Date of Degree

9-2019

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Luis Quadri

Committee Members

Theodore Muth

Nicolas Biais

Edward Kennelly

Kathleen McDonough

Subject Categories

Biochemistry | Microbiology | Pharmaceutics and Drug Design

Abstract

Mycobacterium tuberculosis (Mtb) is a resilient, obligate bacterial pathogen responsible for pulmonary tuberculosis disease (TB), that has upheld a significant impact on global public health throughout history. The World Health Organization (WHO) approximates nearly 10 million new TB cases arose in 2017 alone, accounting for 1.6 million deaths. There has been a notable rise in TB cases produced by multidrug‑resistant (MDR) and extensively drug-resistant (XDR) strains of Mtb. This, along with the intrinsic resistance of Mtb to many standard drugs and poor patient compliance, is deeply impacting global control of TB. Among the several strategies currently in place to progressively reduce TB burden, research into the development of novel anti-TB drugs is vital for both treatment and prevention.

In pursuit of investigating novel drugs against Mtb, the first-in-class nucleoside antibiotic salicyl-AMS (5′-O-[N-salicylsulfamoyl]-adenosine) was characterized. Salicyl-AMS is an inhibitor of the salicylate adenylation enzyme MbtA, required for the initial step towards biosynthesis of the small molecule, iron-scavenging siderophore metabolites known as the mycobactins (MBTS). Salicyl-AMS was shown to inhibit MbtA activity, diminish production of MBTS, and display antimicrobial activity against Mtb. Though salicyl-AMS reduced the burden of Mtb in a mouse model of infection, it demonstrated a poor pharmacological profile.

Here, we evaluated two novel salicyl-AMS analogues to further probe the structure activity relationship (SAR), alongside salicyl-AMS and six of the most potent previously reported analogues, in both biochemical and cell-based studies. In pursuit of this, we optimized the purification of a recombinant MbtA and the conditions of the continuous, spectrophotometric HA-MesG assay to improve the methodological platform for the study of MbtA inhibition. The new recombinant MbtA (H10MbtAopt) protein was used in conjunction with the improved HA-MesG assay to determine enzyme kinetic parameters (Km and kcat) and measure the intrinsic inhibition (Ki) by salicyl-AMS. Biochemical studies of the nine MbtA inhibitors included determination of kinetic parameters (Kiapp, konapp, koff, and tR) and analysis of their mechanism of inhibition. Additionally, we developed a non-pathogenic, fast-growing Mycobacterium smegmatis model engineered for susceptibility to MbtA inhibitors. The model strain was dependent on the MbtA from Mtb for production of MBTS and used to evaluate antimicrobial properties (MIC and PAE) of the nine analogues. In all, these studies expand our understanding of the mechanism of inhibition of MbtA by these inhibitors, expose new SAR insights into this family of nucleoside antibiotics, and highlight several candidates for future preclinical evaluation.

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