Development and Application of a High-Throughput Screening Assay for HIV-1 Integrase Enzyme Activities

John, Sinu P.; Fletcher, Thomas M.; Jonsson, Colleen B.

doi:10.1177/1087057105276318

Cited by 19 publications

(17 citation statements)

References 37 publications

(59 reference statements)

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“…Therefore, identification and development of new HIV-1 inhibitor is still an urgent task for anti-HIV-1 drug research nowadays. Diverse screening strategies were used for the discovery of new HIV-1 agents [20][21][22][23]. Cell-based screening assay offer some advantages in the quest for novel inhibitors because it allows for the screening of multiple targets simultaneously, even in some cases revealing targets not captured in biochemical assays.…”

Section: Hiv-1 High-throughput Screening Inhibitor Virus-cell-basementioning

confidence: 99%

Establishment of a high-throughput screening system for universal anti-HIV targets

et al. 2010

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The process of identifying novel human immunodeficiency virus 1 (HIV-1) inhibitors presents a challenge for industrial and scientific research. Virus-cell-based screening approaches offer some advantages in the quest for novel inhibitors because they include multiple targets in a single screen and in some cases reveal targets not captured in biochemical assays. In this study, a high-throughput screening (HTS) system for HIV-1 inhibitors was developed, which allows the simultaneous screening of all the HIV-1 targets required for replication in the cell culture. HeLa/cluster of differentiation 4 (CD4)/long terminal repeat (LTR) indicator cells, which stably expressed high levels of HIV receptor CD4 and contained the firefly luciferase reporter gene under the control of the HIV-1 LTR promoter, were generated. The expression of CD4 and LTR function in this cell line was validated by Western blot and luciferase assay. MT2 cells, a human T-cell leukemia cell line that support high levels of HIV-1 replication, were infected with HIV-1, and then the infected MT2 cells were co-cultured with HeLa/CD4/LTR cells. In the optimized assay conditions, HIV-1 replication occurs rapidly in the MT2 cells, resulting in the infection of the HeLa/CD4/LTR cells and a significant induction of luciferase signals through LTR, which is activated by the expression of HIV-1 tat gene. The luciferase signals of HeLa/CD4/LTR cells co-cultured with HIV-1 infected MT2 cells were significantly stronger than the signals of noninfected HeLa/CD4/LTR cells (P < 0.001). The inhibitory effects of HIV-1 inhibitors (3′-Azido-3′-deoxythymidine [AZT], efavirenz [EFV], and nevirapine [NVP]) were evaluated with this assay, and the inhibitory concentration 50% (IC 50 ) values of the above three inhibitors were 58, 1.4, and 85 nmol/L, respectively, indicating that the assay provides the necessary sensitivity for identifying antiviral molecules. The Z' factor had a value of 0.563, indicating this is a very robust assay. These results suggested that HIV-1 infection assay represents a novel approach to HIV-1 antiviral screening that allows for the effective execution of HTS campaigns. HIV-1, high-throughput screening, inhibitor, virus-cell-based assayCitation: Yin Q, Zhuang D M, Jiang Y Q, et al. Establishment of a high-throughput screening system for universal anti-HIV targets. Chinese Sci Bull, 2010, 55: 937−942,

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Section: Hiv-1 High-throughput Screening Inhibitor Virus-cell-basementioning

confidence: 99%

Establishment of a high-throughput screening system for universal anti-HIV targets

et al. 2010

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“…, a donor oligonucleotide that mimics 1 of the viral DNA ends, and a target oligonucleotide [11][12][13][14][15][16][17][18][19][20] . Traditionally, IN activity is measured by a radiolabeled substrate assay in which reaction products are separated on polyacrylamide denaturing gels and subjected to autoradiography [1,11,12] .…”

Section: +mentioning

confidence: 99%

“…However, the disadvantages of the method are that: (i) it requires radiolabeled substrates, special equipment, and appropriate handling of hazardous radioactive waste; and (ii) it is inconvenient to process a large amount of reactions due to the lowthroughput format. Compared with the radiolabeled substrate assay, biotin (or digoxin)-labeled microtiter plate assays are safe and high-throughput, and the products of IN reactions are easy to measure with a spectrophotometer [14][15][16] . However, the major drawbacks of these microtiter plate assays are the low signal/noise ratio and intensive labor because of the necessity for plate coating and repeated washing.…”

Section: +mentioning

confidence: 99%

“…It is clear that the increase of fluorescence signal in this assay is specifically due to the IN-mediated 3'-processing reaction. This enzymatic assay for IN 3'-processing activity described here does not strictly correspond to MichaelisMenten conditions as well as most of the enzymatic assays for IN activities [11][12][13][14][15][16][17][18][19][20] . The time-dependent fluorescence intensity for the 3'-processing reaction in this assay displayed 3 distinct phases ( Figure 3A): the lag in phase I lasted about 20-30 min, followed by an apparent linear increase (phase II) for about 1.5 h, and an equilibrium phase (phase III) after 2 h. This phenomenon is similar to the observation made by Smolov et al [20] .…”

Section: Optimization Of 3'-processing Assaymentioning

confidence: 99%

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

Liu

et al. 2007

Acta Pharmacologica Sinica

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Aim: 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 stemloop 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 Mn 2+ . 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 IC 50 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.

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“…The reaction is then initiated by the addition of target dsDNA labelled in some manner, and after an incubation period, the ligated product is quantified. John et al (2005) reported a highly efficient and sensitive high-throughput screen, HIV IN Target SRI Assay for HIV-1 IN activity, using 5' biotin-labelled DNA (5' BIO donor) and 3' digoxygenin-labelled DNA (3' DIG target). Following 3' processing of the 5' BIO donor, strand transfer proceeds with integration of the 5' BIO donor into the 3' DIG target.…”

Section: Hiv-1 Enzyme Targetsmentioning

confidence: 99%

HIV-Screening Strategies for the Discovery of Novel HIV-Inhibitors

Abad¹,

Bedoya²,

Bermejo³

2011

Recent Translational Research in HIV/AIDS

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Development and Application of a High-Throughput Screening Assay for HIV-1 Integrase Enzyme Activities

Cited by 19 publications

References 37 publications

Establishment of a high-throughput screening system for universal anti-HIV targets

Establishment of a high-throughput screening system for universal anti-HIV targets

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

HIV-Screening Strategies for the Discovery of Novel HIV-Inhibitors

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