Computational high-throughput screening and in vitro approaches identify CB-006-3; A novel PI3K-BRAFV600E dual targeted inhibitor against melanoma

Malignant melanoma is characterized by both genetic and molecular alterations that activate phosphoinositide 3-kinase (PI3K), and RAS/BRAF pathways. In this work, through diversity-based high-throughput virtual screening we identified a lead molecule that selectively targets PI3K and BRAF^V600E kinases. Computational screening, Molecular dynamics simulation and MMPBSA calculations were performed. PI3K and BRAF^V600E kinase inhibition was done. A375 and G-361 cells were used for in vitro cellular analysis to determine antiproliferative effects, annexin V binding, nuclear fragmentation and cell cycle analysis. Computational screening of small molecules indicates compound CB-006-3 selectively targets PI3KCG (gamma subunit), PI3KCD (delta subunit) and BRAF^V600E. Molecular dynamics simulation and MMPBSA bases binding free energy calculations predict a stable binding of CB-006-3 to the active sites of PI3K and BRAF^V600E. The compound effectively inhibited PI3KCG, PI3KCD and BRAF^V600E kinases with respective IC₅₀ values of 75.80, 160.10 and 70.84 nM. CB-006-3 controlled the proliferation of A375 and G-361 cells with GI₅₀ values of 223.3 and 143.6 nM, respectively. A dose dependent increase in apoptotic cell population and sub G₀/G₁ phase of cell cycle were also observed with the compound treatment in addition to observed nuclear fragmentation in these cells. Furthermore, CB-006-3 inhibited BRAF^V600E, PI3KCD and PI3KCG in both melanoma cells. Collectively, based on the computational modeling and in vitro validations, we propose CB-006-3 as a lead candidate for selectively targeting PI3K and mutant BRAF^V600E to inhibit melanoma cell proliferation. Further experimental validations, including pharmacokinetic evaluations in mouse models will identify the druggability of the proposed lead candidate for further development as a therapeutic agent for treating melanoma.