3. Electronic Theses and Dissertations (ETDs) - All submissions
Permanent URI for this communityhttps://wiredspace.wits.ac.za/handle/10539/45
Browse
2 results
Search Results
Item Evaluation of the NIH clinical collection to identify potential HIV-1 integrase inhibitors(2014-09-09) Abrahams, ShaakiraHIV-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.Item Generation of soluble, catalytically active covalent HIV-1 subtype C integrase-DNA complexes to identify novel strand transfer inhibitors(2012) Beyleveld, Grant JamesThe HIV-1 integrase (IN) enzyme is an integral part of the viral replication cycle and has no known human homologues, making it an ideal target for antiretroviral therapy. To date, only one inhibitor of IN strand transfer activity (Raltegravir, IsentressTM) is available for human use. However, the inevitable emergence of antiretroviral drug resistance requires ongoing research into new/novel therapies. There are currently no assays to screen for IN inhibitors against HIV-1 subtype C in South Africa (and worldwide), therefore, the overall objective of this study was to generate and characterize locally relevant, soluble, functional recombinant HIV-1 subtype C IN proteins for use in strand transfer assays. Recombinant integrase genes, including a soluble HIV-1 subtype C mutant (05ZAFV6 with C56S, C65S, W131D, F185D and C280S) and HIV-1 subtype C Y143C mutant (05ZAFV6 soluble with Y143C) were designed, generated and cloned in frame into pET15b. Optimal bacterial expression conditions for the expression of these constructs as well as an HIV-1 subtype C wild type (05ZAFV6), subtype B wild type (NL4-3), and subtype B soluble (NL4-3 with F185K and C280S; as controls) IN, in E.coli BL21 cells were determined. All five recombinant IN were successfully purified using nickel affinity chromatography, and subsequently used to establish a strand transfer assay to assess their activity and their response to two well-known integrase inhibitors, L-Chicoric acid and Raltegravir. All five recombinant IN proteins were found to be biologically active, with INY143C (116.67%) showing equivalent activity to INBwt (117.37%), while INCsol (52.96%) was the lowest. The IC50 values of L-Chicoric acid were higher than the expected values for all five recombinant IN, with the subtype B and C IN solubility mutations contributing to an increased resistance to inhibition by L-Chicoric acid. The dose responses to Raltegravir for INCwt and INBsol were as expected, with IC50’s in line with published data, and the INY143C mutant (known mutation conferring resistance to Raltegravir) was resistant to inhibition of strand transfer activity at all Raltegravir concentrations tested except the highest (50 μM). Finally, methods to complex the INY143C mutant to thiolated-DNA were evaluated, however definitive data could not be obtained. Future work should focus on optimization of the purification and characterization of the IN-DNA complexes. Overall, this study has led to the establishment of functional strand transfer assays based on HIV-1 subtype C recombinant IN proteins, and established a framework for screening of novel HIV-1 subtype C IN inhibitors.