CB-RAF600E-1 exerts efficacy in vemurafenib-resistant and non-resistant-melanoma cells via dual inhibition of RAS/RAF/MEK/ERK and PI3K/Akt signaling pathways.

Background And Aim: Predicting novel dual inhibitors to combat adverse effects such as the development of resistance to vemurafenib in melanoma treatment due to the reactivation of MAPK and PI3K/AKT signaling pathways is studied to help in reversal of cancer symptoms.Reversal of cancer symptoms in melanoma associated with vemurafenib resistance is driven by reactivation of MAPK and PI3K/Akt signaling pathways. Novel dual inhibitors targeting these proteins would be beneficial to combat resistance.

Methods: High-throughput virtual screening of the ChemBridge library against B-RAFV600E and Akt was performed using an automated protocol with the AutoDock VINA program. Luminescence and time-resolved fluorescence kits were used to measure enzyme activities. The MTT assay was used to determine proliferation in normal and vemurafenib-resistant A375 cells. Flow cytometry was used to examine apoptosis, cell cycle, and phosphorylation of ERK/Akt signaling pathway.

Results: High-throughput screening from the ChemBridge library identified 15 compounds with high binding energy towards B-RAFV600E; among these, CB-RAF600E-1 had the highest ΔG score -11.9 kcal/mol. The compound also had a high affinity towards Akt, with a ΔG score of -11.5 kcal/mol. CB-RAF600E-1 dose-dependently inhibited both B-RAFV600E and Akt with IC values of 635 nM and 154.3 nM, respectively. The compound effectively controlled the proliferations of normal and vemurafenib-resistant A375 cells, with GI values of 222.3 nM and 230.5 nM, respectively. A dose-dependent increase in the sub G/G phase of the cell cycle and total apoptosis was observed following compound treatment in both normal and vemurafenib-resistant melanoma cells. Treatment with CB-RAF600E-1 decreased the pERK/pAkt dual-positive populations in normal and vemurafenib-resistant A375 cells.

Conclusion: CB-RAF600E-1, identified as a novel dual inhibitor effective against normal and vemurafenib-resistant melanoma cells, requires further attention for development as an effective chemotherapeutic agent for melanoma management.