Pharmaceutical Optimization of Peptide Toxins for Ion Channel Targets: Potent, Selective, and Long-Lived Antagonists of Kv1.3

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.

Prolonged immunomodulation in inflammatory arthritis using the selective Kv1.3 channel blocker HsTX1[R14A] and its PEGylated analog

“…Such a change in affinity was expected as attaching large moieties to ion channel modulating peptides is known to significantly reduce or even eliminate their affinity for their target. For example, fusing OsK1, OdK2, or ShK with Fc fragments of immunoglobulins, with PEG moieties, or with albumin reduced their affinity for Kv1.3 channels to varying degrees [40; 63]. A thioredoxin fusion of ShK or a biotin-tagged ShK bound to streptavidin had no effect on Kv1.3 currents at 100 nM, whereas ShK itself blocks the channel with low picomolar affinity [44; 64].…”

“…Selective conjugation was directed to the N-terminus of the HSTX1[R14A] via reductive alkylation with an activated 30 kD monomethoxy-PEG-aldehyde, similar to methods used to make N-terminally PEGylated ShK [40]. The conjugation reaction was performed at a concentration of 2 mg/mL in 50 mM NaH 2 PO 4 , pH 4.5, with a two-fold excess of 30 kD-monomethoxy-PEG-aldehyde (NOF Laboratories, Tokyo, Japan).…”

Sustained inhibition of the Na V 1.7 sodium channel by engineered dimers of the domain II binding peptide GpTx-1

“…These solvent-exposed sites can be extremely important to the engineering of desirable pharmaceutical properties into peptide toxins. 9 Here we relate our insights and advances via derivatization of sites on the presumed non-binding face of a Na V 1.7 inhibitory peptide.…”

“…10a However, significant pharmacokinetic challenges remain, including an expected short in vivo half-life due to rapid elimination by renal filtration. 9 A variety of strategies has been employed to increase the circulating half-life of therapeutic peptides, such as conjugation to a benign chemical moiety with a higher molecular weight and larger hydrodynamic radius. For any of these techniques to be successful, the peptide warhead must be derivatized without impacting the desired potency.…”

“…For any of these techniques to be successful, the peptide warhead must be derivatized without impacting the desired potency. 9 As a part of initial exploratory SAR for future pharmacokinetic optimization, we report here the identification of a site within GpTx-1 suitable for derivatization. We chose to focus our search on positions within GpTx-1 whose substitution with Ala did not impact Na V 1.7 potency.…”