Browse views: by Year, by Function, by GLF, by Subfunction, by Conference, by Journal

Time-resolved fluorescence resonance energy transfer and surface plasmon resonance-based assays for retinoid and transthyretin binding to retinol-binding protein 4.

Sharif, Orzala and Hu, Huiyong and Klock, Heath and Hampton, Eric and Nigoghossian, Edward and Knuth, Mark and Matzen, Jason and Anderson, Paul and Trager, Richard and Uno, Tetsuo and Glynne, Richard and Azarian, Sassan and Caldwell, Jeremy and Brinker, Achim (2009) Time-resolved fluorescence resonance energy transfer and surface plasmon resonance-based assays for retinoid and transthyretin binding to retinol-binding protein 4. Analytical Biochemistry, 392 (2). pp. 162-168. ISSN 1096-0309

Abstract

Retinol-binding protein-4 (RBP4) is an emerging candidate drug target for type 2 diabetes and lipofuscin-mediated macular degeneration. The retinoic acid derivative fenretinide (N-(4-hydroxyphenyl) retinamide; HPR) exerts therapeutic effects in mouse models of obesity, diabetes, and Stargardt's disease by targeting RBP4. Fenretinide competes with retinoids for RBP4 binding, disrupts RBP4-transthyretin (TTR) complexes, and results in urinary secretion of RBP4 and systemic depletion of retinol. To enable the search for nonretinoid molecules with fenretinide-like activities we developed a HTS-compatible homogeneous TR-FRET assay monitoring the displacement of retinoic acid derivatives from RBP4 in high-density 384-well and 1536-well microtiter plate formats. The retinoid displacement assay proved to be highly sensitive and robust after miniaturization with IC(50)s for fenretinide and retinol ranging around 50 and 100 nM, respectively, and Z'-factors around 0.7. In addition, a surface plasmon resonance (SPR)-based secondary assay was developed to interrogate small molecule RBP4 binders for their ability to modulate the RBP4-TTR interaction. Finally, a 1.6 x 10(6) compound library was screened against the retinoid displacement assay. Several potent retinoid competitors were identified that also appeared to disrupt RBP4-TTR complexes. Some of these compounds could potentially serve as valuable tools to further probe RBP4 biology in the future.

Item Type: Article
Related URLs:
Additional Information: author can archive post-print (ie final draft post-refereeing); Publisher's version/PDF cannot be used
Keywords: RBP4; Fenretinide; Diabetes; Macular degeneration; Binding assay; TR-FRET
Related URLs:
Date Deposited: 14 Dec 2009 13:49
Last Modified: 31 Jan 2013 00:57
URI: https://oak.novartis.com/id/eprint/1195

Search

Email Alerts

Register with OAK to receive email alerts for saved searches.