Optimization of KCa2 Channel Activators as Neuroscience Tools and Potential Drugs

描述（由申请人提供）：小电导钙激活钾通道由KCa2.1-2.3 （= SK1-3）基因编码，最著名的是在神经元超极化电流（mAHP）后的阿帕胺敏感介质中。根据神经元类型的不同，KCa2通道的功能从决定瞬时放电速率、设定强直放电频率到调节突发放电和潜在的儿茶酚胺释放不等。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KCa2通道阻滞剂如蜂毒维生素会增加放电率并诱发啮齿动物的癫痫发作，但KCa2通道激活剂会减缓神经元放电，因此被提议用于治疗以过度兴奋性为特征的中枢神经系统疾病，如癫痫、共济失调和神经性疼痛。然而，这一令人信服的治疗假设目前在很大程度上仍未得到验证，因为现有的KCa2通道激活剂如EBIO （EC50 300 &#956；M）或NS309都不适合在体内使用。使用神经保护药物利鲁唑作为合成模板，我们的实验室最近设计了SKA-31 (ec502um)，这是第一个KCa2通道激活剂，它的效力足以在体内使用，并与NIH抗惊厥药物筛选项目（ASP）合作证明了该化合物及其几种衍生物是有效的抗惊厥药物。不幸的是，SKA-31也激活了在血管内皮上表达的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、手动电生理学的结合，我们打算进一步探索SKA 31和EBIO之间的结构活性关系，以提高KCa2对KCa3.1的选择性、效价和脑渗透。最好的新KCa2激活剂将在一组克隆离子通道上进行选择性评估，并使用海马切片对天然KCa2通道的活性进行表征。化合物选择性激活克隆的和天然的KCa2通道将进一步使用HPLC/MS评估药代动力学性质和大鼠脑渗透。同时，我们将向ASP提交选定的化合物，在那里我们将在急性发作模型中测试这些化合物。有希望的化合物将在杏仁核点燃小鼠和大鼠身上进行测试，这两种模型更能代表人类难治性癫痫。脑渗透和潜在亚型选择性KCa2通道激活剂的设计将有助于验证KCa2通道作为治疗癫痫的新药理学靶点，并将进一步为科学界提供工具化合物来研究KCa2通道在共济失调、神经性疼痛和认知中的作用。

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.