Discovery of novel 4-phenyl-2-(pyrrolidinyl)nicotinamide derivatives as potent Nav1.1 activators.

The voltage-gated sodium channel, Nav1.1, is predominantly expressed in parvalbumin-positive fast spiking interneurons and has been genetically linked to Dravet syndrome. Starting from a high throughput screening hit isoxazole derivative 5, modifications of 5 via combinations of IonWorks and Q-patch assays successfully identified the nicotinamide derivative 4. Its increasing decay time constant (tau) of Nav1.1 currents at 0.03 µM along with significant selectivity against Nav1.2, Nav1.5, and Nav1.6 and acceptable brain exposure in mice was observed. Compound 4 is a promising Nav1.1 activator that can be used to analyze pathophysiological functions of the Nav1.1 channel towards treating various central nervous system diseases.

Chemical engineering and structural and pharmacological characterization of the α-scorpion toxin OD1.

Scorpion α-toxins are invaluable pharmacological tools for studying voltage-gated sodium channels, but few structure-function studies have been undertaken due to their challenging synthesis. To address this deficiency, we report a chemical engineering strategy based upon native chemical ligation. The chemical synthesis of α-toxin OD1 was achieved by chemical ligation of three unprotected peptide segments. A high resolution X-ray structure (1.8 Å) of synthetic OD1 showed the typical ßαßß α-toxin fold and revealed important conformational differences in the pharmacophore region when compared with other α-toxin structures. Pharmacological analysis of synthetic OD1 revealed potent α-toxin activity (inhibition of fast inactivation) at Nav1.7, as well as Nav1.4 and Nav1.6. In addition, OD1 also produced potent ß-toxin activity at Nav1.4 and Nav1.6 (shift of channel activation in the hyperpolarizing direction), indicating that OD1 might interact at more than one site with Nav1.4 and Nav1.6. Investigation of nine OD1 mutants revealed that three residues in the reverse turn contributed significantly to selectivity, with the triple OD1 mutant (D9K, D10P, K11H) being 40-fold more selective for Nav1.7 over Nav1.6, while OD1 K11V was 5-fold more selective for Nav1.6 than Nav1.7. This switch in selectivity highlights the importance of the reverse turn for engineering α-toxins with altered selectivity at Nav subtypes.