ABSTRACT
To realize the medicinal potential of peptide toxins, naturally occurring disulfide-rich peptides, as ion channel antagonists, more efficient pharmaceutical optimization technologies must be developed. Here, we show that the therapeutic properties of multiple cysteine toxin peptides can be rapidly and substantially improved by combining direct chemical strategies with high-throughput electrophysiology. We applied whole-molecule, brute-force, structure-activity analoging to ShK, a peptide toxin from the sea anemone Stichodactyla helianthus that inhibits the voltage-gated potassium ion channel Kv1.3, to effectively discover critical structural changes for 15× selectivity against the closely related neuronal ion channel Kv1.1. Subsequent site-specific polymer conjugation resulted in an exquisitely selective Kv1.3 antagonist (>1000× over Kv1.1) with picomolar functional activity in whole blood and a pharmacokinetic profile suitable for weekly administration in primates. The pharmacological potential of the optimized toxin peptide was demonstrated by potent and sustained inhibition of cytokine secretion from T cells, a therapeutic target for autoimmune diseases, in cynomolgus monkeys.
Subject(s)
Cnidarian Venoms/chemistry , Kv1.3 Potassium Channel/antagonists & inhibitors , Peptides/chemistry , Polyethylene Glycols/chemistry , Animals , CHO Cells , Cnidarian Venoms/pharmacokinetics , Cnidarian Venoms/pharmacology , Cricetulus , Crystallography, X-Ray , Dogs , HEK293 Cells , Humans , Interferon-gamma/blood , Interferon-gamma/metabolism , Interleukin-17/blood , Interleukin-17/metabolism , Interleukin-2/blood , Interleukin-2/metabolism , Kv1.1 Potassium Channel/antagonists & inhibitors , Macaca fascicularis , Male , Mice , Molecular Docking Simulation , Patch-Clamp Techniques , Peptides/pharmacokinetics , Peptides/pharmacology , Rats, Sprague-Dawley , Species Specificity , Stereoisomerism , Structure-Activity Relationship , T-Lymphocytes/drug effects , T-Lymphocytes/metabolismABSTRACT
Clinical human genetic studies have recently identified the tetrodotoxin (TTX) sensitive neuronal voltage gated sodium channel Nav1.7 (SCN9A) as a critical mediator of pain sensitization. Herein, we report structure-activity relationships for a novel series of 2,4-diaminotriazines that inhibit hNav1.7. Optimization efforts culminated in compound 52, which demonstrated pharmacokinetic properties appropriate for in vivo testing in rats. The binding site of compound 52 on Nav1.7 was determined to be distinct from that of local anesthetics. Compound 52 inhibited tetrodotoxin-sensitive sodium channels recorded from rat sensory neurons and exhibited modest selectivity against the hERG potassium channel and against cloned and native tetrodotoxin-resistant sodium channels. Upon oral administration to rats, compound 52 produced dose- and exposure-dependent efficacy in the formalin model of pain.
