Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
Curr Protoc. 2021 Jan;1(1):e17. doi: 10.1002/cpz1.17.
Fungi infect over a billion people worldwide and contribute substantially to human morbidity and mortality despite all available therapies. New antifungal drugs are urgently needed. Decades of study have revealed numerous protein targets of potential therapeutic interest for which potent, fungal-selective ligands remain to be discovered and developed. To measure the binding of diverse small molecule ligands to their larger protein targets, fluorescence polarization (FP) can provide a robust, inexpensive approach. The protocols in this article provide detailed guidance for developing FP-based assays capable of measuring binding affinity in whole cell lysates without the need for purification of the target protein. Applications include screening of libraries to identify novel ligands and the definition of structure-activity relationships to aid development of compounds with improved target affinity and fungal selectivity. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Use of saturation binding curves to optimize tracer and lysate protein concentrations Basic Protocol 2: Establishment of competition binding experiments Support Protocol 1: Preparation of fungal cell lysates Support Protocol 2: Preparation of human HepG2 cell lysate.
真菌在全球范围内感染了超过 10 亿人，尽管有所有可用的治疗方法，但它们仍是导致人类发病率和死亡率的主要原因。急需新型抗真菌药物。数十年的研究揭示了许多具有潜在治疗意义的蛋白质靶标，这些靶标仍有待发现和开发具有强大的真菌选择性配体。为了测量各种小分子配体与较大蛋白质靶标的结合，荧光偏振（FP）可以提供一种强大、廉价的方法。本文中的方案提供了详细的指导，用于开发基于 FP 的测定法，这些测定法能够在无需纯化靶蛋白的情况下在全细胞裂解物中测量结合亲和力。应用包括筛选文库以鉴定新型配体和确定结构-活性关系，以帮助开发具有改善的靶亲和力和真菌选择性的化合物。© 2021 威利父子公司。基本方案 1：使用饱和结合曲线优化示踪剂和裂解物蛋白浓度基本方案 2：建立竞争结合实验支持方案 1：真菌细胞裂解物的制备支持方案 2：人 HepG2 细胞裂解物的制备。
