Discovery And Characterization Of Novel Multidrug Resistance Inhibitors

MDR (mutidrug resistance) are major obstacles for successful cancer chemotherapy. One of the well-known mechanisms of MDR is the over-expression of drug efflux pumps on cancer cell surfaces that actively extrude anticancer drug substrates out of cells and, thus, eliminate the effective intracellular accumulation of these drugs. The two major proteins, P-glycoprotein (Pgp/ABCB1/MDR1) and breast cancer resistance protein (BCRP/ABCG2) are members of a superfamily of ATP-binding cassette (ABC) transporters that function as an energy-dependent durg efflux pump. Both Pgp and BCRP can transport a wide variety of drugs efficently leading to multidrug resistance and changes in drug pharmacokinetics.An attractive approach to overcoming MDR is the inhibition of pumping action of these transporters. Several inhibitors (chemosensitizers) of ABC transporters have been developed, but cytotoxic effects and adverse pharmacokinetics have prohibited their use in clinical trial. It is urgent to uncover the pathogenesis thereby develop potential effective agents. In this research, we try to establish the in vitro assay of, Pgp and BCRP and discover some potential novel small molecule reversal agent targeted to Pgp and BCRP.Objective: To discover some novel lead compounds that can inhibit P-gp and BCRP-mediated MDR and to determine the mechanisms of action of these inhibitors on these two human ABC transporters.Methods: P-gp overexpressing cell lines K562/A02 and MCF-7/ADR and BCRP overexpressing cell lines HEK-293/BCRP and MCF-7/MX were used as in vitro screening model of MDR reversal agent. Diversity analysis was performed on a database containing ~200,000 commercial available compounds (www.specs.net). From different structurally diverse clusters, 187 compounds were selected for biological assay based on prediction of physico-chemical properties, chemical stability, potential toxicity and potential metabolism. 187 compounds (C71-C196, C1-C61) were firstly undertaken to rationally identify potential inhibitors of P-gp and BCRP-mediated drug accumulation by flow cytometry analysis. And then some active compounds were further evaluated by inhibition of fluorescent substrates (rhodamine 123, doxorubicin, mitoxantrone) assay, mRNA and protein expression level assay, cytotoxicity sensitization and ATP hydrolyase activity assay.Results: The drug induced cell lines and transfected cell lines with over-expressing relative drug efflux pumps are ideal screening model. The in vitro assay system of reversal agent targeted to Pgp and BCRP are successfully established. Using this screening system, we found a novel potential inhibitor of Pgp, C95 that has inhibitory effect of Rhodamine 123 efflux and cytotoxicity sensitazation better than the positive control varapamil (VRP). The effects of C95 on MDR1 mRNA and protein expression level were further determined by RT-PCR and Western blot assay. It also seems to be inhibitor but not a substrate of Pgp. For identifying inhibitors of BCRP, these compounds of C84, C130, C138, C145, C188, C53, C55 showed higher MDR reversal activity by inhibition of BCRP-mediated mitoxanctrone efflux. They are more potent than the positive control FTC (Fumitremorgin C, specific inhibitor of BCRP) in cytotoxicity cytotoxic sensitization of drug resistance. Among them, C130, C53, C55 showed the most promising drug candidates.Conclusions: C95 was discovered as an ideal leading compound of P-gp reversal agent. A series of compounds targeted to BCRP are identified in vitro, expecially C130, C53 and C55.