Mano Nariyasu, Sato Koichi, Goto Junichi
Graduate School of Pharmaceutical Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai 980-8578, Japan.
Anal Chem. 2006 Jul 1;78(13):4668-75. doi: 10.1021/ac060213s.
Validation of the targets of candidate drugs is critical for rapid and efficient drug discovery and development and for understanding the pharmacological action and potential toxicities of the prospective therapeutic agent. Due to the nonspecific binding of abundant proteins to small molecule-immobilized gels, it is difficult to identify the protein targets of small molecules from crude biological samples by affinity extraction. To address this problem, we have developed an affinity gel for the specific extraction of small molecule-binding proteins. We immobilized small molecules on the agarose gel through a disulfide linker that is cleavable by mild reduction. This system has allowed specific and noncovalent complex formation between the small molecule and the target protein, keeping the effect of the nonspecific abundant proteins adsorbed on both the linker and gel surface to minimum. By preparing this affinity matrix with deoxycholate as a model small molecule, we captured two independent deoxycholate-binding proteins of different affinities from mouse ascites, anti-deoxycholate antibody, and serum albumin. As other proteins were not captured, this affinity extraction method should contribute significantly to the accurate and rapid drug discovery and development.
候选药物靶点的验证对于快速高效的药物发现与开发以及理解潜在治疗药物的药理作用和潜在毒性至关重要。由于大量蛋白质与固定有小分子的凝胶存在非特异性结合，通过亲和提取从粗生物样品中鉴定小分子的蛋白质靶点具有一定难度。为解决这一问题，我们开发了一种用于特异性提取小分子结合蛋白的亲和凝胶。我们通过一种可经温和还原裂解的二硫键连接子将小分子固定在琼脂糖凝胶上。该系统允许小分子与靶蛋白之间形成特异性的非共价复合物，将吸附在连接子和凝胶表面的非特异性丰富蛋白质的影响降至最低。通过以脱氧胆酸盐作为模型小分子制备这种亲和基质，我们从小鼠腹水中捕获了两种具有不同亲和力的独立的脱氧胆酸盐结合蛋白、抗脱氧胆酸盐抗体和血清白蛋白。由于未捕获到其他蛋白质，这种亲和提取方法应能为准确快速的药物发现与开发做出重大贡献。
