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Mechanistic Basis of Sensitivity/Resistance Towards Anti-cancer Drugs Targeting Topoisomerase II
Annual summary rept. 28 Mar 2003-27 Mar 2006
DUKE UNIV DURHAM NC
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Human topoisomerase llalpha hstopo llalpha is an essential enzyme that is the target of a number of anticancer drugs in clinical use. Clinically, resistance to anticancer drugs develops through various mechanisms, one of which can be found in a class of atypical multidrug resistant mutants at-MDR of hstopo llalpha. Our goal is to clarify the biochemical basis of at-MDR among hstopo 110 mutants to shed more light on the topo II enzymatic mechanism. To this end, we have developed several footprinting techniques for studying the conformational changes of hstopo llalpha. Additionally, we have identified sites of covalent thiol modification of hstopo llalpha by anticancer drugs and chemopreventive agents. To this end, we developed a cysteine footprinting technique using liquid chromatography-electrospray ionization-mass spectrometry LC-ESI-MS to assess the differential reactivities of the cysteine residues of hstopo llalpha and compared their relative solvent accessibilities to a solvent contact surface SOS analysis of a structural model of hstopo llalpha. In the presence of DNA, cofactors and anticancer drugs, we have seen changes in the relative thiol reactivities of hstopo llalpha to thiol-alkylating reagents including monobromobimane and iodoacetamide. These changes, when examined in the context of the structural model of hstopo llalpha, give insight into the conformational changes occurring in hstopo llalpha. Additionally, using LC-ESl-MS, we have located sites of covalent cysteine modification of hstopo 110 by both anticancer drugs and chemopreventive agents, resulting in the first direct evidence of this novel poisoning mechanism by thiol alkylation of hstopo llalpha.
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