The
assay for the detection of HIV-1 integrase strand transfer (ST) inhibitors
was adapted from previously described methods.21 (link) A double-stranded biotinylated donor DNA, corresponding
to the HIV U5 viral DNA end, was added to the wells of Streptavidin-coated
96-well microtiter plates (R&D systems). Following a 1 h incubation
at r.t. and three wash steps with PBS, purified recombinant integrase
(1 μM) was assembled using DTT (dithiothreitol, 1 M) onto the
preprocessed donor DNA through incubation for 30 min at 22 °C.
After the wash step, the test compounds and a positive control inhibitor
(Raltegravir, Merck) were added into individual wells at a final concentration
of 10 μM. The microtiter plates were incubated for 30 min at
37 °C and washed. The strand transfer reaction was initiated
through the addition of FITC-labeled target dsDNA (5′-TGACCAAGGGCTAATTCACT-FITC-3′
and 5′-AGTGAATTAGCCCTTGGTCA-FITC-3′).
The plates were incubated for a period of 1 h at 37 °C followed
by washing as before. An AP (Alkaline S2 phosphatase)-conjugated anti-FITC
secondary antibody (Sigma) was added and the plates were washed. The
chromogenic substrate (BluePhos, KPL) was added to allow for photometric
measurement at 620 nm using a microplate reader (xMark, Biorad). Percentage
inhibition was calculated utilizing the formula: where A620(Comp) = Absorbance at 620 nm with compounds, A620_No_integrase_control = Absorbance at 620 nm containing no enzyme, and A620_No_inhibitor_control = Absorbance at 620 nm containing
no inhibitor. All inhibition values are the average of triplicate
experiments.
assay for the detection of HIV-1 integrase strand transfer (ST) inhibitors
was adapted from previously described methods.21 (link) A double-stranded biotinylated donor DNA, corresponding
to the HIV U5 viral DNA end, was added to the wells of Streptavidin-coated
96-well microtiter plates (R&D systems). Following a 1 h incubation
at r.t. and three wash steps with PBS, purified recombinant integrase
(1 μM) was assembled using DTT (dithiothreitol, 1 M) onto the
preprocessed donor DNA through incubation for 30 min at 22 °C.
After the wash step, the test compounds and a positive control inhibitor
(Raltegravir, Merck) were added into individual wells at a final concentration
of 10 μM. The microtiter plates were incubated for 30 min at
37 °C and washed. The strand transfer reaction was initiated
through the addition of FITC-labeled target dsDNA (5′-TGACCAAGGGCTAATTCACT-FITC-3′
and 5′-AGTGAATTAGCCCTTGGTCA-FITC-3′).
The plates were incubated for a period of 1 h at 37 °C followed
by washing as before. An AP (Alkaline S2 phosphatase)-conjugated anti-FITC
secondary antibody (Sigma) was added and the plates were washed. The
chromogenic substrate (BluePhos, KPL) was added to allow for photometric
measurement at 620 nm using a microplate reader (xMark, Biorad). Percentage
inhibition was calculated utilizing the formula: where A620(Comp) = Absorbance at 620 nm with compounds, A620_No_integrase_control = Absorbance at 620 nm containing no enzyme, and A620_No_inhibitor_control = Absorbance at 620 nm containing
no inhibitor. All inhibition values are the average of triplicate
experiments.
