Tuberculosis is a respiratory infection with over 10 million reported cases each year. This infection rate is responsible for over two million deaths annually which, second only to HIV in fatality among infectious diseases. Despite reported cases drastically decreasing since 1960, recent studies have shown that tuberculosis infection rates have steadily increased over the past few years suggesting that customary antibiotics have become increasingly ineffective. This observation suggests that there is an alarming increase in the prevalence of drug resistant strains of tuberculosis, thus the need for the discovery of novel antitubercular agents. Searching for potential enzymatic pathways for drug discovery requires the pathway to possess minimal overlap with the host cell. The shikimate pathway is a seven step metabolic route that produces aromatic amino acids and other cellular metabolites. This pathway is typically present in a variety of bacterium species and contains no mammalian analog, making any of the enzymes in this pathway desirable targets for screening of potential antitubercular agents. The protein of interest in this project, Mycobacterium tuberculosis Shikimate Kinase (MtSK), catalyzes the 5th step of this pathway, converting shikimate to shikimate 3 phosphate. The objective here is to express and characterize MtSK to screen for potential antitubercular agents. Initial methods included a transformation of XL1 blue competent E. coli cells. This preliminary transformation was performed using a pET21b plasmid with an aroK gene inserted at the multiple cloning site. Successfully transformed XL1 blue cells were cloned. A second transformation of BL21 DE3 competent cells was performed. Small scale expression showed the presence of a band around 20 kDa. The theoretical mass of the enzyme is 19.6 kDa which suggests MtSK was successfully expressed within the transformed cells. Expression analysis for large scale supported data from small scale as a band of 20 kDa once again appeared in the verification SDS-PAGE gel. Purification of MtSK was carried out through nickel affinity chromatography. Subsequently, kinetic characterization and inhibitors studies will be performed using inhibitors like avarone and hymenidin.

Screening for novel anti-tubercular agents against tuberculosis shikimate kinase

ACS Spring Meeting 2021

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