Anti-persistent mycobacterial inhibitors from Streptomycessp. H7763 targeting isocitrate lyase and malate synthase in the glyoxylate shunt of Mycobacterium sp.

Anti-persistent mycobacterial inhibitors from Streptomycessp. H7763 targeting isocitrate lyase and malate synthase in the glyoxylate shunt of Mycobacterium sp.

Globally, two billion people are infected with latent tuberculosis (TB) infection, showing asymptomatic immune response but capable to reactivate into chronic TB in life. Immunocompromised patients such as HIV preinfected people have fueled up the reactivation rate while the drug resistant bacilli w...

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Main Author: Ch'ng, Ai Ying
Format: Thesis
Language:English
Published: 2012
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QR Microbiology
Online Access:https://eprints.ums.edu.my/id/eprint/11592/1/ph000000061.pdf
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spelling my-ums-ep.115922017-11-07T07:04:02Z Anti-persistent mycobacterial inhibitors from Streptomycessp. H7763 targeting isocitrate lyase and malate synthase in the glyoxylate shunt of Mycobacterium sp. 2012 Ch'ng, Ai Ying QR Microbiology Globally, two billion people are infected with latent tuberculosis (TB) infection, showing asymptomatic immune response but capable to reactivate into chronic TB in life. Immunocompromised patients such as HIV preinfected people have fueled up the reactivation rate while the drug resistant bacilli which evolved without proper medications have resulted in a more complicated treatment. During dormancy, the persistent strain of Mycobacterium tuberculosis is non-replicative and recalcitrate to conventional TB drugs which mostly inhibit the biosynthetic cellular process. The persistent strain regulates a switch of metabolism to glyoxylate shunt and uses 2-carbon compounds such as acetate as the primary carbon source to survive. The glyoxylate shunt enzymes namely isocitrate lyase (ICL) and malate synthase (MS) have been structural solved and identified as important virulence and persistence factors. Both ICL and MS thus become attractive targets for anti-persistent TB drug discovery. In the search for persistent TB Inhibitors from soil actinomycetes metabolites, a positive extract was obtained and the hits strain was identified and designated as Streptomyces sp. H7763. Subsequently, compound P1, P2 and P3 were purified from this butanol extract using bioassay-guided chromatography approaches. P2 was elucidated spectroscopically as 2-amino-3-{cyclohexa-1,4-dieny1) propanoic acid, which was detected for the first time to inhibit Mycobacterium sp. As a persistent strain inhibitor, P2 strongly Inhibited the acetate grown Mycobacterium smegmatis mc2155, H8000 with MIC = 0.02±0.00 µg/mL using a modified resazurin based microtiter assay (REMA) targeting the glyoxylate shunt. P2 competitively inhibited MS but not ICL against glyoxylate with Ki = 34.85 mM, showing a comparable potency with the control 3-nitropropionate which acted competitively against the acetyl-coenzyme A with Ki = 36.20 mM. Extensive molecular docking studies of P2 with MS was performed and the interaction Indicated a better affinity of conformation over the glyoxylate, with a predominant salt bridge bonding between amino group of P2 and Asp 633, a bidentate coordination of the carboxylate group to the Mg²�ion which is essential to the catalytic activity and the cyclohexa-1,4 dienyl ring of P2 that experienced an aromatization Inactivation mechanism with Arg 339 which further Interrupts the catalytic mechanism. Desiccated P2 was labile to oxidation and aromatised to yield P1, an amino acid which was not bioactive. However, calcium-alginate encapsulated P2 had succeeded to prolong Its antimycobacterial activity for over 4 weeks and had pre-developed as a slow-release delivery inhibitor to accumulate at the intracellular environment. P2 was not toxic against Arternia salina (LC₅₀>2000 µg/mL) based on a brine shrimp lethality assay. P3 was a non-selective inhibitor, showing unstable anti-mycobacterial activity against M. smegmatis mc²155, H8000 and M. tuberculosis H37Rv, ATCC 25618. In conclusion, a persistent Mycobacterium sp. Inhibitor P2 targeting the MS was successfully isolated from the Streptomyces sp. H7763 culture and may serve as a good lead candidate for further latent TB Infection drug design since the enzyme does not exist in mammals. 2012 Thesis https://eprints.ums.edu.my/id/eprint/11592/ https://eprints.ums.edu.my/id/eprint/11592/1/ph000000061.pdf text en public phd doctoral Universiti Malaysia Sabah School of Science and Technology
institution Universiti Malaysia Sabah
collection UMS Institutional Repository
language English
topic QR Microbiology
spellingShingle QR Microbiology
Ch'ng, Ai Ying
Anti-persistent mycobacterial inhibitors from Streptomycessp. H7763 targeting isocitrate lyase and malate synthase in the glyoxylate shunt of Mycobacterium sp.
description Globally, two billion people are infected with latent tuberculosis (TB) infection, showing asymptomatic immune response but capable to reactivate into chronic TB in life. Immunocompromised patients such as HIV preinfected people have fueled up the reactivation rate while the drug resistant bacilli which evolved without proper medications have resulted in a more complicated treatment. During dormancy, the persistent strain of Mycobacterium tuberculosis is non-replicative and recalcitrate to conventional TB drugs which mostly inhibit the biosynthetic cellular process. The persistent strain regulates a switch of metabolism to glyoxylate shunt and uses 2-carbon compounds such as acetate as the primary carbon source to survive. The glyoxylate shunt enzymes namely isocitrate lyase (ICL) and malate synthase (MS) have been structural solved and identified as important virulence and persistence factors. Both ICL and MS thus become attractive targets for anti-persistent TB drug discovery. In the search for persistent TB Inhibitors from soil actinomycetes metabolites, a positive extract was obtained and the hits strain was identified and designated as Streptomyces sp. H7763. Subsequently, compound P1, P2 and P3 were purified from this butanol extract using bioassay-guided chromatography approaches. P2 was elucidated spectroscopically as 2-amino-3-{cyclohexa-1,4-dieny1) propanoic acid, which was detected for the first time to inhibit Mycobacterium sp. As a persistent strain inhibitor, P2 strongly Inhibited the acetate grown Mycobacterium smegmatis mc2155, H8000 with MIC = 0.02±0.00 µg/mL using a modified resazurin based microtiter assay (REMA) targeting the glyoxylate shunt. P2 competitively inhibited MS but not ICL against glyoxylate with Ki = 34.85 mM, showing a comparable potency with the control 3-nitropropionate which acted competitively against the acetyl-coenzyme A with Ki = 36.20 mM. Extensive molecular docking studies of P2 with MS was performed and the interaction Indicated a better affinity of conformation over the glyoxylate, with a predominant salt bridge bonding between amino group of P2 and Asp 633, a bidentate coordination of the carboxylate group to the Mg²�ion which is essential to the catalytic activity and the cyclohexa-1,4 dienyl ring of P2 that experienced an aromatization Inactivation mechanism with Arg 339 which further Interrupts the catalytic mechanism. Desiccated P2 was labile to oxidation and aromatised to yield P1, an amino acid which was not bioactive. However, calcium-alginate encapsulated P2 had succeeded to prolong Its antimycobacterial activity for over 4 weeks and had pre-developed as a slow-release delivery inhibitor to accumulate at the intracellular environment. P2 was not toxic against Arternia salina (LC₅₀>2000 µg/mL) based on a brine shrimp lethality assay. P3 was a non-selective inhibitor, showing unstable anti-mycobacterial activity against M. smegmatis mc²155, H8000 and M. tuberculosis H37Rv, ATCC 25618. In conclusion, a persistent Mycobacterium sp. Inhibitor P2 targeting the MS was successfully isolated from the Streptomyces sp. H7763 culture and may serve as a good lead candidate for further latent TB Infection drug design since the enzyme does not exist in mammals.
format Thesis
qualification_name Doctor of Philosophy (PhD.)
qualification_level Doctorate
author Ch'ng, Ai Ying
author_facet Ch'ng, Ai Ying
author_sort Ch'ng, Ai Ying
title Anti-persistent mycobacterial inhibitors from Streptomycessp. H7763 targeting isocitrate lyase and malate synthase in the glyoxylate shunt of Mycobacterium sp.
title_short Anti-persistent mycobacterial inhibitors from Streptomycessp. H7763 targeting isocitrate lyase and malate synthase in the glyoxylate shunt of Mycobacterium sp.
title_full Anti-persistent mycobacterial inhibitors from Streptomycessp. H7763 targeting isocitrate lyase and malate synthase in the glyoxylate shunt of Mycobacterium sp.
title_fullStr Anti-persistent mycobacterial inhibitors from Streptomycessp. H7763 targeting isocitrate lyase and malate synthase in the glyoxylate shunt of Mycobacterium sp.
title_full_unstemmed Anti-persistent mycobacterial inhibitors from Streptomycessp. H7763 targeting isocitrate lyase and malate synthase in the glyoxylate shunt of Mycobacterium sp.
title_sort anti-persistent mycobacterial inhibitors from streptomycessp. h7763 targeting isocitrate lyase and malate synthase in the glyoxylate shunt of mycobacterium sp.
granting_institution Universiti Malaysia Sabah
granting_department School of Science and Technology
publishDate 2012
url https://eprints.ums.edu.my/id/eprint/11592/1/ph000000061.pdf
_version_ 1747836424072200192

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