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小分子与活细胞中过度表达的原生蛋白质的结合量化

Yuwen Yin¹, Serena Li Zhao¹, Digamber Rane¹

¹Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, 500 W. 12th Ave., Columbus, Ohio 43210, United States.

Journal of the American Chemical Society

|December 20, 2023

概括

我们开发了一种新的测试方法, 测量药物与活细胞内的原生蛋白质的结合力. 这种光探针细胞结合试验 (FPCBA) 有助于量化药物向相互作用,用于药物发现.

科学领域:

生物化学
细胞生物学
药理学

背景情况:

药物发现依赖于对蛋白质标的小分子亲和力和选择性.
在细胞环境中精确测量这些相互作用对于治疗开发至关重要.

研究的目的:

开发和验证一种用于量化小分子与活细胞内的原生未标记蛋白质的新型试验.
为了确定候选药物的细胞解离常数 (Kd) 和竞争性结合常数 (Ki).

主要方法:

一种光探针细胞结合试验 (FPCBA) 使用化物结合联体被开发出来.
一个内部核糖体进入点 (IRES) 载体促进了目标蛋白和正交光蛋白 (mVenus) 的同时表达.
使用流细胞测量来将探针吸收与蛋白质表达相关联,从而能够测量Kd.

主要成果:

使用蛋白激酶C (PKC) 异酶和已知的合物如博,PDBu和布里奥斯1进行了FPCBA的验证.
对HEK293T细胞表达的全长PKC的相互作用确定了细胞Kd和Ki值.
观察到的一些PKC小分子对的细胞选择性与已知的生物化学值不同,突出显示了细胞环境的重要性.

结论:

FPCBA提供了一种可靠的方法来测量小分子与细胞环境中的原生蛋白质的结合性.
这种测定有助于量化药物向相互作用,有助于优化候选药物.
这些发现强调了细胞环境在确定药物选择性和疗效方面的重要性.

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