ABSTRACT Ion channels are water-filled pores that mediate the passage of ions across cell membranes, thereby contributing to a multitude of essential cellular functions. Though many currently available drugs work via interaction with ion channels, the vast majority of ion channels lack the potent and selective small molecule tool compounds necessary to properly study their therapeutic potential. The Slack channel, encoded by KCNT1, is a member of the Slo family of potassium channels; this family of channels has a diversity of roles throughout the body, notably including the regulation of neuronal activity. Recently, it was discovered that an RNA-binding protein known as Fragile X mental retardation protein (FMRP) binds to and activates Slack channels. Gene silencing of fragile X mental retardation type 1 (FMR1) leads to an absence of FMRP and causes fragile X syndrome (FXS), an X- linked disorder that is the most common known cause of inherited intellectual disability (ID) and monogenic autism spectrum disorder (ASD). Studies in animals have shown that Slack activity is critical for certain types of higher cognitive function. Thus, we hypothesize that diminished Slack activity in FXS patients due to the absence of FMRP contributes to the ID associated with the disease, and normalization of Slack activity via pharmacotherapy represents a potential treatment approach. Unfortunately, there are presently no known potent and selective small molecule Slack activator probes available to investigate this hypothesis. Studies with such probes represent an essential, early step in helping to validate this novel approach to FXS therapy. The proposed research will employ a medicinal chemistry hit optimization approach to develop structure activity relationships (SAR) in two distinct chemical scaffolds that we discovered through a high-throughput screening (HTS) approach. We will assess the Slack activator potency and efficacy of our newly synthesized compounds using an HTS-compatible Tl+ flux assay and the “gold standard” method for measuring ion channel activity, voltage- clamp electrophysiology. We will test the capacity of our compounds to increase the activity of Slack channels in Slack-expressing HEK-293 cells where FMRP expression has been knocked out as well as in neurons isolated from Fmr1-/y mice. We will evaluate the selectivity of compounds versus Slo family members, other ion channels, and ultimately a broad panel of molecular targets. We will also monitor our compounds with regard to solubility, membrane permeability, and potential for cellular efflux to ensure that our probes will not be limited by poor attributes in these areas. To reach our goal efficiently, we have designed a thoughtful critical path that can serve us with compound progression decisions and ensure a holistic evaluation of compounds. Completion of our aims will enable us to reach our end goal of developing high quality Slack activator cell-based probes that may be used by the ...