HIV-1 integrase is an essential enzyme in the HIV replication cycle and is a validated target for antiretroviral drugs. Due to the inevitable emergence of drug resistance of HIV-1 strains to all currently approved FDA antiretroviral drugs, antivirals with new mechanisms of action are continuously investigated. As such, this study aimed to reposition existing drugs as HIV-1 integrase inhibitors by screening the NIH Clinical Collection compound library comprising 727 compounds. Recombinant integrase was expressed in bacterial cells, purified by nickel affinity chromatography, and used to set up a Scintillation Proximity Assay (SPA). The SPA was subsequently amended to an automated system to allow for rapid screening of compounds. The complete compound library was successfully screened using the newly established automated SPA. Overall, only two compounds were identified as HIV-1 IN inhibitors: cefixime trihydrate and a previously identified HIV integrase inhibitor, epigallocatechin gallate. These compounds exerted IC50 values < 10μM in the automated SPA. Cefixime trihydrate was not toxic to mammalian cells (CC50 > 200μM) while no appreciable antiretroviral activity was observed in in vitro phenotypic inhibition assays (23% inhibition of viral replication), thus concluding that this compound was non-selective. By contrast, epigallocatechin gallate was toxic to mammalian cells at the evaluated ranges (CC50 = 23 + 1μM) and therefore could not be validated as an integrase inhibitor in in vitro phenotypic inhibition assays. Overall, this study resulted in the establishment of an automated SPA, the successful screening of 727 compounds, and the availability of a platform to expedite the future screening of potential HIV-1 integrase inhibitors.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/15448 |
| Date | 09 September 2014 |
| Creators | Abrahams, Shaakira |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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