Human DNA topoisomerase I (hTOP1) is a highly effective new molecular target for anticancer drugs such as camptothecins (CPTs). CPTs inhibit (poison) TOP1 by trapping a covalent reaction intermediate, the ternary TOP1 cleavable complex. These TOP1 cleavable complexes represent a new type of cellular lesion highly effective in killing tumor cells. In addition to CPTs, a growing list of compounds (TOP1 poisons) (e.g. indolocarbazoles, nitidines, saintopin, morpholinyl doxorubicin, aclacinomycin A, indeno[1,2-c]isoquinolines, nogalamycin, actinomycin D, protoberberines, dibenzo[c,h]cinnolines, 2-phenylbenzimidazoles and terbenzimidazoles) have been identified to induce TOP1 cleavable complexes. Despite the clear importance of TOP1 cleavable complexes in tumor cell killing, the molecular mechanism underlying tumor cell killing by TOP1 poisons remains unclear. Our preliminary studies have suggested that at least two distinct molecular mechanisms may be involved in trapping TOP1 cleavable complexes by these different TOP1 poisons. CPTs, the prototypic TOP1 poisons, trap TOP1 cleavable complexes by specifically inhibiting the religation step of the TOP1-catalyzed breakage/religation reaction, presumably by binding to the TOP1- DNA complex at the site of DNA cleavage (religation blockers). By contrast, nogalamycin, a DNA binder with both intercalative and minor groove-directed modes of interactions, traps TOP1 cleavable complexes by inducing a distal DNA bend which favors TOP1 binding/cleavage (cleavage enhancers). The identification of two distinct modes of TOP1 poisoning by different TOP1 poisons may offer new opportunities for drug design and development. In the current application, we plan to characterize these two modes of TOP1 poisoning and to probe the differential biological responses induced by different TOP1 poisons.
The specific aims are (1) to establish a DNA curvature model for TOP1 cleavage enhancers using nogalamycin as a model, and (2) to probe the protein conformation and cellular modification of TOP1 cleavable complexes induced by different TOP1 poisons.
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