PDF(Pubmed)
Abstract：
Guanylyl cyclase-A (GC-A), activated by endogenous atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), plays an important role in the regulation of cardiovascular and renal homeostasis and is an attractive drug target. Even though small molecule modulators allow oral administration and longer half-life, drug targeting of GC-A has so far been limited to peptides. Thus, in this study we aimed to develop small molecular activators of GC-A.
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.
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.
摘要：
目的：鸟苷酸环化酶-A（GC-A），内源性心房利钠肽（ANP）和B型利钠肽（BNP）激活，在心血管和肾脏稳态的调节中起着重要作用，是一个有吸引力的药物靶标。即使小分子调节剂允许口服给药和更长的半衰期，迄今为止,GC-A的药物靶向限于肽。因此，在这项研究中，我们旨在开发GC-A的小分子活化剂。
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。
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。
