Selective protection of methionine enkephalin released from brain slices by enkephalinase inhibition.

Methionine enkephalin release was evoked by depolarization of slices from rat striatum with potassium. In the presence of 0.1 microM thiorphan [(N(R,S)-3-mercapto-2-benzylpropionyl)glycine], a potent inhibitor of enkephalin dipeptidyl carboxypeptidase (enkephalinase), the recovery of the pentapeptide in the incubation medium was increased by about 100 percent. A similar effect was observed with the dipeptide phenylalanylalanine, a selective although less potent enkephalinase inhibitor. Inhibition of other known enkephalin-hydrolyzing enzymes--aminopeptidase by 0.1 mM puromycin or angiotensin-converting enzyme by 1 microM captopril--did not significantly enhance the recovery of released methionine enkephalin. These data indicate that enkephalinase is critically involved in the inactivation of the endogenous opioid peptide released from striatal neurons.

Influence of the N- and C-terminal chains on the zinc-binding and conformational properties of the central zinc-finger structure of Moloney murine leukaemia virus nucleocapsid protein: a steady-state and time-resolved fluorescence study.

The nucleocapsid protein NCp10 of the Moloney murine leukaemia virus is a small basic protein characterized by a central Cys26-X2-Cys29-X4-His34-X4-Cys39 zinc-finger domain. Mutants with deletion of either the N- or C-terminal chain (or both) surrounding the central zinc-finger domain were synthesized by a solid-phase approach in order to evaluate the influence of these lateral chains on zinc binding and conformational properties of NCp10. For this purpose, the steady-state and time-resolved fluorescence properties of the single Trp-35 residue of the various NCp10 derivatives were analyzed. The binding properties of the various derivatives suggest that the central zinc-finger domain affinity for zinc is not modified by the N-terminal chain and is only slightly (about one order of magnitude) increased by the C-terminal chain leading to a Kapp of (1.2 +/- 0.2).10(14) M-1 for the whole NCp10. Concerning the conformation of the NCp10 derivatives, fluorescence data are in agreement with structureless polypeptide chains in the absence of zinc. In contrast, in the presence of zinc, the fluorescence intensity decays are in agreement with a unique conformation of the finger motif backbone and a distribution of the Trp-indole moiety into two classes with different local environments. Decay-associated spectra, fluorescence quenching by acrylamide and anisotropy decay data further suggest that the Trp-indole moiety of both classes was highly exposed to solvent and had a high degree of rotational freedom. Finally, in contrast to the C-terminal chain, the N-terminal chain modifies the local environment and the accessibility to external quenchers of both Trp-35 classes, suggesting that it was folded in the vicinity of the Trp-35 residue.

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.

Structure of the complex between the HIV-1 nucleocapsid protein NCp7 and the single-stranded pentanucleotide d(ACGCC).

The nucleocapsid protein NCp7 of HIV-1 Mal contains two successive Zn knuckles of the CX2CX4HX4C type and plays a major role in virion morphogenesis, genomic RNA packaging and viral infectivity, mainly through single-stranded nucleic acid binding. We report here the study by 1H 2D NMR of the complex formed between the (12-53)NCp7, encompassing the two Zn knuckles, and d(ACGCC), a deoxynucleotide sequence analog corresponding to the shortest NCp7 binding site. Ten structures of the (12-53)NCp7/d(ACGCC) complex have been obtained from 607 NOE-derived distance constraints, 28 of which are intermolecular, and from molecular dynamics studies. The oligonucleotide is almost perpendicular to the sequence linking the two Zn knuckles. The Trp37 indole ring is inserted between the C2 and G3 bases and stacked on the latter. The complex is stabilized by hydrophobic interactions and hydrogen bonds, and accounts for the observed loss of virus infectivity induced by mutations in the Zn knuckle domain. Thus, the interaction between d(ACGCC) and the inactive mutant Cys23 (12-53)NCp7 was found by NMR to be completely different from that observed with the wild-type peptide. A mechanism of action for NCp7 in virus morphogenesis and replication is proposed from these results, which could facilitate the design of possible antiviral agents acting by a new mechanism.

Angiotensin III: a central regulator of vasopressin release and blood pressure.

Among the main bioactive peptides of the brain renin-angiotensin system, angiotensin (Ang) II and AngIII exhibit the same affinity for type 1 and type 2 AngII receptors. Both peptides, injected intracerebroventricularly, cause similar increases in vasopressin release and blood pressure. Because AngII is converted in vivo to AngIII, the identity of the true effector is unknown. This review summarizes new insights into the predominant role of brain AngIII in the control of vasopressin release and blood pressure and underlines the fact that brain aminopeptidase A, the enzyme forming central AngIII, could constitute a putative central therapeutic target for the treatment of hypertension.

Differences in transition state stabilization between thermolysin (EC 3.4.24.27) and neprilysin (EC 3.4.24.11).

Important homologies in the topology of the catalytic site and the mechanism of action of thermolysin and neprilysin have been evidenced by site-directed mutagenesis. The determination of differences in transition state stabilization between these peptidases could facilitate the design of specific inhibitors. Thus, two residues of thermolysin which could be directly (Tyr157) or indirectly (Asp226) involved in the stabilization of the transition state and their putative counterparts in neprilysin (Tyr625 and Asp709) have been mutated. The results show that Tyr157 is important for thermolysin activity while Tyr625 has no functional role in neprilysin. Conversely, the mutation of Asp226 induced a slight perturbation of thermolysin activity while Asp709 in neprilysin seems crucial in neprilysin catalysis. Taken together these data seem to indicate differences in the transition state mode of stabilization in the two peptidases.

Binding properties of a highly potent and selective iodinated aminopeptidase N inhibitor appropriate for radioautography.

Aminopeptidase N (APN) is a zinc metallopeptidase involved in the inactivation of biologically active peptides. The knowledge of its precise distribution is crucial to investigate its physiological role. This requires the use of appropriate probes such as the recently developed highly potent and selective radiolabeled APN inhibitor 2(S)-benzyl-3-[hydroxy(1'(R)-aminoethyl)phosphinyl]propanoyl-L-3-[ (12 5)I]iodotyrosine ([(125)I]RB 129). Its binding properties were investigated using rat brain homogenates (K(d)=3.4 nM) or APN expressed in COS-7 cells (K(d)=0.9 nM). The specific binding was 95% at [K(d)], and preliminary autoradiography in intestine is promising. The decreased affinity of [(125)I]RB 129 (=10(-6) M) for the E(350)D APN mutant, supports the critical role of E(350) in the amino-exopeptidase action of APN.

Antinociceptive response induced by mixed inhibitors of enkephalin catabolism in peripheral inflammation.

RB101 (N-((R,S)-2-benzyl-3[(S)(2-amino-4-methylthio)butyl dithio]-1-ox-opropyl)-L-phenylalanine benzyl ester) is a recently developed full inhibitor of the enkephalin-catabolizing enzymes able to cross the blood-brain barrier, whereas RB38A ((R)-3-(N-hydroxycarboxamido-2-benzylpropanoyl)-L-phenylalanine) is as potent as RB101 but almost unable to enter the brain. In this study, we have investigated the effects of systemic administration of morphine, RB101 and RB38A on nociception induced by pressure on inflamed peripheral tissues. Antinociceptive test was performed between 4 and 5 days after injection into the rat left hindpaw of Freund's complete adjuvant to produce localized inflammation. Morphine (1, 2 and 4 mg/kg, i.v.) induced antinociception in inflamed paws at all the doses used, and only at the highest dose in non-inflamed paws. RB101 (10 and 20 mg/kg, i.v.) induced an antinociceptive response only in the inflamed paws. RB38A, also induced an antinociceptive effect in the inflamed paws, but only at the highest dose (20 mg/kg, i.v.). The responses induced by morphine and the inhibitors of enkephalin catabolism were antagonized by the systemic administration of naloxone (1 mg/kg) or methylnaloxonium (2 mg/kg) which acts essentially outside the brain. Central injection (i.c.v.) of methylnaloxonium (2 micrograms) blocked the effect of morphine only in non-inflamed paws, and slightly decreased the response induced by RB101 on inflamed paws. These results indicate that the endogenous opioid peptides, probably enkephalins, are important in the peripheral control of nociception from inflamed tissues.

Paradoxical analgesia induced by low doses of naloxone is not potentiated by complete inhibition of enkephalin degradation.

The involvement of a presynaptic autoinhibition of endogenous enkephalin release has been suggested as a possible explanation for the paradoxical analgesia induced by low doses of naloxone. This hypothesis was investigated by using the systemically active mixed inhibitor of enkephalin degrading enzymes, RB 101. As already described, in both hot plate (55 +/- 0.5 degrees C) and acetic acid (0.6%) abdominal constriction tests in mice, subcutaneous administration of naloxone produced biphasic effects, with antinociceptive responses at very low doses (microgram range) and hyperalgesia at higher dose (mg range). However at concentrations producing an extracellular increase in enkephalin levels and subsequent analgesia, the mixed inhibitor prodrug of the enkephalin-metabolizing enzymes RB 101 (20 or 100 mg/kg i.v. and 5 or 10 mg/kg i.v., in the hot plate test and in the abdominal constriction test, respectively) did not potentiate the paradoxical analgesia induced by naloxone. These results are inconsistent with a negative autoregulation of endogenous enkephalin release and could suggest the involvement of the diffuse noxious inhibitory controls (DNIC). Indeed, the finding that low doses of RB 101 (1 mg/kg i.v. in the hot plate test, and 250 micrograms/kg i.v. in the abdominal constriction test) were able to induce hyperalgesic responses could indicate that the DNIC are tonically activated by endogenous enkephalins accounting for the antinociceptive responses elicited by low doses of naloxone.

Facilitation of enkephalins catabolism inhibitor-induced antinociception by drugs classically used in pain management.

The aim of this study was to investigate the facilitatory effects of subanalgesic or low doses of different drugs (acetylsalicylic acid, ibuprofen and morphine) on the antinociceptive responses induced by the endogenous opioid peptides, enkephalins, protected from their catabolism by the dual enkephalin-degrading enzymes inhibitor RB101. According to the analgesic profile of the three studied compounds different antinociceptive assays were used: the hot plate and formalin tests in mice, and the tail flick and paw pressure tests on inflamed paws in rats and polyarthritic rats. Facilitatory effects of subanalgesic doses of acetylsalicylic acid and ibuprofen on RB101-induced antinociceptive responses were observed in the early and late phases of the formalin test, respectively. In the hot plate, tail flick and paw pressure tests, the dose-dependent analgesic effects of RB101 were strongly potentiated by subanalgesic doses of morphine (0.5 mg/kg), while in these tests, acetylsalicylic acid and ibuprofen were unable to modify the RB101-induced antinociceptive responses. The synergism in antinociceptive effects observed with the combination of RB101 and morphine supported by isobolographic analysis, may have interesting clinical implications, considering both the lack of opiate drawbacks observed with RB101 and the high potentiation of its antinociceptive effects with very low doses of morphine.

Long lasting antinociceptive properties of enkephalin degrading enzyme (NEP and APN) inhibitor prodrugs.

Prodrugs of phosphinic dual inhibitors of the enkephalin degrading enzymes, neutral endopeptidase (NEP) and aminopeptidase N (APN), corresponding to the formula H(3)N(+)CH(R(1))P(O)(OR)CH(2)CH(CH(2)Bip)CONHCH(CH(3))COOCH(2)Ph, with R(1) = CH(3) or Ph and R being a benzyl ester, a S-acyl-2-thioethyl derivative, or an acyloxyalkyl group, were synthesized to improve the poor central bioavailability of their precursors. As expected, these compounds (50 mg/kg, iv or ip) induced long lasting ( approximately 2 h) antinociceptive responses in the hot plate test in mice with a ceiling effect varying between 25 and 42% of analgesia. A very rapid hydrolysis of the carboxylate ester contrasting with a slow deprotection of the phosphinate group (t(1/2) approximately 1 h) was observed in serum while 80% of free drug was obtained after 1 h incubation with brain membranes. These results account for the long duration of action observed with these prodrugs.

Evidence of the preferential involvement of mu receptors in analgesia using enkephalins highly selective for peripheral mu or delta receptors.

In order to study the preferential involvement of mu or delta receptors in the analgesic effects of enkephalins, several peptides which selectively interact with these two kinds of receptors in peripheral organs were synthesized. The inhibitory potency on the electrically stimulated mouse vas deferens (delta receptors) of the short peptide Tyr-D-Ala-Gly-NH-CH(CH3)CH2CH(CH3)2 (6) is 2100 times lower (IC50 = 1220 nM) than that of the longer and more hydrophilic peptide Tyr-D-Ser-Gly-Phe-Leu-Thr (10) (IC50 = 0.58 nM). In contrast, the IC50 values of all the synthesized compounds on the guinea pig ileum assay (mu receptors) are in the same range (100-360 nM). Likewise, their analgesic activities in mice, measured on the hot-plate test after intracerebroventricular injection, are similar. Therefore, the dissociation between antinociceptive properties in mice and potencies on the mouse vas deferens unambiguously reflects a preferential implication of mu receptors in analgesia. The possible involvement of brain delta receptors in behavioral effects is discussed.

1H NMR configurational correlation for retro-inverso dipeptides: application to the determination of the absolute configuration of "enkephalinase" inhibitors. Relationships between stereochemistry and enzyme recognition.

A stereospecific synthesis of thiorphan [N-[2(RS)-(mercaptomethyl)-1-oxo-3-phenylpropyl]glycine] and retro-thiorphan [3-[[1(RS)-(mercaptomethyl)-2-phenylethyl]amino]-3-oxopropanoic acid], two highly potent inhibitors of enkephalinase, a neutral endopeptidase involved in enkephalin metabolism, is reported. Due to a rapid isomerization process, derivatives of retro-thiorphan, which contains a 2-substituted malonyl moiety, cannot be separated by classical methods. However, a separation of the diastereoisomeric mixtures of these retro-thiorphan derivatives was achieved by HPLC. The absolute configuration of each isomer was determined by using an NMR configurational correlation. The inhibitory potency of the various inhibitors indicates that, in the thiorphan series, the affinity for enkephalinase is independent of the stereochemistry of the 2-(mercaptomethyl)-1-oxo-3-phenylpropyl moiety. In contrast, in the retro-thiorphan series a 100-fold difference in the inhibitory activity of the two enantiomers is observed. This indicates that there are large differences in the conformational behavior of the two series of inhibitors at the active site of the enzyme.

Beta-amino-thiols inhibit the zinc metallopeptidase activity of tetanus toxin light chain.

Tetanus neurotoxin is a 150-kDa protein produced by Clostridium tetani, which causes the lethal spastic paralytic syndromes of tetanus by blocking inhibitory neurotransmitter release at central synapses. The toxin light chain (50 kDa) has a zinc endopeptidase activity specific for synaptobrevin, an essential component of the neuroexocytosis apparatus. Previous unsuccessful attempts to block the proteolytic activity of this neurotoxin with well-known inhibitors of other zinc proteases led us to study the design of specific inhibitors as a possible drug therapy to prevent the progressive evolution of tetanus following infection. Starting from the synaptobrevin sequence at the level of the cleavage site by tetanus neurotoxin (Gln76-Phe77), a thiol analogue of glutamine demonstrated inhibitory activities in the millimolar range. A structure-activity relationship performed with this compound led us to determine the requirement for the correct positioning of the thiol group, the primary amino group, and a carboxamide or sulfonamide group on the side chain. This resulted in the design of a beta-amino-(4-sulfamoylphenyl)glycine-thiol, the first significantly efficient inhibitor of tetanus neurotoxin with a Ki value of 35 +/- 5 microM.

New thiol inhibitors of neutral endopeptidase EC 3.4.24.11: synthesis and enzyme active-site recognition.

Selective, as well as mixed, inhibitors of the two zinc metallopeptidases, neutral endopeptidase (NEP) and angiotensin converting enzyme (ACE), are of major clinical interest in the treatment of hypertension and cardiac failure. New thiol inhibitors, corresponding to the general formula HS-CH(R1)-CH2-CH(R2)-CONH-CH(R3)-COOH, were designed in order to explore the putative S1 subsite of the active site of NEP. The inhibitors were also tested on ACE and the most representative on thermolysin (TLN) for comparison. The relatively low inhibitory potencies exhibited by these compounds (IC50S in the 10(-7) M range for NEP and in the 10(-6) M range for ACE) as compared to that of thiorphan (IC50S 2.10 x 10(-9) M on NEP and 1.40 x 10(-7) M on ACE) clearly indicate the absence of the expected energetically favorable interactions with the active site of both peptidases. A 100-fold loss of potency for these inhibitors was also observed for thermolysin as compared to thiorphan. Using the mutated Glu102-NEP, it was possible to demonstrate that the inhibitors do not fit the S1 subsite of NEP but interact with the S'1 and S'2 subsites through binding of their R1 and R2 residues and that the C-terminal amino acid is located outside the active site. These results seem to indicate that these thiol inhibitors are not well adapted for optimal recognition of the S1 subsite of NEP, and probably ACE, and that other zinc-chelating moieties such as carboxylate or phosphonate groups may be preferred for this purpose.

New kelatorphan-related inhibitors of enkephalin metabolism: improved antinociceptive properties.

In order to improve the in vivo protection of enkephalins from enzymatic degradation, a new series of inhibitors derived from kelatorphan [HONHCOCH2CH(CH2Ph)CONHCH(CH3)COOH], the first-described complete inhibitor of enkephalin metabolism, were designed by modification of the C-terminal amino acid. The progressive lengthening of the chain of this residue shows that a beta-alanine seems to be the best basic model for the conception of such types of compounds. On the other hand, the methylation of the amide bond, which is well accepted by aminopeptidase N (EC 3.4.11.2) and dipeptidylaminopeptidase, induced a significant loss of affinity for neutral endopeptidase -24.11. Starting from these data, compounds containing a variously substituted beta-alanine residue and corresponding to the general formula HONHCOCH2CH(CH2Ph)CONHCH(R1)CH(R2)COOH were synthesized. All these molecules inhibit neutral endopeptidase -24.11 and dipeptidylaminopeptidase in the nanomolar range, and those containing an aromatic chain (compound 7A, R1 = CH2Ph,R2 = H, and compound 8A, R1 = Ph, R2 = H) inhibit the biologically relevant aminopeptidase N, with IC50's around 10(-8) M. Intracerebroventricular injection in mice of these multienzyme inhibitors produced an efficient and naloxone-reversible analgesic response (hot plate test): compounds 7A and 8A were shown to be more potent than kelatorphan in increasing the jump latency time, in agreement with their in vitro properties, and these new compounds were found to increase the forepaw lick latency, a reflex considered as a typical morphine response.

Retro-inverso concept applied to the complete inhibitors of enkephalin-degrading enzymes.

Peptide retro-inverso modification was applied to the complete hydroxamate inhibitors of the three zinc metallopeptidases (neutral endopeptidase 24-11 (NEP, EC 3.4.24.11), aminopeptidase N (APN, EC 3.4.11.2), and a dipeptidylaminopeptidase (DAP) involved in the in vitro enkephalin degradation by brain tissues. Compounds corresponding to the general formula RN(OH)CO(CH2)nCH(CH2Ph)NHCOCH(R')COOH (n = 0, 1) were synthesized. In the first series of inhibitors (n = 0), the "retro-inverso" modification induced a large decrease in inhibitory potency for NEP as compared to that of the parent compounds. In contrast, the presence of a methylene group between the hydroxamate and CH alpha in the second series (n = 1) led to derivatives with inhibitory potencies in the nanomolar range, similar to their analogues with a natural amide bond. On the other hand, the retro-inverso modification led to a slight improvement in the inhibition of DAP and APN, in the first series of inhibitors, while the inverse result occurred in the second series. Thus, compounds containing an alpha-amino acid moiety in P'1 position behave as weak inhibitors of the three enzymes, with IC50 values in the micromolar range, and compounds bearing a beta-amino acid moiety in the same position are more specific than the parent compounds for NEP inhibition.

Optimal recognition of neutral endopeptidase and angiotensin-converting enzyme active sites by mercaptoacyldipeptides as a means to design potent dual inhibitors.

An interesting approach for the treatment of congestive heart failure and chronic hypertension could be to avoid the formation of angiotensin II by inhibiting angiotensin converting enzyme (ACE) and to protect atrial natriuretic factor by blocking neutral endopeptidase 24.11 (NEP). This is supported by recent results obtained with potent dual inhibitors of the two zinc metallopeptidases, such as RB 105, HSCH2CH(CH3)PhCONHCH(CH3)COOH (Fournié-Zaluski et al. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 4072-4076), which reduces blood pressure in experimental models of hypertension, independently of the salt and renin angiotensin system status. In order to develop new dual inhibitors with improved affinities, long duration of action, and/or better bioavailabilities, various series of mercaptoacyldipeptides corresponding to the general formula HSCH(R1)CONHCH(R1')CON(R)CH(R2')COOH have been synthesized. The introduction of well-selected beta-branched chains in positions R1 and R1', associated with a tyrosine or a cyclic amino acid in the C-terminal position, led to potent dual inhibitors of NEP and ACE such as 21 [N-[(2S)-2-mercapto-3-methylbutanoyl]-Ile-Tyr] and 22 [N-[(2S)-2-mercapto-3-phenylpropanoyl]Ala-Pro] which have IC50 values in the nanomolar range for NEP and subnanomolar range for ACE. These compounds could have different modes of binding to the two peptidases. In NEP, the dual inhibitors seem to interact only with the S1' and S2' subsites, whereas additional interactions with the S1 binding subsite of ACE probably account for their subnanomolar inhibitory potencies for this enzyme. The localization of the Pro residue of 22 outside the NEP active site is supported by biochemical data using (Arg102,Glu)NEP and molecular modeling studies with thermolysin used as model of NEP. One hour after oral administration in mice of a single dose (2.7 x 10(-5) mol/kg), 21 inhibited 80% and 36% of kidney NEP and lung ACE, respectively, while 22 inhibited 40% of kidney NEP and 56% of lung ACE.

Exploration of neutral endopeptidase active site by a series of new thiol-containing inhibitors.

With the aim of characterizing the active site of the neutral endopeptidase [EC 3.4.24.11 (NEP)] and especially its putative S1 subsite, two series of new thiol inhibitors designed to interact with the S1, S'1, and S'2 subsites of the enzyme have been synthesized. These molecules correspond to the general formula HSCH(R1)CH(R2)CONHCH(R3)COOH (series I) and HSCH(R1)CH(R2)CONHCH(R3)CONHCH(R4)COOH (series II). Due to the synthetic pathway used, these inhibitors were obtained as mixtures of four stereoisomers. HPLC separation of the stereoisomers of 17 HSCH[CH2CH(CH3)2]CH(CH2Ph)CONHCH(CH3)COOH allowed the stereochemical dependence of the inhibitory potency to be determined. The most active isomer 17b (IC50 = 3.6 nM) is assumed to have the S,S,S stereochemistry, as deduced from both NMR and HPLC data. Although none of the inhibitors obtained were significantly more active than thiorphan, HSCH2CH(CH2Ph)CONHCH2COOH (IC50 = 4 nM), which interacts only with the S'1 and S'2 subsites of NEP, their enhanced hydrophobicity is expected to improve their pharmacokinetic properties. All these compounds displayed low affinities for ACE (IC50s > 1 microM). The determination of the IC50s of two inhibitors of series II for NEP and for a mutated enzyme in which Arg102 was replaced by Glu102 allowed their mode of binding to the active site of NEP to be characterized. The R2 and R3 chains fit the S'1-S'2 subsites, while the R4 group is probably located outside the active site. Taken together these results indicate that the R1 chain of these inhibitors creates no additional stabilizing interactions with the active site of NEP. Two hypotheses may account for this: there is no hydrophobic S1 subsite in NEP or the inhibitors have structures which are too constrained for optimized interactions with the active site.

Metallopeptidase inhibitors of tetanus toxin: A combinatorial approach.

The bacterial protein tetanus toxin (TeNt), which belongs to the family of zinc endopeptidases, cleaves synaptobrevin, an essential synaptic protein component of the neurotransmitter exocytosis apparatus, at a single peptide bond (Gln76-Phe77). This protease activity is a particularly attractive target for designing potent and selective synthetic inhibitors as a possible drug therapy for tetanus. beta-Aminothiols mimicking Gln76 of synaptobrevin have been previously shown to inhibit the tetanus neurotoxin enzymatic activity in the 35-250 microM range. These compounds have now been modified to interact with S' subsites of the TeNt active site, with the aim of increasing their inhibitory potencies. Combinatorial libraries of pseudotripeptides, containing an ethylene sulfonamide or an m-sulfonamidophenyl moiety as the P1 side chain and natural amino acids in P1' and P2' positions, were synthesized. The best inhibitory activity was observed with Tyr and His as P1' and P2' components, respectively. This led to new inhibitors of TeNt with Ki values in the 3-4 microM range. These molecules are the most potent inhibitors of TeNt described so far.