Gilles Gacel

D-Tyr—Ser—Gly—Phe—Leu—Thr, a highly preferential ligand for δ-opiate receptors

Binding studies of radiolabeled enkephalins with brain homogenates [l-3] suggest that these peptides interact with at least two distinct classes of binding sites called p and S receptors. Enkephalins bind to 6 receptors which constitute almost 20% of the total binding with a high affinity (Kd _ 0.5 nM and to &f receptors with a lower af~nity (Kd 5 nM) [ 1,2]. Furthermore, displacement experiments with antagonists show that morphine and synthetic opiates interact strongly with g receptors whereas peptides exhibit a high preference for 6 receptors [ 1,2,4]. Such results were also found in peripheric organs and attributed to the preponderance of g receptors in the guinea pig ileum (GPI) and 6 receptors in the mouse vas deferens (MVD) [l-4]. Therefore, the difference in the pharmacological action of any compound on the two organs was used to evaluate its p and 6 specificity [4]. The biological significance of the presence of +U and 6 receptors in the brain remains still unknown although it seems that p receptors could be involved in analgesia [4] whereas 6 receptors might be implicated in behavioral effects [S] . A possible dissociation of the pharmacological responses associated to multiple opiate receptors have incited a great number of studies in vivo [6.173 and in vitro [3,7] for the last months. However. these studies were perfo~ed with D-Ala’-D-LeuS enkephalin (DADL), a compound which exhibits only a partial specificity for 6 receptors 141. Therefore it was of great importance to search for a much higher specific ligand, in order to avoid multiple biological responses arising from simultaneous binding to the two kinds of states. Starting from conformational data and structure activity relationships in the enkephalin series, such a compound was prepared. This new hexapeptide Tyr-D-SerGly-Phe-Leu-Thr exhibits a very high specificity for 6 receptor, since its potency is 620-times greater in MVD (f& = 0.58 nM) than in GPI (K’,, = 360 nM).

Rational design of enkephalinase inhibitors: Substrate specificity of enkephalinase studied from inhibitory potency of various dipeptides

Abstract The inhibitory potency (IC 50 ) of a variety of dipeptides regarding enkephalinase (enkephalin carboxydipeptidase) activity from mouse striatum indicates that the substrate specificity of this enzyme can be clearly distinguished from that of other metalloproteases. The importance of an aromatic ring on the antepenultimate residue and of a small C-terminal aminoacid differing from proline is stressed. These structure-activity relationships can be extended to larger peptides and have led to the rational design of potent and specific inhibitors like Thiorphan, (DL-3-mercapto-2-benzylpropanoyl)-glycine.

Different effects of μ and δ opiate agonists on respiration

Abstract The involvement of different opiate receptor subtypes in opiate-induced respiratory depression was studied in the unanaesthetized rat. Synthetic opioid agonists, specific for μ or δ receptors, were administered intraperitoneally in freely moving rats while respiratory parameters were recorded by means of the whole body plethysmographic method. TRIMU-4 (Tyr-D-Ala-Gly-NH-CH(CH 3 )-CH 2 -CH(CH 3 ) 2 ), a specific agonist of the μ receptor, reduced the tidal volume and did not change the respiratory frequency. DSLET (Tyr-D-Ser-Gly-Phe-Leu-Thr), a relatively specific agonist of the δ receptor subtype, reduced respiratory frequency and was significantly less effective on tidal volume than was TRIMU-4. It is concluded that the respiratory depression occurring after the administration of opiates in clinical practice is a dual complementary effect involving μ and δ receptors.

Inhibitory potency of various peptides on enkephalinase activity from mouse striatum.

A variety of peptides chemically unrelated to enkephalins are relatively good inhibitors (IC50 in the micromoLar range) of “enkephalinase” activity i.e. of the peptidase releasing Tyr-Gly-Gly from Leu-enkephalin. Its specificity has been also reinvestigated with a series of Met-enkephalin analogues. The poor recognition of several analogues by this inactivating enzyme might account for their enhanced biological activity.

Pharmacological identification of δ and μ opiate receptors on bulbar respiratory neurons

Abstract The opiate receptors of central neurons related to the generation of the respiratory rhythm were identified using microiontophoresis of the synthetic opioid peptides DSLET (Tyr-D-Ser-Gly-Phe-Leu-Thr) and TRIMU-4 (Tyr-D-Ala-Gly-NH-CH (CH 3 )-CH 2 -CH(CH 3 ) 2 ), exhibiting a high selectivity for δ and μ receptors respectively. Both agonists induced depressions of spontaneous and L-glutamate-induced discharges; the effects were antagonized by naloxone and not mimicked by a related almost inactive peptide DSLLET (Tyr-D-Ser-Gly-Leu-Leu-Thr). The effect of DSLET had a faster time course than that of TRIMU-4 and persisted after prolonged applications of TRIMU-4. It is concluded that δ and μ receptor subtypes are distinct and are both present on central respiratory-related neurons.

conformation and biological activities of hexapeptides related to enkephalins: Respective roles fo the ammonium and hydroxyl groups of tyrosine

Abstract In order to explore the role of the additional Arg or Tyr residue in the activity of Arg-Methionine-enkephalin (Arg-Met-E) and Tyr-Met-E, a series of analog were prepared and their biological activities in the inhibition of 3 H-Leu-E binding to mouse striatum or the contraction of guineapig ileum and mouse vas deferens were analyzed. From these studies the following general conclusions can be drawn i) the presence of an ammonium group located on the CHα of the N-terminal aminoacid is absolutely required for activity whereas its position relative to the backbone can be somewhat modified, ii) the presence of an OH group strictly on Tyr 1 of E is an absolute requirement. NMR studies indicate that the characteristic conformation of E backbone is retained in the analogs ruling out that the observed changes in activity are related to modified conformation of this part of the molecule. A model for the interaction of enkephalins with their receptors, involving electrostatic and Van der Waals forces, is proposed.

Fluorescent enkephalin derivatives with biological activity

Abstract Fluorescent dansylated derivatives of enkephalins were prepared, with the dansyl group either at the N- or C-terminal part of methionine-enkephalin (Met-E) or its peptidase-resistant analogue D-Ala 2 -Met-E. An energy transfer, from the Tyr to the dansyl group, was found for all compounds and spectral properties were dependent upon the environment polarity. Met-E-(CH 2 ) 2 dansyl II and D-Ala 2 -Met-E-(CH 2 ) 2 dansyl III exhibit biological activities on guinea-pig ileum and binding affinities on striatal membranes, similar to Met-E. III exhibits significant analgesic activity in mice after i.v. administration (about 35 % that of morphine). Fluorescent enkephalins potentially represent useful probes for the exploration of opiate receptors, studies of ligand-receptor interactions and evaluation of enkephalins cleaving peptidases in various tissues.