D-Maurocalcine, a Pharmacologically Inert Efficient Cell-penetrating Peptide Analogue

Cathy Poillot,Kaouthar Dridi,Hicham Bichraoui,Julien Pêcher,Sebastien Alphonse,Badreddine Douzi,Michel Ronjat,Hervé Darbon,Michel De Waard

doi:10.1074/jbc.m110.104919

Abstract

Maurocalcine has been the first demonstrated animal toxin acting as a cell-penetrating peptide. Although it possesses competitive advantages, its use as a cell-penetrating peptide (CPP) requires that analogues be developed that lack its characteristic pharmacological activity on ryanodine-sensitive calcium channels without affecting its cell-penetrating and vector efficiencies. Here, we present the synthesis, three-dimensional (1)H NMR structure, and activity of D-maurocalcine. We demonstrate that it possesses all of the desired features for an excellent CPP: preserved structure, lack of pharmacological action, conserved vector properties, and absence of cell toxicity. This is the first report of a folded/oxidized animal toxin in its D-diastereomer conformation for use as a CPP. The protease resistance of this new peptide analogue, combined with its efficient cell penetration at concentrations devoid of cell toxicity, suggests that D-maurocalcine should be an excellent vector for in vivo applications.

Highlights

Over the last 15 years, several peptides have been described as possessing the property of accumulating inside cells
If direct translocation is used, the peptide accumulates in the cytoplasm and reaches the nucleus, whereas, if endocytosis is the main pathway of cell entry, peptides end up in late endosomes
While searching for pharmacological agents regulating the activity of ryanodine receptors (RyR), our group came across a cationic toxin of 33-mer, maurocalcine (L-MCa), which was originally isolated and purified from a Tunisian scorpion, Scorpio maurus palmatus [10]

Summary

EXPERIMENTAL PROCEDURES

Products—N-␣-Fmoc-L-amino acid, N-␣-Fmoc-D-amino acid, Wang-Tentagel resin, and reagents used for peptide synthesis were obtained from Iris Biotech. The purity of D-MCa is illustrated by analytical C18 reversed phase HPLC, and the folding/oxidation process is witnessed by the shift in elution time (Fig. 1B). Determination of the Three-dimensional Solution Structure of D-MCa—NMR resonance assignment and structure calculation were performed for D-MCa. The spin systems were identified on the basis of both COSY and TOCSY spectra. To compare the native structure with the D-configuration described, we recalculated the L-configuration by using the previously published NMR data set [32] with the same protocol as for the D-configuration (Fig. 3C) This newly calculated structure of L-MCa is pretty close to the previously published one (r.m.s. deviation: 0.51 Å on the ordered regions) [32].

Other ppm

Hydrogen bonds

Findings

DISCUSSION