title: Time-resolved fluorescence resonance energy transfer and surface plasmon resonance-based assays for retinoid and transthyretin binding to retinol-binding protein 4.
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.
authors.affiliation: Genomics Institute of the Novartis Research Foundation (GNF), San Diego, CA 92121, USA.
authors.forename: Orzala
authors.initials: O
authors.lastname: Sharif
authors.forename: Huiyong
authors.initials: H
authors.lastname: Hu
authors.forename: Heath
authors.initials: H
authors.lastname: Klock
authors.forename: Eric N
authors.initials: EN
authors.lastname: Hampton
authors.forename: Edward
authors.initials: E
authors.lastname: Nigoghossian
authors.forename: Mark W
authors.initials: MW
authors.lastname: Knuth
authors.forename: Jason
authors.initials: J
authors.lastname: Matzen
authors.forename: Paul
authors.initials: P
authors.lastname: Anderson
authors.forename: Richard
authors.initials: R
authors.lastname: Trager
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Uno
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Glynne
authors.forename: Sassan M
authors.initials: SM
authors.lastname: Azarian
authors.forename: Jeremy S
authors.initials: JS
authors.lastname: Caldwell
authors.forename: Achim
authors.initials: A
authors.lastname: Brinker
mesh_terms: D004353:Drug Evaluation, Preclinical; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D015394:Molecular Structure; D011228:Prealbumin / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D012176:Retinoids / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D054839:Retinol-Binding Proteins, Plasma / Q000378:metabolism*; D020349:Surface Plasmon Resonance / Q000379:methods*; D013997:Time Factors
chemical_list: D011228:Prealbumin; C101326:RBP4 protein, human; D012176:Retinoids; D054839:Retinol-Binding Proteins, Plasma