Ph.D. / Despite nearly three decades of intensive research, the HIV/AIDS pandemic remains a major challenge to modern medicine. The discovery and development of antiretroviral agents acting against various essential viral processes and enzymatic targets have greatly enhanced the quality of life for infected individuals, but no cure or preventative vaccine is available as yet and HIV infection is currently considered irreversible. Furthermore, the emergence of viral resistance to every class and type of antiretroviral treatment agent necessitates the continued discovery of antiretroviral agents with novel mechanisms of action. The first antiretroviral agent targeting the retroviral integrase enzyme (InsentressTM, Raltegravir) received regulatory approval from the United States Food and Drug Administration during 2007, validating HIV-1 integrase as a therapeutic target and providing a much-needed second- or third-line treatment option for treatment experienced patients. This enzyme was selected as a target for the current work. As limited data were available on the primary and secondary structure of the biologically relevant HIV-1 integrase enzyme, a first step in the present work was the construction of monomeric, dimeric and tetrameric models of the enzyme with biologically relevant catalytic centres incorporating both viral and host co-factors and DNA. The models were constructed to identify potential inhibitors of the strandtransfer reaction of HIV-1 integrase and were based on observations and interactions reported in the literature and on crystal structure data of HIV-1 integrase sub-domains and related structures available in the Protein Data Bank. The monomeric model was used as the macromolecular target in docking studies with “drug-like” compound databases, identifying the pyrrolidinone compound class as an in silico hit candidate for further development. Initial activity screening of a number of commercially available pyrrolidinone analogues against recombinant HIV-1 subtype B integrase in direct enzyme assays confirmed the predicted potential for strand transfer inhibition of the compound class, and provided initial support in the further development of this compound class as inhibitors of HIV-1 integrase that target the strand-transfer step. Retrosynthetic analysis of the pyrrolidinone hit candidates provided a facile one-pot, three-component synthetic pathway from readily available starting materials, which generally gave the proposed products cleanly and in acceptable yields. A range of closely related analogues were designed and synthesised. The analogues making up this series generally differed by only one functional group, in order to enable initial structureactivity relationship investigations during later stages of the project. Foreword Page XVI The synthesised pyrrolidinone analogues were screened through a range of direct and cell-based in vitro assays to determine the toxicity and strand-transfer activity of each. In general, the pyrrolidinone compounds proved well-tolerated in PM1 cell culture, with clear potential to further develop the strandtransfer inhibition of the compound family in second- and further-generation optimisation stages. Furthermore, the aqueous solubility and membrane permeability of each compound were determined in vitro, providing initial biological profiles of each test compound. As no in vivo testing was performed with any of the compounds during this first round of drug discovery and optimisation, several computational models were employed to extrapolate the in vitro and structural data to possible in vivo scenarios. Two pyrrolidinone analogues (11.6 and 15.2) were identified as low micro-molar strand-transfer inhibitors of wildtype-equivalent HIV-1 integrase, with low toxicity in cell culture and favourable solubility and permeability profiles. Resistance screening of these two compounds against four mutant HIV-1 integrases (Q148H; Q148H/G140S; N155H and N155H/E92Q) has shown some promise, with compound 15.2 retaining a measure of activity against the Raltegravir-resistant N155-mutants. These hit candidates will form the basis of structure-activity relationship optimisations in second- and further generation design stages.
|05 November 2012
|South African National ETD Portal
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