Anticancer Quinolinol Small Molecules Target Multiple Pathways to Promote Cell Death and Eliminate Melanoma Cells Resistant to BRAF Inhibitors

Abstract

Small molecule inhibitors that target the E3 ligase activity of MDM2-MDM4 have been explored to inhibit the oncogenic activity of MDM2-MDM4 complex. MMRi62 is a small molecule that was identified using an MDM2-MDM4 E3 ligase-based high throughput screen and a cell-death-based secondary screen. Our previous studies showed that MMRi62 promotes MDM4 degradation in cells and induces p53-independent apoptosis in cancer cells. However, MMRi62 activity in solid tumor cells such as melanoma cells, especially in BRAF inhibitor resistant melanoma cells, have not been explored. Although its promotion of MDM4 degradation is clear, the direct MMRi62 targets in cells are unknown. In this report, we show that MMRi62 is a much more potent p53-independent apoptosis inducer than conventional MDM2 inhibitors in melanoma cells. A brief structure-activity study led to development of SC-62-1 with improved activity. SC-62-1 potently inhibits and eliminates clonogenic growth of melanoma cells that acquired resistance to BRAF inhibitors. We developed a pair of active and inactive SC-62-1 probes and profiled the cellular targets of SC-62-1 using a chemical biology approach coupled with IonStar/nano-LC/MS analysis. We found that SC-62-1 covalently binds to more than 15 hundred proteins in cells. Pathways analysis showed that SC-62-1 significantly altered several pathways including carbon metabolism, RNA metabolism, amino acid metabolism, translation and cellular response to stress. This study provides mechanistic insights into the mechanisms of action for MMRi62-like quinolinols. This study also suggests multi-targeting compounds like SC-62-1 might be useful for overcoming resistance to BRAF inhibitors for improved melanoma treatment.

Keywords: E3 ligase; MDM2-MDM4; MMRi62; cell death; chemical robe; quinolinol; targets.

Figure 1

Westen Blot analysis of MMRi62 and AMG232 for the effect on MDM2, MDM4, p53 and cleaved PARP induction. (A,B) Dose response curves in the presence of MMRi62 (A) or AMG232 (B) for A375 and shp53-A375 cells. IC₅₀s are shown at the bottom. The representative data of three independent experiments were shown and the IC₅₀ values were obtained from three independent experiments. (C,D) WB analysis of MDM2, MDM4, p53 and cleaved PARP (cPARP) induction in A375 and shp53-A375 cells treated with MMRi62 (C) or AMG232 (D) for 24 h (E). WB analysis of MDM2, MDM4, p53 and cleaved PARP (cPARP) induction in A375 treated with AMG232 or MMRi62 for 48 h and 72 h (F), WB analysis of cleaved PARP (cPARP) and p53 in shp53A375 cells treated with AMG232 or MMRi62 for 48 h and 72 h. Tubulin is the protein loading control. The intensities of cPARP and tubulin bands in (C,F) were quantified by ImageJ1.54P software and fold increases in cPARP over untreated control shown under cPARP WB were obtained after normalization against tubulin.

Figure 2

Chemical structure and antiproliferative activity of MMRi62, SC-62-1 and their inactive counterparts. Top: Chemical structures of MMRi62, MMRi62Me, SC-62-1 and SC-62-1Me. Bottom: The IC₅₀ of MMRi62 and SC-62-1 in A375 cells.

Figure 3

Effect of SC-62-1 and its combinations with Vemurafinib (Vem) on survival and proliferation of melanoma cells and Vem-resistant cells in vitro. (A) Microscopy images of crystal violet stained A375 cells after 2 week exposure to 1, 2, 4 μM Vem, SC-62-1 or their combinations. (B) Dose response curves of Vem-resistant A375 cells (A375VemR) with acquired resistance. (C) Microscopy images (top) and whole plates of crystal violet stained A375VemR cells after 8 day exposure to 4, 8, and 16 μM Vem, SC-62-1 or their combinations. (D) Combination index diagram obtained for SC-62-1-Vem combinations. CI < 1, synergism, CI = 1, additive, CI > 1, antagonism. Arrowheads indicate that the higher the CI values above 1, the more antagonistic they are, while the smaller the CI values below 1 toward zero, the more synergistic they are.

Figure 4

Synthesis of SC-62-1 and azide probes, hypothetical mechanisms of action of SC-62-1 and molecular characterization of azide probes. (A) Synthesis of quinolinol Betti base SC-62-1. (B) Hypothetical MOA for SC-62-1 as covalent inhibitors of target proteins via quinone methide to alkylate target proteins. (C) Synthesis routes for SC-62-16 and SC-62-16Me. (D) Anti-proliferation assays in A375 cells treated with SC-62-16 and SC-62-16Me for 72h. IC_50s values of the indicated compounds are shown. (E) Western blot analysis of SC-62-16 and SC-62-16Me effect on MDM4/MDM2 degradation and apoptotic PARP cleavage induction in treated A375 cells for 24 h. GAPDH is the protein loading control.

Figure 4

Synthesis of SC-62-1 and azide probes, hypothetical mechanisms of action of SC-62-1 and molecular characterization of azide probes. (A) Synthesis of quinolinol Betti base SC-62-1. (B) Hypothetical MOA for SC-62-1 as covalent inhibitors of target proteins via quinone methide to alkylate target proteins. (C) Synthesis routes for SC-62-16 and SC-62-16Me. (D) Anti-proliferation assays in A375 cells treated with SC-62-16 and SC-62-16Me for 72h. IC_50s values of the indicated compounds are shown. (E) Western blot analysis of SC-62-16 and SC-62-16Me effect on MDM4/MDM2 degradation and apoptotic PARP cleavage induction in treated A375 cells for 24 h. GAPDH is the protein loading control.

Figure 5

Identification of SC-62-16 bound proteins and targeted pathways. (A) Diagram for the optimized procedure of pulldown of SC-62-1-bound proteins. (B) Number of SC-62-16-bound proteins and their distributions in different categories. (C) Results of pathway analysis of the SC-62-16-bound proteins. The top 11 significantly altered pathways in using KEGG and REACTOME database are shown. (D) List of 11 pathways that are significantly impacted by SC-62-1.

Figure 6

Proposed model for MOA of SC-62-1 in induction of cancer cell death. SC-62-1’s anticancer activity is via production of quinone methide which alkylates many target proteins. Its downstream effect includes induction of proteasomal degradation of MDM4 protein and lysosomal degradation of FTH1 and significant alteration of the pathways including RNA metabolism, carbon metabolism, mitochondrial function, protein translation and protein degradation. Collectively, induction of changes in these pathways by SC-62-1 creates overwhelming cellular stress that cannot be resolved, thus leading to p53-independent cell death.