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.

Fig. 1

MCa analogues, MCa_b-Abu and MCa_b-Abu E12A. A. Ribbon representation of the 3D solution structure of MCa illustrating the positions of the three disulfide bridges C₁-C₄, C₂-C₅ and C₃-C₆. S-S bonds are shown in blue. The positions of positively charged lysines, essential for cell penetration, are shown in red. B. Differences in side chain between cysteine residues and 2-aminobutyric acid (Abu) used for substitution of all cysteine residues in MCa amino acid sequence. C. Amino acid sequences of three different MCa analogs used in this study. A fourth analog is shown in Fig. 7A. D. Amino acid sequence of MTX_b-Abu, an analog of MTX in which all cysteine residues are replaced by Abu, and an extra biotinylated lysine residue added at the N-terminus. Note that MTX contains six basic amino acid residues in its sequence.

Fig. 2

Determination of the secondary structures of MCa_b, MCa_b-Abu and MCa_b-Abu E12A by circular dichroism. Each spectrum presented is the mean of three independent acquisitions taken at a concentration of 50 μM in pure water at 20°C.

Fig. 3

Effect of 1 μM MCa_b, MCa_b-Abu or MCa_b-Abu E12A on [³H]-ryanodine binding onto heavy SR vesicles. Specific [³H]-ryanodine binding was measured as described under Materials and Methods. Control binding has been performed in the absence of MCa analog.***, p ≤ 0.01. Note the loss of effect upon cysteine replacement by Abu derivatives. The experiment was repeated three times with similar results.

Fig. 4

Effects of MCa_b, MCa_b-Abu and MCa_b-Abu E12A on voltage-activated sarcoplasmic reticulum Ca²⁺ release. A. Indo-1 Ca²⁺ records in response to membrane depolarizations of increasing duration in a control fiber and in fibers dialyzed, respectively, with MCa_b (100 μM), MCa_b-Abu (200 μM) and MCa_b- Abu E12A (200 μM). B, C. Corresponding mean values for peak Δ[Ca²⁺], and final Δ[Ca²⁺] at the end of the record in control fibers (n=11) and in fibers dialyzed with either MCa_b (n=3), MCa_b-Abu (n=10) or MCa_b-Abu E12A (n=3). Note the loss of pharmacological consequences of replacing cysteine by Abu derivatives.

Fig. 5

Distribution of MCa_b/, MCa_b-Abu/and MCa_b-Abu E12A/Strep-Cy5 in CHO cells. A. Confocal images showing the cell penetration of Strep-Cy5 (2 hrs incubation) in the absence or presence of 4 μM MCa_b, MCa_b-Abu or MCa_b-Abu E12A in CHO cells. Colors: blue (Strep-Cy5), red (nuclei, DHE) and green (plasma membrane, concanavalin A). Note the lack of differences in cell distribution between Strep-Cy5 and the MCa analogues used. The punctate distribution is linked to the use of streptavidin as cargo. Scale bars: 10 μm. B. Confocal image of CHO cells showing that 4 μM MTX_b-Abu is unable to deliver Strep-Cy5 inside cells (2 hrs incubation). Color code as in A. Scale bar: 15 μm.

Fig. 6

Mean cell fluorescence intensities (MFI) as a function of the concentration of cell penetrating complexes for each MCa analog. Indicated concentrations are for Strep-Cy5 (10 nM to 2.5 μM). The ratio MCa analog/Strep-Cy5 was 4/1. Data were fitted by a sigmoid equation of the type MFI = MFI_max/(1 + exp(−(x-PC₅₀)/b)) where MFI_max = 180 ± 6 a.u. (MCa_b), 73 ± 4 a.u. (MCa_b-Abu) and 129 ± 8 a.u. (MCa_b-Abu E12A), b = 200 ± 18 (MCa_b), 301 ± 38 (MCa_b-Abu) and 344 ± 64 (MCa_b-Abu E12A), and half-penetration concentration values PC₅₀ = 669 ± 27 nM (MCa_b), 910 ± 51 nM (MCa_b-Abu) and 1042 ± 89 nM (MCa_b-Abu E12A). a.u.: arbitrary units. The MFI values are obtained from a fit of the FACS histograms (n=10,000 events in each case). Representative example of n=3 experiments. Experiments could not be averaged because the different photomultiplier settings were not calibrated.

Fig. 7

Design of a cell-penetrating analog for the efficient coupling and delivery of a variety of cargoes. A. primary amino acid sequence of the Cys-MCa-Abu analog. This analogue is identical to MCa_b-Abu except that the N-terminal K(biot) has been replaced by an N-terminal cysteine residue. B. Cell penetration of FITC-Gpep-Cys when covalently linked to Cys-MCa-Abu (right panel, 1 μM concentration, 2 hrs incubation with CHO cells). No penetration is observed for 1 μM FITC-Gpep-Cys alone (left panel). Code colors: red, concanavalin-A-rhodamine, and green, FITC label. C. Cell penetration of 3 μM doxorubicin or the covalently linked complex, doxorubicin-linker-Cys-MCa-Abu, in MDA-MB-231 cells. Note that doxorubicin alone goes to the nucleus (red, left panel), whereas coupled to Cys-MCa-Abu it concentrates in the cytoplasm. Green: concanavalin-A-FITC for plasma membrane staining. D. Cell penetration of 50 nM quantum dots (QD) alone (left panel) or coupled after maleimide modification of QDs to Cys-MCa-Abu in CHO cells (right panel). Code colors: red, nuclei (DHE) and blue, QDs. From B to D, note the diffuse cytoplasmic staining of the cargoes when coupled to Cys-MCa-Abu.

Fig. 8

Neuronal toxicity of MCa analogues. Neuronal survival after 24 hrs incubation with 10 μM of each MCa analogue was assessed with the MTT assay. No significant differences in neuronal survival were observed between the analogues.***, survival affected with mean + 3 S.D. < 100%. Average of 6 data points.