Design and development of novel DNA Topoisomerase inhibitors

DNA topoisomerase (topo) inhibitors are amongst the most widely used and effective anticancer drugs that target either type I or type II enzymes, however, their clinical application is severely restricted by dose-limiting side effects and the development of drug resistance, commonly the result of diminished expression of the target enzyme and evolution of multidrug resistance (MDR) phenomena. The motivation for the present study was the hypothesis that dual inhibitors that target type I and type II proteins may offer the prospect of circumventing acquired altered-topoisomerase resistance associated with downregulation of a single protein, with consequential improvements in therapeutic index. A fully characterised [principally by nmr and mass spectrometry], extensive library of >100 novel, spacer-linked anthraquinone-amino acid conjugates [code-named NU:UB] was synthesised as putative dual inhibitors of topo I and II. Key conjugates, exemplified by the proline conjugate, NU:UB 31 (208), were shown to co-target topo I and the individual (o. and P) isoforms of human topo II in vitro in enzyme inhibition assays using gel electrophoretic methods; furthermore, their pattern of cell-kill correlated with topo levels in a panel of animal and human cancer cell lines. Lead compounds were identified and subsequently shown to have notable antitumour activity in MAC15A experimental colon cancer which is refractory to standard agents. This research programme has made a significant contribution to an understanding of the structural requirements for dual enzyme inhibition and has provided new potential anticancer drugs that have progressed to pre-clinical evaluation. [PART A] In another aspect, the chemistry of spacer-linked anthraquinone-amino acid conjugates was extended to the synthesis and characterisation of oligopeptide derivatives that were designed as matrix metalloproteinase (MMP)-activated prodrugs, in a new approach to tumour-selective drug targeting. A prototype oligopeptide prodrug PLI [(a D-ala-ala-ala-leu-gly-leu hexapapetide derivative (293)] of the in vivo-active dual topo inhibitor NU:UB 31, containing MMP-9 sensitive cleavage 'hotspots' in the peptide motif, underwent selective initial cleavage by human recombinant enzyme and by homogenates of a highly MMP-9 expressing HTI080 human fibrosarcoma. Preliminary data suggests that it may be feasible to selectively deliver potent cytotoxic agents to the site of a tumour by exploiting the proteolytic capacity of over-expressed MMPs in the tumour environment. [PART B]