title: A homogeneous time-resolved fluorescence-based high-throughput screening for discovery of inhibitors of Nef-sdAb19 interaction.
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.
authors: Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, Jinan University, Guangzhou 510632, P.R. China.
authors: Xiaoqin
authors: X
authors: Fan
authors: Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, Jinan University, Guangzhou 510632, P.R. China.
authors: Jinmei
authors: J
authors: Wei
authors: Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, Jinan University, Guangzhou 510632, P.R. China.
authors: Haiting
authors: H
authors: Xiong
authors: Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, Jinan University, Guangzhou 510632, P.R. China.
authors: Xiaohui
authors: X
authors: Liu
authors: Cochin Institute, INSERM U1016, Centre National de la Recherche Scientifique UMR8104, Université Paris-Descartes, Paris, France.
authors: Serge
authors: S
authors: Benichou
authors: Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, Jinan University, Guangzhou 510632, P.R. China.
authors: Xuejuan
authors: X
authors: Gao
authors: Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, Jinan University, Guangzhou 510632, P.R. China.
authors: Langxia
authors: L
authors: Liu
mesh_terms: D019380:Anti-HIV Agents; D000914:Antibodies, Viral; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D061905:Single-Domain Antibodies; D054311:nef Gene Products, Human Immunodeficiency Virus
chemical_list: D019380:Anti-HIV Agents; D000914:Antibodies, Viral; D061905:Single-Domain Antibodies; D054311:nef Gene Products, Human Immunodeficiency Virus