Victoria University

Inhibition and reaction mechanism of Mycobacterium tuberculosis anthranilate phosphoribosyltransferase: A potential target for novel drug design

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dc.contributor.advisor Parker, Emily
dc.contributor.advisor Tyler, Peter
dc.contributor.author Kundu, Preeti
dc.date.accessioned 2020-06-30T03:58:05Z
dc.date.available 2020-06-30T03:58:05Z
dc.date.copyright 2020 en_NZ
dc.date.issued 2020
dc.identifier.uri http://researcharchive.vuw.ac.nz/handle/10063/8945
dc.description.abstract Tuberculosis (TB), which is estimated to affect 2 billion individuals worldwide, is an infection predominately caused by Mycobacterium tuberculosis(M. tuberculosis). Of particular concern is the increasing prevalence of TB, which is becoming resistant to the treatments currently available. Anthranilate phosphoribosyltransferase (AnPRT) catalyses the formation of N-(5’-phosphoribosyl)anthranilate (PRA) from 5-phospho-α-ribose-1-diphosphate (PRPP) and anthranilate and plays an important role in the synthesis of an essential amino acid in M.tuberculosis. A strain with a genetic knockout of the trpD gene, which encodes for the AnPRT enzyme, was unable to cause disease, even in immune-deficient mice. Therefore, this enzyme is a potential drug target for the development of new treatments against TB and other infectious diseases. This research explores the synthesis of different substrates and potential transition state analogues in order to understand catalysis and inhibition of AnPRT enzymes to aid novel drug design. The first part of this study utilises “bianthranilate-like” phosphonate inhibitors that display effective inhibition of the AnPRT enzyme, with the lowest Ki value being 1.3 μM. It was found strong enzymatic inhibition increases with an increased length of the phosphonate linker that occupies multiple anthranilate binding sites within the anthranilate binding channel of the enzyme. Crystal studies of the enzyme in complex with the inhibitors were carried out in order to expose the binding interactions. The second part of this study investigates several new compounds that target the active site of M. tuberculosis AnPRT, based on a virtual screening approach. This approach identified a strong AnPRT inhibitor, which displays an apparent Ki value of 7.0 ± 0.4 μM with respect to both substrates. This study also exposed a conformational change at the active site of the enzyme that occurs on inhibitor binding. The observed conformational changes of the enzyme active site diminish the binding of the substrate PRPP. These pieces of information provide future inhibitor design strategies to aid the development of novel anti-TB agents that target the AnPRT enzyme. To elucidate the reaction mechanism of M. tuberculosis AnPRT, the third part of this study explores the substrate binding sites in detail. This study uses structural analysis, complemented by differential scanning fluorimetry (DSF) and isothermal titration calorimetry (ITC), to reveal detailed information of the substrate and inhibitor binding sites. The final part of this thesis presents the synthesis of various PRPP analogues and potential transition state mimics that were designed based on the likely reaction mechanism of the enzyme. This set of inhibitors includes a number of iminoribitol analogues that were developed to capture the geometry of the flattened ribose ring and include a nitrogen atom within the ring to mimic the positive charge characteristics that are expected in the oxocarbenium-ion-like transition state predicted for M. tuberculosis AnPRT. Additionally, we were able to solve the structure of M. tuberculosis AnPRT in complex with one of the potential transition state mimics, which was observed to bind at the active site of the enzyme. This structure provides new insight into the catalytic mechanism of the enzyme and creates an opportunity to develop more specific inhibitors against the M. tuberculosis AnPRT enzyme. en_NZ
dc.language.iso en_NZ
dc.publisher Victoria University of Wellington en_NZ
dc.publisher Victoria University of Wellington en_NZ
dc.rights Author retains copyright en_NZ
dc.subject inhibition en_NZ
dc.subject reaction mechanism en_NZ
dc.subject drug design en_NZ
dc.title Inhibition and reaction mechanism of Mycobacterium tuberculosis anthranilate phosphoribosyltransferase: A potential target for novel drug design en_NZ
dc.type Text en_NZ
vuwschema.contributor.unit School of Chemical and Physical Sciences en_NZ
vuwschema.type.vuw Awarded Doctoral Thesis en_NZ
thesis.degree.discipline Chemistry en_NZ
thesis.degree.grantor Victoria University of Wellington en_NZ
thesis.degree.level Doctoral en_NZ
thesis.degree.name Doctor of Philosophy en_NZ
vuwschema.subject.anzsrcfor 030402 Biomolecular Modelling and Design en_NZ
vuwschema.subject.anzsrcfor 030403 Characterisation of Biological Macromolecules en_NZ
vuwschema.subject.anzsrcfor 030406 Proteins and Peptides en_NZ
vuwschema.subject.anzsrcfor 030499 Medicinal and Biomolecular Chemistry not elsewhere classified en_NZ
vuwschema.subject.anzsrcfor 030503 Organic Chemical Synthesis en_NZ
vuwschema.subject.anzsrcfor 060107 Enzymes en_NZ
vuwschema.subject.anzsrcfor 060112 Structural Biology (incl. Macromolecular Modelling) en_NZ
vuwschema.subject.anzsrctoa 3 Applied Research en_NZ


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