Differential recognition of "enkephalinase" and angiotensin-converting enzyme by new carboxyalkyl inhibitors

New carboxylalkyl compounds derived from Phe-Leu and corresponding to the general formula C6H5-CH2-CH(R)CO-L.Leu with R = -COOH, 3, R = -CH2-COOH, 4, R = -NH-CH2-COOH, 5, R = -NH-(CH2)2-COOH, 6, have been found to inhibit the breakdown of the Gly3-Phe4 bond of [3H] Leu-enkephalin or [3H]D.Ala2-Leu-enkephalin resulting from the action of the mouse striatal metallopeptidases: "enkephalinase" or angiotensin-converting enzyme (A.C.E.). The carboxyl coordinating ability of the Zn atom seems to be significantly higher in ACE than in "enkephalinase". Moreover, IC50 values against "enkephalinase" were found in the same range whatever the length of the chain bearing the carboxyl group whereas a well-defined position of this group with respect to the Zn atom is required for strong ACE inhibition. These features suggest a larger degree of freedom of the carboxyalkyl moieties within the active site of "enkephalinase". Therefore the differential recognition of active sites of both peptidases leads to: i) N-(carboxymethyl)-L-Phe-L-Leu, 5, a competitive inhibitor of "enkephalinase" (KI = 0.7 microM) and ACE (KI = 1.2 microM) which could be used as mixed inhibitor for both enzymes; ii) N-[(R,S)-2-carboxy, 3-benzylpropanoyl]-L-Leucine, 3, a full competitive inhibitor of "enkephalinase" (KI = 0.34 microM) which does not interact with ACE (IC50 greater than 10,000 microM). This compound can be considered as the first example of a new series of highly potent and specific "enkephalinase" inhibitors.     

Neuronal localization of the neutral endopeptidase ''enkephalinase'' in rat brain revealed by lesions and autoradiography.

The cellular localization of rat brain enkephalinase was studied after induction of selective unilateral lesions using in vitro quantitative autoradiography of the specific binding of the enzyme inhibitor [3H]-N-[(2RS)-3-hydroxyaminocarbonyl-2-benzyl-1-oxopropyl]glycine ([3H]HACBO-Gly). Twenty-one days following injection of kainic acid in the caudate-putamen (CP) [3H]HACBO-Gly binding was locally decreased by 52% with a concomitant reduction of 67 and 78% in the ipsilateral substantia nigra (SN) and globus pallidus (GP), respectively. Inhibition of axonal transport in the CP by unilateral stereotaxic injection of colchicine induced a large (30-60%) and progressive decrease in enkephalinase labelling within the ipsilateral GP and SN. Taken together these results strongly suggest that in the CP a large fraction of enkephalinase is localized on intrinsic striatal neurones, and that the enzyme present both in the GP and the SN is partly localized on nerve terminals originating from neurones in the CP. No change in [3H]HACBO-Gly binding was observed in the CP following injection of 6-hydroxydopamine in the nigrostriatal bundle, contrasting with the 30% depletion in opioid receptors. This would indicate that enkephalinase is present in only very low amounts, if at all, on striatal dopaminergic nerve terminals.     

Enantiomers of [R,S]-thiorphan: dissociation of analgesia from enkephalinase A inhibition

The [R] and [S] enantiomers of the enkephalinase A inhibitor [R,S]-thiorphan have been prepared by asymmetric synthesis. The [S] isomer is principally responsible for the angiotensin converting enzyme inhibitory activity of [R,S]-thiorphan, whereas there were only small differences in the ability of the [R] and [S] isomers to inhibit enkephalinase both in vivo and in vitro. In contrast, the in vivo analgesic activity of [R,S]-thiorphan resided principally in the [R] isomer. These data indicate a surprising dissociation of enkephalinase inhibition from analgesic activity. The fact that the two enantiomers of [R,S]-thiorphan were effective inhibitors of enkephalinase, yet the [R] isomer had substantially greater analgesic activity, indicates that factors other than enkephalinase inhibition may be important for [R, S]-thiorphan's analgesic properties.     

Decrease in Enkephalinase a Number in Kidney Membranes from Hypercholesterolemic and Hypertensive Rats

Abstract

The variation of enkephalinase A number on the hypertensive and hypercholesterolemia rats kidney membranes is studied using the [³H]-acetorphan, a potent inhibitor of enkephalinase A to label the protease in rat kidney. The binding of [³H]-acetorphan to kidney membrane determined in vitro with both equilibrium and kinetic methods is saturable and reversible involving a single class of sites with a dissociation constant of 4-5.3 nM. The [³H]-acetorphan binding capacity is identical, Bmax ～ 51 pmoles per mg of proteins, for kidney membranes from Sprague Dawley and Wistar Kyoto rats. In contrast, the enkephalinase A number is decreased in the pathological states studied: 20 % for hypertensive rats and 50% for hypercholesterolemic rats. Such pharmacological results provide a great deal of information about the modification appeared in the metabolism of peptidic substrates of enkephalinase A in hypercholesterolemia and hypertension.     

Degradation of cholecystokinin octapeptide by the neutral endopeptidase EC 3.4.24.11 and design of proteolysis-resistant analogues of the peptide

Inactivation of cholecystokinin octapeptide in vitro involves a metalloendopeptidase (EC 3.4.24.11) also called enkephalinase that inactivated the peptide both by a sequential pathway of hydrolysis (removal of Phe-NH₂ followed by cleavage of Trp-Met-Asp) and by an endopeptidase action (production of the tetrapeptides).

As enkephalinase cleaved CCK-8 at the Gly⁴-Trp⁵, Trp⁵-Met⁶ and Asp⁷-Phe⁸ bonds, we investigated the stability of analogues having: (1) substitutions of amino acids by a stereoisomer, (2) a substitution of Asp⁷ by a β Ala residue and (3) modifications of the Trp residue obtained by replacing the nitrogen atom in the indol ring by either an oxygen ([Bfa⁵]CCK-8) or a sulphur atom ([Bta⁵]CCK-8). Among these different CCK derivatives, [βAla⁷], [ Met⁶] and [ Trp⁵]CCK-8 were not hydrolyzed by enkephalinase: [ Ala^d]CCK-8 was rapidly cleaved by the enzyme. [Bta⁵] and [Bfa⁵]CCK-8 did not prove to be quite resistant; however the C-terminal tetrapeptides having the same modifications on the Trp residue were not cleaved although they interacted with the enzyme binding site. The stability and biological activity of the peptidase-resistant analogues of CCK-8 remain to be determined in vivo.     

Effect of inhibitors of enkephalin degradation in the isolated guinea-pig ileum

Bestatin and high concentration of puromycin increase the depressing effect of [Met] enkephalin on the twitch response of the electrically stimulated guinea-pig ileum. Thiorphan (enkephalinase A inhibitor) is hardly effective, but phelorphan (mercapto-acetyl-Phe-Phe) a newly synthesized enzyme-inhibitor which effectively inhibits the enkephalinase A, enkephalinase B and soluble aminopeptidase activity, potentiates the effect of enkephalin dose-dependently and in low concentrations (0.01-1 microM). Enkephalinase A, though present in these tissues, is not functional under the conditions of the test, because it is inhibited by the physiological buffer itself. These results demonstrate that enkephalinase B and the membrane bound aminopeptidase, but not the soluble aminopeptidase or enkephalinase A hydrolyse enkephalins in the isolated guinea-pig ileum.     

Enkephalin-degrading enzymes and angiotensin-converting enzyme in human and rat meninges

The neutral endopeptidase NEP 24.11 (enkephalinase) has been visualized in human spinal cord by in vitro autoradiography using [3H]HACBO-Gly as a radiolabelled probe. The specific binding was present in the substantia gelatinosa and particularly dense in meninges surrounding the spinal cord. Enzymatic studies using [3H][D-Ala2, Leu]enkephalin as substrate confirmed the presence of NEP in dura and pia mater of human tissue. In addition, the human meninges were shown to contain high concentrations of angiotensin-converting enzyme (ACE) and aminopeptidases. The three enzymes have also been detected in rat tissues but their distribution pattern differs from that of human tissue. In dura mater, 45% of the [Leu]enkephalin hydrolysis was due to enkephalinase and 38% to bestatin-sensitive aminopeptidases. In contrast in pia mater aminopeptidases were more efficient in hydrolyzing enkephalin. The possible role of these enzymes in the meninges could be to maintain the homeostatic concentration of neuropeptides in the central nervous system.     

Comparison of dipeptidyl carboxypeptidase and endopeptidase activities in the three enkephalin-hydrolysing metallopeptidases: "angiotensin-converting enzyme", thermolysin and "enkephalinase"

Angiotensin-converting enzyme (ACE), thermolysin and "enkephalinase", three metallopeptidases cleaving the Gly3-Phe4 amide bond of enkephalins, were compared regarding substrate specificity and effects of butanedione, an arginyl-directed reagent. The hydrolysis of enkephalins and analogues was more affected by the nature of P1 and P2 residues in the case of thermolysin than in those of ACE or "enkephalinase"; amidation of the C-terminal carboxylate decreased drastically the hydrolysis by ACE but only marginally by thermolysin and the effect was intermediate for "enkephalinase". With adequate model substrates, the ratio of dipeptidylcarboxypeptidase to tripeptidylcaroxypeptidase (endopeptidase) activities were of 25 for ACE, 3 for "enkephalinase" and only 0.3 for thermolysin. Finally a butanedione treatment increased thermolysin activity, but abolished ACE activity; it reduced "enkephalinase" activity by 80% when measured with a free C-terminal carboxylate enkephalin analogue but only slightly with the corresponding amidated derivative. A critical role of an Arg residue in ACE and, to a lesser extent, in "enkephalinase" (but not in thermolysin) is suggested to be responsible for the preferential dipeptidylcarboxypeptidase activity of these two enzymes.     

Enkephalinase inhibitor potentiates substance P- and electrically induced contraction in ferret trachea

To determine the role of endogenous enkephalinase (EC 3.4.24.11) in regulating peptide-induced contraction of airway smooth muscle, we studied the effect of the enkephalinase inhibitor, leucine-thiorphan (Leu-thiorphan), on responses of isolated ferret tracheal smooth muscle segments to substance P (SP) and to electrical field stimulation (EFS). Leu-thiorphan shifted the dose-response curve to SP to lower concentrations. Atropine or the SP antagonist [D-Pro2,D-Trp7,9]SP significantly inhibited SP-induced contractions in the presence of Leu-thiorphan. Leu-thiorphan increased the contractile responses to EFS dose dependently, an effect that was significantly inhibited by the SP antagonist [D-Pro2,D-Trp7,9]SP. SP, in a concentration that did not cause contraction, increased the contractile responses to EFS. This effect was augmented by Leu-thiorphan dose dependently and was not inhibited by hexamethonium or by phentolamine but was inhibited by atropine. Because contractile responses to acetylcholine were not significantly affected by SP or by Leu-thiorphan, the potentiating effects of SP were probably on presynaptic-postganglionic cholinergic neurotransmission. Captopril, bestatin, or leupeptin did not augment contractions, suggesting that enkephalinase was responsible for the effects. These results suggest that endogenous tachykinins modulate smooth muscle contraction and endogenous enkephalinase modulates contractions produced by endogenous or exogenous tachykinins and tachykinin-induced facilitation of cholinergic neurotransmission.     

Propioxatins A and B, new enkephalinase B inhibitors. IV. Characterization of the active site of the enzyme using synthetic propioxatin analogues

Propioxatins A and B are inhibitors of enkephalinase B, which hydrolyzes enkephalin at the Gly-Gly bond. In order to clarify the structure-activity relationships of propioxatin, several compounds were synthesized and their inhibitory activity for not only enkephalinase B but also enkephalinase A was examined. The hydroxamic acid group in propioxatin was primarily essential for coordinating the metal ion in the active site of the enzyme. Among devalyl propioxatin A derivatives, the proline-containing compounds inhibited enkephalinase B and others inhibited both enzymes. An alteration of the character of the P3' amino acid valine in propioxatin A, e.g. amidation of carboxylic acid or replacement of the side chain, caused a 2 to 400-fold decrease of the inhibitory activity for enkephalinase B or an appearance of enkephalinase A inhibition with Ki values in the micromolar range. Substitution of the proline by alanine also resulted in a 1,000-fold loss of inhibitory activity for enkephalinase B. Propioxatin A was the most potent and specific inhibitor of enkephalinase B among the synthesized compounds. These potent and specific inhibitory effects were caused by the P2' proline residue, the P3' valine side chain and its free carboxylic acid. Each of the S1', S2', and S3' subsites in an enkephalinase B active site has a large and hydrophobic pocket, but the arrangement might be unique. The results could explain why enkephalinase B does not hydrolyze longer peptides.     

The effect of N-formyl-methionyl-leucyl-phenyl-alanine on cholinergic neurotransmission and its modulation by enkephalinase in rabbit airway smooth muscle

N-formyl-methionyl-leucyl-phenylalanine (FMLP), a synthetic analogue of bacterial chemotactic peptide, may play a role in airway hyperresponsiveness, and is cleaved by neutral endopeptidase-24.11 (enkephalinase). To determine the effect of FMLP on parasympathetic contraction of airway smooth muscle and its modulation by endogenous enkephalinase, we studied isolated rabbit tracheal ring segments under isometric conditions in vitro. FMLP did not cause muscle contraction, but it potentiated the contractile response to electrical field stimulation (EFS) in a dose-dependent fashion, with the maximal increase from the baseline response being 59.8 +/- 6.2% (mean +/- S.E.M., P less than 0.001), an effect that was abolished by t-Boc-Phe-Leu-Phe-Leu-Phe, partially inhibited by pyrilamine, but not by phentolamine or [D-Pro2,D-Trp7,9]substance P. In contrast, the contractile response to administered acetylcholine was not affected by FMLP. Pretreatment of tissues with thiorphan, an enkephalinase inhibitor, further potentiated the effect of FMLP on the EFS-induced contraction. These results suggest that FMLP facilitates cholinergic neurotransmission in rabbit airway smooth muscle probably by increasing acetylcholine release, and that this effect may be modulated by enkephalinase in the airway.     

Mechanism of action of ethanol on enkephalinase A activity in the rat brain

The influence of ethanol, its metabolites and some opiates on enkephalinase A activity was studied in rat experiments in vitro after acute and chronic administration of ethanol. It was demonstrated that addition of ethanol to the reaction mixture activated enkephalinase A of the midbrain and hypothalamus of intact rats, the maximal effect being attained at an ethanol concentration of 10(-3) M. Multiple washings with buffer of the ethanol-preincubated membranous fraction of these brain structures in the control rats did not lead to a significant reduction in the activating effect of ethanol on enkephalinase A. No activation was recorded upon the use of an enzymatic preparation of the brain from chronically alcoholized animals. Morphine, naltrexon, beta-carbolines, salsolinol (10(-4) M) and acetaldehyde (10(-8)-10(-2) M) did not activate the enzyme. It is suggested that enkephalinase A activation in rats given ethanol is determined by a direct action of ethanol on the enzyme.     

Multiple cleavage sites of cholecystokinin heptapeptide by "enkephalinase"

Degradation of Boc CCK7 (Boc Tyr1 (SO3H)-Met2-Gly3-Trp4-Met5-Asp6-Phe7-NH2), a fully active analog of CCK8, by purified rabbit kidney neutral metalloendopeptidase (enkephalinase) was studied as a basis for the rational design of potent peptidases-resistant analogs of cholecystokinin. Characterization of the metabolites was performed by HPLC using several elution procedures. Three cleavage sites were evidenced: one major at the Asp6-Phe7 bond and two minor at Gly3-Trp4 and Trp4-Met5 bonds. All cleavages were fully inhibited by thiorphan, a potent inhibitor of enkephalinase. The relative importance of the different cleavages was established using several cholecystokinin analogs. At 25 degrees C the half-disappearance time was 18 min for Boc CCK7, Boc[diNle2,5]CCK7 and 70 min for Boc[diNle2,5 D.Asp6]CCK7. Although, half-life of Boc CCK7 and Boc[diNle2,5]CCK7 were identical, the replacement of Met by Nle, a more hydrophobic aminoacid, greatly favoured the cleavage at the Trp4-Nle5 bond which became the major breakdown. This feature was exemplified by the substitution of L.Asp by D.Asp, preventing the Trp4-Nle5 cleavage, which gave rise to the most enkephalinase-resistant analog in this series.     

Inhibition of apomorphine-induced yawning and penile erection by neurotensin.

The yawns and penile erection elicited in rats by apomorphine (100 micrograms/kg SC) are dose-dependently suppressed by the enkephalinase-resistant analog of NT, [D-Trp11]NT, intracerebroventricularly (ICV) injected (10-120 ng per rat). This antagonistic effect was shared by NT (0.75-3 micrograms per rat) administered ICV. The yawns induced by pilocarpine (2 mg/kg IP) were similarly antagonized by [D-Trp11]NT (30-120 ng per rat). The enkephalinase inhibitor acetorphan (5 mg/kg IV) reduced in a naloxone (2 mg/kg, SC)-resistant manner the apomorphine-induced penile erection or yawning.     

Changes in levels of the tripeptide Tyr-Gly-Gly as an index of enkephalin release in the spinal cord: effects of noxious stimuli and parenterally-active peptidase inhibitors

The tripeptide Tyr-Gly-Gly (YGG), representing the product of enkephalin hydrolysis by enkephalinase (EC 3.4.24.11), was characterized and its levels measured in spinal cord perfusates of halothane-anaesthetized rats. During noxious pinching of the muzzle, which is known to trigger enkephalin release, YGG levels were enhanced more markedly and for longer than were those of [Met5]enkephalin (YGGFM), in the same samples. By contrast, neither YGG nor YGGFM levels were affected by pinching the tail. Treatment with carbaphethiol, a parenterally-active aminopeptidase inhibitor, markedly increased YGG levels and lengthened the duration of the increase produced by pinching the muzzle. Treatment with acetorphan, a parenterally-active enkephalinase inhibitor, given alone or in combination with carbaphethiol, completely prevented the rise in YGG triggered by noxious stimulation. By contrast, [Met5]enkephalin levels in the perfusates were increased by the combined administration of the two peptidase inhibitors but these levels were not further enhanced by noxious stimulation. Thus, spinal cord YGG appears to be formed under the influence of enkephalinase and to constitute a sensitive index of enkephalin release.     

Enkephalin dipeptidyl carboxypeptidase (enkephalinase) activity: selective radioassay, properties, and regional distribution in human brain

Abstract: The compound [³H-Tyr¹,D-Ala²,Lcu-OH⁵]enkephalin has been synthesised as a potentially selective substrate for enkephalin dipeptidyl carboxypcptidase ( enkephalinase ) activity in brain. lncubations in the presence of homogenates and particulate fractions from rodent and human brain result in the formation of [³H]Tyr-D-Ala-Gly, which can be conveniently isolated by polystyrene bead column chromatography. The enzyme activity responsible for the hydrolysis of the Gly³-Phe⁴ amide bond of this substrate displays close resemblance to that hydrolysing the natural enkephalins at the same level. In addition, enkephalinase activity characterised in postmortem human brain is closely similar to that in rodent brain, with regard to optimal pH and apparent affinities of various substrates and inhibitors, including the potent compound thiorphan. Enkephalinase activity is distributed in a highly heterogeneous fashion among regions of human brain, the highest levels being found in globus pallidus and pars reticulata of the substantia nigra. This distribution is poorly correlated with that of opiate receptor binding sites but displays some resemblance to that of reported Met5-enkephalin levels.     

Molecular cloning and amino acid sequence of human enkephalinase (neutral endopeptidase)

We have isolated a cDNA clone encoding human enkephalinase (neutral endopeptidase, EC 3.4.24.11) in a lambda gt10 library from human placenta, and present the complete 742 amino acid sequence of human enkephalinase. The human enzyme displays a high homology with rat and rabbit enkephalinase. Like the rat and rabbit enzyme, human enkephalinase contains a single N-terminal transmembrane region and is likely to be inserted through cell membranes with the majority of protein, including its carboxy-terminus, located extracellularly.     

Expression of enzymatically active enkephalinase (neutral endopeptidase) in mammalian cells

A cDNA encoding the rat enkephalinase protein (neutral endopeptidase; EC 3.4.24.11) has been constructed from overlapping lambda gt10 cDNA clones. This cDNA was inserted into an expression plasmid containing the cytomegalovirus enhancer and promoter. When transfected with this plasmid, Cos 7 cells transiently expressed the enkephalinase protein in a membrane-bound state. Recombinant enkephalinase recovered in solubilized extracts from transfected Cos 7 cells was enzymatically active and displayed properties similar to those of the native enzyme with respect to sensitivity to classical enkephalinase inhibitors.     

Novel inhibitors of enkephalin-degrading enzymes. III: 4-Carboxymethylamino-4-oxo-3 (phenylamino) butanoic acids as enkephalinase inhibitors.

4-Carboxymethylamino-4-oxo-3-(4'-aminophenylamino) butanoic acid (25), its ethyl ester (26) and the corresponding unsubstituted-aryl analogues (17) and (16) are fairly potent inhibitors of enkephalinase (neutral endopeptidase; EC 3.4.24.11), Ki = 0.14-0.39 microM, with weak inhibitory potency, Ki = 15-75 microM, towards aminopeptidase MII. In the mouse abdominal constriction test, the esters (26) and (16) showed systemic inhibitory (antinociceptive) activity with ED50 values 62 +/- 3.05 and 81 +/- 1.74 mg/kg respectively. In the mouse tail immersion test, both (26) and (16) exhibited antinociceptive activity when administered intracerebroventricularly and (26) also exhibited a systemic effect which was only partially reversed by naltrexone. The antinociceptive effect seen with (26) reflects its ranking in vitro as an inhibitor of enkephalinase (Ki = 0.14 microM) but it is possible that this effect is not totally opioid-mediated. Compounds (26) and (16) represent the first combined inhibitors of enkephalinase and aminopeptidase MII.     

Enkephalinase from rat kidney. Purification, characterization, and study of substrate specificity

"Enkephalinase," a membrane-bound peptidase hydrolyzing the Gly3-Phe4 amide bond of enkephalins, initially characterized in brain, was purified from a rat kidney microsomal fraction. After differential solubilization with Triton X-100, the use of DEAE-Sephadex, concanavalin A, and hydroxylapatite chromatography led to a 2000-fold purification, close to homogeneity. Renal enkephalinase appears to be a glycoprotein Mr = 92,000-95,000 with catalytic properties and sensitivity to chelating agents and inhibitors (Thiorphan, phosphoramidon) very similar to those of the cerebral enzyme. The enzyme co-purified until the final step with "renal brush-border neutral proteinase" (EC 3.4.24.11) activity assayed with 125I-insulin B chain as substrate and displaying similar sensitivity to inhibitors. The specificity of the purified enkephalinase has been studied using either peptides derived from the enkephalins or model peptides of general formula (Ala)m-Tyr-(Ala)n as substrates. In all cases the bond cleaved was that involving the amino group of an aromatic residue, specificity being also defined by the nature of the neighboring residue on the COOH-terminal side. A free carboxyl in the latter residue was essential in the two series of substrates, indicating that enkephalinase more efficiently functions as a dipeptidyl carboxypeptidase than as an endopeptidase.