figshare
Browse

Restricted Access

Reason: Access restricted by the author. A copy can be requested for private research and study by contacting your institution's library service. This copy cannot be republished

Structural and Functional Characterisation of Novel Potential Drug Targets in Helicobacter pylori

thesis
posted on 2017-02-07, 04:54 authored by Joyanta Kumer Modak
Helicobacter pylori colonises the human gastric mucosa. H. pylori infection may cause diseases of the upper gastrointestinal tract, such as chronic gastritis, ulcers and gastric cancer. The success rate of the current H. pylori treatment regimen has declined over time, mainly due to antibiotic resistance. Therefore, there is a clear need for identification of novel targets that can be used in the development of alternative treatment strategies for H. pylori infections. To address this, the present study focuses on two enzymes from H. pylori that represent novel potential targets for drug design: M17 aminopeptidase (HpM17AP) and α-carbonic anhydrase (HpαCA). HpM17AP plays a role in bacterial defence against human innate immune response and contributes to the mechanism of drug resistance, whereas HpαCA is important for buffering periplasmic pH of H. pylori close to neutral as a means of survival in the harsh acidic environment of human stomach. Due to their crucial role in colonisation and survival within the host, HpM17AP and HpαCA need to be evaluated as potential drug targets.
 
   In order to understand the structural basis of catalysis and inhibition of HpM17AP, the crystal structures of HpM17AP and its complex with its inhibitor have been determined and analysed. In addition, the specificity of HpM17AP towards the N-terminal amino acid of its peptide substrates has been established. The inhibitor-bound structure of HpM17AP revealed that the D-phenylalanine moiety of the inhibitor binds in the S1 subsite of the enzyme. At the end of the S1 subsite, HpM17AP was found to harbor a hydrophilic pocket, which is a unique feature of the enzyme compared to very similar homologues from other bacteria. This pocket is flanked by a sodium ion and its coordinating water molecules. Furthermore, structural analysis revealed that variable loops at the entrance to and in the middle of the substrate-binding channel are important determinants of the substrate specificity of M17 aminopeptidases. In fact, the study has demonstrated that HpM17AP has broad substrate specificity and, in contrast to most characterised M17 aminopeptidase, HpM17AP displays preference for L-Arg over L-Leu residues in the peptide substrate.
 
   In order to understand the molecular details of catalysis and the structural basis for the inhibition of HpαCA by sulfonamides, the crystal structures of HpαCA in complex with a series of sulfonamide inhibitors have been determined and analysed. This study revealed that HpαCA and human carbonic anhydrase likely follow the same catalytic mechanism, where HCO3- is generated from CO2 via a nucleophilic attack on CO2 by the zinc-bound hydroxide ion. In addition, the study has demonstrated that sulfonamides act as site-directed inhibitors by mimicking the transition state of the CO2 hydration reaction. Analysis of the structures revealed that the binding mode of sulfonamide correlates well with their inhibitory activities. Furthermore, the study has identified two pockets near the active site in HpαCA that are distinctly different from the corresponding regions in the structure of human carbonic anhydrase II. Thus, our analysis identified major structural features that can be exploited in the design of selective and more potent inhibitors of HpαCA that may lead to novel antimicrobials.
 
   Finally, this study has shown that sulfonamides RSO2NH2 kill H. pylori via mechanisms that are different from the mechanisms of action of common antibiotics used to treat H. pylori infections. Despite having been chosen for this study due to their known activity as inhibitors of carbonic anhydrases, whose function is essential at acidic pH, these compounds also displayed bactericidal activity at neutral pH. As neutral pH approximates the conditions under which H. pylori persists in the stomach, we have analysed the mechanisms by which spontaneous mutations give rise to sulfonamide resistance at pH close to 7.0. The carbonic anhydrase inhibitors acetazolamide, methazolamide and ethoxzolamide produce a complex phenotype, likely involving the combination of different mechanisms which involve modifications in cellular proteins and systems other than α- and β-carbonic anhydrases themselves. The frequency of spontaneous mutations resulting in H. pylori resistance to sulfonamides was 2.5×10-8 or less, indicating that resistance does not develop easily. Furthermore, this and other studies have shown that the antimicrobial activity of sulfonamides is specific to H. pylori and several other bacteria, including Streptococcus pneumoniae, Neisseria spp. Brucella suis. This suggests that this class of sulfonamides can be developed into selective anti-H. pylori agents with a novel mechanism of action, which can be used for H. pylori infections management.

History

Principal supervisor

Anna Roujeinikova

Additional supervisor 1

Terry Kwok-Schuelein

Year of Award

2017

Department, School or Centre

Biomedical Sciences (Monash Biomedicine Discovery Institute)

Additional Institution or Organisation

Microbiology

Campus location

Australia

Course

Doctor of Philosophy

Degree Type

DOCTORATE

Faculty

Faculty of Medicine Nursing and Health Sciences

Usage metrics

    Faculty of Medicine, Nursing and Health Sciences Theses

    Exports

    RefWorks
    BibTeX
    Ref. manager
    Endnote
    DataCite
    NLM
    DC