Objective

Activated effector memory T-cells (TEM) have been implicated in the pathogenesis of a range of autoimmune diseases including multiple sclerosis and psoriasis. Activated TEM cells express high levels of the voltage-gated potassium channel Kv1.3, which functions to control cell excitability/membrane potential and are upregulated in inflammatory conditions. Inhibition of Kv1.3 reduces the release of pro-inflammatory mediators, inhibits TEM cell proliferation and migration to inflamed tissues, and has been shown to ameliorate disease symptoms in preclinical animal models and human clinical trials. Small molecule Kv1.3 inhibitors have failed to deliver a successful drug, in part due to lack of potency and selectivity. An alternative approach based on identification and peptide engineering of venom toxins has proven more successful, yielding lead compounds with nM potency, greater ion channel selectivity, and improved PK profiles. One such example is ShK-186 (Dalazatide), an optimised analogue of the native Stichodactyla helianthus (ShK) sea anemone neurotoxin originally identified as a potent (but not selective) Kv1.3 inhibitor. ShK-186 is the first in-class Kv1.3 channel inhibitor to show clinical safety and efficacy in a Phase 1b psoriatic arthritis trial run by Kineta. However, most Kv1.3 toxins, including ShK, are only moderately selective over other Kv1.x and KCa family members known to be expressed in T-cells. Using Venomtech’s proprietary Targeted-Venom Discovery Array platform (T-VDA™) and Metrion’s high quality patch clamp electrophysiology assays, we collaborated to identify novel potent and selective Kv1.3 peptide inhibitors as new drug discovery leads for autoimmune and inflammatory disease. Toxin hits could be used as templates for optimisation via mutagenesis and protein engineering (e.g. Murray et al., 2015) or small molecule peptidomimetics approaches, as well as serving as new tools for ligand binding studies to identify new small molecule blockers. From the T-VDA library, 370 venom fractions (5 – 10 peptides) were collected using Venomtech’s protocols and fractionated by HPLC from a variety of spider, scorpion and snake families and tested for their inhibitory effect against human Kv1.3 channels expressed in Chinese hamster ovary cells using QPatchHT automated patch clamp system. Crude venom samples lyophilised in sucrose (5 µM) were reconstituted in water before subsequent serial dilution in extracellular recording solution to a final on cell 500 ng/ml test concentration (~20 – 200 nM based on range of peptide sizes), crude fractions were tested in the presence of the carrier BSA (0.1%) to minimise non-specific binding. ShK (50 pM) and Sucrose (5 µM) were used as positive and negative controls, respectively. The initial screen showed that only scorpion fractions, from Scorpionidae, Buthidae and Caraboctonidae families, were active (> 40% inhibition of Kv1.3 current), with a 1.4% hit rate for all sample species and 26% within the scorpion samples. The active scorpion species were further sub fractionated (1 – 3 peptides) yielding 74 samples of which 6 showed strong inhibition (>80%) of Kv1.3 current at 100 ng /ml test concentration. These samples were submitted for potency determination and assessment of Kv1.x gene family selectivity. Scorpion peptides showed a range of Kv1.3 potency (IC50 = 1.5 – 150 ng /ml) across different families that were similar to ShK-186 (1.59 ± 0.32 ng / ml) with up to 100-fold selectivity against the Kv1.x gene family. In conclusion, Metrion’s validated automated patch clamp electrophysiology assays were able to reliably detect ion channel activity in venom peptide fractions within Venomtech’s T-VDA library. Based on their potency and selectivity these scorpion peptides may offer novel starting points for new therapeutic ligands to modulate Kv1.3 channels in human T-cell disease.