RESUMEN
A current clinical challenge in cancer is multidrug resistance (MDR) mediated by ABC transporters. Breast cancer resistance protein (BCRP) or ABCG2 transporter is one of the most important ABC transporters implicated in MDR and the use of inhibitors is a promising approach to overcome the resistance in cancer. This study aimed to characterize the molecular mechanism of ABCG2 inhibitors identified by a repurposing drug strategy using antiviral, anti-inflammatory and antiparasitic agents. Lopinavir and ivermectin can be considered as pan-inhibitors of ABC transporters, since both compounds inhibited ABCG2, P-glycoprotein and MRP1. They inhibited ABCG2 activity showing IC50 values of 25.5 and 23.4 µM, respectively. These drugs were highly cytotoxic and not transported by ABCG2. Additionally, these drugs increased the 5D3 antibody binding and did not affect the mRNA and protein expression levels. Cell-based analysis of the type of inhibition suggested a non-competitive inhibition, which was further corroborated by in silico approaches of molecular docking and molecular dynamics simulations. These results showed an overlap of the lopinavir and ivermectin binding sites on ABCG2, mainly interacting with E446 residue. However, the substrate mitoxantrone occupies a different site, binding to the F436 region, closer to the L554/L555 plug. In conclusion, these results revealed the mechanistic basis of lopinavir and ivermectin interaction with ABCG2. See also the Graphical abstract(Fig. 1).
RESUMEN
De novo synthesis of fatty acids is essential to maintain intensive proliferation of cancer cells. Unlike normal cells that utilize food-derived circulating lipids for their fuel, cancer cells rely on heightened lipogenesis irrespective of exogenous lipid availability. Overexpression and activity of the multidomain enzyme fatty acid synthase (FASN) is crucial in supplying palmitate for protumorigenic activity. Therefore, FASN has been proposed as an attractive target for drug development. As an effort to set up an effective toolkit to study FASN inhibitors in human and rodent tissues, we validated activity-based protein profiling (ABPP) as a viable approach to unveil inhibitors targeting FASN thioesterase domain (FASN-TE). ABPP was combined with multi-well plate-assays designed for classical substrate-based FASN activity analysis together with powerful monitoring of cancer cell proliferation using IncuCyte® Live Cell Analyzing System. FASN-TE inhibitors were identified by competitive ABPP using HEK293 cell lysates in a screen of in-house compounds (200+) designed to target serine hydrolase (SH) family. The identified compounds were tested for their inhibitor potencies in vitro using a substrate-based activity assay monitoring FASN-dependent NADPH consumption in LNCaP prostate cancer cell preparation, in parallel with selected reference inhibitors, including orlistat (THL), GSK2194069, GSK837149A, platensimycin and BI-99179. LNCaP lysate supernatant was validated as a reliable native preparation to monitor FASN-dependent NADPH consumption as opposed to human glioma GAMG cells, whereas FASN enrichment was a prerequisite for accurate assays. While inhibitor pharmacology was identical between human prostate and glioma cancer cell FASN preparations, notable differences were revealed between human and rodent FASN preparations, especially for inhibitors targeting FASN-TE. ABPP combined with substrate-based assays facilitated identification of pan thiol-reactive inhibitor scaffolds, exemplified by the 1,2,4-thiadiazole moiety. Finally, selected compounds were evaluated for their antiproliferative efficacy in situ using GAMG cells. These studies revealed that while the tested compounds acted as potent FASN inhibitors in vitro, only a few showed antiproliferative efficacy in situ. To conclude, we describe a versatile toolkit to study FASN inhibitors in vitro and in situ using human cancer cells and reveal dramatic pharmacological differences between human and rodent FASN preparations.
