An exploration of the anti-infective potential of plant-sourced natural products

Soraya Leedham

Emerging and re-emerging infectious viruses have consistently threatened the public health domain, the most notable and recent of which is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Viral pandemics are not the only emerging microbe-based public health crisis, with the ongoing emergence, spread and persistence of multidrug resistant bacteria posing a serious global threat. To combat the twin threats of novel viral emergence and antimicrobial resistance, an increase in treatment options, namely antivirals and antimicrobials, and an increase in infection prevention controls, such as disinfectants, are two important components in reducing the risk of infection and preventing transmission. Plant-sourced natural products are promising leads for the discovery of new antivirals and disinfectants, providing an immense library of novel compounds for exploration, characterised by enormous scaffold diversity and structural complexity when compared to synthetic molecules. This thesis aims to highlight the important roles plant-sourced natural products can play in the fields of antiviral and disinfectant development. The first aim of this project was to develop a simple, cost effective, high-throughput screening method which could be applied to the screening of natural product libraries for anti-coronavirus activity. This assay utilised HCoV-OC43 as a low biosafety surrogate for pathogenic human coronaviruses, as well as a combination of acoustically-focused compound dispensing, resazurin reduction assays and qRT-PCR to form an inexpensive, streamlined compound screening pipeline. The assay was validated with remdesivir, with an overall Z score of >0.5 validating it as an effective high throughput assay. Further validation was conducted through the screening of 246 natural compounds, with six compounds identified as potential leads in initial screening with the resazurin reduction assay. These results were corroborated with dose-response analysis, utilising qRT-PCR, with two compounds found to have IC50 values <10 μM. The second aim of the project was to demonstrate proof-of-concept by applying this screening method to a library of Western Australian plant extracts, utilising it in a bio-assay guided fractionation process for the identification of lead antiviral compounds. Two of the most active extracts from the initial screening process were investigated further using this method, resulting in the isolation of two antiviral compounds from a native plant species. The IC50 values of these two compounds against HCoV-OC43 were 2.99 ± 0.67 μM and 0.79 ± 0.03 μM, respectively, though further testing showed that these compounds were not efficacious against SARS-CoV-2. Finally, this project aimed to highlight the broad-spectrum, anti-infective potential of plant-sourced natural products by investigating the antimicrobial activity of GS-2, an ammonium carboxylate salt of the natural plant-sourced compound capric acid and L-arginine. Suspension-based testing of GS-2 revealed potent activity against a variety of gram-positive and gram-negative organisms as well as fungicidal activity against Candida albicans, and antiviral activity against HCoV-OC43. Further disinfectant-based testing revealed bactericidal activity against Escherichia coli and Streptococcus suis with exposure times of two minutes, and bactericidal activity against Staphylococcus aureus with exposure times of 24 hours. Overall, this thesis has outlined the development of a rapid, cost-effective compound screening system which can be modified to target any number of viral pathogens within a short timeframe, making it ideal for screening large libraries in the face of emerging outbreaks. In addition, this project has illustrated the potential of plant-sourced natural products, identifying four potential antiviral compounds from native Australian plants, and providing a significant body of evidence to support the broad-spectrum potential of the modified plant-sourced natural product, GS-2.

An exploration of the anti-infective potential of plant-sourced natural products

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