BACKGROUND: In addition to activities needed to catalyse integration, retroviral integrases exhibit non-specific endonuclease activity that is enhanced by certain small compounds, suggesting that integrase could be stimulated to damage viral DNA before integration occurs. METHODS: A non-radioactive, plate-based, solution phase, fluorescence assay was used to screen a library of 50,080 drug-like chemicals for stimulation of non-specific DNA nicking by HIV-1 integrase. RESULTS: A semi-automated workflow was established and primary hits were readily identified from a graphic output. Overall, 0.6% of the chemicals caused a large increase in fluorescence (the primary hit rate) without also having visible colour that could have artifactually caused this result. None of the potential stimulators from this moderate-size library, however, passed a secondary test that included an inactive integrase mutant that assessed whether the increased fluorescence depended on the endonuclease activity of integrase. CONCLUSIONS: This first attempt at identifying integrase stimulator compounds establishes the necessary logistics and workflow required. The results from this study should encourage larger scale high-throughput screening to advance the novel antiviral strategy of stimulating integrase to damage retroviral DNA.
BACKGROUND: In addition to activities needed to catalyse integration, retroviral integrases exhibit non-specific endonuclease activity that is enhanced by certain small compounds, suggesting that integrase could be stimulated to damage viral DNA before integration occurs. METHODS: A non-radioactive, plate-based, solution phase, fluorescence assay was used to screen a library of 50,080 drug-like chemicals for stimulation of non-specific DNA nicking by HIV-1 integrase. RESULTS: A semi-automated workflow was established and primary hits were readily identified from a graphic output. Overall, 0.6% of the chemicals caused a large increase in fluorescence (the primary hit rate) without also having visible colour that could have artifactually caused this result. None of the potential stimulators from this moderate-size library, however, passed a secondary test that included an inactive integrase mutant that assessed whether the increased fluorescence depended on the endonuclease activity of integrase. CONCLUSIONS: This first attempt at identifying integrase stimulator compounds establishes the necessary logistics and workflow required. The results from this study should encourage larger scale high-throughput screening to advance the novel antiviral strategy of stimulating integrase to damage retroviral DNA.
Authors: Oyebisi Jegede; John Babu; Roberto Di Santo; Damian J McColl; Jan Weber; Miguel Quiñones-Mateu Journal: AIDS Rev Date: 2008 Jul-Sep Impact factor: 2.500
Authors: G Natsoulis; P Seshaiah; M J Federspiel; A Rein; S H Hughes; J D Boeke Journal: Proc Natl Acad Sci U S A Date: 1995-01-17 Impact factor: 11.205