Early investigational drugs that target epidermal growth factor receptors for the treatment of head and neck cancer.

INTRODUCTION: Squamous-cell carcinoma of the head and neck (SCCHN) remains a challenging clinical problem, due to the persistent high rate of local and distant failures and the occurrence of secondary primaries. For locally advanced SCCHN, a combination of chemotherapy (CT), radiation or surgery is often used, but there are limitations, which may reduce compliance. Molecular targeted therapies, namely anti-EGFR treatments, are in development with the aim of improving clinical outcomes and mitigating treatment-related toxicities. AREAS COVERED: This review provides an overview of early investigational drugs that target EGFR for the treatment of SCCHN and discusses the ongoing trials in this domain. EXPERT OPINION: Targeted therapies are increasingly used in oncology, especially in SCCHN. Cetuximab has demonstrated a significant improvement in the treatment outcome, both as a curative treatment in combination with radiation therapy and as a palliative treatment in combination with CT; however, it failed to show any benefit in combination with concomitant chemoradiotherapy. Presently, there are many new agents, including monoclonal antibodies and small-molecule tyrosine kinase inhibitors, which are either currently under investigation for or which warrant further investigation for treating SCCHN. The discovery of predictive factors that help to identify patients most likely to respond to EGFR inhibitors as well as patient-customized therapies would help to improve patient outcomes in the future.

Deletion of the RNA-binding proteins ZFP36L1 and ZFP36L2 leads to perturbed thymic development and T lymphoblastic leukemia.

ZFP36L1 and ZFP36L2 are RNA-binding proteins (RBPs) that interact with AU-rich elements in the 3' untranslated region of mRNA, which leads to mRNA degradation and translational repression. Here we show that mice that lacked ZFP36L1 and ZFP36L2 during thymopoiesis developed a T cell acute lymphoblastic leukemia (T-ALL) dependent on the oncogenic transcription factor Notch1. Before the onset of T-ALL, thymic development was perturbed, with accumulation of cells that had passed through the beta-selection checkpoint without first expressing the T cell antigen receptor beta-chain (TCRbeta). Notch1 expression was higher in untransformed thymocytes in the absence of ZFP36L1 and ZFP36L2. Both RBPs interacted with evolutionarily conserved AU-rich elements in the 3' untranslated region of Notch1 and suppressed its expression. Our data establish a role for ZFP36L1 and ZFP36L2 during thymocyte development and in the prevention of malignant transformation.

Expression and role of BORIS/CTCFL in human cancer stem cells

Le cancer est défini comme la croissance incontrôlée des cellules dans le corps. Il est responsable de 20 % des décès en Europe. Plusieurs expériences montrent que les tumeurs sont issues et se développent grâce à un petit nombre de cellules, que l'on appelle cellules souches cancéreuses (CSC). Ces CSC sont également responsables de l'apparition de métastases et de la résistance aux médicaments anticancéreux. De ce fait, l'identification des gènes qui contribuent aux propriétés de ces CSC (comme la survie des tumeurs, les métastases et la résistance aux médicaments) est nécessaire pour mieux comprendre la biologie des cancers et d'améliorer la qualité des soins des patients avec un cancer. A ce jour, de nombreux marqueurs ont été proposés ainsi que de nouvelles thérapies ciblées contre les CSC. Toutefois, et malgré les énormes efforts de la recherche dans ce domaine, la quasi-totalité des marqueurs de CSC connus à ce jour sont aussi exprimés dans les cellules saines. Ce projet de recherche visait à trouver un nouveau candidat spécifique des CSC. Le gène BORIS (pour Brother of Regulator of Imprinted Sites), nommé aussi CTCFL (CTCF-like), semble avoir certaines caractéristiques de CSC et pourrait donc devenir une cible prometteuse pour le traitement du cancer. BORIS/CTCFL est une protéine nucléaire qui se lie à l'ADN, qui est exprimée dans les tissus normaux uniquement dans les cellules germinales et qui est réactivée dans un grand nombre de tumeurs. BORIS est impliqué dans la reprogrammation épigénétique au cours du développement et dans la tumorigenèse. En outre, des études récentes ont montré une association entre l'expression de BORIS et un mauvais pronostic chez des patients atteints de différents types de cancers. Nous avons développé une nouvelle technologie basée sur les Molecular Beacon pour cibler l'ARNm de BORIS et cela dans les cellules vivantes. Grâce à ce système expérimental, nous avons montré que seule une toute petite sous-population (0,02 à 5%) de cellules tumorales exprimait fortement BORIS. Les cellules exprimant BORIS ont pu être isolées et elles présentaient les caractéristiques de CSC, telles qu'une forte expression de hTERT et des gènes spécifiques des cellules souches (NANOG, SOX2 et OCT4). En outre, une expression élevée de BORIS a été mise en évidence dans des populations enrichies en CSC ('side population' et sphères). Ces résultats suggèrent que BORIS pourrait devenir un nouveau et important marqueur de CSC. Dans des études fonctionnelles sur des cellules de cancer du côlon et du sein, nous avons montré que le blocage de l'expression de BORIS altère largement la capacité de ces cellules à former des sphères, démontrant ainsi un rôle essentiel de BORIS dans l'auto- renouvellement des tumeurs. Nos expériences montrent aussi que BORIS est un facteur important qui régule l'expression de gènes jouant un rôle clé dans le développement et la progression tumorale, tels le gène hTERT et ceux impliqués dans les cellules souches, les CSC et la transition épithélio-mésenchymateuse (EMT). BORIS pourrait affecter la régulation de la transcription de ces gènes par des modifications épigénétiques et de manière différente en fonction du type cellulaire. En résumé, nos résultats fournissent la preuve que BORIS peut être classé comme un gène marqueur de cellules souches cancéreuse et révèlent un nouveau mécanisme dans lequel BORIS jouerait un rôle important dans la carcinogénèse. Cette étude ouvre de nouvelles voies pour mieux comprendre la biologie de la progression tumorale et offre la possibilité de développement de nouvelles thérapies anti-tumorales et anti-CSC avec BORIS comme molécule cible. - Cancer is defined as the uncontrolled growth of cells in the body. It causes 20% of deaths in the European region. Current evidences suggest that tumors originate and are maintained thanks to a small subset of cells, named cancer stems cells (CSCs). These CSCs are also responsible for the appearance of metastasis and therapeutic resistance. Consequently, the identification of genes that contribute to the CSC properties (tumor survival, metastasis and therapeutic resistance) is necessary to better understand the biology of malignant diseases and to improve care management. To date, numerous markers have been proposed to use as new CSC- targeted therapies. Despite the enormous efforts in research, almost all of the known CSCs markers are also expressed in normal cells. This project aimed to find a new CSC-specific candidate. BORIS (Brother of Regulator of Imprinted Sites) or CTCFL (CTCF-like) is a DNA binding protein involves in epigenetic reprogramming in normal development and in tumorigenesis. Recent studies have shown an association of BORIS expression with a poor prognosis in different types of cancer patients. Therefore, BORIS seems to have the same characteristics of CSCs markers and it could be a promising target for cancer therapy. BORIS is normally expressed only in germinal cells and it is re-expressed in a wide variety of tumors. We developed a new molecular beacon-based technology to target BORIS mRNA expressing cells. Using this system, we showed that the BORIS expressing cells are only a small subpopulation (0.02-5%) of tumor cells. The isolated BORIS expressing cells exhibited the characteristics of CSCs, with high expression of hTERT and stem cell genes (NANOG, SOX2 and OCT4). Furthermore, high BORIS expression was observed in the CSC-enriched populations (side population and spheres). These results suggest that BORIS might be a novel and powerful CSCs marker. In functional studies, we observed that BORIS knockdown significantly impairs the capacity to form spheres in colon and breast cancer cells, thus demonstrating a critical role of BORIS in the self-renewal of tumors. The results showed in the functional analysis indicate that BORIS is an important factor that regulates the expression of key-target genes for tumor development and progression, such as hTERT, stem cells, CSCs markers and EMT (epithelial mesenchymal transition)-related marker genes. BORIS could affect the transcriptional regulation of these genes by epigenetic modification and in a cell type dependent manner. In summary, our results support the evidence that BORIS can be classified as a cancer stem cell marker gene and reveal a novel mechanism in which BORIS would play a critical role in tumorigenesis. This study opens new prospective to understand the biology of tumor development and provides opportunities for potential anti-tumor drugs.

Wipe sampling procedure coupled to LC-MS/MS analysis for the simultaneous determination of 10 cytotoxic drugs on different surfaces.

A simple wipe sampling procedure was developed for the surface contamination determination of ten cytotoxic drugs: cytarabine, gemcitabine, methotrexate, etoposide phosphate, cyclophosphamide, ifosfamide, irinotecan, doxorubicin, epirubicin and vincristine. Wiping was performed using Whatman filter paper on different surfaces such as stainless steel, polypropylene, polystyrol, glass, latex gloves, computer mouse and coated paperboard. Wiping and desorption procedures were investigated: The same solution containing 20% acetonitrile and 0.1% formic acid in water gave the best results. After ultrasonic desorption and then centrifugation, samples were analysed by a validated liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in selected reaction monitoring mode. The whole analytical strategy from wipe sampling to LC-MS/MS analysis was evaluated to determine quantitative performance. The lowest limit of quantification of 10 ng per wiping sample (i.e. 0.1 ng cm(-2)) was determined for the ten investigated cytotoxic drugs. Relative standard deviation for intermediate precision was always inferior to 20%. As recovery was dependent on the tested surface for each drug, a correction factor was determined and applied for real samples. The method was then successfully applied at the cytotoxic production unit of the Geneva University Hospitals pharmacy.

Photoaffinity labeling of the T cell receptor on cloned cytotoxic T lymphocytes by covalent photoreactive ligand.

The interaction of the T cell antigen receptor with a photoreactive antigenic peptide derivative bound covalently to the H-2Kd (Kd) molecule was studied by photoaffinity labeling on cloned, CD8 positive cytotoxic T lymphocytes. The Kd-restricted Plasmodium berghei circumsporozoite peptide 253-260 (YIPS-AEKI) was conjugated with iodo-4-azidosalicylic acid at the N terminus and with 4-azidobenzoic acid at the T cell receptor residue Lys-259. Cell-associated or soluble Kd molecules were photoaffinity-labeled with the peptide derivative by selective photoactivation of the N-terminal photoreactive group. Incubation of cell-associated or soluble covalent Kd-peptide derivative complexes (ligands) with cytotoxic T lymphocytes that recognized this peptide derivative and activation of the orthogonal photoreactive group resulted in specific photoaffinity labeling of the T cell receptor. The labeling was inhibitable by an anti-Kd antibody and was absent on Kd-restricted cytotoxic T lymphocytes of different specificity. The binding of the soluble ligand reached a maximum after 2-4 min at 37 degrees C, after 30 min at 18 degrees C, and after 3 h at 4 degrees C. In contrast, binding of the cell-associated ligand reached a transient maxima after 50 and 110 min at 37 and 18 degrees C, respectively. The degree of binding at 37 degrees C was approximately 30% lower than that at 18 degrees C. No binding took place at 4 degrees C. Inhibition studies with antibodies and drugs indicated that the binding of the cell-associated, but not the soluble ligand, was highly dependent on T cell-target cell conjugate formation, whereas the binding of the soluble ligand was greatly dependent on CD8.

Tumor necrosis factor and transforming growth factor β regulate clock genes by controlling the expression of the cold inducible RNA-binding protein (CIRBP).

The circadian clock drives the rhythmic expression of a broad array of genes that orchestrate metabolism, sleep wake behavior, and the immune response. Clock genes are transcriptional regulators engaged in the generation of circadian rhythms. The cold inducible RNA-binding protein (CIRBP) guarantees high amplitude expression of clock. The cytokines TNF and TGFβ impair the expression of clock genes, namely the period genes and the proline- and acidic amino acid-rich basic leucine zipper (PAR-bZip) clock-controlled genes. Here, we show that TNF and TGFβ impair the expression of Cirbp in fibroblasts and neuronal cells. IL-1β, IL-6, IFNα, and IFNγ do not exert such effects. Depletion of Cirbp is found to increase the susceptibility of cells to the TNF-mediated inhibition of high amplitude expression of clock genes and modulates the TNF-induced cytokine response. Our findings reveal a new mechanism of cytokine-regulated expression of clock genes.

Amniotic fluid insulin-like growth factor binding-protein 3 concentration as early indicator of fetal growth restriction

Rapport de synthèse : Introduction : La croissance foetale infra-utérine dépend d'un grand nombre de facteurs maternels, placentaires et foetaux. Une inadéquation d'un ou plusieurs de ces facteurs peut induire un retard de croissance infra-utérin (RCIU) ou au contraire une macrosomie. Les principales causes de RCIU comprennent les infections maternelles, l'éclampsie, les cardiovasculopathies maternelles, la toxicomanie, les malformations foetales et les insuffisances placentaires. Les facteurs endocriniens constituent un petit pourcentage des causes de RCIU, mais méritent que l'on s'y intéresse de plus près. Les facteurs hormonaux les plus importants pour la croissance fatale sont l'insuline et les insuline-like growth factors (IGFs) et non l'hormone de croissance (GH) qui joue un rôle majeur dans la croissance postnatale. Notre attention s'est portée sur IGF-1 qui joue un rôle important dans la croissance intrautérine. Sa biodisponibilité dépend de plusieurs protéines plasmatiques, les IGF-binding proteins (IGFBP 1 à 9). IGFBP-3 est la principale de ces IGFBPs, autant d'un point de vue quantitatif que fonctionnel. Nous avons cherché à déterminer si les concentrations d'IGF-1 et d'IGFBP-3 dans le liquide amniotique au début du deuxième trimestre étaient prédictives de la croissance infra-utérine. Les gènes codant pour IGF-1 et IGFBP-3 contenant certaines séquences polymorphiques, nous avons également étudié leur influence sur la croissance foetale. L'analyse du liquide amniotique présente l'avantage de pouvoir être effectuée dès la 14ème semaine d'aménorrhée alors que la biométrie foetale échographique ne permet pas à ce stade de déceler des anomalies de la croissance infra-utérine. Méthode : Nous avons analysé des échantillons de liquide amniotique prélevés entre la 14ème et la 18ème semaine de grossesse chez 196 patientes. Les concentrations d'IGF-1 et d'IGFBP-3 ont été dosées par ELISA, les polymorphismes analysés par PCR. Ces résultats ont été ensuite analysés en fonction du poids de naissance des nouveaux-nés, répartis en trois groupes normal pour l'âge gestationnel (AGA), petit pour l'âge gestationnel (SGA) et grand pour l'âge gestationnel (LGA). Résultats : Les concentrations d'IGFBP3 dans le liquide amniotique sont significativement plus élevées (p = 0.030) dans le groupe SGA par rapport au groupe AGA, d'autant plus quand les taux sont ajustés en fonction de paramètres tels que l'âge gestationnel lors de l'amniocentèse (ANCOVA analysis : p = 0.009). La distribution du polymorphisme VNTR (variable number of tandem repeat) dans la région promotrice d'IGF-1 au sein du groupe SGA est significativement différente de celle du groupe AGA (p = 0.029). En effet, la fréquence de l'association allélique 19CA/20CA est diminuée dans le groupe SGA. Nous n'avons pas identifié de différence de distribution des séquences polymorphiques d'IGFBP-3 entre les différents groupes. Conclusion : Une concentration élevée d'IGFBP-3 dans le liquide amniotique au début du deuxième trimestre est associée à un risque plus élevé de retard de croissance alors que l'association allélique 19CA/20CA dans la région polymorphique IGF-1 VNTR est un facteur protecteur.

Study of common database feeding with results coming from different analytical methods in the framework of illicit drugs chemical profiling

Analytical results harmonisation is investigated in this study to provide an alternative to the restrictive approach of analytical methods harmonisation which is recommended nowadays for making possible the exchange of information and then for supporting the fight against illicit drugs trafficking. Indeed, the main goal of this study is to demonstrate that a common database can be fed by a range of different analytical methods, whatever the differences in levels of analytical parameters between these latter ones. For this purpose, a methodology making possible the estimation and even the optimisation of results similarity coming from different analytical methods was then developed. In particular, the possibility to introduce chemical profiles obtained with Fast GC-FID in a GC-MS database is studied in this paper. By the use of the methodology, the similarity of results coming from different analytical methods can be objectively assessed and the utility in practice of database sharing by these methods can be evaluated, depending on profiling purposes (evidential vs. operational perspective tool). This methodology can be regarded as a relevant approach for database feeding by different analytical methods and puts in doubt the necessity to analyse all illicit drugs seizures in one single laboratory or to implement analytical methods harmonisation in each participating laboratory.

Characterization of the ligand-binding site of the serotonin 5-HT3 receptor: the role of glutamate residues 97, 224, AND 235.

Ligand-gated ion channels of the Cys loop family are receptors for small amine-containing neurotransmitters. Charged amino acids are strongly conserved in the ligand-binding domain of these receptor proteins. To investigate the role of particular residues in ligand binding of the serotonin 5-HT3AS receptor (5-HT3R), glutamate amino acid residues at three different positions, Glu97, Glu224, and Glu235, in the extracellular N-terminal domain were substituted with aspartate and glutamine using site-directed mutagenesis. Wild type and mutant receptor proteins were expressed in HEK293 cells and analyzed by electrophysiology, radioligand binding, fluorescence measurements, and immunochemistry. A structural model of the ligand-binding domain of the 5-HT3R based on the acetylcholine binding protein revealed the position of the mutated amino acids. Our results demonstrate that mutations of Glu97, distant from the ligand-binding site, had little effect on the receptor, whereas mutations Glu224 and Glu235, close to the predicted binding site, are indeed important for ligand binding. Mutations E224Q, E224D, and E235Q decreased EC50 and Kd values 5-20-fold, whereas E235D was functionally expressed at a low level and had a more than 100-fold increased EC50 value. Comparison of the fluorescence properties of a fluorescein-labeled antagonist upon binding to wild type 5-HT3R and E235Q, allowed us to localize Glu235 within a distance of 1 nm around the ligand-binding site, as proposed by our model.

Mutations inducing divergent shifts of constitutive activity reveal different modes of binding among catecholamine analogues to the beta(2)-adrenergic receptor.

1. We compared the changes in binding energy generated by two mutations that shift in divergent directions the constitutive activity of the human beta(2) adrenergic receptor (beta(2)AR). 2. A constitutively activating mutant (CAM) and the double alanine replacement (AA mutant) of catechol-binding serines (S204A, S207A) in helix 5 were stably expressed in CHO cell lines, and used to measure the binding affinities of more than 40 adrenergic ligands. Moreover, the efficacy of the same group of compounds was determined as intrinsic activity for maximal adenylyl cyclase stimulation in wild-type beta(2)AR. 3. Although the two mutations had opposite effects on ligand affinity, the extents of change were in both cases largely correlated with the degree of ligand efficacy. This was particularly evident if the extra loss of binding energy due to hydrogen bond deletion in the AA mutant was taken into account. Thus the data demonstrate that there is an overall linkage between the configuration of the binding pocket and the intrinsic equilibrium between active and inactive receptor forms. 4. We also found that AA mutation-induced affinity changes for catecholamine congeners gradually lacking ethanolamine substituents were linearly correlated to the loss of affinity that such modifications of the ligand cause for wild-type receptor. This indicates that the strength of bonds between catechol ring and helix 5 is critically dependent on the rest of interactions of the beta-ethanolamine tail with other residues of the beta(2)-AR binding pocket.

Population PK-guided simulation study in children exposed to drugs in human milk

Risks of significant infant drug exposure through human milk arepoorly defined due to lack of large-scale PK data. We propose to useBayesian approach based on population PK (popPK)-guided modelingand simulation for risk prediction. As a proof-of-principle study, weexploited fluoxetine milk concentration data from 25 women. popPKparameters including milk-to-plasma ratio (MP ratio) were estimatedfrom the best model. The dose of fluoxetine the breastfed infant wouldreceive through mother's milk, and infant plasma concentrations wereestimated from 1000 simulated mother-infant pairs, using randomassignment of feeding times and milk volume. A conservative estimateof CYP2D6 activity of 20% of the allometrically-adjusted adult valuewas assumed. Derived model parameters, including MP ratio were consistentwith those reported in the literature. Visual predictive check andother model diagnostics showed no signs of model misspecifications.The model simulation predicted that infant exposure levels to fluoxetinevia mother's milk were below 10% of weight-adjusted maternal therapeuticdoses in >99% of simulated infants. Predicted median ratio ofinfant-mother serum levels at steady state was 0.093 (range 0.033-0.31),consistent with literature reported values (mean=0.07; range 0-0.59).Predicted incidence of relatively high infant-mother ratio (>0.2) ofsteady-state serum fluoxetine concentrations was <1.3%. Overall, ourpredictions are consistent with clinical observations. Our approach maybe valid for other drugs, allowing in silico prediction of infant drugexposure risks through human milk. We will discuss application of thisapproach to another drug used in lactating women.

Transcriptional activation of the macrophage migration-inhibitory factor gene by the corticotropin-releasing factor is mediated by the cyclic adenosine 3',5'- monophosphate responsive element-binding protein CREB in pituitary cells.

Macrophage migration-inhibitory factor (MIF) has recently been identified as a pituitary hormone that functions as a counterregulatory modulator of glucocorticoid action within the immune system. In the anterior pituitary gland, MIF is expressed in TSH- and ACTH-producing cells, and its secretion is induced by CRF. To investigate MIF function and regulation within pituitary cells, we initiated the characterization of the MIF 5'-regulatory region of the gene. The -1033 to +63 bp of the murine MIF promoter was cloned 5' to a luciferase reporter gene and transiently transfected into freshly isolated rat anterior pituitary cells. This construct drove high basal transcriptional activity that was further enhanced after stimulation with CRF or with an activator of adenylate cyclase. These transcriptional effects were associated with a concomitant rise in ACTH secretion in the transfected cells and by an increase in MIF gene expression as assessed by Northern blot analysis. A cAMP-responsive element (CRE) was identified within the MIF promoter region which, once mutated, abolished the cAMP responsiveness of the gene. Using this newly identified CRE, DNA-binding activity was detected by gel retardation assay in nuclear extracts prepared from isolated anterior pituitary cells and AtT-20 corticotrope tumor cells. Supershift experiments using antibodies against the CRE-binding protein CREB, together with competition assays and the use of recombinant CREB, allowed the detection of CREB-binding activity with the identified MIF CRE. These data demonstrate that CREB is the mediator of the CRF-induced MIF gene transcription in pituitary cells through an identified CRE in the proximal region of the MIF promoter.

Specific binding of antigenic peptides to cell-associated MHC class I molecules.

T lymphocytes recognize antigen in the form of peptides that associate with specific alleles of class I or class II major histocompatibility (MHC) molecules. By contrast with the clear MHC allele-specific binding of peptides to purified class II molecules purified solubilized class I molecules either bind relatively poorly or show degenerate specificity. Using photo-affinity labelling, we demonstrate here the specific interaction of peptides with cell-associated MHC class I molecules and show that this involves metabolically active processes.

Beta(1)-selective agonist (-)-1-(3,4-dimethoxyphenetylamino)-3-(3,4-dihydroxy)-2-propanol [(-)-RO363] differentially interacts with key amino acids responsible for beta(1)-selective binding in resting and active states.

(-)-1-(3,4-Dimethoxyphenetylamino)-3-(3,4-dihydroxy)-2-propanol [(-)-RO363] is a highly selective beta(1)-adrenergic receptor (beta(1)AR) agonist. To study the binding site of beta(1)-selective agonist, chimeric beta(1)/beta(2)ARs and Ala-substituted beta(1)ARs were constructed. Several key residues of beta(1)AR [Leu(110) and Thr(117) in transmembrane domain (TMD) 2], and Phe(359) in TMD 7] were found to be responsible for beta(1)-selective binding of (-)-RO363, as determined by competitive binding. Based on these results, we built a three-dimensional model of the binding domain for (-)-RO363. The model indicated that TMD 2 and TMD 7 of beta(1)AR form a binding pocket; the methoxyphenyl group of N-substituent of (-)-RO363 seems to locate within the cavity surrounded by Leu(110), Thr(117), and Phe(359). The amino acids Leu(110) and Phe(359) interact with the phenyl ring of (-)-RO363, whereas Thr(117) forms hydrogen bond with the methoxy group of (-)-RO363. To examine the interaction of these residues with beta(1)AR in an active state, each of the amino acids was changed to Ala in a constitutively active (CA)-beta(1)AR mutant. The degree of decrease in the affinity of CA-beta(1)AR for (-)-RO363 was essentially the same as that of wild-type beta(1)AR when mutated at Leu(110) and Thr(117). However, the affinity was decreased in Ala-substituted mutant of Phe(359) compared with that of wild-type beta(1)AR. These results indicated that Leu(110) and Thr(117) are necessary for the initial binding of (-)-RO363 with beta(1)-selectivity, and interaction of Phe(359) with the N-substituent of (-)-RO363 in an active state is stronger than in the resting state.

A coiled coil trigger site is essential for rapid binding of synaptobrevin to the SNARE acceptor complex.

Exocytosis from synaptic vesicles is driven by stepwise formation of a tight alpha-helical complex between the fusing membranes. The complex is composed of the three SNAREs: synaptobrevin 2, SNAP-25, and syntaxin 1a. An important step in complex formation is fast binding of vesicular synaptobrevin to the preformed syntaxin 1.SNAP-25 dimer. Exactly how this step relates to neurotransmitter release is not well understood. Here, we combined different approaches to gain insights into this reaction. Using computational methods, we identified a stretch in synaptobrevin 2 that may function as a coiled coil "trigger site." This site is also present in many synaptobrevin homologs functioning in other trafficking steps. Point mutations in this stretch inhibited binding to the syntaxin 1.SNAP-25 dimer and slowed fusion of liposomes. Moreover, the point mutations severely inhibited secretion from chromaffin cells. Altogether, this demonstrates that the trigger site in synaptobrevin is crucial for productive SNARE zippering.