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Background: Type IA family of topoisomerases is preserved in all living organisms. The lack of a proven druggable pocket in type IA topoisomerases may be one of the core reasons behind the fact that there are no clinically approved inhibitors of these enzymes yet. A cysteine residue introduced into a rational position of Mycobacterium tuberculosis topoisomerase I (MtbTOP1) may serve as a model for parasitic TOP3β and a target for covalent inhibitors with reactive warheads. Structural determination of a covalent complex containing an inhibitor would aid the identification of a druggable pocket in parasitic type IA topoisomerases. Methods: A mutant bacterial topoisomerase I, MtbTOP1-V195C, is used as a target for compounds with acrylamide warheads. The compounds are tested against the wild-type MtbTOP1 and MtbTOP1-V195C for inhibition of relaxation of negatively supercoiled DNA. The selective compounds are then investigated to confirm that they form a covalent adduct with MtbTOP1-V195C. Hit compounds will be then tested against Giardia lamblia TOP3β, a parasitic type IA topoisomerase. Result: Among 162 compounds with cysteine-reactive warheads, five are found to be more active against MtbTOP1-V195C in three experimental replicates. When assayed against human type IB topoisomerase, only two of them (namely NCI 43 and NCI 86), are observed to exhibit specificity for type IA enzyme. Pre-incubation with the enzyme for 30 minutes, lowered the IC50 value of NCI 43 supporting the idea of covalent adduct formation between MtbTOP1-V195C and NCI 43. Some structural analogs of these compounds were tested to get more potent and specific inhibition. Conclusion: Certain compounds with acrylamide warheads have the potential to function as covalent inhibitors of the mutant MtbTOP1-V195C and structural optimization of the possible hits may be needed to identify more potent inhibitors for parasitic TOP3β and other type IA topoisomerases. However, this search in turn may shed light on a drug-binding pocket for the development of novel inhibitors of type IA topoisomerases. This research is supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R35GM139817.
Chadni et al. (Fri,) studied this question.