Discovery and Structure-Based Optimization of Benzimidazole-Derived Activators of Slack Potassium Channels

Purpose: Fragile X syndrome (FXS) is a genetic disorder caused by the absence of Fragile X Mental Retardation Protein (FMRP) in neurons resulting in intellectual disability, behavioral, and learning challenges. FMRP is an RNA-binding protein that also interacts with numerous cytoplasmic and nuclear proteins. Preclinical studies have shown that FMRP binds the C-terminus of the sodium-activated potassium channel Slack, to modulate electrical activity in the brain. This finding was validated by biochemical and electrophysiological studies which confirmed that sodium-activated potassium currents in neurons are decreased in Fmr1-knockout versus Slack WT mice.

Thus, evidence suggests that the deficiency of FMRP in FXS may affect the physiological role of Slack in regulating neuronal excitability, contributing to cognitive dysfunction associated with FXS. We therefore hypothesize that Slack dysfunction can be restored with small molecules in the treatment of intellectual disability associated with neurodevelopmental disorders.

Our research has identified the hit compound VU0519388 (VU388), as a moderately potent Slack activator. Using medicinal chemistry strategies, we seek to generate analogs with enhanced Slack activity, which may serve as valuable tools to characterize the pathophysiological role of Slack in FXS.

Method: Our approach involved identifying multiple regions of VU388that could be readily diversified and designing short efficient synthetic routes used to produce small libraries of analogs. Structure and purity of all analogs were confirmed using spectra obtained from a Bruker Fourier 300HD NMR spectrometer and an Agilent 6230 time-of-flight LC/MS. Cellular activity was then evaluated using a Tl+ flux assay in HEK-293 cells that stably express wild-type (WT) Slack channels.

Results: Each region of the VU388 scaffold proved tolerant of modification to some degree. Substitution with a variety of electron withdrawing and donating groups at various positions on the western benzimidazole ring and eastern aryl ring produced analogs with superior potency relative to VU388. Monosubstitution on the eastern aryl ring was well tolerated compared to disubstitution.

Conclusion: Our systematic optimization plan has identified clear structure-activity relationships and multiple Slack activator analogs with improved activity relative to VU388. Multiple regions of the scaffolds are amenable to SAR development, which greatly enhances the probability of reaching our goal of highly optimized probes. Additional modifications that combine optimal features may provide additional SAR and analogs with optimal potency for use as a molecular probe.