Regulation of arterial diameter through specific sensing of endogenous steroids and novel nonsteroidal analogs by BK channel subunits

通过 BK 通道亚基对内源性类固醇和新型非类固醇类似物的特异性感应来调节动脉直径

• 批准号: 9894850
• 负责人: ALEX M. DOPICO
• 金额: $ 59.97万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-20 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9894850
• 关键词: Address; Affect; Aging; Agonist; Aldosterone; Amino Acids; Animal Disease Models; Area; Arteries; Award; Binding; Biological; Biological Assay; Blood; Blood Circulation; Blood Vessels; Blood flow; Brain; Caliber; Cerebrovascular Spasm; Cerebrum; Chemicals; Cholesterol; Complement; Complex; Computer Models; Computing Methodologies; Consumption; Coupled; Data; Databases; Development; Dilator; Disease; Disease model; Docking; Electrophysiology (science); Endothelium; Engineering; Ensure; Epigenetic Process; Estradiol; Evaluation; Exhibits; Feedback; Genetically Engineered Mouse; Genomics; Health; Hormonal; Hormones; Human; Hydrogen Bonding; Hypertension; Hypertensive Encephalopathy; Impairment; In Vitro; Inbred SHR Rats; Ion Channel; Ion Channel Protein; Knowledge; Lead; Ligands; Lipids; Mammals; Manuals; Mediating; Membrane; Mesenteric Arteries; Mesentery; Methods; Minor; Modeling; Modeling of Functional Interactions; Modification; Molecular; Muscle Contraction; New Agents; Oranges; Organ; Pathologic; Pathology; Perfusion; Peripheral; Pharmaceutical Chemistry; Pharmaceutical Preparations; Pharmacology; Physiological; Physiology; Potassium Channel; Pregnanolone; Process; Progesterone; Protein Subunits; Proteins; Publications; Publishing; Recombinants; Regulation; Robotics; Science; Signal Transduction; Site; Smooth Muscle; Smooth Muscle Myocytes; Steroid Receptors; Steroids; Stroke; Structural Models; Structure; Tail; Testing; Therapeutic; Therapeutic Intervention; Time; Tissues; Translations; Transmembrane Domain; Vascular Smooth Muscle; Vascular resistance; Vasodilation; Vasodilator Agents; Voltage-Gated Potassium Channel; analog; base; basilar artery; biophysical chemistry; cerebrovascular pathology; dehydroepiandrosterone; design; differential expression; experimental study; in vivo; kinetic model; large-conductance calcium-activated potassium channels; mRNA Differential Displays; middle cerebral artery; mutant; neurosteroids; non-genomic; novel; paracrine; patch clamp; pharmacophore; receptor; relating to nervous system; response; screening; selective expression

Rapid, nongenomic effects of endogenous steroids (STs), such as regulation of local artery diameter, have been classically attributed to steroid receptors coupled to downstream signaling. Whether endogenous steroids that reach nM-μM levels in circulation (hormones) or locally (paracrine neurosteroids) can regulate artery diameter by steroid-ion channel protein direct interactions remains largely unknown. Ion channel function often results from concerted actions of channel-forming and regulatory subunits. Smooth muscle (SM)-abundant β1 subunits associate with channel-forming α to increase the Ca2+-sensitivity of Ca2+/voltage-gated K+ channels (BK), which allows arterial SM BK to feedback on depolarization-driven Ca2+ influx and promote artery dilation. Aim 1 (lipid Molecular Pharmacology and vascular resistance Physiology) tests this central hypothesis: endogenous vasoactive STs regulate arterial diameter independently of ST receptors but through direct sensing of ST by specific sites in α and β1 subunits. Based on previous publications with cholesterol and lithocholate, as well as preliminary data, we will apply computational modeling and patch-clamp on recombinant channels expressed in SM cells from genetically engineered mice, to identify the molecular interactions between selected STs and specific BK amino acids, plus diameter evaluation in vessels of pathophysiological relevance: middle cerebral, basilar and mesenteric arteries, which express different levels of β1. In parallel, Aim 2 (ion channel Biophysics and Medicinal Chemistry) combines pharmacophore optimization, compound prioritization+ functional assays with robotic patch-clamp, complex gating analysis, and computational docking to obtain two distinct classes of novel β1-targeting nonsteroidal analogs (NSTAs) that present different structural basis of interaction with the protein and thus either activate β1-containing BK or oppose this action. Finally, knowledge obtained with aims 1 and 2 will be integrated into Aim 3 (Translation Science), where we will demonstrate that the novel NSTAs that act as either agonists or antagonists on β1-containing BK respectively behave either as endothelium-independent, artery dilators or oppose this action, with different arteries displaying differential sensitivity based on β1 expression. We will further integrate the aims by showing that selective, new agents either counteract or synergize the vascular actions of endogenous STs. Finally, the efficacy of the new agents will be tested in a model of disease where the arterial tone is increased, i.e., the spontaneously hypertensive rat. Our focus on brain and peripheral arteries is most relevant to the therapy of human conditions that affect both vessels (e.g., hypertensive encephalopathy). β1-based ligands that act independently of the endothelium will be particularly useful when endothelial function is impaired, such as with ageing or stroke. The combination of computational methods with electrophysiology on different point mutants as a strategy opposed to time-consuming structural methods, the use of high-throughput patch-clamp, and the feasibility of all experiments in our hands ensure the successful evaluation of several sites and ligands in the timeframe of an R01 award.

内源性类固醇（STS）的快速，非原位作用，例如局部动脉直径的调节 经典归因于类固醇受体与下游信号传导结合的。在循环中达到Nm-μm水平的内源性类固醇是否能否通过类固醇-ION通道蛋白直接相互作用调节动脉直径，这在很大程度上尚不清楚。离子通道函数通常是由于通道形成和调节亚基的一致作用而产生的。平滑肌（SM）丰富的β1亚基与通道形成的α相关，以增加Ca2+/电压门控的K+通道（BK）的Ca2+ - 敏感性，这使动脉SMK可以反馈对沉积驱动的CA2+ CA2+影响和促进动脉词典。 AIM 1（脂质分子药理学和血管耐药性生理）检验了此中心假设：内源性血管活性STS调节动脉直径，独立于ST受体，而通过α和β1亚基中特定位点的ST直接敏感性。基于先前的胆固醇和岩石盐的出版物以及初步数据，我们将应用计算建模和斑块夹在从基因工程小鼠的SM细胞中表达的重组通道上，以识别所选STS和特定BK氨基酸之间的分子相互作用，以及在碱性含量和质量质量相关的碱性和转移的碱性相关的碱性含量： β1。 In parallel, Aim 2 (ion channel Biophysics and Medicinal Chemistry) combines pharmacophore optimization, compound prioritization+ functional assays with robotic patch-clamp, complex gating analysis, and computational docking to obtain two distinct classes of novel β1-targeting nonsteroidal analogs (NSTAs) that present different structural basis of interaction with the protein and thus either activate β1-containing BK or oppose this action.最后，用目标1和2获得的知识将集成到AIM 3（翻译科学）中，我们将证明，在含β1的BK的激动剂或拮抗剂上，新颖的NSTA在含β1的BK上分别表现出独立的内皮，动脉扩张剂，或反对这种动作，或反对这种动脉，并以不同的动脉表现出基于β1表达的不同动脉。我们将通过证明选择性的新代理抵消或协同内源性ST的血管作用来进一步整合目标。最后，新药物的有效性将在疾病模型中进行测试，在该模型中，动脉张力增加，即赞助者样高血压大鼠。我们对大脑和周围动脉的关注与影响两种血管的人类疾病的治疗最相关（例如高血压性脑病）。当hothothothium的功能受损时，例如衰老或中风，基于β1的基于独立于原始植物的配体将特别有用。在不同点突变体上的计算方法与电生理学的组合是一种策略，该策略与耗时的结构方法，使用高通量贴片夹的使用以及所有实验在我们手中的可行性，确保在R01奖的时限中成功评估几个地点和配体。