Development of a dimer-based screening system for dimerization inhibitor of HIV-1 protease

I Dewa Agung Panji Dwipayana(1*), Yana Maolana Syah(2), Reza Aditama(3), Feraliana Feraliana(4), Azzania Fibriani(5)

(1) Biology Department, School of Life Science and Technology, Bandung Institute of Technology (ITB), Ganesha street 10, 40132, Bandung, Jawa Barat-Indonesia
(2) Chemistry Department, Faculty of Mathematics and Natural Sciences, Bandung Institute of Technology (ITB), Ganesha street 10, 40132, Bandung, Jawa Barat-Indonesia
(3) Chemistry Department, Faculty of Mathematics and Natural Sciences, Bandung Institute of Technology (ITB), Ganesha street 10, 40132, Bandung, Jawa Barat-Indonesia
(4) Biology Department, School of Life Science and Technology, Bandung Institute of Technology (ITB), Ganesha street 10, 40132, Bandung, Jawa Barat-Indonesia
(5) Biology Department, School of Life Science and Technology, Bandung Institute of Technology (ITB), Ganesha street 10, 40132, Bandung, Jawa Barat-Indonesia
(*) Corresponding Author

Abstract


An in vitro dimer-based screening system (DBSS) for selecting new HIV-1 protease dimerization inhibitor candidates from natural compounds had been established. This system utilizes a fusion between HIV-1 protease and dimer binding domain of AraC protein (proteaseHIV1-AraCDBD) where fluorescence signal will be emitted in the presence of HIV-1 protease inhibitor. However, this screening system had not been evaluated. Therefore, this study was aimed to evaluate it in recombinant Escherichia coli culture. The expression of proteaseHIV1-AraCDBD fusion gene was observed for 18 hours. Its crude lysate isolation was done once every 3 hours and analyzed using SDS PAGE. To test the DBSS, darunavir was used as positive control, and Nigella sativa extract (JH3) was used as the test compound. The results of SDS PAGE analysis on crude lysates presented a ~24.2 kDa band, which was the predicted size of the proteaseHIV1-AraCDBD fusion protein based on its amino acid sequence. The growth curve and protein expression profiles revealed that the 15 hours was the optimum culture age to be used in the screening system. Darunavir testing in DBSS showed an increase in fluorescence signal compared to the negative control. The same increase in fluorescence signal was also obtained from the JH3 compound test. In conclusion, DBSS could be used as an assay to screen for new HIV-1 protease inhibitors, and the JH3 compound demonstrated the ability to inhibit HIV-1 protease dimerization.

Keywords


dimer based screening system, HIV, protease

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References


Abagyan R, Totrov M, Kuznetsov D. 1994. ICM—a new method for protein modeling and design: applications to docking and structure prediction from the distorted native conformation. Journal of computational chemistry 15(5): 488-506. DOI:10.1002/jcc.540150503.

Aoki M, Das D, Hayashi H, Aoki-Ogata H, Takamatsu Y, Ghosh AK, Mitsuya H. 2018. Mechanism of darunavir (DRV)’s high genetic barrier to HIV-1 resistance: a key V32I substitution in protease rarely occurs, but once it occurs, it predisposes HIV-1 to develop DRV resistance. MBio 9(2): e02425-17. DOI: 10.1128/mBio.02425-17.

Cuevas JM, Geller R, Garijo R, López-Aldeguer J, Sanjuán R. 2015. Extremely high mutation rate of HIV-1 in vivo. PLoSBiol 13(9): e1002251. DOI: 10.1371/ journal.pbio.1002251.

Fibriani A, Feraliana, Steven N, Rahmita M, Rachman EG. 2018. Plasmid construction for development of a high throughput system selection of new anti-HIV drugs derived from biological resources Indonesia. Biotechnol Ind J 14(3): 166.

Lascar RM, Benn P. 2009. Role of darunavir in the management of HIV infection. HIV AIDS (Auckland, NZ) 1: 31-39. DOI: 10.2147/HIV.S5397.

Okada A, Gotoh Y, Watanabe T, Furuta E, Yamamoto K, Utsumi R. 2007. Targeting two‐component signal transduction: a novel drug discovery system. Methods in Enzymology 422: 386-395. DOI:10.1016/S0076-6879(06)22019-6.

Raush E, Totrov M, Marsden BD, Abagyan R. 2009. A new method for publishing three-dimensional content. PLoS One 4(10): e7394. DOI: 10.1371/journal.pone.0007394.

Roy A, Yang J, Zhang Y. 2012. COFACTOR: an accurate comparative algorithm for structure-based protein function annotation. Nucleic Acids Research 40(W1): W471-W477. DOI: 10.1093/nar/gks372.

Studier FW, Moffatt BA. 1986. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. Journal of Molecular Biology 189(1): 113-130. DOI: 10.1016/0022-2836(86)90385-2.

Studier FW. 1990. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185: 60-89. DOI: 10.1016/0076-6879(90)85008-c.

Trott O, Olson AJ. 2010. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. Journal of Computational Chemistry 31(2), 455-461. DOI: 10.1002/jcc.21334.

UNAIDS (Joint United Nations Programme on HIV and AIDS). 2019. Country factsheets INDONESIA | 2018. https://www.unaids.org/en/regionscountries/countries/indonesia (accessed 1 August 2020).

UNAIDS (Joint United Nations Programme on HIV and AIDS). 2020. GLOBAL HIV STATISTICS. https://www.unaids.org/sites/default/files/media_asset/UNAIDS_FactSheet_en.pdf (accessed 1 August 2020).

Wang Y, Liu Z, Brunzelle J.S, Kovari IA, Dewdney TG, Reiter SJ, Kovari LC. 2011. The higher barrier of darunavir and tipranavir resistance for HIV-1 protease. Biochemical and Biophysical Research Communications 412(4): 737-742. DOI: 10.1016/j.bbrc.2011.08.045.

Yang J, Zhang Y. 2015. I-TASSER server: new development for protein structure and function predictions. Nucleic Acids Research 43(W1): W174-W181. DOI: 10.1093/nar/gkv342.

Zhang Y, Chang YCE, Louis JM, Wang YF, Harrison RW, Weber IT. 2014. Structures of darunavir-resistant HIV-1 protease mutant reveal atypical binding of darunavir to wide open flaps. ACS Chemical Biology 9(6): 1351-1358. DOI: 10.1021/cb4008875

Zhang Y. 2009. I‐TASSER: Fully automated protein structure prediction in CASP8. Proteins: Structure, Function, and Bioinformatics 77(S9): 100-113. DOI: 10.1002/prot.22588.




DOI: https://doi.org/10.37604/jmsb.v2i2.42

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