Hongqiu He

Protein–protein docking with binding site patch prediction and network-based terms enhanced combinatorial scoring†

Protein–protein docking has made much progress in recent years, but challenges still exist. Here we present the application of our docking approach HoDock in CAPRI. In this approach, a binding site prediction is implemented to reduce docking sampling space and filter out unreasonable docked structures, and a network‐based enhanced combinatorial scoring function HPNCscore is used to evaluate the decoys. The experimental information was combined with the predicted binding site to pick out the most likely key binding site residues. We applied the HoDock method in the recent rounds of the CAPRI experiments, and got good results as predictors on targets 39, 40, and 41. We also got good results as scorers on targets 35, 37, 40, and 41. This indicates that our docking approach can contribute to the progress of protein–protein docking methods and to the understanding of the mechanism of protein–protein interactions. Proteins 2010.

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

AbstractAim: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 (IC50) 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.

High-throughput real-time assay based on molecular beacons for HIV-1 integrase 3'-processing reaction.

AbstractAim:To develop a high-throughput real-time assay based on molecular beacons to monitor the integrase 3′-processing reaction in vitro and apply it to inhibitor screening.Methods:The recombinant human immunodeficiency virus (HIV)-1 integrase (IN) is incubated with a 38 mer oligonucleotide substrate, a sequence identical to the U5 end of HIV-1 long terminal repeats (LTR). Based on the fluorescence properties of molecular beacons, the substrate is designed to form a stem-loop structure labeled with a fluorophore at the 5′ end and a quencher at the 3′ end. IN cleaves the terminal 3′-dinucleotide containing the quencher, resulting in an increase in fluorescence which can be monitored on a spectrofluorometer. To optimize this assay, tests were performed to investigate the effects of substrates, enzyme and the metal ion concentrations on the IN activity and optimal parameters were obtained. Moreover, 2 IN inhibitors were employed to test the performance of this assay in antiviral compound screening.Results:The fluorescent intensity of the reaction mixture varies linearly with time and is proportional to the velocity of the 3′-processing reaction. Tests were performed and the results showed that the optimal rate was obtained for a reaction mixture containing 50 mg/L recombinant HIV-1 IN, 400 nmol/L substrate, and 10 mmol/L Mn2+. The IN 3′-processing reaction under the optimal conditions showed a more than 18-fold increase in the fluorescence intensity compared to the enzyme-free control. The IC50 values of the IN inhibitors obtained in our assay were similar to the values obtained from a radiolabeled substrate assay.Conclusion:Our results demonstrated that this is a fast, reliable, and sensitive method to monitor HIV IN 3′-processing reaction and that it can be used for inhibitor screening.

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

Both HIV-1 integrase (IN) and the central catalytic domain of IN (IN-CCD) catalyze the disintegration reaction in vitro. In this study, IN and IN-CCD proteins were expressed and purified, and a high-throughput format enzyme-linked immunosorbent assay (ELISA) was developed for the disintegration reaction. IN exhibited a marked preference for Mn2+ over Mg2+ as the divalent cation cofactor in disintegration. Baicalein, a known IN inhibitor, was found to be an IN-CCD inhibitor. The assay is sensitive and specific for the study of disintegration reaction as well as for the in vitro identification of antiviral drugs targeting IN, especially targeting IN-CCD.

DNA Length and Cationic Cofactor Dependent Strand Transfer Activity of HIV-1 Integrase

HIV-1 integrase (IN)-catalyzed strand transfer is a divalent cationic cofactor dependent reaction which involves INDNA interaction. In this work, viral DNA substrates with different lengths were used. The strand transfer activity of IN with these DNA substrates in the presence of Mn and Mg were investigated, as well as some other reaction characters related with IN-DNA interaction. It is found that the strand transfer activity is both divalent cation and viral DNA length dependent. The reaction conditions for the wild type IN are stricter than those of soluble mutant IN. Moreover, IN significantly prefers E strand over A strand as the target strand, and both the recessed and blunt end viral DNA have similar strand transfer activity in the presence of either metal ion. This work provides valuable insight for the understanding of IN-DNA interaction and will be helpful in the anti-IN drug development. HIV-1; integrase; strand transfer; IN-DNA interaction