples were resolved on 4% to 12% Bis-Tris Gels, and Western blotting was performed using antibodies against ARAF (Abcam,# ab200653), BRAF (Santa Cruz Biotechnology, #sc-5284), CRAF (BD #610152), MEK1 (Cell Signaling Technology, #2352), phospho-MEK (S217/221; Cell Signaling Technology, #9154), phospho-ERK (T402/Y404; Cell Signaling Technology, #4370), phospho-p90 RSK (Abcam,# ab32413), and vinculin (Millipore Sigma, #V9131).

Techniques: Binding Assay, Immunoprecipitation, Control, Mutagenesis, Titration

Journal: Cancer Discovery

Article Title: 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

doi: 10.1158/2159-8290.CD-24-0139

Figure Lengend Snippet: 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.

Article Snippet: Immunoprecipitation samples and input samples were resolved on 4% to 12% Bis-Tris Gels, and Western blotting was performed using antibodies against ARAF (Abcam,# ab200653), BRAF (Santa Cruz Biotechnology, #sc-5284), CRAF (BD #610152), MEK1 (Cell Signaling Technology, #2352), phospho-MEK (S217/221; Cell Signaling Technology, #9154), phospho-ERK (T402/Y404; Cell Signaling Technology, #4370), phospho-p90 RSK (Abcam,# ab32413), and vinculin (Millipore Sigma, #V9131).

Techniques: Activity Assay, Mutagenesis, Western Blot