A designer ligand specific for Kv1.3 channels from a scorpion neurotoxin-based library

Abstract

Venomous animals immobilize prey using protein toxins that act on ion channels and other targets of biological importance. Broad use of toxins for biomedical research, diagnosis, and therapy has been limited by inadequate target discrimination, for example, among ion channel subtypes. Here, a synthetic toxin is produced by a new strategy to be specific for human Kv1.3 channels, critical regulators of immune T cells. A phage display library of 11,200 de novo proteins is designed using the ␣-KTx scaffold of 31 scorpion toxin sequences known or predicted to bind to potassium channels. Mokatoxin-1 (moka1) is isolated by affinity selection on purified target. Moka1 blocks Kv1.3 at nanomolar levels that do not inhibit Kv1.1, Kv1.2, or KCa1.1. As a result, moka1 suppresses CD3/28-induced cytokine secretion by T cells without cross-reactive gastrointestinal hyperactivity. The 3D structure of moka1 rationalizes its specificity and validates the engineering approach, revealing a unique interaction surface supported on an ␣-KTx scaffold. This scaffold-based/target-biased strategy overcomes many obstacles to production of selective toxins.mokatoxin ͉ moka1 ͉ phage display ͉ peptide toxin ͉ animal venom

Designer and natural peptide toxin blockers of the KcsA potassium channel identified by phage display

“…To originally test the strategy of combining structure-based peptide design and the power of phage display, we considered the case of Kv1.3, a channel with known natural peptide toxin ligands that were inadequately specific. Construction and sorting of a library with ∼10 4 native and de novo peptides sharing a scorpion α-KTx scaffold allowed isolation of Moka1, a specific Kv1.3 blocker (1).…”

“…This state of affairs is easily understood; the small amounts of toxins isolated from natural sources makes target identification a challenge and their purpose in the wild does not favor target specificity. Here, we advance our approach to overcoming these problems, that is, creation of expression libraries of toxins allowing cloning based on target binding (1), by seeking a specific, high-affinity ligand for an orphan channel receptor.…”

“…As a proof of concept, we previously addressed a case of inadequate target discrimination by known natural toxins using a library of ∼11,200 peptides designed to share the fold in α-KTx scorpion toxins and a specific ligand for the human voltage-gated potassium channel Kv1.3 was isolated (1). Moka1, composed of domains from three scorpion species, blocks Kv1.3 with nanomolar affinity, allowing it to suppress T-cell-mediated immune responses, and is without unwanted side effects on gastrointestinal motility seen with natural toxins because it does not cross-inhibit Kv1.1 and Kv1.2.…”

Selective Inhibition of CCR7− Effector Memory T Cell Activation by a Novel Peptide Targeting Kv1.3 Channel in a Rat Experimental Autoimmune Encephalomyelitis Model

“…Currently, the newly designed Kv1.3 blocker peptides (e.g. Moka-1 (15), ShK (L5) (5), etc.) were synthesized to target the Kv1.3 channel as a new therapy for MS.…”

Rational design of a Kv1.3 channel-blocking antibody as a selective immunosuppressant

“…Moka1 and Vm24 toxin peptides were synthesized in solid phase by InnoPep. Peptide folding, HPLC purification, and LC-MS validation were performed based on previously published procedures (28,35).…”