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postgraduate thesis: Overcoming antibiotic resistance : bismuth compounds as potent inhibitors of class B1 metallo-[beta]-lactamases

TitleOvercoming antibiotic resistance : bismuth compounds as potent inhibitors of class B1 metallo-[beta]-lactamases
Authors
Advisors
Advisor(s):Sun, H
Issue Date2018
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Lai, T. [黎梓培]. (2018). Overcoming antibiotic resistance : bismuth compounds as potent inhibitors of class B1 metallo-[beta]-lactamases. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR.
AbstractBacteria with antibiotic resistance poses a huge threat to human health. Particularly, the notorious resistant determinant metallo-β-lactamases (MBLs), e.g. imipenemases (IMPs), Verona integron-encoded metallo-β-lactamases (VIMs) and New Delhi metallo-β-lactamases (NDMs) are Zn(II) hydrolytic enzymes that cleave the β-lactam ring of common β-lactam antibiotics, conferring bacterial resistance to the last resort carbapenems and even many bicyclic β-lactams. No inhibitors specifically against MBLs are available for clinical application up to now. Bismuth-based drugs, i.e. colloidal bismuth subcitrate (CBS) and ranitidine bismuth citrate (RBC) are highly effective metallodrugs which exhibit excellent antimicrobial activities towards bacteria such as H. pylori and are relatively non-toxic to the human body. To reveal the in vitro and in vivo inhibitory effects of Bi(III) to MBLs, a panel of Bi(III) compounds were tested against selected class B1 MBLs including NDM-1, VIM-2 and IMP-4. Bi(III) compounds are found to irreversibly inhibit these MBLs with IC50 values of 0.70-14.48 µM. Synergism of a combination of meropenem and Bi(III) compounds against MBL-positive E. coli was demonstrated with FIC index of 0.047-0.313. These results conclude that Bi(III) potently inhibits class B1 MBLs both in vitro and in vivo. To understand the molecular mechanism of the inhibition, biochemical studies were carried out to understand the interactions of Bi(III) with NDM-1. A unique mechanism, involving one Bi(III) displacing two Zn(II) ions was observed, leading to the release of Zn(II) cofactors and ultimately enzyme inhibition. Mutagenesis studies revealed that Cys208 at the active site serves a pivotal role for Bi(III) binding. Binding of Bi(III) to Cys208 results in an increase in UV absorption at 340 nm, due to a characteristic ligand-to-metal-charge transfer (LMCT) for Bi-S coordination bond. No changes in the oligomeric state of NDM-1 were observed upon Bi(III) binding, as determined from gel filtration studies. Restoration of Zn(II) to Bi-NDM-1 only recovered ~25% of enzymatic activity, indicative of irreversible enzymatic inhibition. Binding of Bi(III) was demonstrated in vivo using gel electrophoresis-coupled ICP-MS by comparing the corresponding intensity of Bi(III) peaks for wild-type, C208A as well as C208S NDM-1. Structure of NDM-1 in the Bi(III)-bound form was determined by X-ray crystallography through soaking the crystals of native-form of NDM-1 with Bi(III) solution. The best structure was solved to 1.40 Å resolution. Two anomalous peaks were observed in the difference electron density map (mFo-DFc), corresponding to the Bi1 and Bi2 conformers of the Bi-binding site of NDM-1, with occupancy of 0.55 and 0.10 respectively. Bi1 coordinates to His120, His122, Asp124 (in a bidentate mode), Cys208 and a water molecule, completing a trigonal prismatic coordination geometry. The bond distances were measured to be 2.65-3.02 Å. The crystallographic study leads to the visualization of the Bi(III) coordination site in NDM-1, consistent with the biochemical studies. The study in this thesis may open a new horizon for the rational design of effective broad-spectrum MBL inhibitors as well as revolutionize the treatment of infection caused by MBL-positive bacteria with a combination of β-lactam antibiotic and a metallodrug, with negligible drug-resistant phenomenon.
DegreeDoctor of Philosophy
SubjectBeta lactamases - Inhibitors
Beta lactam antibiotics
Dept/ProgramChemistry
Persistent Identifierhttp://hdl.handle.net/10722/266309

 

DC FieldValueLanguage
dc.contributor.advisorSun, H-
dc.contributor.authorLai, Tsz-pui-
dc.contributor.author黎梓培-
dc.date.accessioned2019-01-18T01:51:59Z-
dc.date.available2019-01-18T01:51:59Z-
dc.date.issued2018-
dc.identifier.citationLai, T. [黎梓培]. (2018). Overcoming antibiotic resistance : bismuth compounds as potent inhibitors of class B1 metallo-[beta]-lactamases. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR.-
dc.identifier.urihttp://hdl.handle.net/10722/266309-
dc.description.abstractBacteria with antibiotic resistance poses a huge threat to human health. Particularly, the notorious resistant determinant metallo-β-lactamases (MBLs), e.g. imipenemases (IMPs), Verona integron-encoded metallo-β-lactamases (VIMs) and New Delhi metallo-β-lactamases (NDMs) are Zn(II) hydrolytic enzymes that cleave the β-lactam ring of common β-lactam antibiotics, conferring bacterial resistance to the last resort carbapenems and even many bicyclic β-lactams. No inhibitors specifically against MBLs are available for clinical application up to now. Bismuth-based drugs, i.e. colloidal bismuth subcitrate (CBS) and ranitidine bismuth citrate (RBC) are highly effective metallodrugs which exhibit excellent antimicrobial activities towards bacteria such as H. pylori and are relatively non-toxic to the human body. To reveal the in vitro and in vivo inhibitory effects of Bi(III) to MBLs, a panel of Bi(III) compounds were tested against selected class B1 MBLs including NDM-1, VIM-2 and IMP-4. Bi(III) compounds are found to irreversibly inhibit these MBLs with IC50 values of 0.70-14.48 µM. Synergism of a combination of meropenem and Bi(III) compounds against MBL-positive E. coli was demonstrated with FIC index of 0.047-0.313. These results conclude that Bi(III) potently inhibits class B1 MBLs both in vitro and in vivo. To understand the molecular mechanism of the inhibition, biochemical studies were carried out to understand the interactions of Bi(III) with NDM-1. A unique mechanism, involving one Bi(III) displacing two Zn(II) ions was observed, leading to the release of Zn(II) cofactors and ultimately enzyme inhibition. Mutagenesis studies revealed that Cys208 at the active site serves a pivotal role for Bi(III) binding. Binding of Bi(III) to Cys208 results in an increase in UV absorption at 340 nm, due to a characteristic ligand-to-metal-charge transfer (LMCT) for Bi-S coordination bond. No changes in the oligomeric state of NDM-1 were observed upon Bi(III) binding, as determined from gel filtration studies. Restoration of Zn(II) to Bi-NDM-1 only recovered ~25% of enzymatic activity, indicative of irreversible enzymatic inhibition. Binding of Bi(III) was demonstrated in vivo using gel electrophoresis-coupled ICP-MS by comparing the corresponding intensity of Bi(III) peaks for wild-type, C208A as well as C208S NDM-1. Structure of NDM-1 in the Bi(III)-bound form was determined by X-ray crystallography through soaking the crystals of native-form of NDM-1 with Bi(III) solution. The best structure was solved to 1.40 Å resolution. Two anomalous peaks were observed in the difference electron density map (mFo-DFc), corresponding to the Bi1 and Bi2 conformers of the Bi-binding site of NDM-1, with occupancy of 0.55 and 0.10 respectively. Bi1 coordinates to His120, His122, Asp124 (in a bidentate mode), Cys208 and a water molecule, completing a trigonal prismatic coordination geometry. The bond distances were measured to be 2.65-3.02 Å. The crystallographic study leads to the visualization of the Bi(III) coordination site in NDM-1, consistent with the biochemical studies. The study in this thesis may open a new horizon for the rational design of effective broad-spectrum MBL inhibitors as well as revolutionize the treatment of infection caused by MBL-positive bacteria with a combination of β-lactam antibiotic and a metallodrug, with negligible drug-resistant phenomenon. -
dc.languageeng-
dc.publisherThe University of Hong Kong (Pokfulam, Hong Kong)-
dc.relation.ispartofHKU Theses Online (HKUTO)-
dc.rightsThe author retains all proprietary rights, (such as patent rights) and the right to use in future works.-
dc.rightsThis work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.-
dc.subject.lcshBeta lactamases - Inhibitors-
dc.subject.lcshBeta lactam antibiotics-
dc.titleOvercoming antibiotic resistance : bismuth compounds as potent inhibitors of class B1 metallo-[beta]-lactamases-
dc.typePG_Thesis-
dc.description.thesisnameDoctor of Philosophy-
dc.description.thesislevelDoctoral-
dc.description.thesisdisciplineChemistry-
dc.description.naturepublished_or_final_version-
dc.date.hkucongregation2018-
dc.identifier.mmsid991044069404203414-

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