Structural studies by 1H NMR and molecular modeling of peptide substrates of thermolysin in relation with its proteasic activity in water and glycerol.

In an attempt to explain the relationship between conformations of peptide substrates of thermolysin in natural form and the experimental enzymatic cleavages, five peptides of various length were studied in two solvents H2O and glycerol, which may mimic the catalytic environmental conditions. As NMR failed to define sufficiently rough constraints to ensure a convergence of a refinement process for such short and flexible peptides, the conformational space was first searched using the MCMM method. The generated structures were then clustered in families using a 0.3A rmsd criterion and the derived structural characteristics were compared to the experimental NMR parameters. In a first approach, the NMR consistent conformations were compared with the structure of a thermolysin bound peptidic inhibitor ZG(P)LL to characterize the free-ligand predisposition to be cleaved. Further molecular dynamic calculations were performed at 300 K on the conformations corresponding to families in agreement with the ZG(P)LL structure in order to obtain information on their stability and on the trajectories of the torsion angles involved in the active site recognition. In conclusion, for four studied peptides, some conformations were found to be in agreement with 5 of the 8 cleavages experimentally observed.

Investigation of the structural parameters involved in the delta-opioid selectivity of several families of opioid peptides.

Three series of highly delta-opioid selective peptides are now available, and each family is used as template to investigate the structural parameters involved in delta-receptor recognition and in the modulation of the selectivity of the parent peptide. The first series includes cyclic derivatives such as Tyr-D-Pen-Gly-Phe-D-Pen(DPDPE) and Tyr-D-Pen-Gly-Phe-Pen(DPLPE); the second are the synthetic linear constrained peptides [Tyr-D-Ser(OtBu)-Gly-Phe-Leu-Thr(DSTBULET), Tyr-D-Ser(OtBu)-Gly-Phe-Leu-Thr (OtBu)(BUBU) and especially Tyr-D-Cys(StBu)-Gly-Phe-Leu-Thr(OtBu) (BUBUC)] and the last one the natural peptides [Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2 (deltorphin or dermenkephalin) and Tyr-D-Ala-Phe-Asp-Val-Val-GlyNH2 ([D-Ala2] deltorphin I)]. In the present study, the possibly of transposing some of the decisive factors of delta-selectivity evidenced in the two other families, to the linear constrained peptides series was examined. With this aim in view, residues such as Phe3, pClPhe4 or Asp were introduced in the sequence of DSTBULET, BUBU or BUBUC. Direct comparison between the biochemical profiles of the [pClPhe4] analogs of the linear constrained peptides and their parent compounds shows that the addition of an electronegative atom on the Phe4 residue of enkephalin sequences is not an absolute parameter for delta-selectivity improvement. The hydrophobic delta-receptor subsite seems able to receive a range of molecular volumes and electronegativities. By contrast, this subsite cannot interact with a Phe3 aromatic ring introduced in this series of peptides. Moreover, the results obtained with linear peptides including additional negatively charged residues demonstrate that the proposed location of the delta-receptors in a cationic membrane environment is not adequate to explain the selectivity profile of a number of compounds.

Analgesic potency of TRIMU-5: a mixed mu 2 opioid receptor agonist/mu 1 opioid receptor antagonist.

TRIMU-5 (Tyr-D-Ala-Gly-NH-(CH2)2CH(CH3)2) is a potent enkephalin analog with analgesic actions. Detailed studies show high affinity for both mu 1 and mu 2 sites, with poor affinity for delta, kappa 1 and kappa 3 receptors. Of all the mu ligands examined in binding assays, TRIMU-5 was the most mu-selective. In mice, TRIMU-5 administered either intracerebroventricularly (i.c.v.) or intrathecally elicited analgesia which was readily reversed by the mu-selective antagonist beta-funaltrexamine (beta-FNA). However, the analgesia observed following i.c.v. injections differed from traditional mu ligands: (1) the dose of drug required for analgesic activity i.c.v. was 100-fold greater than those following intrathecal administration; (2) although sensitive to beta-FNA, the analgesia was not antagonized by naloxonazine; and (3) the analgesia was reversed by an opioid antagonist given intrathecally (i.t.) but not i.c.v. Thus, TRIMU-5 analgesia appeared to be mediated spinally through mu 2 receptors. TRIMU-5 did have supraspinal actions, inhibiting gastrointestinal transit, another mu 2 action. In binding studies TRIMU-5 had high affinity for mu 1 sites, but pharmacological studies revealed antagonist actions at this receptor. In mice, the analgesia produced by morphine given i.c.v. was antagonized by coinjection of a low TRIMU-5 dose which was inactive alone. Similarly, TRIMU-5 coadministered with morphine into the periaqueductal gray of rats reversed the analgesia seen with morphine alone. Thus, TRIMU-5 is a highly selective mixed mu 2 opioid receptor agonist/mu 1 opioid receptor antagonist.

Kappa 3 opiate receptor binding in the mouse and rat.

Previous work examining the binding of [3H]naloxone benzoylhydrazone (NalBzoH) in calf brain has identified a novel binding site, kappa 3. In mice and rats NalBzoH elicits an analgesic response which can clearly be differentiated from classical mu, delta or kappa 1 mechanisms and which is pharmacologically consistent with a kappa 3 receptor mechanism of action. In the current studies we demonstrate the presence of kappa 3 sites in both mouse and rat brains. The selectivity of the kappa 3 sites for opioids clearly discriminated it from traditional mu, kappa 1, kappa 2 or delta receptors. In the rat, the density of kappa 3 sites increased 2.5-fold from age 2 days to 21 days, after which it remained relatively stable. Among a number of brain regions in the rat, the density of kappa 3 sites varied dramatically. Highest levels were present in the hypothalamus, thalamus, striatum and midbrain with very low levels in the cerebellum. Intermediate levels were present in cortex, brain stem and spinal cord. Together, these studies support the presence of kappa 3 receptors in both mouse and rat brain which are very similar to those previously reported in calf brain.

Synthesis, biochemical and pharmacological properties of BUBUC, a highly selective and systemically active agonist for in vivo studies of delta-opioid receptors.

Based on the results of conformational studies of linear and cyclic delta-opioid peptides such as BUBU [Tyr-D-Ser(OtBu)-Gly-Phe-Leu-Thr(OtBu)] and DPLPE c[Tyr-D-Pen-Gly-Phe-Pen], a new enkephalin-related peptide, Tyr-D-Cys(StBu)-Gly-Phe-Leu-Thr(OtBu) (BUBUC) was synthesized and tested for its opioid activity and selectivity at both the peripheral and central levels. Amongst all the synthetic compounds described so far, BUBUC appears to be the most highly delta-selective probe [KI (mu) = to 2980 nM, KI (delta): 2.9 nM, KI (mu)/KI (delta) approximately 1000]. This selectivity was confirmed by the results of pharmacological studies, including measurements of supraspinal analgesia and behavioral changes in mice. In the later test, BUBUC was shown to increase the rearing activity after IV administration at very low concentrations (0.1 mg/kg) and this effect was reversed by the delta-selective antagonist naltrindole. No antinociceptive response was observed at a 10-fold higher concentration. Thanks to its enzymatic stability and its hydrophobicity. BUBUC is the first systemically active, highly selective delta agonist and should therefore be useful to characterize the physiological role of delta-opioid receptors.