Abstract Description

Tuberculosis (TB) is the disease caused by Mycobacterium tuberculosis (Mtb). Mtb is a very lethal agent, with high incidence and mortality rates globally. As a result, in 2023, there were 10.8 million incidents and 1.25 million deaths. The anti-TB first-line drugs (Isoniazid, Rifampicin, Pyrazinamide, and Ethambutol) have a high success rate for drug-susceptible TB. However, Mtb acquires resistance to anti-TB drugs through mutations that alter the coding sequence of drug targets and influence protein folding and stability. Molecular chaperone systems maintain proteostasis in cells and have specific functions linked to stabilisation of client proteins, such as intrinsic holdase activity, ATPase activity, and refolding activity. MtbDnaK, the Hsp70 molecular chaperone of bacteria, is a promising drug target because it is essential and vulnerable. We identified structural and sequence differences between Human Hsp70 (HSPA1A) and MtbDnaK we which aim to exploit to identify compounds that selectively inhibit MtbDnaK and not HSPA1A. Using in silico computational analysis we predicted the selective binding of the compounds to MtbDnaK and HSPA1A proteins in the APO, ADP, and ATP-bound states. Using differential scanning fluorimetry (DSF) we determined the binding of the compounds to MtbDnaK by changes in the melting temperature (Tm) in the APO, ADP, and ATP-bound states. We also used partial proteolysis of MtbDnaK to see if the compounds would alter the MtbDnaK conformation. Next, we investigated the aggregation suppression of Malate Dehydrogenase (MDH) by MtbDnaK (which measures intrinsic holdase activity) in the presence and absence of the compounds. Our data so far showed that, despite having activity against Mtb cultures, the compounds do not affect the Tm of MtbDnaK nor protect it from protease K in the APO, ADP, and ATP-bound state. Lastly, the compounds showed that they do not affect the holdase activity of MtbDnaK.