Design of a Disulfide-less, Pharmacologically Inert, and Chemically Competent Analog of Maurocalcine for the Efficient Transport of Impermeant Compounds into Cells

Abstract

Maurocalcine is a 33-mer peptide initially isolated from the venom of a Tunisian scorpion. It has proved itself valuable as a pharmacological activator of the ryanodine receptor and has helped the understanding of the molecular basis underlying excitation-contraction coupling in skeletal muscles. Because of its positively charged nature, it is also an innovative vector for the cell penetration of various compounds. We report a novel maurocalcine analog with improved properties: (i) the complete loss of pharmacological activity, (ii) preservation of the potent ability to carry cargo molecules into cells, and (iii) coupling chemistries not affected by the presence of internal cysteine residues of maurocalcine. We did this by replacing the six internal cysteine residues of maurocalcine by isosteric 2-aminobutyric acid residues and by adding an additional N-terminal biotinylated lysine (for a proof of concept analog) or an N-terminal cysteine residue (for a chemically competent coupling analogue). Additional replacement of a glutamate residue by alanyl at position 12 further improves the potency of these analogues. Coupling to several cargo molecules or nanoparticles are presented to illustrate the cell penetration potency and usefulness of these pharmacologically inactive analogs. Maurocalcine (MCa)3 is a highly basic 33-mer peptide isolated from the venom of the scorpion Scorpio maurus palmatus. It efficiently binds to the ryanodine receptor of skeletal muscles (RyR1 isoform) (1) and promotes channel opening to promote calcium release from the sarcoplasmic reticulum (SR). This pharmacological effect of MCa can be indirectly monitored through the stimulation it exerts on [ 3 H]ryanodine binding (2). In muscle fibers, MCa produces a transient loss of voltage control of Ca 2ϩ release from RyR1 channels. This effect is due to an alteration of repolarization-induced closure of RyR1 channels, a process normally under the control of voltage-dependent dihydropyridine (DHP)-sensitive calcium channels (3). This function of MCa is due to a partial sequence homology between MCa and a cytoplasmic loop of the DHP-sensitive channel (2). These observations explain why MCa, along with other members of the same family of toxins such as imperatoxin 1A (4), hemicalcin (5), and opicalcin 1 and 2 (6) are useful both for their pharmacological properties and for deciphering fine molecular details of the excitation-contraction coupling process.Recently, MCa has also proven of interest for its property of efficiently crossing the plasma membrane, either alone or when coupled to a membrane-impermeant cargo protein (2, 7). MCa is a highly charged peptide with 12 basic residues out of 33, and a net global positive charge of ϩ8. Most of these residues are on one face of the molecule, the opposite face being mostly hydrophobic in nature. The rich content of basic amino acid residues of MCa is reminiscent of that of all cell-penetrating peptides (CPPs) characterized so far (Tat, penetratin, and poly-R). Hence, MCa can be classified within an eme...

Small Efficient Cell-penetrating Peptides Derived from Scorpion Toxin Maurocalcine

“…This may represent an inhibitory region because of the presence of Glu 12 and Asp 15 , two negatively charged residues. The finding that mutation of Glu 12 to Ala enhances cell penetration of both MCa F (18) and MCa UF (11) further supports this conclusion. MCa UF25-33 -C turned out to have CPP properties also, even though this sequence did not confer a competitive advantage to other MCa CPP analogues as shown in Fig.…”

“…1B). This peptide loses its secondary structures (11). Because this project aims at identifying shorter CPP sequences based on MCa UF1-33 sequence by the delivery of Cy5 cargo, we first determined where at the N terminus (C-MCa UF1-33 ) or C terminus (MCa UF1-33 -C) the cargo could be best grafted after addition of an extra cysteine residue (C) (Fig.…”

“…Also, the use of peptides with internal disulfide bridges, despite have advantageous features in term of stability in vivo, makes chemical coupling of these CPPs to cargoes more complicated (difficulty to add extra Cys residues to the peptides for instance without interfering with the correct folding process). The third strategy that was used to circumvent one of this criticism was the chemical synthesis of an MCa analog in which all internal Cys residues were replaced by isosteric 2-aminobutyric acid residues (11). The resulting peptide was still 33-mer long but one step in production was saved by avoiding the folding process.…”

Administration de molécules actives dans les cellules

“…Par ailleurs, la maurocalcine partage en commun avec les autres CPP la propriété d'être un peptide basique dont de nombreux acides aminés sont chargés positivement (11 sur 33). De plus, la plupart des acides aminés basiques sont localisés sur une même face de la molécule en accord avec la distribution asymétrique observée sur des molécules telles que Tat et pénétratine [9]. Le Tableau I recense des exemples de divers peptides de pénétration cellulaire, et la Figure 1 illustre l'origine de quelques-uns d'entre eux.…”

Multiple actions of φ-LITX-Lw1a on ryanodine receptors reveal a functional link between scorpion DDH and ICK toxins

“…In addition, calcines are membrane permeable, and this property is attractive for broad therapeutic use and may be exploited to deliver drugs intracellularly. Indeed, a pharmacologically inert, disulfideless analog of maurocalcine has been used as a carrier molecule to transport compounds into cells (24).…”