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• W1990324073 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 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) 3The abbreviations used are: MCa, maurocalcine; CD, circular dichroism; CHO, Chinese Hamster Ovary cells; CPP, cell-penetrating peptide; DHE, dihydroethidium; DHP, dihydropyridine; DMSO, dimethylsulfoxide; FITC, fluorescein isothiocyanate; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide; FACS, fluorescence-activated cell sorter; FDB, flexor digitorum brevis; PBS, phosphate-buffered saline; QD, quantum dot; RyR1, ryanodine receptor type I; SR, sarcoplasmic reticulum; Strep-Cy5, streptavidin-cyanine5; Abu, 2-aminobutyric acid. 3The abbreviations used are: MCa, maurocalcine; CD, circular dichroism; CHO, Chinese Hamster Ovary cells; CPP, cell-penetrating peptide; DHE, dihydroethidium; DHP, dihydropyridine; DMSO, dimethylsulfoxide; FITC, fluorescein isothiocyanate; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide; FACS, fluorescence-activated cell sorter; FDB, flexor digitorum brevis; PBS, phosphate-buffered saline; QD, quantum dot; RyR1, ryanodine receptor type I; SR, sarcoplasmic reticulum; Strep-Cy5, streptavidin-cyanine5; Abu, 2-aminobutyric acid. 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.Altafaj X. Cheng W. Esteve E. Urbani J. Grunwald D. Sabatier J.M. Coronado R. De Waard M. Ronjat M. J. Biol. Chem. 2005; 280: 4013-4016Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) 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 [3H]ryanodine binding (2.Esteve E. Smida-Rezgui S. Sarkozi S. Szegedi C. Regaya I. Chen L. Altafaj X. Rochat H. Allen P. Pessah I.N. Marty I. Sabatier J.M. Jona I. De Waard M. Ronjat M. J. Biol. Chem. 2003; 278: 37822-37831Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). In muscle fibers, MCa produces a transient loss of voltage control of Ca2+ 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.Pouvreau S. Csernoch L. Allard B. Sabatier J.M. De Waard M. Ronjat M. Jacquemond V. Biophys. J. 2006; 91: 2206-2215Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). This function of MCa is due to a partial sequence homology between MCa and a cytoplasmic loop of the DHP-sensitive channel (2.Esteve E. Smida-Rezgui S. Sarkozi S. Szegedi C. Regaya I. Chen L. Altafaj X. Rochat H. Allen P. Pessah I.N. Marty I. Sabatier J.M. Jona I. De Waard M. Ronjat M. J. Biol. Chem. 2003; 278: 37822-37831Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). These observations explain why MCa, along with other members of the same family of toxins such as imperatoxin 1A (4.el-Hayek R. Lokuta A.J. Arevalo C. Valdivia H.H. J. Biol. Chem. 1995; 270: 28696-28704Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), hemicalcin (5.Shahbazzadeh D. Srairi-Abid N. Feng W. Ram N. Borchani L. Ronjat M. Akbari A. Pessah I.N. De Waard M. El Ayeb M. Biochem. J. 2007; 404: 89-96Crossref PubMed Scopus (64) Google Scholar), and opicalcin 1 and 2 (6.Zhu S. Darbon H. Dyason K. Verdonck F. Tytgat J. Faseb. J. 2003; 17: 1765-1767Crossref PubMed Scopus (145) Google Scholar) 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.Esteve E. Smida-Rezgui S. Sarkozi S. Szegedi C. Regaya I. Chen L. Altafaj X. Rochat H. Allen P. Pessah I.N. Marty I. Sabatier J.M. Jona I. De Waard M. Ronjat M. J. Biol. Chem. 2003; 278: 37822-37831Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 7.Boisseau S. Mabrouk K. Ram N. Garmy N. Collin V. Tadmouri A. Mikati M. Sabatier J.M. Ronjat M. Fantini J. De Waard M. Biochim. Biophys. Acta. 2006; 1758: 308-319Crossref PubMed Scopus (52) Google Scholar). MCa is a highly charged peptide with 12 basic residues out of 33, and a net global positive charge of +8. 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 emerging family of toxin CPPs that have no structural homologies apart from their content in basic residues. Recently, a new toxin, crotamin, has been purified from the venom of a South American snake. It also behaves as a CPP but apparently with a preference for dividing cells (8.Nascimento F.D. Hayashi M.A. Kerkis A. Oliveira V. Oliveira E.B. Radis-Baptista G. Nader H.B. Yamane T. Tersariol I.L. Kerkis I. J. Biol. Chem. 2007; 282: 21349-21360Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The great diversity in CPP sequences observed so far suggests that designing new MCa CPP analogues should be easy. This tolerance in sequence variation is possibly due to the diverse nature of membrane receptors implicated in CPP cell translocation. These receptors nevertheless possess a single point in common: they all appear to interact with CPPs on the basis of electrostatic interactions. In contrast, the structural characteristics of the interaction between MCa and RyR1 appear far more constrained. A single mutation within MCa, the replacement of an Arg residue by an alanyl, abolishes the pharmacological effect of MCa but has only a mild effect on its cell penetration efficacy (9.Mabrouk K. Ram N. Boisseau S. Strappazzon F. Rehaim A. Sadoul R. Darbon H. Ronjat M. De Waard M. Biochim. Biophys. Acta. 2007; 1768: 2528-2540Crossref PubMed Scopus (30) Google Scholar). Nevertheless, segregating the pharmacological properties from the cell-penetrating properties proved more complex than expected on the sole basis of amino acid substitutions of MCa (9.Mabrouk K. Ram N. Boisseau S. Strappazzon F. Rehaim A. Sadoul R. Darbon H. Ronjat M. De Waard M. Biochim. Biophys. Acta. 2007; 1768: 2528-2540Crossref PubMed Scopus (30) Google Scholar). This appears to be due to structural imperatives of the molecule because it has four functions to fulfill: first, possess the attributes of a CPP; second, conserve sequence homology with the DHP-sensitive calcium channel; third, bind to RyR1; and fourth, activate this latter channel. Many residues contribute both to the pharmacological effects and the cell penetration properties, namely some basic residues that make the functional segregation difficult with simple amino acid substitutions. To both circumvent these difficulties and take advantage of the flexibility in structural constraints required for MCa cell penetration, we sought a novel strategy for the design of an MCa analogue that would lose its pharmacological activity while retaining most of its cell penetration efficacy. As determined by 1H NMR, MCa folds along an inhibitor cystine knot motif with a disulfide bridge pattern of Cys3–Cys17, Cys10–Cys21, and Cys16–Cys32. It contains three β-strands that run from amino acid residues 9–11 (strand 1), 20–23 (strand 2), and 30–33 (strand 3), with β-strands 2 and 3 forming an anti-parallel β-sheet (10.Mosbah A. Kharrat R. Fajloun Z. Renisio J.G. Blanc E. Sabatier J.M. El Ayeb M. Darbon H. Proteins. 2000; 40: 436-442Crossref PubMed Scopus (80) Google Scholar). In earlier studies on another toxin, maurotoxin (MTX), active on voltage-gated potassium channels, it was found that the disulfide bridges of the peptide play an essential role in the three-dimensional structure of the toxin and thus on its activity (11.di Luccio E. Matavel A. Opi S. Regaya I. Sandoz G. M'Barek S. Carlier E. Esteve E. Carrega L. Fajloun Z. Rochat H. Loret E. de Waard M. Sabatier J.M. Biochem. J. 2002; 361: 409-416Crossref PubMed Scopus (11) Google Scholar). We therefore adopted a similar strategy for the design of a novel analogue of MCa in which we replaced all native cysteine residues, engaged in the three disulfide bridges, by isosteric 2-aminobutyric acid residues. The goal was to obtain a structurally altered analogue displaying a complete loss of pharmacological activity but preserving the positively charged nature of the peptide, a property required for the efficient cell penetration of a CPP. To further validate this analogue, three derivatives were produced that comprise either an N-terminal biotinylated lysine residue with or without an alanyl substitution of Glu12, previously shown to favor cell penetration (9.Mabrouk K. Ram N. Boisseau S. Strappazzon F. Rehaim A. Sadoul R. Darbon H. Ronjat M. De Waard M. Biochim. Biophys. Acta. 2007; 1768: 2528-2540Crossref PubMed Scopus (30) Google Scholar), or an N-terminal cysteine residue for coupling chemistries to various cargoes. This chemical synthesis was accompanied by pharmacological assays, toxicity experiments, and proof of concept that cargo penetration is fully conserved. The data indicate that cysteine replacement within MCa can produce potent cell-penetrating analogues of MCa devoid of pharmacological activity. Reagents—Streptavidin-Cy5 (Strep-Cy5) was from Amersham Biosciences, dihydroethidium (DHE) from Molecular Probes, and rhodamine- and FITC-conjugated concanavalin A were from AbCys and Sigma, respectively. Doxorubicin was from Alexis Biochemicals. The TK705-amino-(polyethyleneglycol) quantum dots (QD) with surface-activated amine groups were purchased from Invitrogen. [3H]Ryanodine was from PerkinElmer Life Sciences. Peptide Synthesis—MCab was synthesized as previously described (7.Boisseau S. Mabrouk K. Ram N. Garmy N. Collin V. Tadmouri A. Mikati M. Sabatier J.M. Ronjat M. Fantini J. De Waard M. Biochim. Biophys. Acta. 2006; 1758: 308-319Crossref PubMed Scopus (52) Google Scholar). MCab-Abu, MCab-Abu E12A, and FITC-Gpep-Cys were purchased from the Department of Pharmaceutical Sciences, University of Ferrara (Italy). Cys-MCa-Abu was synthesized by NeoMPS. MTXb-Abu was assembled by the group of Dr. J. M. Sabatier. Formation of MCab/, MCab-Abu/, MCab-Abu E12A/, or MTXb-Abu/Strep-Cy5 Complexes—Soluble Strep-Cy5 was mixed with four molar equivalents of MCab, MCab-Abu, MCab-Abu E12A, or MTXb-Abu for 2 h at 37 °C in the dark in phosphate-buffered saline (PBS, in mm: NaCl 136, Na2HPO4 4.3, KH2PO4 1.47, KCl 2.6, CaCl2 1, MgCl2 0.5, pH 7.2). Conjugation of Cys-MCa-Abu to Various Cargoes—Cys-MCa-Abu was conjugated to a FITC-Gpep-Cys molecule (sequence derived from Gβ1: FITC-β-AGITSVAFSRSGRLLLAGYDDFN-Abu-NIWDAMKGDRAC-OH) according to the method used by Davidson et al. (12.Davidson T.J. Harel S. Arboleda V.A. Prunell G.F. Shelanski M.L. Greene L.A. Troy C.M. J. Neurosci. 2004; 24: 10040-10046Crossref PubMed Scopus (198) Google Scholar). Briefly, an equimolar mixture of Cys-MCa-Abu and FITC-Gpep-Cys was heated to 65 °C for 15 min and then incubated at 37 °C for 1 h. The complex was purified by fast protein liquid chromatography. Cys-MCa-Abu was also conjugated to doxorubicin. Briefly, doxorubicin.HCl (1 mg/ml) was suspended in PBS, pH 8.0, and conjugated to Cys-MCa-Abu using succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Pierce) according to the protocol of Liang and Yang (13.Liang J.F. Yang V.C. Bioorg. Med. Chem. Lett. 2005; 15: 5071-5075Crossref PubMed Scopus (79) Google Scholar). Successful coupling was followed by a 16.5% SDS-PAGE and UV detection of the resulting conjugated doxorubicin-linker-Cys-MCa-Abu peptide. QD were also conjugated to Cys-MCa-Abu. The amino groups of the QD (≈100–120 functions per particle) were first converted into a maleimide coating as described (14.Cai W. Shin D.W. Chen K. Gheysens O. Cao Q. Wang S.X. Gambhir S.S. Chen X. Nano. Lett. 2006; 6: 669-676Crossref PubMed Scopus (878) Google Scholar), thereby yielding QDM. Briefly, QD (1 nmol) were incubated for 4 h in the dark at room temperature in PBS in the presence of 4-maleimidobutyric acid N-hydrosuccinimide ester (1.8 μmol, in anhydrous dimethyl sulfoxide (DMSO)), then purified using NAP-5 columns (Amersham Biosciences). 20 μl of Tris-(2-carboxyethyl)phosphine hydrochloride solution 0.5 m was added to 100 nmol of the Cys-MCa-Abu in 80 μl of water for 30 min, and then incubated overnight at room temperature with QDM in the presence of 1 mm EDTA, pH 7.4. Non-reacted maleimide groups were quenched for 20 min by adding 2-mercapthoethanol in excess (500 nmol). The QDM-Cys-MCa-Abu conjugates were purified using NAP-5 columns. Surface modifications of QD were detected by 1% agarose gel electrophoresis in a TAE buffer pH 8, and imaged by fluorescence using a 633-nm excitation wavelength and collecting emitted light above 700 nm. The hydrodynamic diameter of the QDM-Cys-MCa-Abu nanoparticles measured using a Zetasizer Nano (Malvern Instruments) were 25 nm in PBS (20.5 nm for non-modified QD). The QDM-Cys-MCa-Abu concentration was calculated using 532 nm absorbance measurements (QD molar extinction coefficient ϵ = 2.1 × 106m-1 cm-1 at 532 nm). Cell Culture—Wild-type Chinese Hamster Ovary (CHO-K1) cells (from ATCC) were maintained at 37 °C in 5% CO2 in F-12K nutrient medium (Invitrogen) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Invitrogen) and 10,000 units/ml streptomycin and penicillin (Invitrogen). MDA-MB-231 cells from ATCC were grown in Leibovitz L15 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum and 10,000 units/ml streptomycin and penicillin. Media used for the culture of cerebellar granule neurons was based on Dulbecco's modified Eagle's medium (Invitrogen) containing 10 unit/ml penicillin, 10 μg/ml streptomycin, 2 mm l-glutamine, and 10 mm HEPES, 25 mm KCl, and 10% fetal bovine serum. Primary cultures were prepared from 6-day-old S/IOPS NMRI mice (Charles River Laboratories), as described previously (15.Gallo V. Kingsbury A. Balazs R. Jorgensen O.S. J. Neurosci. 1987; 7: 2203-2213Crossref PubMed Google Scholar). Preparation of Heavy SR Vesicles—Heavy SR vesicles were prepared following the method of Kim et al. (16.Kim D.H. Ohnishi S.T. Ikemoto N. J. Biol. Chem. 1983; 258: 9662-9668Abstract Full Text PDF PubMed Google Scholar). Protein concentration was measured by the Biuret method. Isolation and Preparation of Flexor Digitorum Brevis Muscle Fibers—Experiments were performed on single skeletal fibers isolated from the flexor digitorum brevis (FDB) muscles from 4–8-week-old OF1 mice (Charles River Laboratories) in accordance with the guidelines of the French Ministry of Agriculture (87/848) and of the European Community (86/609/EEC). Procedures for enzymatic isolation of single fibers and partial insulation of the fibers with silicone grease were as previously described (17.Collet C. Allard B. Tourneur Y. Jacquemond V. J. Physiol. 1999; 520: 417-429Crossref PubMed Scopus (61) Google Scholar, 18.Jacquemond V. Biophys. J. 1997; 73: 920-928Abstract Full Text PDF PubMed Scopus (64) Google Scholar). In brief, mice were killed by cervical dislocation after halothane (Sigma-Aldrich) inhalation before removal of the muscles. Muscles were treated with 2 mg/ml collagenase type I (Sigma-Aldrich) in Tyrode solution for 60 min at 37 °C. Single fibers were then isolated by triturating the muscles in the experimental chamber. The major part of a single fiber was electrically insulated with silicone grease (Rhodia Siliconi Italia, Treviolo, Italia) so that whole-cell voltage-clamp could be achieved on a short portion of the fiber extremity. All experiments were performed at room temperature (20–22 °C). Structural Analyses of MCab, MCab-Abu, and MCab-Abu E12A by Circular Dichroism—Circular dichroism (CD) spectra were recorded on a Jasco 810 dichrograph using 1-mm thick quartz cells. Spectra were recorded between 180 and 260 nm at 0.2 nm/min and were averaged from three independent acquisitions. The spectra were corrected for water signal and smoothed using a third-order least squares polynomial fit. [3H]Ryanodine Binding Assay—Heavy SR vesicles (1 mg/ml) were incubated at 37 °C for 3 h in an assay buffer composed of 5 nm [3H]ryanodine, 150 mm NaCl, 2 mm EGTA, 2 mm CaCl2 (pCa = 5), and 20 mm HEPES, pH 7.4. 1 μm MCab, MCab-Abu, or MCab-Abu E12A was added to the assay buffer just prior to the addition of heavy SR vesicles. [3H]Ryanodine bound to heavy SR vesicles was measured by filtration through Whatman GF/B glass filters followed by three washes with 5 ml of ice-cold washing buffer composed of 150 mm NaCl, 20 mm HEPES, pH 7.4. Filters were then soaked overnight in 10 ml of scintillation mixture (Cybscint, ICN) and bound radioactivity determined by scintillation spectrometry. Nonspecific binding was measured in the presence of 20 μm cold ryanodine. Each experiment was performed in triplicate and repeated three times. All data are presented as mean ± S.D. Fluorescent Measurements under Voltage-Clamp Conditions— An RK400 patch-clamp amplifier (BioLogic) was used in whole-cell configuration as previously described (18.Jacquemond V. Biophys. J. 1997; 73: 920-928Abstract Full Text PDF PubMed Scopus (64) Google Scholar). Voltage-clamp was performed with a microelectrode filled with the intracellular-like solution (in mm: 120 K glutamate, 5 Na2-ATP, 5 Na2-phosphocreatine, 5.5 MgCl2, 5 d-glucose, 5 HEPES adjusted to pH 7.2 with KOH). Indo-1 (Molecular Probes) was present in this solution at 0.2 mm for fluorescence measurements under voltage-clamp conditions. The extracellular solution contained (in mm): 140 TEA-methanesulphonate, 2.5 CaCl2, 2 MgCl2, 0.002 tetrodotoxin, 10 HEPES, pH 7.2. The tip of the microelectrode was inserted through the silicon, within the insulated part of the fiber. Membrane depolarizations were applied every 30 s from a holding command potential of -80 mV. For the present set of measurements, the cytoplasm was dialyzed with the microelectrode solution, which contained the calcium dye Indo-1 and a given peptide to be tested (200 μm for MCab-Abu and MCab-Abu E12A, and 100 μm for MCab). To facilitate intracellular dialysis, the electrode tip was broken within the silicon-insulated portion of the fiber by pushing it back and forth a few times toward the bottom of the chamber. Under these conditions, intracellular equilibration of the solution was awaited for 30 min. Equilibration was followed from the time course of increase of indo-1 fluorescence in the tested portion of the fiber. Indo-1 fluorescence was measured on an inverted Nikon Diaphot epifluorescence microscope equipped with a commercial optical system allowing the simultaneous detection of fluorescence at 405 nm (F405) and 485 nm (F485) by two photomultipliers (IonOptix, Milton, MA) upon 360-nm excitation. Background fluorescence at both emission wavelengths was measured next to each fiber tested and was then subtracted from all measurements. In an earlier study, we showed that 10–100 μm levels of MCa were necessary to affect voltage-activated Ca2+ release in intact mammalian skeletal muscle fibers (3.Pouvreau S. Csernoch L. Allard B. Sabatier J.M. De Waard M. Ronjat M. Jacquemond V. Biophys. J. 2006; 91: 2206-2215Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). For this reason, and despite the cell penetration properties of MCa and of its derivatives, it is easier and less costly to apply these compounds intracellularly through the voltage-clamp electrode rather than in the extracellular medium. Calibration of the Indo-1 Response and [Ca2+]intra Calculation—The standard ratio method was used with the parameters: r = F405/F485, with Rmin, Rmax, KD, and β having their usual definitions. Results were either expressed in terms of Indo-1% saturation or in actual free calcium concentration (18.Jacquemond V. Biophys. J. 1997; 73: 920-928Abstract Full Text PDF PubMed Scopus (64) Google Scholar, 19.Csernoch L. Bernengo J.C. Szentesi P. Jacquemond V. Biophys. J. 1998; 75: 957-967Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). In vivo values for Rmin, Rmax, and β were measured using procedures previously described (17.Collet C. Allard B. Tourneur Y. Jacquemond V. J. Physiol. 1999; 520: 417-429Crossref PubMed Scopus (61) Google Scholar). No correction was made for Indo-1 Ca2+ binding and dissociation kinetics. MTT Assay—Primary cultures of cerebellar granule neurons were seeded into 96-well microplates at a density of ∼8 × 104 cells/well. After 4 days of culture, the cells were incubated for 24 h at 37 °C with 10 μm MCab, MCab-Abu, Cys-MCa-Abu, or MCab E12A-Abu. Control wells containing cell culture medium alone or with cells, both without peptide addition, were included in each experiment. The cells were then incubated with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) for 30 min. Conversion of MTT into purple-colored MTT formazan by the living cells indicates the degree of cell viability. The crystals were dissolved in DMSO, and the optical density was measured at 540 nm using a microplate reader (Biotek ELx-800, Mandel Scientific Inc.) for quantification of cell viability. All assays were run in triplicate. Flow Cytometry—MCab/, MCab-Abu/, MCab-Abu E12A, or MTXb-Abu/Strep-Cy5 complexes were incubated for 2 h with CHO cells to allow cell penetration. Control condition was represented by an incubation of cells with Strep-Cy5 alone. The cells were then washed twice with PBS to remove the excess extracellular complexes. Next, the cells were treated with 1 mg/ml trypsin (Invitrogen) for 10 min at 37 °C to remove remaining membrane-associated extracellular cell surface-bound complexes. The cell suspension was centrifuged at 500 × g and resuspended in PBS. Flow cytometry analyses were performed with live cells using a Becton Dickinson FACSCalibur flow cytometer (BD Biosciences). Data were obtained and analyzed using CellQuest software (BD Biosciences). Live cells were gated by forward/side scattering from a total of 10,000 events. Mean fluorescence values were determined from Gaussian fits of the resulting histograms and plotted as a function of complex concentration. Mean values of intracellular fluorescence for Strep-Cy5 alone incubation (less than 1% of the fluorescence observed for the lowest concentration of any of the various MCa/Strep-Cy5 complexes) were also subtracted. Analysis of the Subcellular Localization of Various MCa/or MTX/Cargo Complexes by Confocal Microscopy—For Strep-Cy5 complexes, 4 μm MCab, MCab-Abu, MCab-Abu E12A, or MTXb-Abu were coupled to 1 μm Strep-Cy5 as described above. CHO cells were incubated with the resulting complexes for 2 h, and then washed with Dulbecco's modified Eagle's medium alone. Immediately after washing, the nucleus was stained with 1 μg/ml DHE for 20 min, and then washed again with DMEM. After this step, the plasma membrane was stained with 5 μg/ml of FITC-conjugated concanavalin A for 3 min. Cells were washed once more, but with PBS. For the FITC-Gpep-Cys-Cys-MCa-Abu complex, 1 μm conjugate was incubated with CHO cells for 2 h, the plasma membrane stained with 5 μg/ml of rhodamine-conjugated concanavalin A. For the QDM-Cys-MCa-Abu complex, 50 nm of the complex was incubated with CHO cells for 2 h, and the nuclei stained with 1 μg/ml DHE for 20 min. For the doxorubicin-linker-Cys-MCa-Abu complex, 3 μm of the conjugate was incubated with MDA-MB-231 for 2 h, followed by staining of the plasma membrane with 5 μg/ml of FITC-conjugated concanavalin A for 3 min. In all experiments, live cells were immediately analyzed by confocal laser scanning microscopy using a Leica TCS-SP2. Alexafluor-488 (excitation at 488 nm), rhodamine and doxorubicin (excitation at 543 nm), or Cy5 and QDM (excitation at 642 nm) were sequentially excited and emission fluorescence collected in z-confocal planes of 10–15-nm steps. Images were merged in Adobe Photoshop 7.0. Synthesis of Disulfide-less Analogs of MCa—Fig. 1A illustrates the three-dimensional solution structure of MCa with three disulfide bridges. The aim of this study was to design a MCa analog for which convenient chemical coupling could be performed by using an N-terminal additional cysteine residue. As shown, MCa already contains six cysteine residues that contribute to the folding of the peptide to form an inhibitor cysteine knot motif. Adding an additional cysteine residue at the N terminus may significantly change the normal folding of the peptide and the classical disulfide bridge arrangement (C1-C4, C2-C5, and C3-C6) in an unpredicted manner. In turn, this could significantly affect the pharmacological activity and cell-penetration properties of the resulting molecule(s). To facilitate chemical coupling strategies of MCa to cargo molecules, we investigated the requirement of disulfide bridges on MCa pharmacology and cell penetration properties. Several analogs were synthesized, each with an N-terminal biotinylated lysine for easy binding to fluorescent streptavidin molecules (our reporter cargo for this study): MCab, intact with the native disulfide bridges, MCab-Abu in which we replaced all internal cysteine residues by isosteric 2-aminobutyric acid (Abu, Fig. 1B), and MCab-Abu E12A, an analog of MCab-Abu in which Glu12 was replaced by alanyl, a substitution known to improve cell penetration efficacy of MCab (9.Mabrouk K. Ram N. Boisseau S. Strappazzon F. Rehaim A. Sadoul R. Darbon H. Ronjat M. De Waard M. Biochim. Biophys. Acta. 2007; 1768: 2528-2540Crossref PubMed Scopus (30) Google Scholar) (Fig. 1C). We would expect that removing the disulfide bridges of MCa might affect its pharmacology more than its cell penetration properties for two reasons. First, disulfide bridge patterns are known to contribute to the pharmacological activity of toxins (20.Fajloun Z. Ferrat G. Carlier E. Fathallah M. Lecomte C. Sandoz G. di Luccio E. Mabrouk K. Legros C. Darbon H. Rochat H. Sabatier J.M. De Waard M. J. Biol. Chem. 2000; 275: 13605-13612Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 21.Fajloun Z. Mosbah A. Carlier E. Mansuelle P. Sandoz G. Fathallah M. di Luccio E. Devaux C. Rochat H. Darbon H. De Waard M. Sabatier J.M. J. Biol. Chem. 2000; 275: 39394-39402Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Second, the structural requirements for the pharmacological activity of MCa were shown to be more stringent than those required for cell penetration as probed by alanine scanning of MCa (9.Mabrouk K. Ram N. Boisseau S. Strappazzon F. Rehaim A. Sadoul R. Darbon H. Ronjat M. De Waard M. Biochim. Biophys. Acta. 2007; 1768: 2528-2540Crossref PubMed Scopus (30) Google Scholar). Finally, we synthesized MTXb-Abu, a biotinylated version of MTX that has no cell-penetrating properties on its own and that acts on voltage-dependent potassium channels (Fig. 1D). This peptide, in which we also replaced six internal cysteine residues by Abu derivatives, as for the MCab-Abu peptide, was used as a negative control in cell penetration experiments. Removing Disulfide Bridges in MCa Disrupts the Secondary Structure of the Peptide—Circular dichroism" @default.
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