Synthesis of an iberiotoxin derivative by chemical ligation: A method for improved yields of cysteine-rich scorpion toxin peptides

Abstract

Automated and manual solid phase peptide synthesis techniques were combined with chemical ligation to produce a 37-residue peptide toxin derivative of iberiotoxin which contained: (i) substitution of Val 16 to Ala, to facilitate kinetic feasibility of native chemical ligation, and; (ii) substitution of Asp 19 to orthogonally protected Cys-4-MeOBzl for chemical conjugate derivatization following peptide folding and oxidation. This peptide ligation approach increased synthetic yields approximately 12-fold compared to standard linear peptide synthesis. In a functional inhibition assay, the ligated scorpion toxin derivative, iberiotoxin V16A/D19-Cys-4-MeOBzl, exhibited 'native-like' affinity (K d = 1.9 nM) and specificity towards the BK Ca 2+ -activated K + Channel (K Ca 1.1). This was characterized by the rapid association and slow dissociation rates (k on = 4.59 × 10 5 M −1 s −1 ; k off = 8.65 × 10 −4 s −1 ) as determined by inhibition of macroscopic whole-cell currents of cloned human K Ca 1.1 channel. These results illustrate the successful application of peptide chemical ligation to improve yield of cysteine-rich peptide toxins over traditional solid phase peptide synthesis. Native chemical ligation is a promising method for improving production of biologically active disulfide containing peptide toxins, which have diverse applications in studies of ion-channel function.

KCa3.1 channels facilitate K+ secretion or Na+ absorption depending on apical or basolateral P2Y receptor stimulation

“…Previous studies of K + secretion in colon and renal epithelia have identified K Ca 1.1 (BK) channels as a predominant Ca 2+ ‐activated conductive pathway mediating K + efflux across the apical membrane (Sausbier et al 2006; Palmer & Frindt, 2007; Heitzmann & Warth, 2008; Sorensen et al 2010). To determine whether K Ca 1.1 channels were responsible for the I sc response evoked by UTP/ATP‐γ‐S in HME cells, the effects of two well‐characterized toxins (iberiotoxin and CTX) known to block BK channels were tested (Castle et al 1989; Kaczorowski et al 1996; Sorensen et al 2008; Bingham et al 2009). The results showed that iberiotoxin had no significant effect on the decrease in I sc , whereas CTX inhibited more than 90% of the P2Y receptor‐activated current.…”

t‐boc synthesis of huwentoxin‐i through native chemical ligation incorporating a trifluoromethanesulfonic acid cleavage strategy

“…Upon peptidyl‐resin reduction, the resin was flow‐washed with DMF (1 min, ×2) and coupled with Boc‐Cys(4‐MeOBzl) (40 min), as described above. The resulting pre‐loaded Boc‐Cys(4‐MeOBzl) thioester linker resin (Boc‐Cys(4‐MeOBzl)‐MPAL resin) was then used in the production of N‐ terminal peptide fragments for NCL at ∼0.5 mMole scale …”

Conotoxins and their Regulatory Considerations

“…A prominent example of this is ω-conotoxin MVIIA, which has been cited as a probe in over 2,000 scientific publications [16]. The potential for activity and structural investigation is being fostered by the ligation of visually traceable media such as biotin and fluorophores [41]. Many examples of the usefulness of conotoxins as probes exist in literature and include receptor subtypes in calcium channels, potassium channels, sodium channels, 5-HT 3 receptors, NMDA receptors and more [6,42].…”