|
|
|
| A Fluorescent Screening Assay for HIV-1 Integrase Inhibitors Targeting Strand Transfer |
| LIU Bin1, LIU Xin1, LI Shan1, HE Hong-qiu2, ZHANG Xiao-yi1, TAN Jian-jun1, CHEN Wei-zu1, WANG Cun-xin1 |
1. College of Life Science and Bioengineering, Beijing University of Technology, Beijing 100124, China;
2. Chongqing Center for Biomedicines and Medical Equipment, Chongqing Academy of Science and Technology, Chongqing 401123, China |
|
|
|
Abstract HIV-1 integrase is an ideal target for anti-HIV-1 drug discovery. The aim of the present study was to develop a highly effective and more convenient screening assay for HIV-1 integrase inhibitors targeting strand transfer. First, the recombinant expression vector pNL-IN, which contains the HIV-1 integrase gene, was transformed into E. coli BL21 (DE3) competent cells for prokaryotic expression. The recombinant integrase protein was purified by affinity chromatography. It was validated that the recombinant integrase protein was pure and active for screening assay development. Then, the biotin-labeled donor DNA and the FITC-labeled target DNA were synthesized and applied in the assay, and streptavidin-coated magnetic beads were used to capture the product DNA in the reaction system. Finally, the fluorescence signal was detected by a fluorescence microplate reader for the calculation of sample activity. Two reported integrase inhibitors, S-1360 and MK-0518, were tested to validate the screening assay, and the results are in accordance with previous studies, which indicated that the screening assay could be used for the screening of integrase inhibitor targeting strand transfer. The screening assay for HIV-1 integrase inhibitors developed in the present study is more convenient, time-saving and cost-effective than previous screening assays.
|
|
Received: 26 October 2012
Published: 25 January 2013
|
|
|
|
|
[1] Engelman A, Mizuuchi K, Craigie R. HIV-1 DNA integration: mechanism of viral DNA cleavage and DNA strand transfer. Cell, 1991, 67 (6): 1211-1221.
[2] Pluymers W, De C E, Debyser Z. HIV-1 integration as a target for antiretroviral therapy: a review. Current Drug Targets - Infectious Disorders, 2001, 1 (2): 133-149.
[3] Lataillade M, Kozal M J. The hunt for HIV-1 integrase inhibitors. AIDS Patient Care STDS, 2006, 20 (7): 489-501.
[4] 李敬云. HIV-1的耐药基因突变. 中国艾滋病性病, 2010, 16 (2): 195-197. Li J Y. HIV-1 drug resistance mutations. Chinese Journal of AIDS & STD, 2010, 16 (2): 195-197.
[5] Tang M W, Shafer R W. HIV-1 antiretroviral resistance: scientific principles and clinical applications. Drugs, 2012, 72 (9): e1-25.
[6] Hazuda D J. HIV integrase as a target for antiretroviral therapy. Current Opinion in HIV and AIDS, 2012, 7 (5): 383-389.
[7] Wainberg M A, Mesplède T, Quashie P K. The development of novel HIV integrase inhibitors and the problem of drug resistance. Current Opinion in Virology, 2012, 2 (5): 656-662.
[8] 王欣, 程绍辉. HIV整合酶抑制剂的应用及耐药研究进展. 中国艾滋病性病, 2012, 18 (6): 429-431. Wang X, Cheng SH H. Research progress in application of HIV-1 integrase inhibitor and drug resistance. Chinese Journal of AIDS & STD, 2012, 18 (6): 429-431.
[9] Chiu T K, Davies D R. Structure and function of HIV-1 integrase. Current Topics in Medicinal Chemistry, 2004, 4 (9): 965-977.
[10] Zeinalipour-Loizidou E, Nicolaou C, Nicolaides A, et al. HIV-1 integrase: from biology to chemotherapeutics. Current HIV Research, 2007, 5 (4): 365-388.
[11] Hazuda D J, Felock P, Witmer M, et al. Inhibitors of strand transfer that prevent integration and inhibit HIV-1 replication in cells. Science, 2000, 287 (5453): 646-650.
[12] He H Q, Ma X H, Liu B, et al. A novel high-throughput format assay for HIV-1 integrase strand transfer reaction using magnetic beads. Acta Pharmacologica Sinica, 2008, 29 (3): 397-404.
[13] 何红秋, 程绍辉, 刘斌, 等. 用荧光方法检测 HIV-1整合酶的体外活性. 光散射学报, 2009, 21 (2): 185-189. He H Q, Cheng SH H, Liu B, et al. Detection of HIV-1 integrase in vitro activities using fluorescent technology. The Journal of Light Scattering, 2009, 21 (2): 185-189.
[14] Zhang J H, Chung T D Y, Oldenburg K R. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. Journal of Biomolecular Screening, 1999, 4 (2): 67-73.
[15] Inglese J, Johnson R L, Simeonov A, et al. High-throughput screening assays for the identification of chemical probes. Nature Chemical Biology, 2007, 3 (8): 466-479.
[16] Billich A. S-1360 Shionogi-GlaxoSmithKline. Current Opinion in Investigational Drugs, 2003, 4 (2): 206-209.
[17] Hombrouck A, Van Remoortel B, Michiels M, et al. Preclinical evaluation of 1H-benzylindole derivatives as novel human immunodeficiency virus integrase strand transfer inhibitors. Antimicrobial Agents and Chemotherapy, 2008, 52 (8): 2861-2869.
[18] 李芳琼, 丁倩, 詹金彪. HIV-1整合酶及其抑制剂的研究进展. 中国生物工程杂志, 2008, 28 (1): 80-86. Li F Q, Ding Q, Zhan J B. Progress in research on HIV-1 integrase and its inhibitors. China Biotechnology, 2008, 28 (1): 80-86.
[19] 郭涤亮, 刘冠男, 周宇, 等. HIV整合酶抑制剂的研究进展. 有机化学, 2010, 30 (4): 477-485. Guo D L, Liu G N, Zhou Y, et al. Research progress in HIV integrase inhibitors. Chinese Journal of Organic Chemistry, 2010, 30 (4): 477-485.
[20] Tan J J, Liu C, Sun X H, et al. Perspectives on developing small molecule inhibitors targeting HIV-1 integrase. Mini Reviews in Medicinal Chemistry, 2012, 12 (9): 875-889.
[21] He H, Liu B, Zhang X, et al. Development of a high-throughput assay for the HIV-1 integrase disintegration reaction. SCIENCE CHINA Life Sciences, 2010, 53 (2): 241-247.
[22] 何红秋, 贾渝跃. HIV-1整合酶核心区可溶性表达及抑制剂筛选. 中国生物工程杂志, 2012, 32 (3): 14-19. He H Q, Jia Y Y. Soluble expression and inhibitor screening of the central core domain of HIV- 1 integrase. China Biotechnology, 2012, 32 (3): 14-19. |
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
| |
Shared |
|
|
|
|
| |
Discussed |
|
|
|
|
