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 ev...