The mechanism of the all-or-none release of the contents of phospholipid vesicles induced by the antimicrobial peptide cecropin A was investigated. A detailed experimental study of the kinetics of dye release showed that the rate of release increases with the ratio of peptide bound per vesicle and, at constant concentration, with the fraction of the anionic lipid phosphatidylglycerol in neutral, phosphatidylcholine membranes. Direct measurement of the kinetics of peptide binding and dissociation from vesicles revealed that the on-rate is almost independent of vesicle composition, whereas the off-rate decreases by orders of magnitude with increasing content of anionic lipid. A simple, exact model fits all experimental kinetic data quantitatively. This is the first time that an exact kinetic model is implemented for a peptide with an all-or-none mechanism. In this model, cecropin A binds reversibly to vesicles, which at a certain point enter an unstable state. In this state, a pore probably opens and all vesicle contents are released, consistent with the all-or-none mechanism. This pore state is a state of the whole vesicle, but does not necessarily involve all peptides on that vesicle. No peptide oligomerization on the vesicle is involved; alternative models that assume oligomerization are inconsistent with the experimental data. Thus, formation of well-defined, peptide-lined pores is unlikely.

NOP receptor stimulation can provide significant albeit mild anti-dyskinetic effect at doses not causing sedation. The therapeutic window, however, varies across compounds. AT-403 could be a potent and selective tool to investigate the role of NOP receptors in vivo.

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Nociceptin/orphanin FQ (N/OFQ) regulates several biological functions via selective activation of the N/OFQ receptor (NOP), a member of the opioid receptor family. We recently identified a new high affinity and highly selective NOP agonist AT‐403. In this study, we characterized the functional profile of AT‐403 and compared it to other known nonpeptide NOP agonists Ro 65‐6570, Ro 2q, SCH‐221510, MCOPPB, AT‐202 and SCH‐486757, using the following assays: GTP γ[35S] stimulated binding, calcium mobilization assay in cells‐expressing human NOP or classical opioid receptors and chimeric G proteins, bioluminescence resonance energy transfer (BRET) based assay for studying NOP receptor interaction with G protein and arrestin, and the electrically stimulated mouse vas deferens bioassay. All compounds behaved as NOP full agonists consistently showing the following rank order of potency MCOPPB > AT‐403 > Ro 65‐6570 = Ro 2q > SCH‐221510 >  AT‐202 > SCH‐486757. AT‐403 and MCOPPB displayed the highest NOP selectivity both at human and murine receptors. Interestingly, while all the other nonpeptide NOP agonists displayed bias toward G protein‐mediated signaling in the BRET assay, AT‐403, similar to the natural ligand N/OFQ, behaved as an unbiased agonist, activating G‐protein‐mediated function as well as arrestin recruitment. AT‐403 may be a useful nonpeptide tool compound to study the pharmacology of NOP activation in disease states.

experiments. V.B.J. designed the computational studies and analyzed the data. V.B.J. designed the compounds, performed the chemical synthesis and SAR analysis. C.E.H. and N.B. contributed to the chemical synthesis. Z.F., Y.W., and S.W. constructed the homology model and conducted the MD simulations and docking experiments. S.R. conducted the Ca 2+ imaging studies. S.G.-R., A.F.-C., and G.F.-B. carried out the in vivo assays. J.K.H. carried out the electrophysiology assays. V.B.J. designed and coordinated the work.