Structure-Activity Relationship Studies in a Series of Xanthine Inhibitors of SLACK Potassium Channels.

Gain-of-function mutations in the KCNT1 gene, which encodes the sodium-activated potassium channel known as SLACK, are associated with the rare but devastating developmental and epileptic encephalopathy known as epilepsy of infancy with migrating focal seizures (EIMFS). The design of small molecule inhibitors of SLACK channels represents a potential therapeutic approach to the treatment of EIMFS, other childhood epilepsies, and developmental disorders. Herein, we describe a hit optimization effort centered on a xanthine SLACK inhibitor (8) discovered via a high-throughput screen. Across three distinct regions of the chemotype, we synthesized 58 new analogs and tested each one in a whole-cell automated patch-clamp assay to develop structure-activity relationships for inhibition of SLACK channels. We further evaluated selected analogs for their selectivity versus a variety of other ion channels and for their activity versus clinically relevant SLACK mutants. Selectivity within the series was quite good, including versus hERG. Analog 80 (VU0948578) was a potent inhibitor of WT, A934T, and G288S SLACK, with IC50 values between 0.59 and 0.71 µM across these variants. VU0948578 represents a useful in vitro tool compound from a chemotype that is distinct from previously reported small molecule inhibitors of SLACK channels.

Design, synthesis, and biological evaluation of a novel series of 1,2,4-oxadiazole inhibitors of SLACK potassium channels: Identification of in vitro tool VU0935685.

Malignant migrating partial seizure of infancy (MMPSI) is a devastating and pharmacoresistant form of infantile epilepsy. MMPSI has been linked to multiple gain-of-function (GOF) mutations in the KCNT1 gene, which encodes for a potassium channel often referred to as SLACK. SLACK channels are sodium-activated potassium channels distributed throughout the central nervous system (CNS) and the periphery. The investigation described here aims to discover SLACK channel inhibitor tool compounds and profile their pharmacokinetic and pharmacodynamic properties. A SLACK channel inhibitor VU0531245 (VU245) was identified via a high-throughput screen (HTS) campaign. Structure-activity relationship (SAR) studies were conducted in five distinct regions of the hit VU245. VU245 analogs were evaluated for their ability to affect SLACK channel activity using a thallium flux assay in HEK-293 cells stably expressing wild-type (WT) human SLACK. Selected analogs were tested for metabolic stability in mouse liver microsomes and plasma-protein binding in mouse plasma. The same set of analogs was tested via thallium flux for activity versus human A934T SLACK and other structurally related potassium channels, including SLICK and Maxi-K. In addition, potencies for selected VU245 analogs were obtained using whole-cell electrophysiology (EP) assays in CHO cells stably expressing WT human SLACK through an automated patch clamp system. Results revealed that this scaffold tolerates structural changes in some regions, with some analogs demonstrating improved SLACK inhibitory activity, good selectivity against the other channels tested, and modest improvements in metabolic clearance. Analog VU0935685 represents a new, structurally distinct small-molecule inhibitor of SLACK channels that can serve as an in vitro tool for studying this target.

Structure-activity relationship studies in a new series of 2-amino-N-phenylacetamide inhibitors of Slack potassium channels.

In this Letter we describe structure-activity relationship (SAR) studies conducted in five distinct regions of a new 2-amino-N-phenylacetamides series of Slack potassium channel inhibitors exemplified by recently disclosed high-throughput screening (HTS) hit VU0606170 (4). New analogs were screened in a thallium (Tl+) flux assay in HEK-293 cells stably expressing wild-type human (WT) Slack. Selected analogs were screened in Tl+ flux versus A934T Slack and other Slo family members Slick and Maxi-K and evaluated in whole-cell electrophysiology (EP) assays using an automated patch clamp system. Results revealed the series to have flat SAR with significant structural modifications resulting in a loss of Slack activity. More minor changes led to compounds with Slack activity and Slo family selectivity similar to the HTS hit.

Heterozygous expression of a Kcnt1 gain-of-function variant has differential effects on SST- and PV-expressing cortical GABAergic neurons.

More than twenty recurrent missense gain-of-function (GOF) mutations have been identified in the sodium-activated potassium (KNa) channel gene KCNT1 in patients with severe developmental and epileptic encephalopathies (DEEs), most of which are resistant to current therapies. Defining the neuron types most vulnerable to KCNT1 GOF will advance our understanding of disease mechanisms and provide refined targets for precision therapy efforts. Here, we assessed the effects of heterozygous expression of a Kcnt1 GOF variant (Y777H) on KNa currents and neuronal physiology among cortical glutamatergic and GABAergic neurons in mice, including those expressing vasoactive intestinal polypeptide (VIP), somatostatin (SST), and parvalbumin (PV), to identify and model the pathogenic mechanisms of autosomal dominant KCNT1 GOF variants in DEEs. Although the Kcnt1-Y777H variant had no effects on glutamatergic or VIP neuron function, it increased subthreshold KNa currents in both SST and PV neurons but with opposite effects on neuronal output; SST neurons became hypoexcitable with a higher rheobase current and lower action potential (AP) firing frequency, whereas PV neurons became hyperexcitable with a lower rheobase current and higher AP firing frequency. Further neurophysiological and computational modeling experiments showed that the differential effects of the Y777H variant on SST and PV neurons are not likely due to inherent differences in these neuron types, but to an increased persistent sodium current in PV, but not SST, neurons. The Y777H variant also increased excitatory input onto, and chemical and electrical synaptic connectivity between, SST neurons. Together, these data suggest differential pathogenic mechanisms, both direct and compensatory, contribute to disease phenotypes, and provide a salient example of how a pathogenic ion channel variant can cause opposite functional effects in closely related neuron subtypes due to interactions with other ionic conductances.

Negative allosteric modulators of group II metabotropic glutamate receptors: A patent review (2015 - present).

INTRODUCTION: Group II metabotropic glutamate (mGlu) receptors have emerged as an attractive potential target for the development of novel CNS therapeutics in areas such as Alzheimer's disease (AD), anxiety, cognitive disorders, depression, and others. Several small molecules that act as negative allosteric modulators (NAMs) on these receptors have demonstrated efficacy and/or target engagement in animal models, and one molecule (decoglurant) has been advanced into clinical trials. AREAS COVERED: This review summarizes patent applications published between January 2015 and November 2020. It is divided into three sections: (1) small molecule nonselective mGlu2/3 NAMs, (2) small molecule selective mGlu2 NAMs, and (3) small molecule selective mGlu3 NAMs. EXPERT OPINION: Much progress has been made in the discovery of novel small molecule mGlu2 NAMs. Still, chemical diversity remains somewhat limited and room for expansion remains. Progress with mGlu3 NAMs has been more limited; however, some promising molecules have been disclosed. The process of elucidating the precise role of each receptor in the diseases associated with group II receptors has begun. Continued studies in animals with selective NAMs for both receptors will be critical in the coming years to inform researchers on the right compound profile and patient population for clinical development.

VU0606170, a Selective Slack Channels Inhibitor, Decreases Calcium Oscillations in Cultured Cortical Neurons.

Malignant migrating partial seizures of infancy is a rare, devastating form of epilepsy most commonly associated with gain-of-function mutations in the potassium channel, Slack. Not only is this condition almost completely pharmacoresistant, there are not even selective drug-like tools available to evaluate whether inhibition of these overactivated, mutant Slack channels may represent a viable path forward toward new antiepileptic therapies. Therefore, we used a high-throughput thallium flux assay to screen a drug-like, 100 000-compound library in search of inhibitors of both wild-type and a disease-associated mutant Slack channel. Using this approach, we discovered VU0606170, a selective Slack channel inhibitor with low micromolar potency. Critically, VU0606170 also proved effective at significantly decreasing the firing rate in overexcited, spontaneously firing cortical neuron cultures. Taken together, our data provide compelling evidence that selective inhibition of Slack channel activity can be achieved with small molecules and that inhibition of Slack channel activity in neurons produces efficacy consistent with an antiepileptic effect. Thus, the identification of VU0606170 provides a much-needed tool for advancing our understanding of the role of the Slack channel in normal physiology and disease as well as its potential as a target for therapeutic intervention.