Enzymatic and structural insights into the mycobacterial alkylhydroperoxide reductase subunit C and repurposing of an antimalaria compound inhibiting the mycobacterial cytochrome BCC oxidase for tuberculosis disease

• Main Author: Chong, Sherilyn Shi Min
• Other Authors: Gerhard Gruber
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2020
• Subjects: Science::Biological sciences; Science::Chemistry::Organic chemistry
• Online Access: https://hdl.handle.net/10356/143914
• Institution: Nanyang Technological University

Tuberculosis (TB) is one of the leading contagious diseases that results in mortality. Treatment with antibiotics has been applied for TB however, with new forms of antibiotic resistance emerging, difficulty in treatment of TB was detected. In this study, alkylhydroperoxide reductase subunit C (MtAhpC), involved in the antioxidant defense system of mycobacterial species against reaction oxygen species (ROS), and cytochrome bcc, complex III of the electron transport chain (ETC), where most endogenous ROS is generated, were investigated. MtAhpC possesses several distinctive features, allowing it to serve as a good drug target. The importance of MtAhpC has been highlighted in isoniazid-resistant strains, the primary antibiotic for TB treatment, where an increased expression in MtAhpC was revealed. One unique feature of MtAhpC was discovered from its crystal structure, where a cavity within MtAhpC was observed, not present in its bacterial counterparts. This was postulated to be a result of a helical displacement. Here, the importance of the helical movement was demonstrated by performing mutagenesis studies, along with enzymatic assays. Additional biophysical assays provided evidence into the oligomeric state of the mutants. To gain structural insights into the catalytic efficiency of the protein, the crystallographic structure of the MtAhpC mutant was determined at 3.3 Å. The reported crystal data provided insights into the effect of the mutation on the helix, which consequently impacts the oligomeric interface. Cytochrome bcc plays a major role in the production of the proton motive force (PMF) for ATP synthesis, as well as ROS. With the concept of drug repurposing, SCR0911, an antimalaria compound, was envisioned for use in mycobacterial species as it was postulated to bind to cytochrome bcc, and more specifically, the Qi site. Previous studies have revealed that inhibitors at the Qi site expedite the formation of endogenous superoxides. The synthesis of SCR0911 was carried out and subjected to antimicrobial susceptibility testing and on-target activity assays. Towards improving the activity of SCR0911, docking studies along with synthesis of analogs were performed. These results provide valuable insights into the helical displacement of MtAhpC, thus allowing the establishment of a foothold in drug design, for the application of molecular docking to identify a ligand that can target MtAhpC specifically. Additionally, SCR0911 was identified to possess anti-TB properties. This enables future works into an improved binding of SCR0911 analog at the Qi site of cytochrome bcc, which could stimulate Mtb to produce an excess of endogenous superoxides.