3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Allosteric effects of chicoric acid on human immunodeficiency virus type 1 integrase(2018) Fish, Muhammad QasimHuman immunodeficiency virus (HIV) integrase (IN) is an essential viral protein involved in the integration of the viral DNA into the host genome. Although having a specific catalytic function, it is apparent from mutagenesis studies that IN is pleiotropic and affects the viral life cycle at multiple points other than integration. Compounds that bind to allosteric sites on IN typically disrupt its ordered multimerization and stalls the viral life cycle at various points. Chicoric acid (CA) is a well-known IN inhibitor, however, its mechanism does not follow a conventional active site inhibition, yet it presents with antiviral activity. We thus, hypothesised that CA has an allosteric inhibitory mechanism. To test the hypothesis we aimed to determine alloteric effects of CA on HIV IN. Site directed mutagenesis was used to develop IN mutants resistant to conventional IN inhibitors and these proteins were purified. These were used in an enzyme-linked immunosorbent assay (ELISA) for comparative resistance profiling of CA and raltegravir (RAL). The ELISA also compared magnesium (Mg2+) and manganese (Mn2+) dependent differences on CA inhibition and to determine the importance of order of addition of assay components. An AlphaScreen assay was developed to test for the disruption of the IN/LEDGF interaction. Crosslinking assays and size exclusion chromatography (SEC) was performed to determine the multimeric state of IN in the presence of CA. Surface Plasmon resonance (SPR) was used to confirm binding of CA to IN and determine the kinetics. In silico docking onto the IN catalytic core (CCD) structures was used to identify a possible binding mode of CA at the allosteric binding site. Resistance profiling depicted a clear distinction between CA and RAL. IN resistance mutants: INQ148H, INN155H, ING140S/Q148H and INE92Q/N155H, showed a fold change in IC50 (FCIC50) of 3.42, 1.09, 1.43 and 2.90 for CA respectively. While the same mutants showed an FCIC50 of 947.99, 392.44, 1262.41 and 583.92 for RAL respectively. Additionally, cooperativity trends between the compound’s inhibition profiles were different. It was concluded that CA and RAL have different resistance profiles. Metal dependent differences show that the IN soluble mutant is resistant to CA inhibition only in the presence of Mg2+. This indicates that metal specific structural differences may play a role in resistance. Order of addition indicated that if DNA was present after CA incubation with IN the inhibition was cooperative, while if DNA was added before incubation with CA the inhibition was non-cooperative. This posits that CA may bind to a single site, possibly only the allosteric site, when DNA is already present in the IN active site. When DNA is absent CA binds to multiple sites, possibly both the active site and allosteric sites. The AlphaScreen indicated that the IN/LEDGF interaction is disrupted by CA with IC50 of 237nM (±27nM). Also the disruption is dependent on the order of addition of assay components. Multimerization of IN was increased in the presence of CA as shown by crosslinking assays as well as SEC. Not only did CA induce multimerization of free IN, a nonreducing gel electrophoresis indicated that virus assembled in the presence of CA had increased multimerization of IN, similar to a control. SPR indicated that CA binds to the full length IN with similar kinetics compared to the catalytic core domain (INCCD) indicating that this domain likely contains the CA binding site. The kinetics indicated a slow kon and koff rate for CA binding. These slow binding kinetics are indicative of a high barrier to resistance. Finally, in sillico molecular docking of CA to the active site as well as the allosteric site indicated that it potentially has a dual binding mode on the IN apo-enzyme. The results show that CA has allosteric effects on IN and may provide a good pharmacophore for further development of allosteric IN inhibitors.Item Development of an HIV-1 intergrase enzyme strand transfer assay(2012-01-30) Fish, Muhammad QasimThe Human Immunodeficiency Virus type 1 (HIV-1) integrase is an essential enzyme required for viral replication. Integrase forms part of an ensemble of proteins known as the preintegration complex and functions by a two-step process. Firstly, the cleaving of the 3’ ends of the viral cDNA genome, known as 3’-end processing. The second step is the insertion of these ends into host DNA by esterification, known as strand transfer. There is no human homologue to integrase which makes it an ideal drug target. However, the strand transfer inhibitor raltegravir is currently the only antiretroviral treatment available that inhibits integrase. The aims of this study were two-fold: firstly to characterise a cohort of South African patients so as to determine the viability of introducing raltegravir as a new treatment option, and secondly, to set up high-throughput integrase inhibitor screening assays (testing integrase enzymatic functionality). An HIV-1 subtype C specific RT-PCR and PCR assay was established for integrase genotyping using 51 integrase inhibitor-naïve patient plasma samples and 22 antiretroviral drug-naive primary viral isolates from South Africa. Seventy-one of the 73 samples were classified as HIV-1 subtype C and two samples were unique AC and CG recombinants in integrase. Amino acid sequence analysis revealed there were no primary mutations (Y143R/C/H, Q148H/R/K, and N155H/S) associated with reduced susceptibility to the integrase inhibitor raltegravir. However, one sample had the T97A mutation, three samples had the E157Q and V165I mutations, and the majority of samples contained the polymorphic mutation, V72I. The expected finding of no major integrase mutations conferring resistance to integrase inhibitors suggests that this new antiretroviral drug class will be effective in our region where HIV-1 subtype C predominates. However, the impact of E157Q and other naturally occurring polymorphisms warrants further phenotypic investigation. The integrase sequence of viral isolate, FV3, was closest to the consensus sequence, and thus chosen for preintegration complex isolation for use in strand transfer assays. Isolation of preintegration complexes following FV3 infections of several cell lines was unsuccessful as determined by western blot analysis. Subsequently, the focus was changed to isolation of HIV-1 subtype B recombinant integrase and its functional evaluation. Expression of native integrase (INwt) and soluble integrase (INsol) was induced in E. coli, and both proteins were purified by nickel chelating chromatography. The purified recombinant proteins were used to develop three assays to test for strand transfer activity, of which two were successfully established. Furthermore, only INsol showed strand transfer activity in the high-throughput microtitre plate assay and scintillation proximity assay (SPA)-bead strand transfer assay. Activity of INsol was shown to be inhibited by the control compound, chicoric acid with an IC50 of 101.5nM in the high-throughput microtitre plate assay, whereas INsol activity as well as a dose response to chicoric acid with an IC50 of 248.5nM was recorded in the SPA-bead strand transfer assay. Visualization of radiolabelled enzymatic products of strand transfer by polyacrylamide gel electrophoresis of urea sequencing gels was unsuccessful. Overall, the high-throughput microtitre plate and SPA-bead strand transfer assays have been successfully established in our laboratories, and are available to screen compound libraries for potential antiretroviral drug candidates targeting integrase strand transfer.