P-glycoprotein (P-gp) encoded by the MDR1 gene, causes multidrug resistance (MDR) to unrelated natural product drugs in human tumor cells, and functions as an ATP-dependent efflux pump. The overall project goals are (1) to unravel the molecular structure of P-gp drug binding sites, (2) to investigate the molecular mechanisms of MDR modulation, and (2) to provide rational approaches to synthesize and develop MDR reversing agents.
The specific aims are (1) investigate the modes of interaction of taxol and vinblastine (VBL) with cyclosporin A (CsA) analogs, tamoxifen (TAM) and related agents, and other MDR modulators for binding to P-gp, and determine whether phosphorylation affects this binding; (2) based on the results of Specific Aim 1, evaluate whether combinations of two MDR modulators enhance drug accumulation and increase the sensitivity of MDR cells to taxol and VBL additively or synergistically; (3) synthesize and use photoaffinity analogs of taxol, CsA and TAM, characterize their covalent binding to P-gp, and determine whether other MDR modulators inhibit their binding to P-gp; (4) identify and map the binding sites of taxol, CsA and TAM by peptide mapping and site-directed antibodies, and determine whether they interact with the VBL binding site; and (5) identify the amino acid sequences of the taxol, CsA and TAM binding sites by peptide sequencing, site-directed mutagenesis of the MDR1 gene, and 3- dimensional molecular modeling analysis. MCF-7 breast cancer cells transfected with the MDR1 gene or MDR1 protein kinase C-alpha (PKC) genes, or an MDR variant, MCF-7/ADR, will be used. Kinetic analysis will be performed to determine whether these agents interact competitively or noncompetitively, and whether phosphorylation affects drug binding to P-gp by examining the effects of PKC alone, PKC activators, PKC inhibitors and phosphorylation phosphatase inhibitors. Based on the kinetic data, the applicant will analyze the modulation of taxol and VBL accumulation and cytotoxicity using combinations of potent MDR modulators, and determine whether their additive or synergistic effects correlate with their competitive or noncompetitive interaction at the cytotoxic drug binding sites, and whether structure-activity relationships can be made. To identify the P-gp drug binding sites, (1) photoactive analogs of taxol, VBL, CsA and TAM will be synthesized and used to characterize their covalent binding to P-gp, (2) the photolabeled P-gp will be immunoprecipitated, digested, and resolved by electrophoresis, and (3) P-gp site-directed anti-peptide antibodies will be used for domainal mapping of the drug-bound peptides. To unravel the molecular architecture and spatial arrangements of the drug binding domains, (1) purified drug-bound peptide fragments will be sequenced and the position(s) of radioactive amino acids will be identified, (2) site-directed mutagenesis of the MDR1 gene will be performed to make single amino acid changes and determine whether such mutations differently affect the P-gp binding of the photoaffinity probes vs. parent drugs, and (3) molecular modeling analysis of the drug binding sites will be performed.
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