We will examine how the biological properties of anthracycline antibiotics, and some of their underlying biophysical and biochemical characteristics, are modulated by replacing the basic amino group of doxorubicin with a hydroxyl moiety. Of particular interest will be to determine the exact manner in which the deamination process affects drug interactions with DNA, topoisomerase II and P-glycoprotein (PGP), three macromolecular species widely recognized as key cellular targets for anticancer drug action. Hydroxyrubicin is the 3'-deaminated and configurationally identical analogue of doxorubicin. Interestingly, our preliminary studies indicate that the deamination process reduces cardiotoxicity while significantly improving antitumor activity against several multidrug-resistant (MDR) phenotypes. Higher levels of activity were found to correlate with higher levels of drug accumulation and retention in MDR cells, suggesting that deamination may result in less efficient drug interactions with the PGP target. To test this hypothesis, the effect of deamination on anthracycline efflux rates from MDR cells will be evaluated in single cell microinjection studies. Quantitative fluorescence microscopic monitoring of cells will be employed to study drug efflux in both parental cell lines as well as MDR phenotype or cells transfected with the mdr1 gene. Anthracyclines interactions with PGP will then be explored in photoaffinity labeling experiments, and the structure-dependent affinity of anthracyclines will be explored. Through this work we also hope to identify new, nontoxic anthracyclines with high affinity for PGP to serve as competitive inhibitors of efflux, potentially useful in reversing MDR. Deamination of doxorubicin is expected to significantly affect its interaction with DNA. The DNA binding affinity, the DNA site and sequence specificity, and related thermodynamic parameters will be assessed for the deaminated anthracyclines. In vitro cytotoxicity of the deaminated analogues will be determined and compared with that of parent anthracyclines. Both drug-sensitive and drug-resistant phenotypes with different degrees of resistance will be studied. It will be of special interest to determine whether or not progressive resistance to doxorubicin in the various MDR phenotypes (MCF-7, KB, 8226, NIH 3T3) under study correlate with efficiency of the PGP pump mechanism and topoisomerase II-mediated DNA cleavage. As a result of these studies we expect to better understand mechanistically the improved biological activities of deaminated anthracyclines, as well as the mechanisms which underlay the development of MDR. Identification of significantly different mechanism of action of 3'-hydroxy anthracyclines would provide strong justification for the development of selected deaminated congeners for clinical trials.
