Small peptides patterned after the N-terminus domain of SNAP25

inhibit SNARE complex assembly and regulated exocytosis

Clara Blanes-Mira,* Jaime M. Merino,� Elvira Valera,� Gregorio Fernandez-Ballester,*Luis M. Gutierrez,� Salvador Viniegra,� Enrique Perez-Paya§ and Antonio Ferrer-Montiel*

*Instituto de Biologıa Molecular y Celular, Universidad Miguel Hernandez, Alicante, Spain

�Departamento de Bioquımica y Biologıa Molecular, Facultad de Ciencias, Universidad de Extremadura, Badajoz, Spain

�Instituto de Neurociencias-CSIC, Universidad Miguel Hernandez, San Juan de Alicante, Spain

§Fundacion Valenciana de Investigaciones Biomedicas-CSIC, Valencia, Spain

Abstract

Synthetic peptides patterned after the C-terminus of syna-

ptosomal associated protein of 25 kDa (SNAP25) efficiently

abrogate regulated exocytosis. In contrast, the use of

SNAP25 N-terminal-derived peptides to modulate SNAP

receptors (SNARE) complex assembly and neurosecretion

has not been explored. Here, we show that the N-terminus of

SNAP25, specially the segment that encompasses 22Ala-

44Ile, is essential for the formation of the SNARE complex.

Peptides patterned after this protein domain are potent

inhibitors of SNARE complex formation. The inhibitory activity

correlated with their propensity to adopt an a-helical secon-

dary structure. These peptides abrogated SNARE complex

formation only when added previous to the onset of aggregate

assembly. Analysis of the mechanism of action revealed that

these peptides disrupted the binary complex formed by

SNAP25 and syntaxin. The identified peptides inhibited Ca2+-

dependent exocytosis from detergent-permeabilized excitable

cells. Noteworthy, these amino acid sequences markedly

protected intact hippocampal neurones against hypoglycae-

mia-induced, glutamate-mediated excitotoxicity with a potency

that rivaled that displayed by botulinum neurotoxins. Our

findings indicate that peptides patterned after the N-terminus

of SNAP25 are potent inhibitors of SNARE complex formation

and neuronal exocytosis. Because of their activity in intact

neurones, these cell permeable peptides may be hits for

antispasmodic and analgesic drug development.

Keywords: combinatorial chemistry, drug discovery, neuro-

secretion, protein–protein interactions, synaptic transmission,

vesicle fusion.

J. Neurochem. (2004) 88, 124–135.

Neurotransmitter release is a highly regulated cascade that

proceeds through an orchestrated sequence of protein–

protein interactions that culminate in the fusion of neuro-

transmitter-loaded vesicles in response to Ca2+ influx in

synaptic junctions (DeBello et al. 1993, 1995; Jahn et al.

2003). This is a complex process mediated by the so-called

SNAP receptors (SNARE) proteins, which include the

membrane-associated proteins syntaxin and synaptosomal

associated protein of 25 kDa (SNAP25), and the vesicle-

associated membrane protein (VAMP; Metha et al. 1996;

Brunger 2001; Bruns and Jahn 2002). These proteins directly

govern vesicle docking and fusion through the formation of

an sodium dodecyl sulphate (SDS)-resistant, ternary complex

referred to as the SNARE complex (Chen and Scheller

2001). The physiological relevance of SNARE proteins in

regulated exocytosis is underscored by the discovery that

they are the targets of botulinum and tetanus neurotoxins, a

family of naturally occurring neurotoxins that potently block

neurosecretion (Schiavo et al. 2000). Furthermore, it has

Received July 2, 2003; revised manuscript received September 3, 2003;

accepted September 14, 2003.

Address correspondence and reprint requests to A. Ferrer-Montiel,

Instituto de Biologıa Molecular y Celular, Universidad Miguel Hernan-

dez, Avenue Del Ferrocarril s/n, 03202 Elche, Alicante, Spain. E-mail:

aferrer@umh.es

Abbreviations used: BSS, basic saline solution; CD, circular dichro-

ism; DTT, dithiothreitol; GST, glutathione-S-transferase; HPLC, high-

performance liquid chromatography; OG, octyl-b-D-glucopyranoside;SDS–PAGE, sodium dodecyl sulphate polyacrylamide gel electrophor-

esis; SNAP25, synaptosomal associated protein of 25 kDa; SNARE,

SNAP receptors; VAMP, vesicle-associated membrane protein (syna-

ptobrevin).

Journal of Neurochemistry, 2004, 88, 124–135 doi:10.1046/j.1471-4159.2003.02133.x

124 � 2003 International Society for Neurochemistry, J. Neurochem. (2004) 88, 124–135

been shown that SNARE proteins represent the minimal

fusion machinery, as evidenced by the catalysis of vesicle

fusion by SNAREs reconstituted in synthetic liposomes

(Weber et al. 1998).

The SNARE complex is formed by a coiled coil structure.

High-resolution structural analysis of the SNARE complex

core shows a parallel four helix bundle formed by the

interaction of two helices from SNAP25, one from syntaxin

and one from VAMP (Fasshauer et al. 1997; Sutton et al.

1998; Xiao et al. 2001). The core of the four helix bundle is

composed of well-defined layers formed by interacting side-

chains from each of the four a-helices (Sutton et al. 1998).This compact structural arrangement is consistent with the

tremendous resistance of the SNARE complex to denatura-

tion by SDS (Hayashi et al. 1994). Mutation of the inner

interacting layers results in the destabilization of the protein

complex (Chen and Scheller 2001; Jahn et al. 2003).

Similarly, botulinum neurotoxins that cleave the SNARE

proteins and peptides that mimic their amino acid sequence

notably interfere with the stability and formation of the SDS-

resistant protein complex (DeBello et al. 1993; Gutierrez

et al. 1995a, 1995b; Martin et al. 1996; Metha et al. 1996;

Ferrer-Montiel et al. 1998a; Apland et al. 1999; Schiavo

et al. 2000; Melia et al. 2002).

Several studies have shown that small £20 mer peptidespatterned after the C-terminus of SNAP25 inhibit complex

assembly and neurotransmitter release (Cornille et al. 1995;

Gutierrez et al. 1995a, 1997; Apland et al. 1999). The

biological activity of these peptides was specific as random

sequences of the same amino acid composition were inert

modulating exocytosis (Gutierrez et al. 1995a, 1997). The

inhibitory activity of these amino acids sequences further

corroborated an essential role of the C-terminus of SNAP25

in complex formation. These observations, along with the

finding that clostridial neurotoxins BoNTs A and E cleave a

portion of the SNAP25 carboxy region, highlighted the need

of an unaltered membrane-proximal C-terminus domain of

the SNARE complex (Blasi et al. 1993; Lawrence et al.

1997; Schiavo et al. 2000). In marked contrast, the role of

the SNAP25 N-terminus remains more elusive, although it

has been reported that antibodies that target this domain

affect complex assembly and neuronal exocytosis (Xu et al.

1999).

Here, we have further addressed this issue by using a step-

wise deletion strategy in conjunction with the synthesis and

assay of small peptides modelled after N-terminus segment

encompassing 1Met-44Ile. We chose this protein domain

because it lies N-terminal of the conserved ionic layer in the

centre of the SNARE complex (Sutton et al. 1998). Deletion

of this SNAP25 domain prevented the assembly of the core

complex. Interestingly, removal of the segment 1Met-21Leu

did not impede complex formation, whereas deletion of the

22Ala-44Ile region fully deterred the interaction of the

SNARE proteins. Similarly, a synthetic peptide mimicking

the amino acid sequence of the 22Ala-44Ile domain blocked

complex formation and Ca2+-dependent exocytosis more

potently than that mirroring the 1Met-21Leu segment. The

inhibitory activity of the most potent peptide was only

detected if they were added prior to the onset of complex

formation, consistent with the proposed zippering model

from N- to C-terminus for assembly (Lin and Scheller 1997).

Collectively, our observations highlight a critical role for the

N-terminus domain of SNAP25, especially of the segment

comprising 22Ala-44Ile, in the stability of the SNARE

complex. Noteworthy, peptides patterned after this protein

domain protected neurones against excitotoxicity, presuma-

bly by inhibiting the release of L-glutamate in intact

neurones. In support of this tenet, these peptides abrogated

catecholamine release from detergent-permeabilized chrom-

affin cells. Thus, sequences mimicking the 22Ala-44Ile

region provide new pharmacological tools that may help to

further our understanding of the mechanism of neuronal

exocytosis.

Experimental procedures

Materials

Recombinant proteins and peptides cDNA plasmids encoding the

VAMP cytosolic domain (kindly provided by Dr R. Jahn), syntaxin

cytosolic portion (kindly provided by Dr R. Scheller) and full-

length SNAP-25 (kindly provided by M.C. Wilson) were cloned

into the pGEX-KG vector to obtain a glutathione-S-transferase

(GST) fusion constructs containing a thrombin cleavage site after

the GST.

Peptides SNAP25_N1 (Ac-MAEDADMRNELEEMQRRADQL-

NH2), SNAP25_N2 (Ac-ADESLESTRRMLQLVEESKDAGI-

NH2), SNAP25_N3 (Ac-ELEEMQRRADQLA-NH2), SNAP25_N4

(Ac-LESTRRMLQLVEE-NH2), and fluorescein-SNAP25_N4 were

purchased from DiverDrugs SL (Barcelona, Spain). Peptide purity

was ‡ 95%. Peptide identity was confirmed by matrix-assisted laserdesorption ionization time of flight (MALDI–TOF) spectrometry.

Deletion of SNAP25

Deletions were obtained by one-step inverse PCR with the proof-

reading Pfu turbo DNA polymerase and two restriction digestions

(Cabedo et al. 2002). Externally oriented primers with SacII unique

restriction sites were used to amplify the whole vector except the

region to be deleted. The following primers were used: (a) D[1–21]sense 5¢-GATTCCGCGGGCTGATGAGTCCCTGGG-3¢, antisense5¢-CTAA-CCGCGGGGAGTCTAGAATTCCACCACC-3¢; (b)

D[22–44] sense 5¢-GATTCCGCGGAGGACTTTGGTTATGTTGG-ATG-3¢, and antisense 5¢-CATACCGCGGCAGCTGGTCAGCCCT-CCTC-3¢; (c) D[1–44], sense 5¢GATTCCGCGGAGGACTTTGG-TTATGTTGGATG-3¢ and antisense 5¢-CTAACCGCGGGGAGTC-TAGAATTCCACCACC-3¢. PCR products were digested (in theamplification buffer) with 20 U of DpnI overnight at 37�C toremove the methylated plasmid templates, subsequently purified,

digested with SacII and ligated. Designed deletions were verified by

restriction digestion with SacII and by automatic sequencing.

Peptides inhibitors of neuronal exocytosis 125

� 2003 International Society for Neurochemistry, J. Neurochem. (2004) 88, 124–135

Expression and purification of recombinant SNARE proteins

Recombinant bacterially expressed SNARE proteins were obtained

as described (Blanes-Mira et al. 2001, 2002). Briefly, GST-fusion

proteins were expressed in the BL21DE3 strain. Protein expression

was induced with 1 mM isopropyl-b-D-thiogalactopyranoside (Sig-ma, St Louis, MO, USA) for 5 h at 30�C. Bacterial cultures werepelleted, washed with lysis buffer (10 mM phosphate pH 7.4,

136 mM NaCl, 2.7 mM KCl), digested with 0.1 mg/mL lysozime

(Sigma) for 10 min at 22�C in lysis buffer, supplemented with 2 mMphenymethylsulphonyl fluoride (Sigma), 5 mM iodoacetamide (Sig-

ma), 5 mM EDTA and sonicated (3 · 45 s) in a Branson 250 sonifierat 4�C. Lysates were solubilized with 1% Triton X-100 for 20 min at4�C and cleared by centrifugation at 20 000 g for 30 min at 4�C.SNAP25 and VAMP were purified from the sonicated bacteria

supernatant by affinity chromatography on glutathione agarose

(Pharmacia, Uppsala, Sweden) following manufacturer instructions.

Purification of recombinant proteins was carried out in 20 mM

HEPES pH 7.4, 100 mM NaCl, 0.05% n-octyl-b-D-glucopyranoside(OG), 5 mM dithiothreitol (DTT) and cleaved with thrombin for 3 h

at 23�C and dialysed against 20 mM HEPES pH 7.0, 80 mM KCl,20 mM NaCl, 0.1% OG. Syntaxin was obtained from the bacterial

pellet by washing the precipitated with 50 mM Tris–HCl pH 8.0,

10 mM EDTA, 100 mM NaCl, 1.0% Triton X-100 in a polytron.

Protein was recovered from inclusion bodies by incubating the

solubilized pellet with 50 mM Tris–HCl pH 8.0, 10 mM EDTA,

100 mM NaCl, 1.0% N-lauroyl-sarcosine, at 4�C overnight. Extrac-ted protein was diluted 1 : 10 in washing buffer (10 mM HEPES

pH 7.4, 0.1% OG) and loaded on to glutathione-agarose resin.

Bound material was purified in washing buffer and cleaved with

thrombin for 3 h at room temperature. Purified proteins were stored

at )80�C. Concentration was assayed with the BCA kit (Pierce,Rockford, IL, USA), and purity verified by sodium dodecyl sulphate

polyacrylamide gel electrophoresis (SDS–PAGE) analysis.

In vitro reconstitution of the SDS-resistant SNARE complex

Syntaxin and VAMP were incubated at final concentrations of

3.0 lM in the presence/absence of the peptides at the indicatedconcentrations for 2 h at 4�C. Thereafter, SNAP25 (3.0 or 0.3 lM,as indicated) was added and the reactions (V ¼ 15 lL) proceed in20 mM HEPES pH 7.4, 100 mM NaCl, 1.0% OG, 2.0 mM DTT at

4�C for the indicated times, and were stopped by the addition ofSDS–PAGE sample buffer. Peptide blockade activity was evaluated

on SDS–PAGE (12%) by the disappearance of the 75 kDa band

corresponding to SDS-resistant ternary core complex. Gels were

digitized and quantified as described (Blanes-Mira et al. 2001).

Circular dichroism spectroscopy

Circular dichroism (CD) was carried out in a JASCO J-810

Spectropolarimeter equipped with a computer-controlled tempera-

ture cuvette holder. CD data for the Far-UV CD spectra (region 195–

250 nm) were recorded with a 1-mm path length cell containing

100 lM peptides in 20 mM Tris–HCl pH 8.0. All spectra wererecorded at 25�C and at 50 nm/min (response time of 1 s), averaged(five scans), and corrected for the buffer contribution. CD signals (in

mdegrees) were converted to mean ellipticity (h, mdegreescm2/dmol) using the relationship hm ¼ 100 · CD signal/

(C · N · l), were C denotes the peptide concentration, N the

number of residues and l the path length. Secondary structure

elements were inferred by fitting the CD spectra as described

(Blanes-Mira et al. 2001).

Chromaffin cell cultures

Chromaffin cell cultures were prepared from bovine adrenal glands

by collagenase digestion and further separated from debris and

erythrocytes by centrifugation on Percoll gradients as described

(Gomis et al. 1994). Cells were maintained in monolayer cultures at

a density of 625 000 cells/cm2 and were used between the third and

sixth day after plating. All the experiments were performed at room

temperature.

Determination of catecholamine release from detergent-

permeabilized chromaffin cells

Secreted noradrenaline and adrenaline was determined in digitonin-

permeabilized cells as described (Gutierrez et al. 1995b). Briefly,

monolayers were washed 4 times with a Krebs/HEPES basal solution

(containing in mM: 15 HEPES pH 7.4 with 134 NaCl, 4.7 KCl, 1.2

KH2PO4, 1.2 MgCl2, 2.5 CaCl2, 0.56 ascorbic acid and 11 glucose).

Cell permeabilization was accomplished with 20 lM digitonin in20 mM Pipes, pH 6.8 with 140 mM monosodium glutamate, 2 mM

MgCl2, 2 mM Mg-ATP and 5 mM EGTA. This incubation was

carried out in the absence or presence of peptides. Following

permeabilization, media were discarded and cells were incubated for

10 min in digitonin-free medium in presence or absence of peptides.

Basal secretion was measured in 5 mM EGTA, whereas stimulated

secretion was measured in a medium containing 10 lM bufferedCa2+ solution. Media were collected and released catecholamines as

well as the total cell content were determined by high-performance

liquid chromatography (HPLC) using an electrochemical detector.

Statistical significance was calculated using Student’s t-test with data

from four or more independent experiments.

Hippocampal cultures

Mixed hippocampal neuronal/glial cultures were prepared as des-

cribed (Ferrer-Montiel et al. 1998b; Valera et al. 2002). Briefly,

hippocampi were dissected from E17–E19 rat embryos, and

incubated at 37�C for 15 min in basic saline solution (BSS,containing: 137 mM NaCl, 3.5 mM KCl, 0.4 mM KH2PO4, 0.33 mM

Na2HPO4Æ7 H2O, 5 mM TES pH 7.4 and 10 mM glucose) with0.025% trypsin (Invitrogen, Carlsbad, CA, USA). Trypsin was

diluted by rinsing the tissue three times for 5 min each with BSS.

Tissue was then dissociated by several passes through a siliconized

Pasteur pipette, first unpolished and then with a fire-polished pipette.

Cells were centrifuged for 5 min at 200 g and pellets resuspended in

BSS. Cells were plated at 2 · 105 viable cells/cm2 in poly-L-lysine-coated (0.2 mg/mL, Sigma) dishes. The culture medium had the

following composition: minimum essential medium (Earle’s salts,

Invitrogen) supplemented with 10% (v/v) heat-inactivated horse

serum (Sigma), 10% (v/v) heat-inactivated fetal bovine serum

(Sigma), 1 mM glutamine and 22 mM glucose. Cultures were

maintained at 37�C in a 5% CO2 atmosphere and half of the mediumwas renewed every 2–3 days. At day 5 after plating, glial proliferation

was inhibited by the addition of 80 lM 5-fluoro-2¢-desoxiuridine.

Glucose deprivation induced cell death

Neurones cultured at 14 days in vitro were used. Culture medium

was removed and neurones were rinsed with fresh medium without

126 C. Blanes-Mira et al.

� 2003 International Society for Neurochemistry, J. Neurochem. (2004) 88, 124–135

glucose and added sucrose to maintain osmolarity (Ferrer-Montiel

et al. 1998b; Valera et al. 2002). Plates were then returned to the

incubator for 3 h. Botulinum neurotoxins E and B (Sigma) were

added 24 h before the excitotoxic insult. Peptides (100 lM) wereadded 2 h before the glucose deprivation and were present during

the insult. Glucose deprivation was terminated by changing the

cultures to a glucose-containing medium (22 mM glucose) plus

10 lM MK-801 to prevent additional NMDA receptor activation.Cell death was blindly assessed 18–24 h post-insult using the trypan

blue (0.4%) exclusion assay.

Results

The N-terminus of SNAP25 modulates SNARE complex

assembly

To identify peptides in the N-terminal half of SNAP25 that

modulate the stability of the SNARE complex, we first

investigated the role of the N-terminus of SNAP25 in the

formation of the SNARE aggregate using a step-wise

deletion approach of segment 1Met-44Ile. The rationale

was based in the differential propensity of specific regions

within this segment to form coiled-coil structures and to

adopt an a-helical structure. As shown in Fig. 1(a), thesegment encompassing residues 1Met-21Leu displays a

100% prediction for coiled-coil formation and 70% probab-

ility to fold into an a-helix, while the region 22Ala-44Iledoes not show significant probability to adopt a coiled-coil

arrangement nor a-helical secondary structure. Hence, wedeleted these two regions, and evaluated the ability of the

deleted SNAP25 species in the assembly of the SNARE

complex in vitro (Fig. 1b). For this task, the SNARE

complex was reconstituted in vitro by incubating equimolar

amounts of the three recombinant SNARE proteins, and its

formation was analysed by SDS–PAGE. Figure 1(b) shows

that incubation of SNAP25, VAMP and syntaxin at 4�Covernight leads to the formation of an SDS-resistant complex

of 75 kDa (control lane), that is sensitive to temperatures

‡ 90�C (90�C lane). Replacement of SNAP25 by D[1–21] didnot affect the formation of the SDS-resistant complex

(Figs 1b and c), although it significantly reduced the thermal

(a) (b)

(c) (d)

Fig. 1 Deletion of domain 1Met-44Ile in the N-terminus half of

SNAP25 notably affects the stability of the SNARE complex. (a)

Coiled-coil and a-helix secondary predictions of the N-terminal half of

SNAP25. Analysis was performed with the programs Agadir (http://

www.embl-heidelberg.de) and Coils (http://www.ch.embnet.org). (b)

Schematic representation of the deleted regions and effect of SNAP25

deletion species D[1–21], D[1–44] and D[22–44] on the in vitro

assembly of the SNARE complex. Complex formation was accom-

plished by incubating equimolar amounts of the SNARE proteins at

4�C overnight, and analyzed by SDS–PAGE. (c) Protein gels were

digitized and quantified as described (Blanes-Mira et al. 2001).

Complex formation was normalized with respect to that obtained for

SNAP25 wild type (control). Data are mean ± SEM with n ¼ 2. (d)

SNARE complex formation as a function of the temperature. Ternary

complexes formed by SNAP25 or D[1–21] were heated to the specified

temperatures for 5 min before SDS–PAGE analysis. The extent of

complex assembly was normalized with respect to that formed at

25�C. Experimental points were fitted to a logistic equation

CF

CFm�ax¼ 1

1þ TT0:5

h ip

where CF is the fraction (%) of SNARE complex formation, T0.5 is the

denaturing temperature and p the slope of the curve. Denaturating

temperatures were 76 ± 0.2�C for SNAP25 wild type, and 72 ± 0.4�Cfor deletion D[1–21] (mean ± SEM, n ¼ 3).

Peptides inhibitors of neuronal exocytosis 127

� 2003 International Society for Neurochemistry, J. Neurochem. (2004) 88, 124–135

stability of the SNARE complex (Fig. 1d). In contrast,

removal of amino acids 1Met-44Ile or 22Ala-44Ile from

SNAP25 fully prevented the assembly of the SDS-resistant

complex (Figs 1c and d, D[1–44] and D[22–44] lanes).Collectively, these results demonstrate that the region 22Ala-

44Ile of SNAP25 is important for the assembly of the

SNARE complex, while segment comprising 1Met-21Leu

affects the stability of the ternary complex, consistent with

observations reported by others (Chapman et al. 1994; Yang

et al. 2000).

Peptides patterned after the N-terminus domain of

SNAP25 inhibit SNARE complex formation

Because truncation of internal protein segments may signi-

ficantly alter the protein structure, we questioned whether

the dramatic effect of the 22Ala-44Ile region on SNARE

complex assembly was specific or it was the consequence

of the resulting protein structure. To address this issue, we

proposed the use of peptides patterned after these SNAP25

regions as specific pharmacological tools (Fig. 2a). Peptides

SNAP25_N1 (Ac-MAEDADMRNELEEMQRRADQL-NH2)

and SNAP25_N2 (Ac-ADESLESTRRMLQLVEESKDAGI-

NH2) were synthesized by solid phase and their effect on the

stability of the reconstituted SNARE complex was evaluated.

To favour the inhibitory activity of the peptides, the SNAP25

concentration was reduced 10-fold (to 0.3 lM) with respect to

that of VAMP and syntaxin. In these experiments, peptides

were incubated with syntaxin and VAMP at 4�C for 2 hbefore the assembly of the SNARE complex was initiated by

addition of SNAP25. As depicted in Fig. 2(b), the three

SNARE proteins assembled into a ternary complex in both

the absence and presence of either peptide. Notice, however,

that the amount of SNARE complex formed was lower

in the presence of 1 mM of either peptide, as evidenced

by the lighter intensity of the �75 kDa protein band and theappearance of a �27 kDa band corresponding to SNAP25.Quantification of these observations indicate that

SNAP25_N1 inhibited a �25% of complex formation, whilethe extent of complex inhibition for SNAP25_N2 increased to

�50% (Fig. 2c). These results support the finding that thesegment 22Ala-44Ile, in the middle of the N-terminal half of

the SNAP25, plays a critical role in the assembly of the

SNARE complex.

Analysis of the content of secondary structure elements by

circular dichroism, showed that both sequences exhibited a

25% content in a-helix in aqueous buffer that was signifi-cantly increased by addition of TFE, as evidenced by the

appearance of CD minima at 207 and 220 nm (Figs 3a and

b). As illustrated in Fig. 3(c), the secondary structure of

SNAP25_N1 increased to 60% in the presence of �50%TFE, while that of SNAP25_N2 was augmented to �77%.Thus, the inhibitory activity of both peptides appears to

Fig. 2 Peptides SNAP25_N1 and SNAP25_N2 encompassing the

domain 1Met-44Ile of SNAP25 N-end inhibit the in vitro assembly of

the SNARE complex. (a) Amino acid sequence of peptides

SNAP25_N1 and SNAP25_N2 encompassing domains 1Met-21Leu

and 22Ala-44Ile, respectively. (b) Effect of SNAP25_N1 and

SNAP25_N2 peptides on SNARE complex assembly. Peptides (1 mM)

were incubated with 3 lM VAMP and 3 lM syntaxin at 4�C for 2 h

before SNARE complex formation by addition of 0.3 lM SNAP25. The

reaction proceed at 4�C overnight. Complex assembly was analyzed

by SDS–PAGE. Control denotes the formation of the protein aggre-

gate in the absence of peptides. (c) Quantification of inhibitory effect of

peptides on the formation of SNARE complex. Gels were digitized and

quantified as described (Blanes-Mira et al. 2001). Data are mean ±

SEM, n ¼ 2.

128 C. Blanes-Mira et al.

� 2003 International Society for Neurochemistry, J. Neurochem. (2004) 88, 124–135

correlate with their propensity to adopt an a-helix secondarystructure.

Identification of core sequences that inhibit SNARE

complex assembly

We next investigated whether shorter sequences that modu-

late the stability of the SNARE complex could be identified.

For this purpose, peptides SNAP25_N3 (Ac-ELEE-

MQRRADQLA-NH2) corresponding to the a-helical regiondetected in the N-terminus (Fig. 1), and SNAP25_N4

(Ac-LESTRRMLQLVEE-NH2) that encompasses the core

of the 22Ala-44Ile region were synthesized and assayed

(Fig. 4a). Because short peptides exhibit lower potency

destabilizing coiled coil complexes, the in vitro reconstitu-

tion of SNARE complexes was modified to accomplish

peptide : SNAP25 ratios greater than 5000 : 1. Specifically,

we used in vitro translated [35S]SNAP25 instead of the

bacterially produced protein. Incubation of [35S]SNAP25

with recombinant VAMP and syntaxin gave rise to the

assembly of the SNARE complex, which was readily detected

as a radioactive band of 75 kDa that disappeared upon heating

the samples to 90�C (Fig. 4b, control and 90�C lanes). Pre-incubation of syntaxin and VAMP with 3 mM SNAP25_N3 or

SNAP25_N4 significantly inhibited the formation of the

SNARE complex (Fig. 4b). Consistent with the stronger

potency of peptide SNAP25_N2, peptide SNAP25_N4

exhibited higher inhibitory activity than peptide SNAP25_N3

(Fig. 4c). The inhibitory activity of both peptides was dose-

dependent. As for larger peptides, CD analysis shows that

both peptides exhibit a conspicuous propensity to adopt a

(a)

(b)

(c)

Fig. 3 Peptides SNAP25_N1 (m) and SNAP25_N2 (d) exhibit signi-

ficant propensity to adopt an a-helical conformation. (a and b) Far-UV

CD spectra of peptides SNAP25_N1 and SNAP25_N2 at increasing

percentages of TFE (0, 5, 10, 20, 30 and 50%). Peptide concentration

was 100 lM in 20 mM Tris pH 8.0. CD spectra represent the average

of five scans, and were corrected for the buffer contribution. (c)

Quantification of the a-helical content as a function of the percentage

of TFE. a-Helical values were inferred as described (Blanes-Mira et al.

2001).

(a)

(b)

(d)

(c)

Fig. 4 Peptides SNAP25_N3 (d) and SNAP25_N4 (m) patterned

after the core segment potently inhibit the formation of the SNARE

complex. (a) Amino acid sequence of peptides SNAP25_N3 and

SNAP25_N4. (b) In vitro reconstituted SNARE complex using

[35S]SNAP25 in the absence (control) and presence of 3 mM

SNAP25_N3 or SNAP25_N4. Control samples heated to 90�C for

5 min are also displayed (90�C). VAMP and syntaxin were used at

3 lM. Complex formation was carried out at 4�C overnight and ana-

lysed by SDS–PAGE. (c) Extent of SNARE complex formation ob-

tained from SDS–PAGE gels by fluorography. Values denote mean ±

SEM, with n ¼ 3. (d) Quantification of a-helical content as a function of

the percentage of TFE. The secondary structure element was calcu-

lated from Far-UV CD spectra of peptides as described (Blanes-Mira

et al. 2001). Peptide concentration was 100 lM in 20 mM Tris pH 8.0.

Peptides inhibitors of neuronal exocytosis 129

� 2003 International Society for Neurochemistry, J. Neurochem. (2004) 88, 124–135

a-helical secondary structure, as evidenced by the strongTFE-inducted a-helical content (Fig. 4d). Taken together,these results substantiate the tenet that peptides mimicking the

22Ala-44Ile segment (Fig. 1a) of the N-terminal half of

SNAP25 are potent inhibitors of SNARE complex formation.

The tendency of the peptides to acquire an a-helical structureseems important for their inhibitory activity, although the

influence of additional properties that contribute to define

their blockade efficacy cannot be ruled out. For instance, it did

not escape to our attention that the first 20 amino acids of

SNAP25 are poorly conserved between different species

while segment 22Ala-44Ile is virtually invariant throughout

evolution (Risinger et al. 1993).

Peptides prevent the formation of the SNARE complex

To gain insights on the inhibitory activity of these peptides, we

compared the kinetics and extent of complex formation in two

conditions: (a) inhibitory peptide SNAP25_N2 was added

prior complex assembly and (b) peptide SNAP25_N2 was

supplied 15 s after the onset of complex formation. The

SNAP25_N2 peptide was selected because of its stronger

efficacy inhibiting complex formation, as evidenced by

dose–response curves that reveal an IC50 of 0.62 ± 0.15 mM

for SNAP25_N2 and 1.5 ± 0.3 mM for SNAP25_N4 (Fig. 5a).

The kinetics of the complex formation was monitored at

4�C (Fig. 5b). As shown, the 75 kDa band was detectable15 s after incubation of the three SNARE proteins, and

complex formation reached steady-state in 30–60 s. Incuba-

tion of syntaxin and VAMP with 2 mM SNAP25_N2 peptide

at 4�C for 10 min before supplying SNAP25 resulted in fullinhibition of SNARE complex formation, at least within the

60 min explored (Fig. 5c). In contrast, the SNAP25_N2 did

not inhibit complex assembly when it was added 15 s after

the mixing of the three SNARE proteins (Fig. 5d). Thus,

peptide SNAP25_N2 prevents complex formation but does

not disrupt pre-assembled complexes.

Mechanistically, peptides patterned after the N-terminus of

SNAP25 may disrupt the binary complex assembled by

SNAP25 and syntaxin, or may impede the interaction of

VAMP with the pre-organized binary aggregate. In an

attempt to address this question, we next studied the effect

of peptide SNAP25_N2 on the arrangement of the binary

complex formed by syntaxin and SNAP25. Assembly of

SNAP25–syntaxin binary complexes was readily monitored

by non-denaturating PAGE (Fig. 6a). Incubation of syntaxin

with 2 mM SNAP25_N2 peptide fully inhibited the consti-

tution of the binary complex with SNAP25 (Fig. 6a, lane 3).

The peptide also disrupted pre-assembled SNAP25-syntaxin

binary complexes suggesting the peptide competes with

SNAP25 for interacting with syntaxin (Fig. 6a, lane 5).

Abrogation of the binary complex by the SNAP25_N2

peptide notably reduced the extent of SNARE complex

assembly, although it did not completely prevent its forma-

tion (Fig. 6a, lanes 4 and 6). In contrast, the interaction of the

(a) (b)

(c)

(d)

Fig. 5 Peptide SNAP25_N2 inhibits the assembly of the SNARE

aggregate if added previous to the onset of complex formation. (a)

Dose–response relationship for the inhibitory activity of the

SNAP25_N2 (j) and SNAP25_N4 (d) peptides. Experimental data

were fitted to a Hill relation

CF

CFm�ax¼ 1

1þ ½compound�IC50

� �nH

where IC50 is the concentration of peptide that prevents the for-

mation of complex to half maximum and nH is an index of the

steepness of the slope. The values obtained were IC50

0.62 ± 0.15 mM and nH ¼ 1.15 ± 0.20 for SNAP25_N2, and IC50

1.5 ± 0.3 and nH ¼ 1.30 ± 0.10 for SNAP25_N4 (mean ± SEM,

n ¼ 2). (b) Kinetics of SNARE complex formation at 4�C in the

absence of SNAP25_N2 peptide. (c) SNAP25_N2 (2 mM) was

incubated with VAMP and syntaxin for 10 min at 4�C. Complex

formation was initiated by addition of SNAP25. (d) SNAP25_N2

peptide was added 15 s after initiation of complex assembly. The

extent of aggregate formation as a function of reaction time was

monitored by SDS–PAGE.

130 C. Blanes-Mira et al.

� 2003 International Society for Neurochemistry, J. Neurochem. (2004) 88, 124–135

three SNARE proteins was not altered when the peptide was

co-incubated with VAMP or added to the pre-assembled

ternary aggregate, which is consistent with the high affinity

and stability of the ternary core complex (Fig. 6a, lanes 7 and

8). Similar results were obtained when the SDS-resistant

SNARE complex was arranged and analyzed under the same

conditions (Fig. 6b). These results demonstrate that peptides

patterned after the N-terminus of SNAP25 prevent the

formation or disrupt the binary complex assembled by

syntaxin and SNAP25, but are unable to affect the consti-

tuted SNARE complex. Thus, the inhibitory activity of

peptides patterned after the N-terminus of SNAP25 is due

primarily to interference with SNARE complex assembly.

Furthermore, they appear to be competitive SNAP25 antag-

onists as a 10-fold increment of the SNAP25 concentration of

SNAP25 virtually abolished the blockade activity of

SNAP25_N2 (data not shown).

Identified peptides potently inhibit Ca2+-evoked

catecholamine release

The inhibitory activity on SNARE complex arrangement

implies that these peptides may modulate the release of

neurotransmitter in excitable cells. To study this issue, we

evaluated the effect of peptides SNAP25_N2 and

SNAP25_N4 on Ca2+-evoked catecholamine release from

detergent-permeabilized chromaffin cells. As illustrated in

Fig. 7, digitonin-treated primary cultures of chromaffin cells

readily release both adrenaline and noradrenaline in response

to a 10-min pulse of 10 lM Ca2+. The extent of the

catecholamine secretion was severely impaired by pre-

incubation of permeabilized chromaffin cells with

SNAP25_N2 and SNAP25_N4 peptides (Fig. 7). Both

peptides block Ca2+-stimulated secretion in a concentra-

tion-dependent manner. The potency blocking catecholamine

release paralleled their activity inhibiting SNARE complex

formation. As depicted in Fig. 7, SNAP25_N2 peptide was

more active than its shorter counterpart SNAP25_N4. Thus,

these observations indicate that peptides patterned after the

N-terminus of SNAP25 block-regulated exocytosis.

Identified peptides are cell-permeable and protect

hippocampal neurones against hypoglycaemia-induced

death

We further investigated the cellular activity of these peptides

in primary neurones from the rat hippocampus. In these

experiments we pursued the topic of whether the peptides

may modulate exocytosis in intact neurones, thus concom-

itantly evaluating the membrane permeability of the peptides.

We used a functional assay that measures the neuronal death

in primary hippocampal cultures induced by glucose-depri-

vation. Hypoglycaemia-evoked neuronal death is due to

excessive exocytosis of L-glutamate (Monyer et al. 1992). As

illustrated in Fig. 8(a), exposure of hippocampal neurones to

glucose-free medium for 3 h evoked neuronal death that was

fully prevented by the presence of the uncompetitive NMDA

receptor antagonist MK-801. This result substantiates the

(a)

(b)

Fig. 6 Peptide SNAP25_N2 abrogates the assembly of the binary

complex formed by SNAP25 and syntaxin. (a) Effect of SNAP25_N2

peptide on the formation of the binary and ternary protein complexes.

Protein interactions were analysed by non-denaturating PAGE. Lane 1

displays SNAP25-syntaxin binary complex; lane 2 shows the ternary

aggregate; lanes 3 and 4, SNAP25_N2 peptide was pre-incubated for

60 min with syntaxin; lanes 5 and 6, peptide SNAP25_N2 was incu-

bated with the pre-formed binary complex; lane 7, peptide was co-

administered with VAMP; lane 8, peptide was added to the assembled

ternary aggregate. Peptide concentration was 2 mM. Binary complex

formation proceed for 2 h at 23�C, while ternary complex assembly

proceed at 4�C overnight. (b) Similar samples as in (a) analysed by

SDS–PAGE. Lane 1, molecular weight standards; lane 2, SNARE

complex; lane 3, SNARE complex heated to 90�C for 5 min; lanes 4, 5,

6 and 7 correspond to lanes 4, 6, 7 and 8 displayed in (a).

Fig. 7 Peptides SNAP25_N2 and SNAP25_N4 inhibit Ca2+-evoked

catecholamine release from digitonin-permeabilized chromaffin cells.

Concentration-dependent inhibition of Ca2+-stimulated release by

peptides SNAP25_N2 and SNAP25_N4. Digitonin permeabilization

lasted 10 min. Net adrenaline and noradrenaline secretion is the

amount evoked by 10 lM Ca2+ minus that stimulated with 5 mM EGTA

for 10 min Net release was normalized with respect to that obtained in

the absence of peptides. Values are mean ± SEM with n ¼ 4.

Experiments were performed at room temperature. NA, nor adrena-

line; A, adrenalin.

Peptides inhibitors of neuronal exocytosis 131

� 2003 International Society for Neurochemistry, J. Neurochem. (2004) 88, 124–135

notion that hypoglycaemia-induced neuronal death is caused

primarily by exposure of neurones to L-glutamate released

into the extracellular medium and emphasizes that blockade

of glutamate exocytosis should prevent neuronal death

(Monyer et al. 1992). As anticipated, pre-treatment of

hippocampal cell cultures with 100 pM BoNT E or BoNT

B resulted in approximately 50% reduction of hypoglycae-

mia-induced neuronal death (Monyer et al. 1992). A dose–

response curve for BoNT E neuroprotective activity showed

an IC50 of 3.0 ± 1.0 pM, and a maximum neuroprotection of

46 ± 5% (Fig. 8b). These results imply that under hypo-

glycaemic conditions, neurones secrete a fraction of the

glutamate by an exocytotic mechanism that involves the

SNARE proteins, and indicate that inhibition of fusion of

synaptic vesicles prevents subsequent neuronal damage.

This finding provides a sensitive assay to investigate the

in vivo inhibitory activity of molecules that affect the

stability or formation of the SNARE complex. As displayed

in Fig. 8(a), incubation of hippocamapal cultures with

100 lM of peptides SNAP25_N1, SNAP25_N2 and

SNAP25_N4 attenuated the neural death triggered by the

hypoglycaemic insult by 15 ± 2%, 40 ± 3.2% and

28 ± 2.8% (mean ± SEM), respectively. Peptide

SNAP25_N3 was not neuroprotectant at this concentration.

Note that the observed neuroprotective activity nicely

paralleled the potency of these peptides inhibiting SNARE

complex formation. A dose–response curve for SNAP25_N2

reveals an IC50 of 1.8 ± 0.3 lM, with a maximum neuro-protective of activity of 40 ± 4% (Fig. 8b). Because these

sequences abrogate SNARE complex assembly and inhibit

Ca2+-dependent exocytosis in permeabilized cells, their

neuroprotective activity in intact neurones imply that they

can translocate through the plasma membrane. To demon-

strate this conclusion, we tagged the N-end of SNAP25_N4

(a) (b)

(c)

Fig. 8 Peptides patterned after the N-terminus of SNAP25 protect

hippocampal neurones from glucose deprivation-evoked death. (a)

Neuroprotection elicited by peptides emulating the N-terminus of

SNAP25, BoNTs E and B and MK801 against 3 h glucose deprivation.

Neuronal viability was assessed blindly 18–24 h postinsult using the

trypan blue exclusion assay. Neurones were incubated with neuro-

toxins 24 h before insult, and with peptides 3 h before glucose depri-

vation. MK-801 (10 lM) and peptides (100 lM) were present during the

3 h glucose deprivation. Values are given as mean ± SD with n ‡ 3000

neurones, and n ‡ 4. (b) Dose–response curves of the BoNT E and

SNAP25_N2 peptide neuroprotective activity against glucose depri-

vation. Solid lines depict the best fit to the equation

D

Dm�ax¼ 1

1þ ½compound�IC50

� �nH

where D denotes normalized cell death, IC50 the concentration of

compound that reduces cell death to half-maximum and nH is the hill

coefficient. The IC50 for BoNT E was 3.0 ± 1 pM, and for SNAP25_N2

was 1.8 ± 0.3 lM. The maximal neuroprotection was 46% ± 5 for

BoNT E and 40% ± 4 for SNAP25_N2. Values are mean ± SEM with

n ¼ 4, and n ¼ 2000 neurones. (c) Illustrative photograph (left)

showing that fluorescently labelled SNAP25_N4 accumulates into the

cytosol of PC12 cells. Fluorescein-conjugated peptide (1 lM) was

incubated for 2 h, cells were extensively washed with phosphate-

buffered saline, and cellular uptake analysed by confocal microscopy.

(Right) Light-transmitted picture of cells shown in the left panel. Scale

bar denotes 10 lm.

132 C. Blanes-Mira et al.

� 2003 International Society for Neurochemistry, J. Neurochem. (2004) 88, 124–135

peptide with fluorescein to monitor its cell permeability.

PC12 cells were incubated with 1 lM fluorescein-

SNAP25_N4 for 2 h, washed and analysed by confocal

microscopy. As shown in Fig. 8(c), the fluorescently labelled

SNAP25_N4 derivative readily accumulated into the cytosol

of the cells. Taken together, these data demonstrate that

peptides patterned after the N-terminus of SNAP25 are cell

permeable peptides that inhibit neuronal exocytosis by

interfering with vesicle fusion.

Discussion

The central aim of this study was to investigate if peptides

derived from the N-terminus of SNAP25 modulate the

stability and function of the SNARE core complex. We

addressed this question because, at variance with the intense

research performed on the role of the SNAP25 C-end

domain, the implication of the N-terminal domain has

remained poorly investigated. As a result, BoNT A and E

and peptides patterned after the C-terminus of SNAP25 have

been used to understand the dynamics of complex formation,

as well as of the neurosecretory cascade (Gutierrez et al.

1997; Ferrer-Montiel et al. 1998b; Apland et al. 1999).

Recently, however, an hexapeptide derived from the N-

terminal domain of SNAP25 was reported to modulate the

stability of the SNARE core complex and to inhibit Ca2+-

evoked neurosecretion in chromaffin cells (Blanes-Mira

et al. 2002). Notably, topical formulations containing this

peptide displayed antiwrinkle activity in humans, similar to

that exhibited by BoNT A (Benedetto 1999; Blanes-Mira

et al. 2002). In addition, deletion of N-terminus domains or

the use of specific antibodies suggest a critical contribution

of this protein segment to SNARE complex assembly and

regulated exocytosis (Chapman et al. 1994; Xu et al. 1999;

Yang et al. 2000).

Analysis of the N-terminal primary structure of SNAP25

revealed the presence of two domains with distinct forecast

propensities to form coiled coils and to adopt an a-helicalsecondary structure. Notably, the segment that exhibited the

lowest predicted propensity to coiled coil and a-helixformation was the most critical for SNARE complex

assembly. Deletion of the region encompassing residues

22Ala-44Ile from SNAP25 completely prevented the in vitro

formation of the core SNARE complex. In marked contrast,

removal of the segment 1Met-21Leu yielded SNARE

complexes that dissociated at a lower temperature. Our

results suggest that the protein domain comprising residues

22Ala-44Ile is critical for the interaction of SNAP25 with the

SNARE proteins, while the 1Met-21Leu segment contributes

to the thermal stability of the complex. These observations

are in agreement with results using antibodies targeting these

two protein domains (Xu et al. 1999). Taken together, our

findings suggest that peptides patterned after these two

protein segments, especially those emulating the segment

22Ala-44Ile, may efficiently modulate the stability and

formation of the SNARE core complex. To substantiate this

notion, we investigated the inhibitory activity of peptides

mimicking domains 1Met-21Leu and 22Ala-44Ile of

SNAP25. Both sequences reduced the in vitro formation of

the SNARE core complex, being the peptide mimicking the

22Ala-44Ile segment the most potent. This conclusion was

further underscored by the inhibitory activity of the 13-mer

peptide SNAP25_N4 comprising the core sequence of the

SNAP25 segment 22Ala-44Ile. Noteworthy, the in vitro

inhibitory activity of SNARE complex formation of these

peptides was paralleled by their efficacy abolishing Ca2+-

Fig. 9 Structural model illustrating the

putative binding site of peptides

SNAP25_N2 on the SNARE complex. (Top)

Putative interaction of SNAP25_N2 peptide

on the SNARE complex preventing the

interaction of the N-terminus domain of

SNAP25. (Bottom) Enlargement showing

the interactions of peptide SNAP25_N4 with

syntaxin (red), the C-terminus of SNAP25

(yellow) and VAMP (blue). Peptide is shown

in magenta. Interactions are highlighted.

Peptides inhibitors of neuronal exocytosis 133

� 2003 International Society for Neurochemistry, J. Neurochem. (2004) 88, 124–135

evoked neurosecretion in chromaffin cells as well as in

neurones. Most significant was the discovery that these

peptides were able to translocate through the plasma

membrane of intact cells, as evidenced by their neuropro-

tective activity of hippocampal neurones against excitotox-

icity. Blockade of regulated exocytosis was observed at a

concentration as low as 1 lM, which is 500-fold lower thanthat required to target the SNARE complex in vitro. This

finding is consistent with the notion that the in vivo

exocytosis-competent SNARE complex is the trans confi-

guration, while in vitro predominates the cis form. It has been

reported that the trans-SNARE complex is energetically less

stable that the cis form and, thus more amenable to

modulation by small molecules (Weber et al. 1998; Brunger

2001; Chen and Scheller 2001; Bruns and Jahn 2002; Melia

et al. 2002).

Mechanistically, peptides mimicking the N-end of

SNAP25 disrupt the interaction of the parental protein with

syntaxin, as clearly evidenced by the full abrogation of the

binary complex formed in vitro by both SNARE protein. The

ternary complex was markedly destabilized when syntaxin

was pre-incubated with the SNAP25_N2 peptide. In contrast,

pre-assembled SNARE complexes were not affected by the

peptide, as demonstrated by the lack of efficacy of the most

potent amino acid sequence. Furthermore, an increment in

the concentration of SNAP25 fully prevented the inhibitory

activity of the peptides. These findings indicate that these

peptides act as competitive antagonists of SNAP25 for the

assembly of the SNARE complex.

This conclusion along with their high propensity to adopt

an a-helical secondary structure, may be used to gaininsights of their binding site by using the three-dimensional

structure of the SNARE complex as a guide. For this task,

the N-terminus of SNAP25 on the structure was trimmed to

leave the sequences of the SNAP25_N2 and SNAP25_N4

(Fig. 9). As illustrated, a model of the peptide SNAP25_N4

docked on syntaxin and the C-terminal domain of SNAP25

shows that this peptide interacts with syntaxin segment

199His-217Met and SNAP25 domain 142Arg-160Leu,

respectively. Notably, the SNAP25_N4 peptide can establish

up to seven interactions with syntaxin and the SNAP25

C-end. As shown, residue 30Arg on the peptide forms a salt

bridge with 145Glu on the C-terminal of SNAP25, while

31Arg and 28Ser interact with 206Glu on syntaxin.

Furthermore, 36Glu on SNAP25_N4 pairs with 213His on

syntaxin. In addition of these interacting pairs, several

hydrophobic interactions notably contribute to peptide

binding (Fig. 9, inset). Similar results are obtained for

peptide SNAP25_N2. In contrast, peptides SNAP25_N1

and SNAP25_N3 are stabilized with fewer interactions,

namely 19Asp on SNAP25_N3 with 142Arg on SNAP25

C-end, and 17Arg on SNAP25_N3 with 196Glu on

syntaxin (not shown). Therefore, the higher number of

interacting surface area exhibited by SNAP25_N2 and

SNAP25_N4 provides a structural explanation of their

higher efficacy and potency precluding the SNARE com-

plex assembly and abrogating neuronal exocytosis.

In conclusion, the most salient contribution of this study is

the discovery of cell-permeable peptides patterned after the

SNAP25 N-terminus that efficiently prevent the formation of

the SNARE complex and potently inhibit regulated exocy-

tosis from excitable cells. These peptides significantly

protected primary neurones against glucose-deprivation

induced neurodegeneration. Therefore, our findings imply

that peptides that target the binary complex assembled by

SNAP25 and syntaxin may be novel therapeutics to

efficiently attenuate dysfunctional exocytosis such as that

characteristic of spasmodic disorders, excitotoxicity and pain

transduction. This notion is further substantiated by the

development of argireline, a hexapeptide encompassing the

12Glu-17Arg amino acid sequence, as an active antiwrinkle

agent for human use (Blanes-Mira et al. 2002). Thus, the

peptides SNAP25_N2 and SNAP25_N4 should be consid-

ered hit compounds for novel antispasmodic and analgesic

drugs that complement BoNT-based therapies.

Acknowledgements

This work was supported by grants from La Fundacion La Caixa

(01/085–00 to AF-M), the Spanish Ministry of Science and

Technology (MCYT) (SAF2000-0142 to AF-M and BMC2002-

00845 to LMG), the Instituto de la Salud Carlos III (FIS-01/1162 to

AF-M), The Generalitat Valenciana (GV01-01 to AF-M), the EU

Biotechnology BIO4-CT97-2086 and the Fundacion Ramon Areces

to EPP.

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