Computational high-throughput screening and in vitro approaches identify CB-006-3;A novel PI3K-BRAF^(V600E) 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.