Discovery, Characterization and Structural Studies of Inhibitors against Mycobacterium Tuberculosis Adenosine Kinase and Biotin Protein Ligase

Abstract

The rapid emergence of drug-resistant Mycobacterium tuberculosis (Mtb) coupled to the high incidence of HIV-Mtb coinfection is of global concern. Consequently, there is a worldwide necessity to develop new drugs with novel mechanisms of action and new molecular targets. In this dissertation, we describe a crystallographic and high-throughput screening (HTS) approach towards the identification and structural characterization of inhibitors against Mycobacterium tuberculosis adenosine kinase (MtbAdoK) and biotin protein ligase (MtbBpL). Parallel studies were also performed to evaluate the in vitro potency and antimycobacterial profile of the compounds. Finally, X-ray crystallography was employed to investigate the structural basis of inhibition and to perform structure-guided drug design.

In the first study, we focused on the biochemical, chemical synthesis and structural characterization of adenosine analogs as inhibitors of MtbAdoK. Here, we adopted a bottom-up structural approach towards the discovery, design, and synthesis of a series of compounds that displayed inhibitory constants ranging from 4.3-121.0 nM against the enzyme. Two of these compounds exhibited low micromolar activity against Mtb with 50.0 % minimum inhibitory concentrations of 1.7 and 4.0 µM. Our selectivity studies showed that the compounds display a higher degree of specificity of MtbAdoK when compared to the human enzyme (hAdoK). Finally, our crystallographic studies revealed the presence of a potentially therapeutically relevant cavity that is unique to the MtbAdoK homodimer.

Next, we describe the discovery, biochemical and structural characterization of novel dihydro spiro compounds as inhibitors of MtbAdoK. Here, we utilized an HTS approach for the identification of the aforementioned compounds. Our enzymatic assays showed that the compounds are selective inhibitors of MtbAdoK when compared hAdoK. In addition, our antimycobacterial studies revealed that the compounds possess nanomolar potency against Mtb (500.0-810.0 nM). Finally, the crystallographic studies revealed that the inhibitors bind in a previously unknown pocket within the enzyme.

Lastly, we explore the potential of MtbBpL as a drug target. Following identification of the compounds via HTS screening, we demonstrate that the inhibitors lacked any activity against human dermal fibroblast but possess antimycobacterial properties. Finally, steady-state kinetic experiments revealed that the compounds are noncompetitive inhibitors of the enzyme suggesting the presence of a previously uncharacterized allosteric site.