Slack channels are sodium-activated potassium channels, and critical regulators of electrical activity in the central nervous system. Slack channels are encoded by the KCNT1 gene, and are a member of the Slo family of potassium channels (Slo2.2). KCNT1 gain of function mutations have been linked to the development of malignant migrating partial seizure of infancy (MMPSI), which is a devastating form of infantile epilepsy. The objective of this study is to develop Slack channel inhibitors for use as in vivo tool compounds via an iterative hit optimization approach. A cell-based high throughput screening campaign was run versus a 110K-member library against wild-type Slack and three MMPSI-associated KCNT1 mutants. Structure-activity relationship development studies were performed around the hit VU0531245 (VU245), to optimize its potency and drug-like properties. Slack inhibitory activity of the resulting compounds was evaluated utilizing a thallium flux assay in HEK-293 cell lines stably expressing Slack. The chemical space around VU245 was methodically scanned, incorporating different bioisosteric replacements into the design of compound libraries. We demonstrated that modifications at the 2-position of the phenyl ring A were well tolerated. Alicyclic, fluorinated, and deuterated alkoxy groups were accommodated as ethoxy replacements, furthermore, they improved some DMPK properties. Azetidine and 1,2,4-oxadiazole rings were optimal for activity at positions B and C, respectively. However, other ring systems were tested and demonstrated acceptable potency and efficacy. A phenyl ring at position D was amenable to multiple substituents, and the 4-fluorophenyl improved the metabolic stability, prompting its use as the basis for further SAR elucidation. Finally, a sulfonamide linker appeared to be optimal for Slack inhibitory activity; however, it may represent a metabolic liability and alternative linkers have been pursued. Overall, good passive permeability with no P-gp-mediated efflux has been observed in the series to date; however, the protein binding remains high (fu ~ 0.01–0.04)

Design and discovery of novel slack potassium channel inhibitor in vivo tools: Continued optimization of the hit compound VU0531245

ACS Spring 2022

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