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: 10090627
• 负责人: ALEX M. DOPICO
• 金额: $ 60.76万
• 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/10090627
• 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; 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; searchable database; 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（ST）的快速非基因组效应，例如调节局部动脉直径，已被证实 传统上归因于与下游信号传导耦合的类固醇受体。在循环（激素）或局部（旁分泌神经类固醇）中达到 nM-μM 水平的内源性类固醇是否可以通过类固醇离子通道蛋白直接相互作用调节动脉直径，目前仍然很大程度上未知。这是通道形成亚基和调节亚基 (SM) 丰富的 β1 亚基与通道形成 α 相关联以增加 Ca2+ 敏感性的协同作用的结果。 Ca2+/电压门控 K+ 通道 (BK) 允许动脉 SM BK 对去极化驱动的 Ca2+ 内流进行反馈并促进动脉扩张。目标 1（脂质分子药理学和血管阻力生理学）测试了这一中心假设：内源性血管活性 ST 调节动脉。直径与 ST 受体无关，但通过 α 和 β1 亚基中的特定位点直接感测 ST 基于先前关于胆固醇和的出版物。石胆酸盐以及初步数据，我们将对基因工程小鼠的 SM 细胞中表达的重组通道应用计算模型和膜片钳，以确定选定的 ST 和特定 BK 氨基酸之间的分子相互作用，以及病理生理学血管的直径评估相关性：大脑中动脉、基底动脉和肠系膜动脉，表达不同水平的 β1 同时，Aim 2（离子通道生物物理学和药物化学）结合了药效团优化、化合物。使用机器人膜片钳、复杂门控分析和计算对接进行优先级+功能测定，以获得两类不同类别的新型β1靶向非甾体类似物（NSTA），它们呈现与蛋白质相互作用的不同结构基础，从而激活含β1的BK或最后，通过目标 1 和 2 获得的知识将被整合到目标 3（翻译科学）中，我们将证明新颖的 NSTA 可以充当激动剂或含有 β1 的 BK 拮抗剂分别表现为不依赖于内皮的动脉扩张剂或对抗这种作用，不同的动脉根据 β1 的表达表现出不同的敏感性，我们将通过证明选择性的新药物可以抵消或协同作用来进一步整合目标。最后，我们将在动脉张力增加的疾病模型（即自发性高血压大鼠）中测试新药物的功效。大脑和外周动脉与影响两个血管的人类疾病（例如高血压脑病）的治疗最相关，当内皮功能受损（例如衰老或中风）时，独立于内皮发挥作用的基于β1的配体将特别有用。计算方法与电生理学对不同点突变体的结合作为一种相对于耗时的结构方法的策略，高通量膜片钳的使用以及我们手中所有实验的可行性确保了成功。在 R01 奖项的时间内评估几个位点和配体。