Chapman Alex M, McNaughton Brian R
Department of Chemistry and ‡Department of Biochemistry & Molecular Biology, Colorado State University , Fort Collins, Colorado 80523, United States.
ACS Chem Biol. 2014 Oct 17;9(10):2223-8. doi: 10.1021/cb5003834. Epub 2014 Aug 21.
Increased cellular levels of protein-protein interactions involving the ankyrin repeat oncoprotein gankyrin are directly linked to aberrant cellular events and numerous cancers. Inhibition of these protein-protein interactions is thus an attractive therapeutic strategy. However, the relatively featureless topology of gankyrin's putative binding face and large surface areas involved in gankyrin-dependent protein-protein interactions present a dramatic challenge to small molecule discovery. The size, high folding energies, and well-defined surfaces present in many proteins overcome some of the challenges faced by small molecule discovery. We used split-superpositive Green Fluorescent Protein (split-spGFP) reassembly to screen a 5×10(9) library of resurfaced proteins that are shape complementary to the putative binding face of gankyrin and identified mutants that potently and selectively bind this oncoprotein in vitro and in living cells. Collectively, our findings represent the first synthetic proteins that bind gankyrin and may represent a general strategy for developing protein basic research tools and drug leads that bind disease-relevant ankyrin repeats.
涉及锚蛋白重复癌蛋白甘菊环蛋白的细胞内蛋白质-蛋白质相互作用增加与异常细胞事件和多种癌症直接相关。因此，抑制这些蛋白质-蛋白质相互作用是一种有吸引力的治疗策略。然而，甘菊环蛋白假定结合面相对无特征的拓扑结构以及与甘菊环蛋白依赖性蛋白质-蛋白质相互作用相关的大表面积对小分子发现构成了巨大挑战。许多蛋白质中存在的大小、高折叠能和明确的表面克服了小分子发现面临的一些挑战。我们使用分裂超正绿色荧光蛋白（split-spGFP）重新组装来筛选一个5×10⁹的表面重塑蛋白质文库，这些蛋白质与甘菊环蛋白的假定结合面形状互补，并鉴定出在体外和活细胞中能有效且选择性地结合这种癌蛋白的突变体。总体而言，我们的发现代表了首批结合甘菊环蛋白的合成蛋白质，可能代表了一种开发与疾病相关锚蛋白重复序列结合的蛋白质基础研究工具和药物先导物的通用策略。
