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

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 (MtAh...

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Main Author: Chong, Sherilyn Shi Min
Other Authors: Gerhard Gruber
Format: Thesis-Doctor of Philosophy
Language:English
Published: Nanyang Technological University 2020
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Online Access:https://hdl.handle.net/10356/143914
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spelling sg-ntu-dr.10356-1439142020-11-01T04:46:27Z 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 Chong, Sherilyn Shi Min Gerhard Gruber Roderick Wayland Bates Interdisciplinary Graduate School (IGS) GGrueber@ntu.edu.sg, Roderick@ntu.edu.sg Science::Biological sciences Science::Chemistry::Organic chemistry 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. Doctor of Philosophy 2020-10-01T02:39:50Z 2020-10-01T02:39:50Z 2020 Thesis-Doctor of Philosophy Chong, S. S. M. (2020). 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. Doctoral thesis, Nanyang Technological University, Singapore. https://hdl.handle.net/10356/143914 10.32657/10356/143914 en AcRF Tier 1 ID889, NRF-CRP18-2017-01) This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0). application/pdf Nanyang Technological University
institution Nanyang Technological University
building NTU Library
continent Asia
country Singapore
Singapore
content_provider NTU Library
collection DR-NTU
language English
topic Science::Biological sciences
Science::Chemistry::Organic chemistry
spellingShingle Science::Biological sciences
Science::Chemistry::Organic chemistry
Chong, Sherilyn Shi Min
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
description 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.
author2 Gerhard Gruber
author_facet Gerhard Gruber
Chong, Sherilyn Shi Min
format Thesis-Doctor of Philosophy
author Chong, Sherilyn Shi Min
author_sort Chong, Sherilyn Shi Min
title 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
title_short 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
title_full 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
title_fullStr 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
title_full_unstemmed 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
title_sort 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
publisher Nanyang Technological University
publishDate 2020
url https://hdl.handle.net/10356/143914
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