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

Author：

Publisher： Spandidos Publications

E-ISSN： 1791-2423|47|4|1485-1493

ISSN： 1019-6439

Source： International Journal of Oncology, Vol.47, Iss.4, 2015-01, pp. : 1485-1493

Disclaimer: Any content in publications that violate the sovereignty, the constitution or regulations of the PRC is not accepted or approved by CNPIEC.

Previous Menu Next

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.