Eps15 (epidermal growth factor receptor pathway substrate 15) homology domain-containing proteins (EHDs) comprise a family of eukaryotic dynamin-related ATPases that participate in various endocytic membrane trafficking pathways. Dysregulation of EHDs function has been implicated in various diseases, including cancer. The lack of small molecule inhibitors which acutely target individual EHD members has hampered progress in dissecting their detailed cellular membrane trafficking pathways and their function during disease. Here, we established a Malachite green-based assay compatible with high throughput screening to monitor the liposome-stimulated ATPase of EHD4. In this way, we identified a drug-like molecule that inhibited EHD4’s liposome-stimulated ATPase activity. Structure activity relationship (SAR) studies indicated sites of preferred substitutions for more potent inhibitor synthesis. Moreover, the assay optimization in this work can be applied to other dynamin family members showing a weak and liposome-dependent nucleotide hydrolysis activity.

中文翻译：

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鉴定针对动力蛋白样 EHD4 的 ATP 酶活性的药物样分子

Eps15（表皮生长因子受体途径底物 15）含同源域蛋白 (EHD) 包含参与各种内吞膜运输途径的真核动力相关 ATP 酶家族。 EHD 功能失调与多种疾病有关，包括癌症。由于缺乏敏锐地针对个体 EHD 成员的小分子抑制剂，阻碍了剖析其详细的细胞膜运输途径及其在疾病期间的功能的进展。在这里，我们建立了一种与高通量筛选兼容的基于孔雀石绿的测定法，以监测 EHD4 的脂质体刺激的 ATP 酶。通过这种方式，我们鉴定了一种抑制 EHD4 脂质体刺激的 ATP 酶活性的药物样分子。结构活性关系（SAR）研究表明了更有效的抑制剂合成的优选取代位点。此外，这项工作中的测定优化可以应用于显示弱且脂质体依赖性核苷酸水解活性的其他动力蛋白家族成员。