Background:The association of a drug with its target protein correlates to its medicinalactivity and the microenvironment plays a key role in this association. The key challenge is to identifymutations which unlikely to respond to designed drugs.Objective:Hsp70 is an anti-apoptotic factor and tumor cells overexpress Hsp70 to survive againstanti-cancer agents. The impact of pathogenic mutations on Hsp70 is unknown. Elucidation of thesealterations is essential to understand the molecular switch mechanism. Thus, critical spots onHsp70 Nucleotide Binding Domain (NBD) are important since mutation-driven sensitivity may beuseful in designing innovative inhibitors.Methods:ATP, AMP-PNP (non-hydrolyzable analog of ATP) along with commercially availablecompounds VER-155008 (ATP analog and competitive inhibitor) and MKT-077 (allosteric inhibitorof ADP bound form) were docked to Hsp70 NBD structure in silico to identify critical aminoacids of inhibition mechanism. Site-directed mutagenesis of the determined critical residues alongwith ATP hydrolysis and luciferase refolding was performed. Wild-type and mutant Hsp70s werecompared to determine the effect on protein functions in the presence or absence of inhibitors.Results:This study identified three mutants that have a loss of function for Hsp70, which may alterthe drug inhibition activity as oncogenic cells have multiple mutations.Conclusion:Two commercial inhibitors employed here that mimic ATP and ADP states, respectively,are not affected by these mutational perturbations and displayed effective interference forHsp70 functions. Designing inhibitors by considering these critical residues may improve drug designand increase drug efficiency.

中文翻译：

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Hsp70 核苷酸结合域中的关键残基应对药物设计挑战

背景：药物与其靶蛋白的关联与其药用活性相关，而微环境在这种关联中起关键作用。关键的挑战是识别不太可能对设计的药物产生反应的突变。目的：Hsp70是一种抗凋亡因子，肿瘤细胞过表达Hsp70以抵抗抗癌剂。致病突变对 Hsp70 的影响尚不清楚。阐明这些变化对于理解分子开关机制至关重要。因此，Hsp70 核苷酸结合域 (NBD) 上的关键点很重要，因为突变驱动的敏感性可能有助于设计创新的抑制剂。方法：ATP，AMP-PNP（不可水解的 ATP 类似物）与市售化合物 VER-155008（ATP 类似物和竞争性抑制剂）和 MKT-077（ADP 结合形式的变构抑制剂）在计算机上与 Hsp70 NBD 结构对接，以识别抑制的关键氨基酸机制。对确定的关键残基进行定点诱变以及 ATP 水解和荧光素酶重折叠。比较野生型和突变型 Hsp70，以确定在存在或不存在抑制剂的情况下对蛋白质功能的影响。结果：本研究确定了三个 Hsp70 功能丧失的突变体，这可能会改变药物抑制活性，因为致癌细胞有多个突变.结论：这里使用的两种商业抑制剂分别模拟 ATP 和 ADP 状态，不受这些突变扰动的影响，并显示出对 Hsp70 功能的有效干扰。通过考虑这些关键残基来设计抑制剂可以改进药物设计并提高药物效率。