A novel high-throughput format assay for HIV-1 integrase strand transfer reaction using magnetic beads

Abstract

Aim: To develop a novel high-throughput format assay to monitor the integrase (IN) strand transfer (ST) reaction in vitro and apply it to a reaction character study and the identification of antiviral drugs. Methods: The donor DNA duplex, with a sequence identical to the U5 end of HIV-1 long terminal repeats, is labeled at its 5' end with biotin (BIO). The target DNA duplex is labeled at its 3' end with digoxin (DIG). IN mediates the integration of donor DNA into target DNA and results in a 5' BIO and 3' DIG-labeled duplex DNA product. Streptavidin-coated magnetic beads were used to capture the product, and the amount of DIG was measured as the ST reaction product. The assay was optimized in 96-well microplate format for high-throughput screening purpose. Moreover, the assay was applied in a ST reaction character study, and the efficiency of the assay in the identification of antiviral compounds was tested. Results: The end-point values, measured as absorbance at 405 nm was approximately 1.5 for the IN-mediated ST reaction as compared with no more than 0.05 of background readings. The ST reaction character and the half maximal inhibitory concentration (IC 50 ) values of 2 known IN inhibitors obtained in our assay were similar to previously reported results using other assays. The evaluation parameter Z´ factor for this assay ranged from 0.6 to 0.9. Conclusion: The assay presented here has been proven to be rapid, sensitive, and specific for the detection of IN ST activity, the reaction character study, as well as for the identification of antiviral drugs targeting IN.

Discovery of potent inhibitors for phosphodiesterase 5 by virtual screening and pharmacophore analysis

“…Both of them were used for improve reliability. The major benefit of CADD in drug discovery is that it costs much less than any biomedical test of inhibition in cells just like in the references from APS [22][23][24][25][26][27][28] . The ligand based method is built on regression analysis for molecular structure and properties against activities, while the protein based method focuses on the docking procedure in which the structure of the protein would be docked with many kinds of inhibitors and the binding energies would be calculated [29,30] .…”

Development of a high-throughput assay for the HIV-1 integrase disintegration reaction

“…Site-directed mutagenesis of K185F/S280C double mutations was conducted by overlapping PCR to construct the wild type IN gene, which was then introduced into pET28b to construct a pWIN recombinant plasmid. After confirmation by sequencing, correctly constructed pWIN was expressed in Escherichia coli BL21 (DE3) as previously described [22]. After IPTG induction, cells were harvested by centrifugation, resuspended in buffer A (20 mmol/L HEPES, pH 7.5, 0.5 mol/L NaCl, 2 mmol/L β-mercaptoethanol, 5 mmol/L imidazole) containing 0.3 g/L lysozyme, and sonicated on ice.…”

“…The plasmid pNL-IN [22] containing the F185K/C280S soluble mutant HIV NL4-3 IN gene was used as the PCR template DNA. Site-directed mutagenesis of K185F/S280C double mutations was conducted by overlapping PCR to construct the wild type IN gene, which was then introduced into pET28b to construct a pWIN recombinant plasmid.…”

“…The pellet was then dissolved in buffer A containing 8 mol/L urea to denature the inclusion bodies. After renaturation, IN was purified by affinity chromatography according to the N-terminal fusion 6-histidine tag following the procedure previously described [22]. The purified IN was dialyzed against buffer B (20 mmol/L HEPES, pH 7.5, 0.5 mol/L NaCl, 0.1 mmol/L EDTA, 1 mmol/L DTT, 10% glycerol) at 4°C overnight and stored at −80°C.…”

“…IN 3P and ST activities were conducted with an ELISA for ST as previously described [22] with the following modification: a blunt DNA with the CAGT-3′ end was used to substitute the post-processed DNA substrate without the GT-3′ end used previously. The disintegration activities of IN and IN-CCD were measured by an ELISA using streptavidin-coated microplates [20].…”