Optimization of KCa2 Channel Activators as Neuroscience Tools and Potential Drugs

描述(申请人提供)：小电导钙激活钾通道由KCa2.1-2.3(=SK1-3)基因编码，最为人所知的是神经元中对阿帕明敏感的中等超极化电流(MAHP)。根据神经元类型的不同，KCa2通道的功能也不同，从确定瞬时放电频率、设置紧张放电频率到调节猝发放电和潜在的儿茶酚胺释放。因此，KCA通道的药理调节提供了显著影响神经元兴奋性的机会。虽然像蜂毒阿帕明这样的钾通道阻滞剂可以增加啮齿动物的放电频率并诱导癫痫发作，但钾钙通道激活剂会减缓神经元的放电，因此已被建议用于治疗以高度兴奋为特征的中枢神经系统疾病，如癫痫、共济失调和神经病理性疼痛。然而，这一令人信服的治疗假说目前仍在很大程度上未经检验，因为现有的钾通道激活剂如eBIO(EC50 300；M)或NS309都不适合在体内使用。以神经保护药物利鲁唑为合成模板，本实验室最近设计了第一个可在体内使用的钾通道激活剂SKA-31(EC50 2 Um)，并与美国国立卫生研究院抗惊厥筛选计划(ASP)合作证明该化合物及其几个衍生物是有效的抗惊厥药物。不幸的是，SKA-31还激活了表达在血管内皮细胞上的KCa3.1通道，从而降低了小鼠的血压。利用经典药物化学和自动化和人工电生理学相结合的方法，我们打算进一步探索SKA31和eBIO周围的构效关系，以提高KCa3.1对KCa2的选择性以及效力和脑渗透。然后，将评估最好的新KCa2激动剂对一组克隆离子通道的选择性，并使用海马片表征其在天然KCa2通道上的活性。选择性地激活克隆的和天然的钾离子通道的化合物将进一步利用高效液相色谱/质谱仪评估其在大鼠体内的药代动力学特性和脑渗透性。同时，我们将把选定的化合物提交给ASP，在那里我们将在急性癫痫模型中测试这些化合物。然后，有希望的化合物将在杏仁核点燃的红藻氨酸诱导的癫痫小鼠和大鼠身上进行测试，这两个模型更能代表人类难治性癫痫。脑渗透性和潜在亚型选择性钾通道激动剂的设计将有助于确认钾通道作为治疗癫痫的新的药理靶点，并将进一步为科学界提供工具化合物来研究钾通道在共济失调、神经病理性疼痛和认知中的作用。 公共卫生相关性：项目叙述钾钙通道在决定神经元兴奋性方面起着重要作用。因此，这些通道的激活剂被认为是治疗癫痫和共济失调等以神经元过度兴奋为特征的疾病的新疗法。在这笔资金的帮助下，我们将尝试设计一种钾离子通道激活剂，它具有足够的效力和选择性，可以用作科学工具化合物，甚至可以开发成药物。

DESCRIPTION (provided by applicant): Small-conductance calcium activated potassium channels are encoded by the KCa2.1-2.3 (= SK1-3) genes and are best known for underlying the apamin-sensitive medium afterhyperpolarization current (mAHP) in neurons. Depending on the type of neuron, the function of KCa2 channels varies from determining instantaneous firing rates, over setting tonic firing frequencies, to regulating burst firing and potentially catecholamine release. Pharmacological modulation of KCa channels therefore offers the opportunity to significantly affect neuronal excitability. While KCa2 channel blockers like the bee venom apamin increase firing rates and induce seizures in rodents, KCa2 channel activators slow down neuronal firing and have therefore been proposed for the treatment of CNS disorders that are characterized by hyperexcitability such as epilepsy, ataxia, and neuropathic pain. However, this compelling therapeutic hypothesis currently remains largely untested because none of the existing KCa2 channel activators such as EBIO (EC50 300 μM) or NS309 are suitable for in vivo use. Using the neuroprotective drug riluzole as a synthetic template, our laboratory recently designed SKA-31 (EC50 2 uM), the first KCa2 channel activator, which is potent enough to be used in vivo, and demonstrated in collaboration with the NIH Anticonvulsant Screening Program (ASP) that the compound and several of its derivatives are effective anticonvulsants. Unfortunately, SKA-31 also activates KCa3.1 channels, which are expressed on vascular endothelium, and thus reduces blood pressure in mice. Using a combination of classical medicinal chemistry and automated and manual electrophysiology we intend to further explore the structure activity relationship around SKA 31 and EBIO in order to improve selectivity for KCa2 over KCa3.1 as well as potency and brain penetration. The best new KCa2 activators will then be evaluated for selectivity over a panel of cloned ion channels and characterized for activity on native KCa2 channels using hippocampal slices. Compounds selectively activating cloned and native KCa2 channels will further be evaluated for pharmacokinetic properties and brain penetration in rats using HPLC/MS. In parallel, we will submit selected compounds to the ASP, where the compounds will we tested in acute seizure models. Promising compounds will then be tested in amygdala kindled mice and rats with kainate-induced epilepsy, two models that are more representative of human refractory epilepsy. The design of brain penetrant and potentially subtype selective KCa2 channel activators would help to validate KCa2 channels as novel pharmacological targets for the treatment of epilepsy and would further provide the scientific community with tool compounds to study the role of KCa2 channels in ataxia, neuropathic pain and cognition. PUBLIC HEALTH RELEVANCE: Project Narrative KCa2 potassium channels play important roles in determining neuronal excitability. Activators of these channels have therefore been suggested as new therapeutics for the treatment of diseases that are characterized by neuronal hyperexcitability such as epilepsy and ataxia. With the help of this grant we will attempt to design a KCa2 channel activator that is potent and selective enough to be used as a scientific tool compound or even to be developed into a drug.