Fluorescently labelled thioacetazone for detecting the interaction with dehydratases HadAB and HadBC

• Published in: Organic & biomolecular chemistry 2022-02, Vol.2 (7), p.1444-1452
• Main Authors: Singh, Bina K, Singha, Monisha, Basak, Shyam, Biswas, Rupam, Das, Amit K, Basak, Amit
• Format: Article
• ISSN: 1477-0520; 1477-0539
• DOI: 10.1039/d1ob02080c

Thioacetazone (TAC) used to be a highly affordable, bacteriostatic anti-TB drug but its use has now been restricted, owing to severe side-effects and the frequent appearance of the TAC resistant M. tuberculosis strains. In order to develop new TAC analogues with fewer side-effects, its target enzymes need to be firmly established. It is now hypothesized that TAC, after being activated by a monooxygenase EthA, binds to the dehydratase complex HadAB that finally leads to a covalent modification of HadA, the main partner involved in dehydration. Another dehydratase enzyme, namely HadC in the HadBC complex, is also thought to be a possible target for TAC, for which definitive evidence is lacking. Herein, using a recently exploited azido naphthalimide template attached to thioacetazone and adopting a photo-affinity based labelling technique, coupled with electrophoresis and in-gel visualization, we have successfully demonstrated the involvement of these enzymes including HadBC along with a possible participation of an alternate mycobacterial monooxygenase MymA. In silico studies also revealed strong interactions between the TAC-probe and the concerned enzymes. Photo-cross-linking experiments demonstrated direct interactions of the drug thioacetazone with its target enzymes, namely both the dehydratases HadAB and HadBC via the monooxygenase EthA in Mycobacterium tuberculosis.