Category: Preclinical Development
Purpose: Tuberculosis (TB) is a global health burden among the top 10 causes of death worldwide and the leading cause of death reported in 2017 from a single infectious agent with 10 million cases and 1.6 million TB-related deaths.1 The current standard regimen for the treatment of drug sensitive and drug-resistant TB requires 6 - 9 months and 20 - 26 months respectively. Moreover, the rise of multidrug-resistant and extensively drug-resistant TB has led to the urgent need for new drugs with novel mechanisms of action. Therefore, our studies have focused on the development of novel anti-TB agents targeting an essential class of enzymes: Methionine aminopeptidases (MetAP). MetAP is a dinuclear metalloprotein, required for bacterial growth and survival. This essential enzyme is responsible for the excision of the initiating N-terminal methionine after protein translation. N-terminal methionine excision is important for localization, stability and post-translational modifications of majority of proteins. The essentiality of MetAP makes this enzyme a promising target for the discovery and development of new anti TB agents. Our hypothesis is that, the development of potent and selective mycobacterial MetAP inhibitors may serve as therapeutic agents against drug-sensitive and drug-resistant TB.
Methods: We overexpressed and purified the recombinant MetAP1c from Mycobacterium tuberculosis (Mtb) using an N-terminal poly-histidine tag and pET100D/TOPO vector in BL21 (DE3) E. coli cells. MetAP1c was efficiently induced, overexpressed and purified to near homogeneity by immobilized metal affinity chromatography using Nickel Talon resins. The enzymatic activities of the purified Mtb MetAP1c were assessed using a chromogenic substrate (L-Methionine p-nitroanilide) in a colorimetric assay and data were analyzed using GraphPad prism software. Previously, we utilized a target based high throughput screening platform and discovered several inhibitors of MtMetAP1c.2 In this study, we characterized the activity of OJT007 as a novel MetAP1c inhibitor using a colorimetric assay at 405nm and determined the minimum inhibitory concentration (MIC) with in vitro assays of Mtb.
Results: The purified recombinant MetAP1c was found to be catalytically active. Here, we report the characterization of OJT007, as an inhibitor of MtMetAP1c with half maximal inhibitory concentration (IC50) of 150 nM and MIC of less than 10 µg/mL against Mtb in vitro. Furthermore, we have developed an optimal co-solvent formulation for OJT007 (comprising of 5% dimethyl sulfoxide, 20% polyethylene glycol 400, 10% tween 80 and 65% normal saline). Preliminary in vivo pharmacokinetic studies for OJT007 are in progress.
Conclusion: The characterization of OJT007 as a potential lead compound to the diminishing anti-TB pipeline could have impact in accelerating the development of new drug candidates for treatment of drug- sensitive, drug-resistant and Latent TB. In addition, these studies will give us more insight into the role of N-terminal protein excision in TB pathogenesis.
References:  World Health Organization (2018) Global tuberculosis report 2018. http://wwwwhoint/tb/publications/ global_report/en/. Olaleye O, Raghunand TR, Bhat S, He J, Tyagi S, Lamichhane G, et al. Methionine Aminopeptidases from Mycobacterium tuberculosis as Novel Antimycobacterial Targets. Chem Biol 2010. doi:10.1016/j.chembiol.2009.12.014.
Collins Onyenaka– Graduate Research Assistant, Texas southern university, Houston, Texas
Omonike Olaleye– PROFESSOR, Texas Southern University, Houston, Texas
Dong Liang– PROFESSOR AND CHAIR, Texas Southern University, Houston, Texas
Sarah John– Houston, Texas