EBI-907

The aim of our study is to investigate in vitro and in vivo MC4R as a novel target in melanoma using the selective antagonist ML00253764 (ML) alone and in combination with vemurafenib, a B-rafV600E inhibitor. The human melanoma B-raf mutated A-2058 and WM 266-4 cell lines were used. An MC4R null A-2058 cell line was generated using a CRISPR/Cas9 system. MC4R protein expression was analysed by western blotting, immunohistochemistry, and immunofluorescence. Proliferation and apoptotic assays were performed with ML00253764, whereas the synergism with vemurafenib was evaluated by the combination index (CI) and Loewe methods. ERK1/2 phosphorylation and BCL-XL expression were quantified by western blot. In vivo experiments were performed in Athymic Nude-Foxn1^nu male mice, injecting subcutaneously melanoma cells, and treating animals with ML, vemurafenib and their concomitant combination. Comet and cytome assays were performed. Our results show that human melanoma cell lines A-2058 and WM 266-4, and melanoma human tissue, express functional MC4R receptors on their surface. MC4R receptors on melanoma cells can be inhibited by the selective antagonist ML, causing antiproliferative and proapoptotic activity through the inhibition of phosphorylation of ERK1/2 and a reduction of BCL-XL. The concomitant combination of vemurafenib and ML caused a synergistic effect on melanoma cells in vitro and inhibited in vivo tumor growth in a preclinical model, without causing mouse weight loss or genotoxicity. Our original research contributes to the landscape of pharmacological treatments for melanoma, providing MC4R antagonists as drugs that can be added to established therapies.

V600E-BRAF kinase is an essential therapeutic target in melanoma and other types of tumors. Because of its resistance to known inhibitors and the adverse effects of some identified inhibitors, investigation of new potent inhibitors is necessary.

In the present work, in silico strategies such as molecular docking simulation, pharmacokinetic evaluation, and density functional theory (DFT) computations were used to identify potential V600E-BRAF inhibitors from a set of 72 anticancer compounds in the PubChem database.

Five top-ranked molecules (12, 15, 30, 31, and 35) with excellent docking scores (MolDock score ≥90 kcal mol^-1, Rerank score ≥60 kcal mol^-1) were selected. Several potential binding interactions were discovered between the molecules and V600E-BRAF. The formation of H-bonds and hydrophobic interactions with essential residues of V600E-BRAF suggested the high stability of these complexes. The selected compounds had excellent pharmacological properties according to the drug likeness rules (bioavailability) and pharmacokinetic properties. Similarly, the energy for the frontier molecular orbitals, such as the HOMO, LUMO, energy gap, and other reactivity parameters, was computed with DFT. The frontier molecular orbital surfaces and electrostatic potentials were investigated to demonstrate the charge-density distributions potentially associated with anticancer activity.

The identified compounds were found to be potent hit compounds for V600E-BRAF inhibition with superior pharmacokinetic properties; therefore, they may be promising cancer drug candidates.