Hameed, Hamza N (2019) An evaluation of the antiparasitic activities of a novel natural product and open-access Pathogen Box libraries. Doctoral thesis, Keele University.

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Abstract

Using a novel library of natural products isolated from temperate zone plants, the antiparasitic activity of 643 Phytopure library compounds were determined against intraerytrocytic P. falciparum, the blood-stream form of T. b brucei and axenic amastigotes of L. mexicana. Twelve compounds with a 50% inhibitory effect (EC50) values of less than 6 μM were detected against P. falciparum, 25 compounds with an EC50 values of less than 2.8 μM against T. b brucei, and 23 compounds with an EC50 values of less than 2.8 μM against L. mexicana. The cytotoxicity effects, and thus their selectivity of action against each parasite, of these selected compounds were determined against a human liver cell line (HepG2) to establish priorities for further work. Here, four structurally-related triterpene compounds (700022, 700107, 700136 and 700240) were shown to have activity against axenic and intramacrophage amastigote stages with reasonable selectivity when compared to the THP-1 and HepG2 human cells.
By exposing promastigote L. mexicana to increasing concentrations over 28 weeks, a 700022 resistant line was generated in vitro. Promastigotes of this resistant cell line were 7.5-fold more resistant to 700022 than compared to the parental wild type line, with axenic promastigotes having a 40-fold increase in resistance. Interestingly, the 700022 resistant promastigotes had a 25% smaller cell surface area and a 85% reduction in flagellum length. The 700022-resistant line was cross resistant to the related triterpenes 700107, 700136 and 700240 and miltefosine (11.8-fold compared to wild type strain). The potential for mutations within genes (LmMT/LmRos3) that encode subunits of the miltefosine transporter complex were investigated. No mutations were associated with LmMT, with three nonsynonymous mutations found in LmRos3. This thesis also reports the evaluation of transgenic L. mexicana expressing a novel NanoLuc luciferase, and a PEST-tagged variant, as a tractable, rapid and sensitive system for antileishmanial compound screening. The validity of this approach is demonstrated by a screen of the MMV Pathogen Box. The opportunity afforded by the transgenic L. mexicana expressing NanoLuc-PEST in an in vitro infected macrophage model is also demonstrated. These transgenic L. mexicana offer an opportunity for high-throughput screening programmes that assess the more clinically-relevant activity against intracellular amastigote parasite without the time, specialist and post-assay processing burdens associated with current high-content imaging techniques.

Item Type: Thesis (Doctoral)
Subjects: Q Science > QH Natural history > QH301 Biology
Divisions: Faculty of Natural Sciences > School of Life Sciences
Depositing User: Lisa Bailey
Date Deposited: 18 Mar 2019 12:10
Last Modified: 18 Mar 2019 12:10
URI: http://eprints.keele.ac.uk/id/eprint/6070

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