Identification of Active Compounds against Melanoma Growth by Virtual Screening for Non-Classical Human DHFR Inhibitors

Abstract

Antifolates such as methotrexate (MTX) have been largely known as anticancer agents because of their role in blocking nucleic acid synthesis and cell proliferation. Their mechanism of action lies in their ability to inhibit enzymes involved in the folic acid cycle, especially human dihydrofolate reductase (hDHFR). However, most of them have a classical structure that has proven ineffective against melanoma, and, therefore, inhibitors with a non-classical lipophilic structure are increasingly becoming an attractive alternative to circumvent this clinical resistance. In this study, we conducted a protocol combining virtual screening (VS) and cell-based assays to identify new potential non-classical hDHFR inhibitors. Among 173 hit compounds identified (average logP = 3.68; average MW = 378.34 Da), two—herein, called C1 and C2—exhibited activity against melanoma cell lines B16 and A375 by MTT and Trypan-Blue assays. C1 showed cell growth arrest (39% and 56%) and C2 showed potent cytotoxic activity (77% and 51%) in a dose-dependent manner. The effects of C2 on A375 cell viability were greater than MTX (98% vs 60%) at equivalent concentrations and times. Our results indicate that the integrated in silico/in vitro approach provided a benchmark to identify novel promising non-classical DHFR inhibitors showing activity against melanoma cells.