Abstract
Tuberculosis Disease (TB) can be treated through a combination drug therapy. However, current treatments are expensive, span several months, and are sometimes ineffective due to Mycobacterium tuberculosis (Mtb) drug resistance. Therefore, alternative therapies should be researched in order to find new cures for TB. The pathogenesis of Mtb is promoted by the protein Hip1, Hydrolase Important for Pathogenesis 1, which may serve as a drug target. Hip1 is found in the cell wall of Mtb and is classified as a serine protease. Hip1 cleaves GroEL2, a putative chaperonin involved in bacterial protein folding, and this mechanism is important for Mtb survival against the human immune system. We developed an Enzyme-Linked Immunosorbent Assay (ELISA) that visualizes Hip1 cleaving the protein’s native substrate GroEL2 as an in vitro method to screen for inhibitors of Hip1. We discovered that Compound 4I can inhibit Hip1 and should be further investigated for pharmaceutical use. In order to gain mechanistic insight of Hip1 activity, we engineered mutant versions of the protein via site-directed mutagenesis, and discovered that a threonine residue (T466) close to the Hip1 catalytic triad (S228, D463, H490) increases proteolytic activity. Furthermore, we found that the presence of magnesium ions and Hip1 purification in an oxygen-reduced environment increases Hip1 catalytic activity. Further experimentation involving the characterization of enzymatic properties and inhibition of the potential drug target, Hip1, can aid in the development of drug therapies for Tuberculosis Disease.