Development of Kv3.1 potentiators for correcting fast-spiking-interneuron hypofunction in schizophrenia and autism spectrum disorder

摘要 损害神经回路连通性发展的基因突变或环境侮辱可能导致 自闭症谱系障碍(ASD)和精神分裂症，它们共同影响生活质量、独立性和 数百万美国人的生产力，每年花费数千亿美元。自闭症与精神分裂症 与兴奋性与抑制性(E/I)突触传递的比率增加有关，增加了 能够恢复E/I平衡的药物可以治疗这两种疾病。GABA能小白蛋白- 表达快脉冲中间神经元(PV-ins)在调节纹状体网络抑制性输出中起关键作用 协调认知、感觉信息处理、运动的高频振荡 行为，以及在自闭症和精神分裂症中经常被干扰的行为。PV-INS的着火能力 高频动作电位(AP)依赖于电压门控钾(K)的表达。 通道KV3.1，其表达在很大程度上仅限于PV-ins。KV3.1中的从头突变与 人类患有自闭症。越来越多的遗传学、数学模型和药理学证据 强烈提示KV3.1通道门控的小分子增强剂/激活剂可促进PV-IN的放电。 抑制性输出和E/I平衡。然而，缺乏有效和特异的KV3.1通道增强剂 合适的药物代谢和药代动力学(DMPK)特性减缓了对 KV3.1对ASD和精神分裂症的治疗潜力在这里，我们建议使用一个分子靶标- 基于药物发现的方法开发2-3种最先进的KV3.1通道增强剂并将其用于 ASD小鼠模型，以评估其恢复PV-IN兴奋性的能力。在目标1中，我们将使用完整的 开发并验证了基于荧光的高通量筛选(HTS)方法，以询问 来自范德比尔特化学生物学研究所图书馆的大约100,000个化合物用于新的KV3.1 增效剂。荧光和自动膜片钳电生理分析将被用来识别有效的， 用于进一步开发的选择性的、化学上易处理的化合物。在目标2中，药物的迭代周期 将使用化学和功能分析来优化DMPK的效力、选择性和体外性质 新型KV3.1增效剂。目标3中概述的研究将使用小鼠脑片电生理学来 评价优化的KV3.1增强剂恢复伏隔核和前核PV-IN兴奋性的能力 额叶皮质。我们将具体描述新开发的KV3.1增强剂对基因的影响 确定了PV-IN电流-电压关系、AP波形和触发频率。这种高风险/高回报 该提案将为从药物上调节PV-IN兴奋性和 抑制输出，批判性评估KV3.1调节E/I平衡的治疗价值，并潜在地 影响ASD和精神分裂症的临床治疗。

SUMMARY Genetic mutations or environmental insults that impair development of neural circuit connectivity can lead to autism spectrum disorder (ASD) and schizophrenia, which together affect the quality of life, independence, and productivity of millions of Americans and cost hundreds of billions of dollars annually. ASD and schizophrenia are associated with an increased ratio of excitatory-to-inhibitory (E/I) synaptic transmission, raising the possibility that drugs that are capable of restoring E/I balance could treat both disorders. GABAergic parvalbumin- expressing fast-spiking interneurons (PV-INs) play critical roles in regulating inhibitory output in striatal networks and coordinating high-frequency oscillations underlying cognition, sensory information processing, motor behavior, and behavior, which are frequently disrupted in ASD and schizophrenia. The ability of PV-INs to fire high-frequency action potentials (APs) is dependent on the expression of the voltage-gated potassium (K+) channel Kv3.1, whose expression is largely restricted to PV-INs. De novo mutations in Kv3.1 are associated with ASD in humans. A growing body of genetic, mathematical modeling, and pharmacological evidence strongly suggests that small molecule potentiators/activators of Kv3.1 channel gating could promote PV-IN firing, inhibitory output, and E/I balance. However, the dearth of potent and specific Kv3.1 channel potentiators with suitable drug metabolism and pharmacokinetic (DMPK) properties has slowed efforts to critically evaluate the therapeutic potential of Kv3.1 in treating ASD and schizophrenia. Here, we propose to employ a molecular target- based drug discovery approach to develop 2-3 state-of-the-art Kv3.1 channel potentiators and then use them in a mouse model of ASD to evaluate their ability to restore PV-IN excitability. In Aim 1, we will employ a fully developed and validated fluorescence-based high-throughput screening (HTS) assay to interrogate approximately 100,000 compounds from the Vanderbilt Institute of Chemical Biology library for novel Kv3.1 potentiators. Fluorescence and automated patch clamp electrophysiology assays will be used to identify potent, selective, and chemically tractable compounds for further development. In Aim 2, an iterative cycle of medicinal chemistry and functional assays will be used to optimize the potency, selectivity, and in vitro DMPK properties of novel Kv3.1 potentiators. The studies outlined in Aim 3 will employ mouse brain slice electrophysiology to evaluate the ability of optimized Kv3.1 potentiators restore PV-IN excitability in the nucleus accumbens and pre- frontal cortex. We will specifically characterize the effects newly developed Kv3.1 potentiators on genetically identified PV-IN current-voltage relationships, AP waveform, and firing frequency. This high-risk/high-reward proposal will create unprecedented opportunities for pharmacologically modulating PV-IN excitability and inhibitory output, critically evaluating the therapeutic value of Kv3.1 for modulating E/I balance, and potentially impacting the clinical treatment of ASD and schizophrenia.