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

  • Int J Oncol. 2015 Oct;47(4):1485-93. doi: 10.3892/ijo.2015.3132.
Xiaoqin Fan  1 ,  Jinmei Wei  1 ,  Haiting Xiong  1 ,  Xiaohui Liu  1 ,  Serge Benichou  2 ,  Xuejuan Gao  1 ,  Langxia Liu  1
Affiliations
  • 1. Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, Jinan University, Guangzhou 510632, P.R. China.
  • 2. Cochin Institute, INSERM U1016, Centre National de la Recherche Scientifique UMR8104, Université Paris-Descartes, Paris, France.
PMID: 26315450 DOI: 10.3892/ijo.2015.3132
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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