Development of deazaflavin analogs as potent and selective MRP1 inhibitors for cancer chemosensitization - Project Summary/Abstract Resistance to chemotherapy is a major contributor to cancer relapse and treatment failure. Of many molecular mechanisms of cancer therapeutic resistance, increased drug efflux, mediated by ATP-binding cassette (ABC) transporters, results in multidrug resistance (MDR) and poses a huge barrier to effective cancer therapy. Small molecule inhibitors of efflux could be used to sensitize cancer cells toward chemotherapeutic treatment and improve treatment quality. Significantly, we have identified a highly potent lead compound, a deazaflavin analog which at single digit nanomolar concentrations drastically increased the intracellular concentration of etoposide (ETP), and to a lesser extent, that of teniposide and pemetrexed, all are important cancer chemotherapeutics. Our prior studies revealed that it is a potent inhibitor of MRP1. Notably, it displayed excellent selectivity over other major ABC transporters, including P-gp, BCRP, MRP2 and MRP3, and fully reversed the MRP1-mediated drug resistance to ETP and doxorubicin in H69AR small cell lung cancer cells. In addition, it exhibited desirable PK in preliminary mice testing without altering distribution of co-administered ETP to normal tissues in healthy mice, and hence may have limited toxicological consequences. These preliminary findings strongly suggest this lead and its analogs could be a highly valuable lead series for developing combination treatments with clinical TOP2 poisons or other anticancer drugs. The objectives of this grant application are to examine a library of deazaflavin analogs for MRP1 inhibiting activity and to further evaluate their potential for cancer chemotherapy sensitization in in vitro and in vivo models, by pursuing these specific aims: Aim 1. to screen and characterize deazaflavin analogs for MRP1 inhibition using membrane vesicle-based and cell-based drug transport assays. In this aim, we will screen an in-house deazaflavin analog library (~30 synthetic analogs) using vesicular transport assay and cell-based calcein retention assay for MRP1 inhibitory activity and characterize their potency, selectivity as well as examine the structure-activity relationship (SAR). We will also perform site-directed mutagenesis on MRP1 to identify residues that may be critical for the inhibitory effect of the analogs. Aim 2. to evaluate the deazaflavin analogs for cancer chemo- sensitization in in vitro and in vivo models. In this aim, we will first evaluate advanced analogs for chemo- sensitization in a panel of lung cancer and neuroblastoma cells toward selected chemotherapeutics and establish correlations to the MRP1 protein expression and inhibition. We will then profile the in vivo toxicity of our top analogs in mice, and further evaluate the anti-tumor effect and toxicological consequence of the analogs in combination with TOP2 poison ETP in a xenograft mouse model of multidrug resistant lung cancer. At the conclusion of the studies outlined in this proposal, a better understanding of the mechanism of action and the chemotherapeutic sensitizing potential of our lead compounds will be gained, which will lay a solid foundation for future preclinical studies.
