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Identification of regions of bacterial topoisomerase I important for cleavage‐religation for the discovery of type IA topoisomerase poisons as novel antibacterial compounds

Type IA and type IIA topoisomerases have little sequence homology but similar mechanisms of action. We are targeting bacterial type IA topoisomerases in our HTS efforts to obtain new leads for discovery of novel antibacterial compounds to combat multi‐drug resistant bacterial pathogens. The goal is to identify small molecules that can act as poisons of bacterial type IA topoisomerases by trapping the covalent intermediate formed between the enzyme and cleaved DNA, similar to the action of fluoroquinolones on bacterial type IIA topoisomerases. We have previously demonstrated that expression of recombinant type IA topoisomerases with the strictly conserved glycine residue in the DXDXXG TOPRIM motif mutated to serine led to accumulation of the covalent cleaved DNA complex and rapid bacterial cell killing. This validates type IA topoisomerases as potential targets for discovery of novel antibacterial drugs acting as type IA topoisomerase poisons. We have isolated additional SOS‐inducing Yersinia pestis and Escherichia coli topoisomerase I mutants and characterized these mutant enzymes biochemically. These mutagenesis results identify regions of the enzyme important for control of the cleavage‐religation equilibrium for potential docking of small molecules to achieve similar bactericidal action as the topoisomerase I mutations.