A homogeneous time-resolved fluorescence-based high-throughput screening for discovery of inhibitors of Nef-sdAb19 interaction

Fan,Xiaoqin; Wei,Jinmei; Xiong,Haiting; Liu,Xiaohui; Benichou,Serge; Gao,Xuejuan; Liu,Langxia

doi:10.3892/ijo.2015.3132

A homogeneous time-resolved fluorescence-based high-throughput screening for discovery of inhibitors of Nef-sdAb19 interaction

Authors:
- Xiaoqin Fan
- Jinmei Wei
- Haiting Xiong
- Xiaohui Liu
- Serge Benichou
- Xuejuan Gao
- Langxia Liu
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Affiliations: Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, Jinan University, Guangzhou 510632, P.R. China, Cochin Institute, INSERM U1016, Centre National de la Recherche Scientifique UMR8104, Université Paris-Descartes, Paris, France
Published online on: August 25, 2015 https://doi.org/10.3892/ijo.2015.3132
Pages: 1485-1493

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Abstract

The human immunodeficiency virus (HIV) protein negative factor (Nef) is important for AIDS pathogenesis. An anti-Nef single-domain antibody (sdAb19) derived from camelids has been previously generated and shown to effectively block the physiological functions of Nef in vitro and in vivo in nef-transgenic mice. However, sdAb19 must be ectopically expressed within the target cell to be able to exert its neutralizing effect on Nef, while the extra-cellular administration method turned out to be ineffective. This might suggest a default of the stability or/and deliverability of sdAb19. The identification of small molecule compounds capable of inhibiting the Nef-sdAb19 interaction and mimicking the neutralizing activity of sdAb19 in vivo would therefore be the means of circumventing the problem encountered with sdAb19. Here we describe the development of a high-throughput screening method combining the homogeneous time-resolved fluorescence (HTRF) and the microscale thermophoresis (MST) techniques for the identification of small-molecule compounds inhibiting the Nef-sdAb19 interaction by binding to Nef protein. Eight small-molecule compounds have been selected for their ability to significantly inhibit the Nef-sdAb19 interaction and to bind to Nef. These molecules could be further assessed for their potential of being the Nef-neutralizing agents in the future.

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1	Greene WC: The brightening future of HIV therapeutics. Nat Immunol. 5:867–871. 2004. View Article : Google Scholar : PubMed/NCBI
2	Foster JL and Garcia JV: HIV-1 Nef: At the crossroads. Retrovirology. 5:842008. View Article : Google Scholar : PubMed/NCBI
3	Kestler HW III, Ringler DJ, Mori K, Panicali DL, Sehgal PK, Daniel MD and Desrosiers RC: Importance of the nef gene for maintenance of high virus loads and for development of AIDS. Cell. 65:651–662. 1991. View Article : Google Scholar : PubMed/NCBI
4	Fackler OT and Baur AS: Live and let die: Nef functions beyond HIV replication. Immunity. 16:493–497. 2002. View Article : Google Scholar : PubMed/NCBI
5	Shugars DC, Smith MS, Glueck DH, Nantermet PV, Seillier-Moiseiwitsch F and Swanstrom R: Analysis of human immunodeficiency virus type 1 nef gene sequences present in vivo. J Virol. 67:4639–4650. 1993.PubMed/NCBI
6	Kwak YT, Raney A, Kuo LS, Denial SJ, Temple BR, Garcia JV and Foster JL: Self-association of the Lentivirus protein, Nef. Retrovirology. 7:772010. View Article : Google Scholar : PubMed/NCBI
7	Cruz NV, Amorim R, Oliveira FE, Speranza FA and Costa LJ: Mutations in the nef and vif genes associated with progression to AIDS in elite controller and slow-progressor patients. J Med Virol. 85:563–574. 2013. View Article : Google Scholar : PubMed/NCBI
8	Foster JL, Denial SJ, Temple BR and Garcia JV: Mechanisms of HIV-1 Nef function and intracellular signaling. J Neuroimmune Pharmacol. 6:230–246. 2011. View Article : Google Scholar : PubMed/NCBI
9	Bouchet J, Basmaciogullari SE, Chrobak P, Stolp B, Bouchard N, Fackler OT, Chames P, Jolicoeur P, Benichou S and Baty D: Inhibition of the Nef regulatory protein of HIV-1 by a single-domain antibody. Blood. 117:3559–3568. 2011. View Article : Google Scholar : PubMed/NCBI
10	Muyldermans S: Single domain camel antibodies: Current status. J Biotechnol. 74:277–302. 2001.PubMed/NCBI
11	Roovers RC, Laeremans T, Huang L, De Taeye S, Verkleij AJ, Revets H, de Haard HJ and van Bergen en Henegouwen PM: Efficient inhibition of EGFR signaling and of tumour growth by antagonistic anti-EFGR Nanobodies. Cancer Immunol Immunother. 56:303–317. 2007. View Article : Google Scholar
12	Gueorguieva D, Li S, Walsh N, Mukerji A, Tanha J and Pandey S: Identification of single-domain, Bax-specific intrabodies that confer resistance to mammalian cells against oxidative-stress-induced apoptosis. FASEB J. 20:2636–2638. 2006. View Article : Google Scholar : PubMed/NCBI
13	Hultberg A, Temperton NJ, Rosseels V, Koenders M, Gonzalez-Pajuelo M, Schepens B, Ibañez LI, Vanlandschoot P, Schillemans J, Saunders M, et al: Llama-derived single domain antibodies to build multivalent, superpotent and broadened neutralizing anti-viral molecules. PLoS One. 6:e176652011. View Article : Google Scholar : PubMed/NCBI
14	Ohmi N, Wingfield JM, Yazawa H and Inagaki O: Development of a homogeneous time-resolved fluorescence assay for high throughput screening to identify Lck inhibitors: Comparison with scintillation proximity assay and streptavidin-coated plate assay. J Biomol Screen. 5:463–470. 2000. View Article : Google Scholar
15	Degorce F: HTRF^®: Pioneering technology for high-throughput screening. Expert Opin Drug Discov. 1:753–764. 2006. View Article : Google Scholar : PubMed/NCBI
16	Jia Y, Quinn CM, Gagnon AI and Talanian R: Homogeneous time-resolved fluorescence and its applications for kinase assays in drug discovery. Anal Biochem. 356:273–281. 2006. View Article : Google Scholar : PubMed/NCBI
17	Chen X, Longgood JC, Michnoff C, Wei S, Frantz DE and Bezprozvanny L: High-throughput screen for small molecule inhibitors of Mint1-PDZ domains. Assay Drug Dev Technol. 5:769–783. 2007. View Article : Google Scholar : PubMed/NCBI
18	Mohamed TM, Zakeri SA, Baudoin F, Wolf M, Oceandy D, Cartwright EJ, Gul S and Neyses L: Optimisation and validation of a high throughput screening compatible assay to identify inhibitors of the plasma membrane calcium ATPase pump - a novel therapeutic target for contraception and malaria. J Pharm Pharm Sci. 16:217–230. 2013.
19	Lemay J, Maidou-Peindara P, Bader T, Ennifar E, Rain JC, Benarous R and Liu LX: HuR interacts with human immunodeficiency virus type 1 reverse transcriptase, and modulates reverse transcription in infected cells. Retrovirology. 5:472008. View Article : Google Scholar : PubMed/NCBI
20	Seidel SA, Dijkman PM, Lea WA, van den Bogaart G, Jerabek-Willemsen M, Lazic A, Joseph JS, Srinivasan P, Baaske P, Simeonov A, et al: Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions. Methods. 59:301–315. 2013. View Article : Google Scholar :
21	Fischer WB, Wang YT, Schindler C and Chen CP: Mechanism of function of viral channel proteins and implications for drug development. Int Rev Cell Mol Biol. 294:259–321. 2012. View Article : Google Scholar : PubMed/NCBI
22	Izzedine H, Launay-Vacher V, Deray G and Hulot JS: Nelfinavir and felodipine: A cytochrome P450 3A4-mediated drug interaction. Clin Pharmacol Ther. 75:362–363. 2004. View Article : Google Scholar : PubMed/NCBI
23	Idriss HT: Questions regarding the role of protein phosphorylation in HIV replication and anti-acquired immune deficiency syndrome therapy. Saudi Med J. 26:7–10. 2005.PubMed/NCBI
24	Jakubiec A and Jupin I: Regulation of positive-strand RNA virus replication: The emerging role of phosphorylation. Virus Res. 129:73–79. 2007. View Article : Google Scholar : PubMed/NCBI
25	Behrend L, Milne DM, Stöter M, Deppert W, Campbell LE, Meek DW and Knippschild U: IC261, a specific inhibitor of the protein kinases casein kinase 1-delta and -epsilon, triggers the mitotic checkpoint and induces p53-dependent postmitotic effects. Oncogene. 19:5303–5313. 2000. View Article : Google Scholar : PubMed/NCBI
26	Yang WS and Stockwell BR: Inhibition of casein kinase 1-epsilon induces cancer-cell-selective, PERIOD2-dependent growth arrest. Genome Biol. 9:R922008. View Article : Google Scholar : PubMed/NCBI
27	Zhou YX, Karle S, Taguchi H, Planque S, Nishiyama Y and Paul S: Prospects for immunotherapeutic proteolytic antibodies. J Immunol Methods. 269:257–268. 2002. View Article : Google Scholar : PubMed/NCBI
28	Evans TR and Kaye SB: Retinoids: Present role and future potential. Br J Cancer. 80:1–8. 1999. View Article : Google Scholar : PubMed/NCBI
29	Kaleagasioglu F, Doepner G, Biesalski HK and Berger MR: Antiproliferative activity of retinoic acid and some novel retinoid derivatives in breast and colorectal cancer cell lines of human origin. Arzneimittelforschung. 43:487–490. 1993.PubMed/NCBI
30	Hua S, Kittler R and White KP: Genomic antagonism between retinoic acid and estrogen signaling in breast cancer. Cell. 137:1259–1271. 2009. View Article : Google Scholar : PubMed/NCBI
31	He JC, Lu TC, Fleet M, Sunamoto M, Husain M, Fang W, Neves S, Chen Y, Shankland S, Iyengar R, et al: Retinoic acid inhibits HIV-1-induced podocyte proliferation through the cAMP pathway. J Am Soc Nephrol. 18:93–102. 2007. View Article : Google Scholar
32	Lu TC, Wang Z, Feng X, Chuang P, Fang W, Chen Y, Neves S, Maayan A, Xiong H, Liu Y, et al: Retinoic acid utilizes CREB and USF1 in a transcriptional feed-forward loop in order to stimulate MKP1 expression in human immunodeficiency virus-infected podocytes. Mol Cell Biol. 28:5785–5794. 2008. View Article : Google Scholar : PubMed/NCBI