Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.27 (thermolysin)
 1,894 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The activation or interruption of the responses induced by regulatory peptides are ensured by ectoenzymes, the most important of them belonging to the group of zinc metallopeptidases. Thus angiotensin converting enzyme (ACE) forms the hypertensive peptide angiotensin II from its inactive precursor AI. This also the case for aminopeptidase N (APN) and neutral endopeptidase 24.11 (NEP, CALLA) which together inactivate the endogenous opioid peptides, enkephalins, whereas only NEP is involved in the metabolism of the atrial natriuretic factor (ANP) at the kidney and vascular levels. The pharmacological effects resulting from the inhibition of these enzymatic processes will appear only in tissues where the peptide substrate is tonically or phasically released. This promising approach is expected to avoid, or at least to minimize, the side effects resulting from excessive and ubiquitous stimulation of peptide receptors by exogenously administered agonists or antagonists. The essential amino acids known to be present in the active site of the bacterial endopeptidase thermolysin from crystallographic studies, have also been found in NEP by using a new program of sequence comparison associated with mutagenesis experiments. Several classes of selective inhibitors of NEP, APN and ACE have been rationally designed by taking into account the structural differences in the active site of these peptidases. Thus, the retro-inversion of the amide bond of the NEP inhibitor thiorphan resulted in the elimination of a residual interaction with ACE. Moreover, we have proposed to associate inhibitory potencies towards two peptidases in the same compound. Thus kelatorphan HONH-CO-CH2-CH(CH2 phi)-CONH-CH(CH3)-COOH and other systemically-active mixed NEP/APN inhibitors were shown capable of completely blocking enkephalin metabolism in vivo. This concept has been extended to mixed NEP/ACE inhibitors with compounds such as HS-CH2-CH(CH2 phi)-CONH-CH(CH2R)-COOH where R = CH-(CH3)2 (ES 34) or -OCH2 phi (ES 37). Only mixed inhibitors of NEP and APN are able to produce potent analgesia after intracerebroventricular or systemic administration without the major side effects of morphine (tolerance and dependence). Thiorphan or its prodrugs acetorphan or sinorphan lead to a increase in natriuresis and diuresis by protection of ANP degradation, but without any significant antihypertensive effect. Contrastingly mixed NEP/ACE inhibitors such as ES34 induce decreases in blood pressure higher than those that produced by the association of selective NEP and ACE inhibitors.
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PMID:[New approach in the research of analgesics and antihypertensive agents]. 184 70

A peptidyl dipeptidase-4 (bacterial PDP-4) was purified to near homogeneity from a supernatant of Pseudomonas maltophilia extracellular medium. Bacterial PDP-4 is a single-polypeptide-chain enzyme, 82 kDa, with an alkaline isoelectric point. Peptides susceptible to hydrolysis by bacterial PDP-4 include angiotensin 1, bradykinin, enkephalins, atriopeptin 2, and smaller synthetic peptides. N-acylated tripeptides are hydrolyzed, but free tripeptides are not. A free carboxy terminus is required for hydrolysis. Peptides with ultimate and penultimate Pro residues are not hydrolyzed. The enzyme does not require an anion for activity. Bacterial PDP-4 was inhibited by EDTA and the dipeptide Phe-Arg. Thiorphan was an inhibitor only at levels well above those required for inhibition of neutral metalloendopeptidase (NEP), an enzyme for which thiorphan is specific. A second NEP and thermolysin inhibitor, phosphoramidon, did not inhibit bacterial PDP-4. The potent angiotensin-converting enzyme inhibitor lisinopril was not inhibitory. Bacterial PDP-4 is distinguished from a similar enzyme from Escherichia coli, which is not susceptible to EDTA inhibition, and one from Corynebacterium equi, which hydrolyzes free tripeptides. These data indicate that the bacterial PDP-4 catalytic site is unlike those of other enzymes that function either wholly or in part as peptidyl dipeptidases.
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PMID:A peptidyl dipeptidase-4 from Pseudomonas maltophilia: purification and properties. 253 48

The three-dimensional structures of (S)-thiorphan and (R)-retro-thiorphan bound to thermolysin have been determined crystallographically and refined to residuals of 0.183 and 0.187 at 1.7-A resolution. Thiorphan [N-[(S)-2-(mercaptomethyl)-1-oxo-3-phenylpropyl]glycine] [HSCH2CH(CH2C6H5)CONHC-H2COOH] and retro-thiorphan [[[(R)-1-(mercaptomethyl)-2-phenylethyl] amino]-3-oxopropanoic acid] [HSCH2CH(CH2C6H5)NHCOCH2COOH] are isomeric thiol-containing inhibitors of endopeptidase EC 24-11 (also called "enkephalinase"). The mode of binding of thiorphan to thermolysin is similar to that of (2-benzyl-3-mercaptopropanoyl)-L-alanylglycinamide [Monzingo, A.F., & Matthews, B.W. (1982) Biochemistry 21, 3390-3394] with the inhibitor sulfur atom coordinated to the active site zinc and the peptide portion forming substrate-like interactions with the enzyme. The isomeric inhibitor retro-thiorphan, which differs from thiorphan by the inversion of an amide bond, utilizes very similar interactions with enzyme. Despite the inversion of the -CO-NH- linkage the carbonyl oxygen and amide nitrogen display very similar hydrogen bonding, as anticipated by B.P. Roques et al. [(1983) Proc. Natl. Acad. Sci. U.S.A. 80, 3178-3182]. These results explain why thermolysin and possibly other zinc endopeptidases such as endopeptidase EC 24-11 fail to discriminate between these retro-inverso inhibitors.
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PMID:Thiorphan and retro-thiorphan display equivalent interactions when bound to crystalline thermolysin. 271 12

With the aim of producing an analgesia physiologically induced by endogenous opioids, several series of inhibitors of the degradation enzymes of enkephalins have been synthetized by using as a model, at the atomic level, the active site of thermolysin, a bacterial endopeptidase similar to enkephalinase. Thiorphan and retro-thiorphan are very potent inhibitors of enkephalinase (KI = 2 nM), but the retro compound is more selective, as it is unable to recognise the angiotensin conversion enzyme. Recently, a series of inhibitors containing a bidentate group were found to be capable of inhibiting the three metallopeptidases which break down the enkephalins. One of these compounds, kelatorphan, totally protects, in vitro and in vivo, Met-enkephalin from enzymatic degradation. Kelatorphan is the first complete inhibitor of enkephalin metabolism and is the only compound to possess an analgesic activity greater than that of a mixture of thiorphan and bestatin (non-specific aminopeptidase inhibitor). A tritiated derivative of kelatorphan has been used to visualise the enkephalinase in the rat brain by means of autoradiography. The enzyme has a heterogeneous distribution with a particularly high concentration in the nigro-striatal system.
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PMID:[Enkephalinase inhibitors and molecular study of the differences between active sites of enkephalinase and angiotensin-converting enzyme]. 299 52

The relationships between various properties of inhibitors of enkephalinase (membrane metalloendopeptidase, EC 3.4.24.11) i.e., enzyme inhibition, protection of endogenous enkephalins, antinociceptive activity and stimulation of locomotor activity was investigated by comparing the relative potencies of the two enantiomers of Thiorphan and acetorphan, its parenterally active prodrug. In vitro (R)- and (S)-Thiorphan were almost equipotent in inhibiting enkephalinase activity (Ki, 1.7 and 2.2 nM, respectively) or thermolysin activity (Ki, 13 and 6 microM, respectively) whereas the (R)-isomer was 44-fold less potent than the (S)-isomer on ACE activity (Ki 4800 and 110 nM, respectively). When tested on slices of rat globus pallidus in the presence of bestatin, to block the aminopeptidase pathway of enkephalin degradation, both Thiorphan enantiomers ensured a complete protection of endogenous (Met5)enkephalin released by depolarization and a suppression of the increase in the extracellular levels of Tyr-Gly-Gly, a characteristic enkephalin metabolite. These two effects occurred at EC50 values of the two enantiomers (10 nM in both cases), consistent with the idea that they were due to enkephalinase inhibition. After i.v. administration of the acetorphan enantiomers to mice, the enkephalinase activity of a rapidly prepared striatal membrane fraction was reduced in a dose-dependent manner with similar "ex vivo" ED50 values (1.0 and 0.3 mg/kg for the (R)- and (S)-isomer, respectively). In contrast the ACE activity of the same preparation was reduced in a significant manner only by (S)-acetorphan (ED50 value of 11 mg/kg).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Enantiomers of thiorphan and acetorphan: correlation between enkephalinase inhibition, protection of endogenous enkephalins and behavioral effects. 347 50

Thiorphan, N-[(R,S)-3-mercapto-2-benzylpropanoyl]glycine is a highly potent inhibitor (Ki = 3.5 nM) of "enkephalinase," a metalloendopeptidase cleaving the Gly-Phe bond (positions 3 and 4) of enkephalins in brain tissue. In accordance with this property, thiorphan displays antinociceptive activity after systemic administration. However, thiorphan also inhibits to a lesser extent (Ki = 140 nM) the widely distributed angiotensin-converting enzyme, a carboxydipeptidase implicated in blood pressure regulation. Therefore, in view of an eventual clinical use of enkephalinase inhibitors, it was very important to develop fully specific compounds. Such derivatives were obtained taking into account that N-methylation of the ultimate amide bond of dipeptides strongly decreases enkephalinase affinity without affecting angiotension-converting enzyme recognition, whereas retro-inversion of the amide bond leads to the inverse effect. Thus, the retro-inverso dipeptide (R)-H2N-CH(CH2 phi)-NHCO-CH2-CO2H exhibits an inhibitory potency on enkephalinase (IC50 approximately equal to 12 muM) close to that of the natural dipeptide L-Phe-Gly (IC50 approximately equal to 3 muM). This result shows the topological analogy between the crucial components involved in enkephalinase recognition both in active dipeptides and structurally related retro-inverso isomers. Taking into account these observations, retro-thiorphan, (R,S)-HS-CH2-CH-(CH2 phi)-NHCO-CH2-COOH, was prepared. As compared to thiorphan, the retro isomer is 50% as potent (Ki = 6 nM) on enkephalinase but displays a drastic loss of potency on angiotension-converting enzyme (IC50 greater than 10,000 nM). This specificity was interpreted as a consequence of differences in the stereochemical constraints involving enzyme-inhibitor hydrogen bonding. This hypothesis is supported by reported crystallographic studies on related enzymes such as thermolysin and carboxypeptidase A. As expected, retro-thiorphan exhibits about the same analgesic potency as thiorphan on the hot plate and writhing tests in mice. Therefore, the topological concept of retro-inverso isomers could be extended to other enkephalinase inhibitors, allowing the design of potent and highly selective compounds occurring as new classes of analgesic and psychoactive agents.
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PMID:Complete differentiation between enkephalinase and angiotensin-converting enzyme inhibition by retro-thiorphan. 630 95

Neurotensin was inactivated by membrane-bound and soluble degrading activities present in purified preparations of rat brain synaptic membranes. Degradation products were identified by HPLC and amino acid analysis. The major points of cleavage of neurotensin were the Arg8-Arg9, Pro10-Tyr11, and Tyr11-Ile12 peptide bonds with the membrane-bound activity and the Arg8-Arg9 and Pro10-Tyr11 bonds with the soluble activity. Several lines of evidence indicated that the cleavage of the Arg8-Arg9 bond by the membrane-bound activity resulted mainly from the conversion of neurotensin1-10 to neurotensin1-8 by a dipeptidyl carboxypeptidase. In particular, captopril inhibited this cleavage with an IC50 (5.7 nM) close to its K1 (7 nM) for angiotensin-converting enzyme. Thiorphan inhibited the cleavage at the Tyr11-Ile12 bond by the membrane-bound activity with an IC50 (17 nM) similar to its K1 (4.7 nM) for enkephalinase. Both cleavages were inhibited by 1,10-phenanthroline. These and other data suggested that angiotensin-converting enzyme and a thermolysin-like metalloendopeptidase (enkephalinase) were the membrane-bound peptidases responsible for cleavages at the Arg8-Arg9 and Tyr11-Ile12 bonds, respectively. In contrast, captopril had no effect on the cleavage at the Arg8-Arg9 bond by the soluble activity, indicating that the enzyme responsible for this cleavage was different from angiotensin-converting enzyme. The cleavage at the Pro10-Tyr11 bond by both the membrane-bound and the soluble activities appeared to be catalyzed by an endopeptidase different from known brain proline endopeptidases. The possibility is discussed that the enzymes described here participate in physiological mechanisms of neurotensin inactivation at the synaptic level.
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PMID:Degradation of neurotensin by rat brain synaptic membranes: involvement of a thermolysin-like metalloendopeptidase (enkephalinase), angiotensin-converting enzyme, and other unidentified peptidases. 630 59