Hits were identified through high-throughput screening and optimized by in silico design. Cyclic GMP was measured in QBIHEK293A cells expressing GC-A, GC-B or chimerae of the two receptors using AlphaScreen technology. Binding assays were performed in membrane preparations or whole cells using 125 I-ANP. Vasorelaxation was measured in aortic rings isolated from Wistar rats.
We have identified small molecular allosteric enhancers of GC-A, which enhanced ANP or BNP effects in cellular systems and ANP-induced vasorelaxation in rat aortic rings. The mechanism of action appears novel and not mediated through previously described allosteric binding sites. In addition, the selectivity and activity depend on a single amino acid residue that differs between the two similar receptors GC-A and GC-B.
We describe a novel allosteric binding site on GC-A, which can be targeted by small molecules to enhance ANP and BNP effects. These compounds will be valuable tools in further development and proof-of-concept of GC-A enhancement for the potential use in cardiovascular therapy.
方法:通过高通量筛选鉴定并通过计算机模拟设计进行优化。在表达GC-A的QBIHEK293细胞中进行环GMP测量,使用AlphaScreen技术的GC-B或两种受体的嵌合体。使用125I-ANP在膜制备物或全细胞中进行结合测定。在从Wistar大鼠分离的主动脉环中测量血管舒张。
结果:我们已经确定了GC-A的小分子变构增强剂,增强ANP或BNP在细胞系统中的作用以及ANP诱导的大鼠主动脉环血管舒张。作用机制似乎是新颖的,不是通过先前描述的变构结合位点介导的。此外,选择性和活性取决于两个相似受体GC-A和GC-B之间不同的单个氨基酸残基。
结论:我们描述了GC-A上的一种新型变构结合位点,可以通过增强ANP和BNP作用的小分子靶向。这些化合物将是进一步开发和验证GC-A增强在心血管治疗中的潜在用途的有价值的工具。