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Biochem. J. (2003) 375 (159–166) (Printed in Great Britain)

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Identification of SNARE complex modulators that inhibit exocytosis from an a-helix-constrained combinatorial library

Clara BLANES-MIRA*, Maria T. PASTOR†, Elvira VALERA‡, Gregorio FERNÁNDEZ-BALLESTER*, Jaime M. MERINO‡, Luis M. GUTIERREZ§, Enrique PEREZ-PAYÁ† and Antonio FERRER-MONTIEL*¹

*Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Av. De la Universidad s/n, 03202 Elche (Alicante), Spain, †Departamento Bioquímica y Biología Molecular, Universidad de Valencia, 46100 Burjassot (Valencia) Spain, ‡Departamento Bioquímica y Biología Molecular, Universidad de Extremadura, Av. Elvas s/n, 06080 Badajoz, Spain, §Instituto Neurociencias-CSIC, Universidad Miguel Hernández, Crta. Alicante-Valencia s/n, 03550 San Juan de Alicante, Spain, and Fundación Valenciana de Investigaciones Biomédicas, CSIC, Amadeo de Saboya, 4, 46010 Valencia, Spain

Synthetic peptides patterned after the proteins involved in vesicle fusion [the so-called SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) proteins] are potent inhibitors of SNARE complex assembly and neuronal exocytosis. It is noteworthy that the identification of peptide sequences not related to the SNARE proteins has not been accomplished yet; this is due, in part, to the structural constraints and the specificity of the protein interactions that govern the formation of the SNARE complex. Here we have addressed this question and used a combinatorial approach to identify peptides that modulate the assembly of the SNARE core complex and inhibit neuronal exocytosis. An a-helix-constrained, mixture-based, 17-mer combinatorial peptide library composed of 137180 sequences was synthesized in a positional scanning format. Peptide mixtures were assayed for their ability to prevent the formation of the in vitro-reconstituted SDS-resistant SNARE core complex. Library deconvolution identified eight peptides that inhibited the assembly of the SNARE core complex. Notably, the most potent 17-mer peptide (acetyl-SAAEAFAKLYAEAFAKG-NH₂) abolished both Ca²⁺-evoked catecholamine secretion from detergent-permeabilized chromaffin cells and L-glutamate release from intact hippocampal primary cultures. Collectively, these findings indicate that amino acid sequences that prevent SNARE complex formation are not restricted to those that mimic domains of SNARE proteins, thus expanding the diversity of molecules that target neuronal exocytosis. Because of the implication of neurosecretion in the aetiology of several human neurological disorders, these newly identified peptides may be considered hits for the development of novel anti-spasmodic drugs.

Key words: combinatorial chemistry, drug discovery, neurosecretion, protein–protein interaction, synaptic transmission, vesicle fusion.

Abbreviations used: Ac, acetyl; BSS, basic saline solution; OG, octyl b-D-glucopyranoside; SNAP25, synaptosomal-associated protein of 25 kDa; SNARE, soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor; VAMP, vesicle-associated membrane protein (synaptobrevin); [O⁶O⁹O¹⁰O¹⁴], shortened peptide nomenclature referring to the amino acids preferred at the combinatorialized positions of the 17-mer (e.g. Ac-SAAEAFAKLYAEAFAKG-NH₂ is referred to as [F⁶L⁹Y¹⁰F¹⁴]).

¹To whom correspondence should be addressed (e-mail aferrer@umh.es).

Received 4 April 2003/25 June 2003; accepted 9 July 2003

Published as BJ Immediate Publication 9 July 2003, DOI 10.1042/BJ20030509

The Biochemical Society, London ©2003