Sharif Orzala, Hu Huiyong, Klock Heath, Hampton Eric N, Nigoghossian Edward, Knuth Mark W, Matzen Jason, Anderson Paul, Trager Richard, Uno Tetsuo, Glynne Richard J, Azarian Sassan M, Caldwell Jeremy S, Brinker Achim
Genomics Institute of the Novartis Research Foundation (GNF), San Diego, CA 92121, USA.
Anal Biochem. 2009 Sep 15;392(2):162-8. doi: 10.1016/j.ab.2009.05.038. Epub 2009 May 29.
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.
视黄醇结合蛋白4（RBP4）是2型糖尿病和脂褐素介导的黄斑变性中一个新出现的候选药物靶点。维甲酸衍生物芬维A胺（N-（4-羟苯基）视黄酰胺；HPR）通过靶向RBP4在肥胖、糖尿病和斯特格黄斑变性小鼠模型中发挥治疗作用。芬维A胺与类视黄醇竞争RBP4结合，破坏RBP4-转甲状腺素蛋白（TTR）复合物，并导致RBP4经尿液分泌以及视黄醇在体内耗竭。为了寻找具有类似芬维A胺活性的非类视黄醇分子，我们开发了一种与高通量筛选兼容的均相时间分辨荧光能量共振转移（TR-FRET）测定法，该方法以高密度384孔和1536孔微量滴定板形式监测类视黄醇衍生物从RBP4上的置换。在小型化后，类视黄醇置换测定法被证明具有高度敏感性和稳健性，芬维A胺和视黄醇的半数抑制浓度（IC50）分别约为50和100 nM，Z'因子约为0.7。此外，还开发了一种基于表面等离子体共振（SPR）的二级测定法，以研究小分子RBP4结合剂调节RBP4-TTR相互作用的能力。最后，针对类视黄醇置换测定法对一个1.6×10⁶化合物库进行了筛选。鉴定出了几种有效的类视黄醇竞争剂，它们似乎也能破坏RBP4-TTR复合物。这些化合物中的一些可能在未来作为进一步探究RBP4生物学特性的有价值工具。
