- Title
- Identification of potential inhibitors of the folate biosynthesis enzymes HPPK of Salmonella enterica and Escherichia coli and pteridine reductase of Trypanosoma brucei through molecular docking and enzyme assays
- Creator
- Gerwel, Tiaan Marc
- Subject
- Uncatalogued
- Date Issued
- 2021-10-29
- Date
- 2021-10-29
- Type
- Master's theses
- Type
- text
- Identifier
- http://hdl.handle.net/10962/192419
- Identifier
- vital:45224
- Description
- Antimicrobial resistance has become a serious threat to the survival of the human species especially those living in rural areas where access to medicine and the knowledge for proper use is scarce. It has been estimated that the number of extreme untreatable resistant infections in Africa will increase to as much as 10 million by the year 2050. Thus the need for novel drugs to act as therapeutic agents is becoming more compelling each year. The subspecies of Trypanosoma brucei (T. brucei) is responsible for the Human African Trypanosomiasis (HAT) also known as sleeping sickness and results in large numbers of deaths and loss of income for many homes. Resistance to current therapeutic agents has been observed and is on the rise increasing the difficulty to treat the infection. Salmonella enterica (S. enterica) serotypes are responsible for acute diarrhoeal disease in humans ranging from invasive typhoid to non-invasive non-typhoid disease, resulting in a large number of deaths, with an estimated 180 million people falling ill annually. The pathogen is spread via the faecal-oral route or through food contaminated with bacterium and prepared in an unsanitary environment. The chance of recovery in rural populations is low. Escherichia coli (E. coli), forming part of commensal gut flora, spread via the faecal-oral route or unhygienic practices, causes a diarrhoeal disease which can progress to a haemorrhagic phase. More than 241 million annual infections are caused by enterotoxigenic E. coli. A common strategy to develop antimicrobial agents is to target the biosynthesis of essential biological molecules, thereby rendering the microbes less viable. One such group of molecules are folates, which are generally synthesised de novo by bacteria. Higher organisms have a scavenger mechanism to obtain their folates from the extracellular environment and in some cases, organisms have both mechanisms. In this study, two enzymes falling into the folate scavenger and de novo synthesis groups were examined to identify potential agents to act as inhibitors. Pteridine reductase 1 (PTR1) is a scavenger enzyme used by a variety of trypanosomes and 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is an enzyme that overlaps the kingdoms of protozoa and bacteria forming part of the de novo biosynthetic pathway. Homology modelling was performed on the HPPK enzyme of S. enterica using Yersinia pestis (Y. pestis) as a template providing a model to use for docking studies. The E. coli HPPK enzyme structure was retrieved from the Protein Data Bank (PDB) and bound molecules were removed to render the enzyme in an apo-state. Docking studies using the generated S. enterica homology model and apo E. coli HPPK was performed using the ZINC 15 database and resulted in 9 hit compounds which showed high binding affinities and binding energy to the enzyme. The HPPK and PTR1 enzyme coding sequences were cloned into pET-28a(+) plasmids and supplied by GenScript, to enable the expression of histidine-tagged proteins in T7 Express lysY competent E. coli cells. Analytical scale expression studies showed the recombinant proteins to be in a soluble form and purification was achieved using nickel-NTA affinity chromatography. The purified PTR1 recombinant protein was used to establish and optimise an NADPH absorbance microplate assay to screen compounds previously identified in docking studies by Kimuda, Laming, Hoppe, & Bishop, (2019). The assay yielded a Z´-factor of above 0.8 indicating an excellent assay to use for screening. An unsuccessful attempt was made to use resazurin reduction as an alternative method to demonstrate PTR1 enzyme activity. HPPK purified recombinant proteins were used to establish and optimise a luminescence microplate assay for the screening of compounds identified in in silico docking studies against the HPPK enzymes of S. enterica and E. coli. The Z´-factor of the luminescence assay was above 0.5, indicative of a functional assay with good separation between enzyme activity signal and negative control of reaction without enzyme. The target-based enzyme screening resulted in the confirmation of compound 3 (3-chloro-N-[(4-oxo-3,4-dihydrophthalazin-1-yl)methyl]benzamide) as an inhibitor of S. enterica HPPK with an IC₅₀ of 10.4 μM. At 50 μM none of the compounds decreased E. coli HPPK enzyme activity by 50%. Further bacterial studies would provide more compelling data to motivate the optimisation of compound 3 as an S. enterica inhibitor. This study demonstrated the effectiveness of using computational methods in the drug discovery process, correlating in silico results with those obtained from target-based assays producing a hit compound that can be used for future drug optimisation.
- Description
- Thesis (MSc (Pharm)) -- Faculty of Pharmacy, Pharmacy, 2021
- Format
- computer
- Format
- online resource
- Format
- application/pdf
- Format
- 1 online resource (176 pages)
- Format
- Publisher
- Rhodes University
- Publisher
- Faculty of Science, Pharmacy
- Language
- English
- Rights
- Gerwel, Tiaan Marc
- Rights
- Use of this resource is governed by the terms and conditions of the Creative Commons "Attribution-NonCommercial-ShareAlike" License (http://creativecommons.org/licenses/by-nc-sa/2.0/)
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| Thumbnail | File | Description | Size | Format | |||
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| View Details Download | SOURCE1 | GERWEL-MSC-TR21-214-EMB_cov.pdf | 2 MB | Adobe Acrobat PDF | View Details Download |