Marie-Claude Fournie-Zaluski

Analgesic effects of kelatorphan, a new highly potent inhibitor of multiple enkephalin degrading enzymes

Kelatorphan, [(R)-3-(N-hydroxy)-carboxamido-2-benzylpropanoyl]-L-alanine, represents the first virtually complete inhibitor of enkephalins metabolism with KI = 1.4 nM against enkephalinase, KI = 2 nM against the Gly2 -Gly3 cleaving dipeptidylaminopeptidase and KI = 7 microM on aminopeptidase activity. The analgesic effect of [Met5]enkephalin was potentiated 50000 times (ED50 approximately 10 ng) by intracerebroventricular co-administration in mice of kelatorphan (50 micrograms). This effect was significantly higher than that produced by bestatin (50 micrograms) + thiorphan (50 micrograms). Kelatorphan alone was at least two-fold more potent as analgesic than the above mixture of inhibitors.

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).

Δ9-tetrahydrocannabinol releases and facilitates the effects of endogenous enkephalins: reduction in morphine withdrawal syndrome without change in rewarding effect

Recent studies have suggested that cannabinoids might initiate the consumption of other highly addictive substances, such as opiates. In this work, we show that acute administration of Δ9‐tetrahydrocannabinol in mice facilitates the antinociceptive and antidepressant‐like responses elicited by the endogenous enkephalins protected from their degradation by RB 101, a complete inhibitor of enkephalin catabolism. This emphasizes the existence of a physiological interaction between endogenous opioid and cannabinoid systems. Accordingly, Δ9‐tetrahydrocannabinol increased the release of Met‐enkephalin‐like material in the nucleus accumbens of awake and freely moving rats measured by microdialysis. In addition, this cannabinoid agonist displaced the in vivo[3H]diprenorphine binding to opioid receptors in total mouse brain. The repetitive pretreatment during 3 weeks of Δ9‐tetrahydrocannabinol in mice treated chronically with morphine significantly reduces the naloxone‐induced withdrawal syndrome. However, this repetitive administration of Δ9‐tetrahydrocannabinol did not modify or even decrease the rewarding responses produced by morphine in the place preference paradigm. Taken together, these behavioural and biochemical results demonstrate the existence of a direct link between endogenous opioid and cannabinoid systems. However, chronic use of high doses of cannabinoids does not seem to potentiate the psychic dependence to opioids.

Antidepressant-type effects of endogenous enkephalins protected by systemic RB 101 are mediated by opioid δ and dopamine D1 receptor stimulation

The role of endogenous enkephalins in behavioural control in mice was investigated by i.v. injection of RB 101 (N-[(R,S)-2-benzyl-3[(S)(2-amino-4- methylthio)butyl dithio]-1-oxopropyl]-L-phenylalanine benzyl ester). RB 101 is a recently reported systemically active mixed inhibitor prodrug of the two enzymes which metabolize the enkephalins neutral endopeptidase 24.11 and aminopeptidase N. RB 101 (2.5-10 mg/kg) induced a dose-dependent long-lasting hyperlocomotion and attenuated the conditioned suppression of motility in mice placed in an environment where they had received footshocks 24 h before. In addition, RB 101 decreased the duration of immobility in the forced swim test. All these actions of RB 101 were antagonized by the selective delta antagonist, naltrindole, supporting the preferential involvement of delta opioid receptors in these enkephalin-controlled behavioural responses. The effects induced by RB 101 were also suppressed by prior administration of the selective dopamine D1 antagonist, SCH 23390, but not by the D2 antagonist, sulpiride. Attenuation of the conditioned suppression of motility was associated with increased striatal dihydroxyphenylacetic acid (DOPAC)/dopamine (DA) and homovanillic acid (HVA)/DA ratios, both effects being antagonized by naltrindole. This latter compound is also efficient to inhibit the effect of imipramine in the mouse forced swim test. Taken together, these results support the occurrence of tonic and phasic controls of mood-related behaviour by endogenous enkephalins through delta and D1 receptor stimulation and suggest a possible future use of these mixed inhibitors as new antidepressants.

First large scale chemical synthesis of the 72 amino acid HIV-1 nucleocapsid protein NCp7 in an active form

The nucleocapsid protein (NC) of the human immunodeficiency virus type 1 plays a crucial role in the formation of infectious viral particles and therefore should be a major target for the development of antiviral agents. This requires an investigation of NC protein structure and of its interactions with both primer tRNA(Lys,3) and genomic RNA. Nucleocapsid protein NCp7, which results from the maturation of NCp15, contains two zinc fingers flanked by sequences rich in basic and proline residues. Here we report the first synthesis of large quantities of NCp7 able to activate HIV-1 RNA dimerization and replication primer tRNA(Lys,3) annealing to the initiation site of reverse transcription. In addition UV spectroscopic analyses performed to characterize the Co2+ binding properties of each zinc finger suggest that the two fingers probably interact in NCp7.

Conformational behaviour of the active and inactive forms of the nucleocapsid NCp7 of HIV-1 studied by 1H NMR

The nucleocapsid protein NCp7 of the human immunodeficiency virus type I (HIV-1) is a 72 amino acid peptide containing two zinc fingers of the type CX2CX4HX4C linked by a short basic sequence 29RAPRKKG35. NCp7 was shown to activate in vitro both viral RNA dimerization and replication primer tRNA(Lys,3) annealing to the initiation site of reverse transcription. In order to clarify the possible structural role of the zinc fingers in the various functions of NCp7, complete sequence specific 1H NMR assignment of the entire protein was achieved by two-dimensional NMR experiments. Moreover, to characterize the role of the peptide linker in NCp7 folding, a synthetic analogue with an inversion of Pro31 configuration was studied by NMR and fluorescence techniques. Several long range NOEs implying amino acid protons from the folded zinc fingers and the spacer, such as Ala25 and Trp37, Phe16 and Trp37, Arg32 and Trp37, Lys33 and Trp37, Cys18 and Lys33 disappeared in the D-Pro31 (12-53)NCp7, confirming the spatial proximity of the two CCHC boxes observed in the (13-51)NCp7. This was also confirmed by iodide fluorescence quenching experiments. The N and C-terminal parts of NCp7 displayed a large flexibility except for two short sequences Tyr56 to Gly58 and Tyr64 to Gly66, which seemed to oscillate between random-coil and helical conformations. The biological relevance of the structural characteristics of NCp7 was studied in vitro and in vivo. Substitution of Pro31 by D-Pro31 in the active (13-64)NCp7 peptide led to a severe reduction of dimerization in vitro. Moreover, site-directed mutagenesis substituting Leu for Pro31 resulted in the formation of non-infectious and immature viral particles. These results suggest that the spatial proximity of the zinc fingers induced by the peptide linker, plays a critical role in encapsidation of genomic RNA and morphogenesis of HIV-1 infectious particles.

Inhibiting the breakdown of endogenous opioids and cannabinoids to alleviate pain

Chronic pain remains unsatisfactorily treated, and few novel painkillers have reached the market in the past century. Increasing the levels of the main endogenous opioid peptides — enkephalins — by inhibiting their two inactivating ectopeptidases, neprilysin and aminopeptidase N, has analgesic effects in various models of inflammatory and neuropathic pain. Stemming from the same pharmacological concept, fatty acid amide hydrolase (FAAH) inhibitors have also been found to have analgesic effects in pain models by preventing the breakdown of endogenous cannabinoids. Dual enkephalinase inhibitors and FAAH inhibitors are now in early-stage clinical trials. In this Review, we compare the effects of these two potential classes of novel analgesics and describe the progress in their rational design. We also consider the challenges in their clinical development and opportunities for combination therapies.

The μ rather than the δ subtype of opioid receptors appears to be involved in enkephalin-induced analgesia

The analgesic activity of some opioid peptides which display a relative selectivity for either the μ-receptor subtype, [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAGO) or the δ-receptor subtype, [D-Ala2,D-Leu5]enkephalin (DADLE), [D-Ser2, Leu5]enkephalyl-Thr (DSLET) and [D-Thr2,Leu5]enkephalyl-Thr (DTLET) is highly correlated with their affinity for central or peripheral μ- but not δ-receptors. Moreover their analgesic effects as well as those elicited by degrading enzyme inhibitors (bestatin + thiorphan) of endogenous enkephalins were easily antagonized by naloxone with similar pA2 values but not by the δ-antagonist ICI 154,129. Therefore the analgesia produced by opioid peptides including endogenous enkephalins is likely connected to μ-receptor stimulation. Finally, there was no obvious potentiation by δ-agonists of the analgesia resulting from either administration of the μ-agonist morphine or endogenous enkephalins. This suggested that in the hot plate test, there is no modulation of the effect resulting from μ-receptor stimulation by a δ-receptor interaction. Likewise, enkephalinergic activity such as that due to thiorphan + bestatin does not appear to be regulated through μ- or δ-receptor stimulation.

In vitro and in vivo effects of kelatorphan on enkephalin metabolism in rodent brain

Biologically relevant assays were used to compare the potency of kelatorphan (N-[3(R)-[(hydroxyamino)carbonyl]-2-benzyl-1-oxopropyl]-L-alanine) as inhibitor of the peptidase-induced metabolism of enkephalins to that of bestatin, a non-specific inhibitor of aminopeptidase and thiorphan, a highly potent blocker of the neutral endopeptidase (EC 3.4.24.11) designated as enkephalinase. Kelatorphan almost completely inhibited the formation of the three metabolites [3H]Tyr, [3H]Tyr-Gly and [3H]Tyr-Gly-Gly produced by incubation of [3H][Tyr1,Met5]enkephalin with rat striatal slices. Co-administered with [Met5]enkephalin in mouse brain, kelatorphan was able to prevent by 80% the degradation of the exogenous peptide. Moreover, a mixture of thiorphan (1 microM) and bestatin (20 microM) or kelatorphan alone (20 microM) induced a 2.2 to 2.5-fold increase in endogenous [Met5]enkephalin overflow after evoked depolarization of superfused rat striatal slices. In this assay, kelatorphan was the only compound to increase by 63% the basal level of released [Met5]enkephalin. Kelatorphan was about 100 times less potent than bestatin to inhibit the total rat striatal aminopeptidases, but as efficient (IC50 = 4 X 10(-7) M) as bestatin to inhibit a minor aminopeptidase activity resembling aminopeptidase M. Therefore the reported enhanced analgesic potency of kelatorphan with regard to the association of bestatin and thiorphan is very likely related to its ability to almost completely inhibit enkephalin-degrading enzymes (including the Tyr-Gly releasing peptidase) and to its better selectivity for the biologically relevant aminopeptidase M. Kelatorphan would be a valuable probe, preferable to the association of bestatin and thiorphan, to investigate the physiological functions regulated by a phasic enkephalinergic activity.

Effects of opioids and non-opioids on c-Fos-like immunoreactivity induced in rat lumbar spinal cord neurons by noxious heat stimulation

This study evaluated Fos-like immunoreactivity in rat lumbar spinal cord neurons following peripheral noxious heat stimulation and the modifications induced by pharmacological agents. Under urethane anaesthesia, the hindpaw was stimulated by dipping it in a regulated temperature bath at various temperatures (44-65 degrees C) and for various durations (5 s to 2 min). There was no Fos-like immunoreactivity in lumbar spinal cord neurons when the paw was stimulated at 44 degrees C for 15 s. From 46 to 52 degrees C, the number of Fos-like immunoreactivity neurons increased with increasing stimulation temperature, but was decreased at 65 degrees C as compared to 52 degrees C. At 52 degrees C, the number of Fos-like immunoreactivity neurons increased with the duration of stimulation. Fos-like immunoreactive neurons in the L4 segment were almost exclusively located in laminae I-II. On the basis of the results of the latter experiments, we chose a stimulation of 52 degrees C for 15 s to perform pharmacological investigations. The number of Fos-like immunoreactive neurons induced by the heat stimulation was significantly decreased by pretreatment with morphine (42, 64 and 75% decrease as compared to control values after 2.5, 5 and 7.5 mg/kg i.v. respectively), and these effects were blocked by naloxone. When various stimulation intensities (46-52 degrees C) were used, the effects of morphine (5 mg/kg i.v.) were most marked when the temperature was highest. In morphine-tolerant rats, morphine (5 mg/kg i.v.) was half as potent in decreasing Fos-like immunoreactivity induced by the heat stimulation than in non-tolerant rats. RB 101, a systemically active mixed inhibitor of enkephalin-metabolising enzymes, significantly decreased Fos-like immunoreactivity induced by heat stimulation (19, 29 and 48% decreases as compared to control values at 10, 20 and 40 mg/kg i.v. respectively) and these effects were blocked by naloxone. Aspirin (150 mg/kg i.v.), proacetaminophen (300 mg/kg i.v.) and tizanidine, a centrally acting myorelaxant (0.25-1 mg/kg i.v.), had no effect on the number of Fos-like immunoreactivity neurons induced by heat stimulation. The use of immunochemistry of the c-Fos protein as a pharmacological test in order to gauge antinociceptive effects at the dorsal horn level is discussed.