Synthesis and in vitro activities of new non-peptidic APA inhibitors.

Aminopeptidase A (APA) is involved in the maturation of angiotensin III, a peptide which seems to be implicated in blood pressure regulation at the brain level. Therefore APA inhibitors are potential new antihypertensive agents with possible novel applications. With the aim of enhancing the bioavailability and potency of EC 33, the APA inhibitor (Ki = 300 nM) initially used in the earlier studies, we have synthesized new non-peptidic inhibitors able to interact with the S1 and S'1 subsites of the targeted enzyme. Compound 10a, (3S,4S)-3-amino-4-mercapto-6-phenyl-hexane-1-sulfonic acid was obtained using an asymmetric synthesis. Inhibitor 10a exhibits a Ki value of 30 nm.

In vivo properties of thiol inhibitors of the three vasopeptidases NEP, ACE and ECE are improved by introduction of a 7-azatryptophan in P2' position.

Three zinc metallopeptidases are implicated in the regulation of fluid homeostasis and vascular tone and represent interesting targets for the treatment of chronic heart failure. We have previously reported the synthesis of a triple inhibitor able to simultaneously inhibit neprilysin (NEP, EC 3.4.24.11), angiotensin-converting enzyme (ACE, EC 3.4.15.1) and endothelin-converting enzyme (ECE-1, EC 3.4.24.71) with nanomolar potency towards NEP and ACE and a lesser affinity for ECE. Here, we report the optimization and biological activities of analogs derived from lead compound 1 (2S)-2-[(2R)-2-((1S)-5-bromo-indan-1-yl)-3-mercapto-propionylamino]-3- (1H-indol-3-yl)-propionic acid by a structural approach. Among several inhibitors, compound 21, (2S)-2-[(2R)-2-((1S)-5-bromo-indan-1-yl)-3-mercapto-propionylamino]-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)-propionic acid was selected by taking into account its good molecular adaptation with the recently published structures of the three vasopeptidases. This optimization procedure led to an improved pharmacologic activity when compared with 1.

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.

First discrete autoradiographic distribution of aminopeptidase N in various structures of rat brain and spinal cord using the selective iodinated inhibitor [125I]RB 129.

The selective and potent aminopeptidase N inhibitor [125I]RB 129 has been used for the radioautographic localization of this enzyme in rat brain, spinal cord and intestine. Brain microvessels and intestine brush-border cells were shown to present a high concentration of aminopeptidase N. Moreover, a labeling of various brain structures was observed. A very high level of binding occurred in the meninges, choroid plexus, pineal gland, paraventricular nucleus and pituitary gland. Moderate to high labeling was also observed in the cortex, caudate-putamen, subthalamic nucleus, central periaqueductal gray, thalamus, as well as in the dorsal and ventral horn of the spinal cord, which are known to contain a high concentration of enkephalins, opioid receptors and neutral endopeptidase. This co-localization confirms the physiological implication of aminopeptidase N in the inactivation of enkephalins accounting for the requirement of dual inhibition of neutral endopeptidase and aminopeptidase N to observe highly significant morphine-like effects induced by the protected endogenous opioid peptides. Aminopeptidase N was also visualized in moderate to high levels in other brain structures such as the hippocampus, nucleus accumbens, substantia nigra, hypothalamus (dorsomedial and ventromedial nuclei), raphe nucleus, pontine nucleus, inferior olive, and in high concentration in the granular layer of cerebellum. In summary, aminopeptidase N has been visualized for the first time in numerous brain areas using the selective inhibitor [125I]RB 129. This iodinated probe could allow the ex vivo and in vivo localization of aminopeptidase N in various tissues to be investigated and may also be used to evaluate quantitative changes in aminopeptidase N expression in pathological situations. Aminopeptidase N, which preferably removes NH2-terminal neutral amino acids from peptides, has probably a host of substrates. Nevertheless, a certain in vivo selectivity could be achieved by the presence of the enzyme in structures where the peptide effector and its receptors are also co-localized.

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.

Acute effect of the dual angiotensin-converting enzyme and neutral endopeptidase 24-11 inhibitor mixanpril on insulin sensitivity in obese Zucker rat.

The aim of this study was to determine whether acute dual angiotensin-converting enzyme (ACE)/neutral endopeptidase 24-11 (NEP) inhibition could improve whole body insulin-mediated glucose disposal (IMGD) more than ACE inhibition alone and whether this effect was mediated by the kinin-nitric oxide (NO) pathway activation. We therefore compared in anaesthetized obese (fa/fa) Zucker rats (ZOs) the effects of captopril (2 mg kg(-1), i.v.+2 mg kg(-1) h(-1)), retrothiorphan (25 mg kg(-1), i.v. +25 mg kg(-1) h(-1)), a selective NEP inhibitor, and mixanpril (25 mg kg(-1), i.v. +25 mg kg(-1) h(-1)), a dual ACE/NEP inhibitor, on IMGD using hyperinsulinaemic euglycaemic clamp technique. The role of the kinin-NO pathway in the effects of mixanpril was tested using a bradykinin B2 receptor antagonist (Hoe-140, 300 microg kg(-1)) and a NO-synthase inhibitor (N(omega)-nitro-L-arginine methyl ester, L-NAME, 10 mg kg(-1) i.v. +10 mg kg(-1) h(-1)) as pretreatments. Insulin sensitivity index (ISI) was lower in ZO controls than in lean littermates. Increases in ISI were observed in captopril- and retrothiorphan-treated ZOs. In mixanpril-treated ZOs, ISI was further increased, compared to captopril- and retrothiorphan-treated ZOs. In ZOs, Hoe-140 and L-NAME alone did not significantly alter and slightly reduced the ISI respectively. Hoe-140 and L-NAME markedly inhibited the ISI improvement induced by mixanpril. These results show that in obese insulin-resistant Zucker rats, under acute conditions, NEP or ACE inhibition can improve IMGD and that dual ACE/NEP inhibition improves IMGD more effectively than does either single inhibition. This effect is linked to an increased activation of the kinin-NO pathway.

Delta9-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 Delta9-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, Delta9-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 Delta9-tetrahydrocannabinol in mice treated chronically with morphine significantly reduces the naloxone-induced withdrawal syndrome. However, this repetitive administration of Delta9-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.

Enhancement of the effects of a complete inhibitor of enkephalin-catabolizing enzymes, RB 101, by a cholecystokinin-B receptor antagonist in diabetic rats.

1. RB 101, a complete inhibitor of enkephalin-catabolizing enzymes, has been previously shown to produce antinociception in normal rats after systemic administration. Moreover, its coadministration with a cholecystokinin-B (CCK-B) receptor antagonist has been shown to strongly enhance its antinociceptive effect in normal rats. In this work, we determined whether RB 101 was able to reduce hyperalgesia and allodynia in diabetic rats, a model of neuropathic pain. The type of opioid receptors (mu or delta) involved was determined using naloxone and naltrindole, respectively, and the interactions between endogenous enkephalins and CCK on nociception control was investigated using coadministration of RB 101 and the CCK-B receptor antagonist CI-988. 2. RB 101 suppressed mechanical hyperalgesia (paw pressure-induced vocalization test), partially alleviated mechanical allodynia (von Frey hair test), and was ineffective in thermal allodynia (tail immersion test). The analgesic effect was completely cancelled by naloxone or naltrindole, suggesting that is requires the availability of mu- and/or delta-opioid receptors. 3. The combination of an inactive dose of CI-988 with the lowest effective dose of RB 101 resulted in a stronger increase in the vocalization threshold comparatively to RB 101 alone. 4. The present study demonstrates that the antinociception generated by RB 101 induced by elevation of extracellular levels of endogenous enkephalins, can be extended to neuropathic pain in diabetic rats and that blockade of CCK-B receptors potentiated antinociceptive effects elicited by RB 101.

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.

Effects of the potent analgesic enkephalin-catabolizing enzyme inhibitors RB101 and kelatorphan on respiration.

We investigated whether the enkephalin-catabolizing enzyme inhibitors RB101 and kelatorphan, which have been shown to be potent analgesics, depress respiration as do opioid analgesics. Ventilation was measured in cats and rodents by the barometric method, in the awake state and during anesthesia. Tissue distribution of the inhibitors was either generalized (RB101, 40-160 mg/kg i.p.), largely restricted by the blood-brain barrier to the periphery (kelatorphan, 0.7-20 mg/kg i.v.), or restricted to the brainstem (i.c.v. injection of RB101 in the fourth ventricle). RB101 did not affect ventilation in any condition tested, and large doses of kelatorphan produced a naloxone-reversible increase in ventilation and breathing frequency. Thus endogenous opioids released during conditions of normal ventilation do not exert any depressant neuromodulatory effect on this function, even when their extracellular concentrations are increased by peptidase inhibitors. The differential effect of these inhibitors on ventilation and nociception is discussed. We conclude that kelatorphan and RB101 are devoid of respiratory-depressant effects and might be interesting pharmacological alternatives to morphine and other opioid agonists.

Aminopeptidase A, which generates one of the main effector peptides of the brain renin-angiotensin system, angiotensin III, has a key role in central control of arterial blood pressure.

Overactivity of the brain renin-angiotensin system (RAS) has been implicated in the development and maintenance of hypertension in several experimental animal models. We have recently reported that, in the murine brain RAS, angiotensin II (AngII) is converted by aminopeptidase A (APA) into angiotensin III (AngIII),which is itself degraded by aminopeptidase N (APN), both peptides being equipotent to increase vasopressin release and arterial blood pressure when injected by the intracerebroventricular (i.c.v.) route. Because AngII is converted in vivo into AngIII, the exact nature of the active peptide is not precisely known. To delineate their respective roles in the central control of cardiovascular functions, specific and selective APA and APN inhibitors are needed to block the metabolic pathways of AngII and AngIII respectively. In the absence of such compounds for APA, we first explored the organization of the APA active site by site-directed mutagenesis. This led us to propose a molecular mechanism of action for APA similar to that proposed for the bacterial enzyme thermolysin deduced from X-ray diffraction studies. Secondly, we developed a specific and selective APA inhibitor, compound EC33 [(S)-3-amino-4-mercaptobutylsulphonic acid], as well as a potent and selective APN inhibitor, PC18 (2-amino-4-methylsulphonylbutane thiol). With these new tools we examined the respective roles of AngII and AngIII in the central control of arterial blood pressure. A central blockade of APA with the APA inhibitor EC33 suppressed the pressor effect of exogenous AngII, suggesting that brain AngII must be converted into AngIII to increase arterial blood pressure. Furthermore, EC33, injected alone i.c.v. but not intravenously, caused a dose-dependent decrease in arterial blood pressure by blocking the formation of brain AngIII but not systemic AngIII. This is corroborated by the fact that the selective APN inhibitor PC18 administered alone via the i.c.v. route increased arterial blood pressure. This pressor response was blocked by prior treatment with the angiotensin type 1 receptor antagonist losartan, showing that blocking the action of APN on AngIII metabolism leads to an increase in endogenous AngIII levels, resulting in arterial blood pressure increase through an interaction with angiotensin type 1 receptors. These results demonstrate that AngIII is a major effector peptide of the brain RAS, exerting a tonic stimulatory control over arterial blood pressure. Thus APA, the enzyme responsible for the formation of brain AngIII, represents a potential central therapeutic target that justifies the development of APA inhibitors, crossing the blood-brain barrier, as central anti-hypertensive agents.

Analgesic doses of the enkephalin degrading enzyme inhibitor RB 120 do not have discriminative stimulus properties.

The systemically active mixed inhibitor of enkephalin metabolism, N-((S)-2-benzyl-3[(S) 2-amino-4-methylthio)butyldithio-]-1-oxopropyl)-L-alanine benzylester (RB 120), alone or in combination with 4-¿[2-[[3-(1H-indol-3-yl))-2-methyl-1-oxo-2-[[(tricyclo[3.3.1.1. ]dec-2-yloxy) carbonyl]amino¿propyl]amino]-1-phenylethyl]amino¿-4-oxo-[R-(R*, R*)]-butanoate N-methyl-D-glucamine (CI 988; CCK(1) receptor antagonist) was investigated for discriminative and morphine generalisation effects using an operant drug discrimination paradigm in rats. Animals dosed with RB 120 (10 mg/kg) failed to develop a discriminative response. Combined CI 988 (0.3 mg/kg) and RB 120 (10 mg/kg) also failed to elicit a discriminative response. Morphine-trained animals (3.0 mg/kg) did not generalise to RB 120 (10 and 20 mg/kg). Similarly, subsequent retraining of the same animals with 1.5 mg/kg of morphine did not elicit generalisation to RB 120 (10 or 20 mg/kg). Combined RB 120 (10 or 20 mg/kg) and CI 988 (0.3 or 3.0 mg/kg) treatment produced no notable drug lever selection in rats able to discriminate morphine (1.5 mg/kg) from saline. These results suggest that RB 120 may have low abuse potential at analgesic doses.

Effects of combined neutral endopeptidase 24-11 and angiotensin-converting enzyme inhibition on femoral vascular conductance in streptozotocin-induced diabetic rats.

1. The successive effects of the angiotensin-converting enzyme inhibitor captopril (CAP, 2 mg kg(-1)+1 mg kg(-1) 30 min(-1) infusion) and the neutral endopeptidase 24-11 inhibitor retrothiorphan (RT, 25 mg kg(-1)+12.5 mg kg(-1) 30 min(-1) infusion) were studied on femoral vascular conductance (FVC) in streptozotocin-induced diabetic (STZ-SD) and control Sprague-Dawley (C-SD) rats. The role of the kinin-nitric oxide (NO) pathway was assessed by (1) using pre-treatments: a bradykinin (BK) B2 receptor antagonist (Hoe-140, 300 microg kg(-1)), a NO-synthase inhibitor (N(omega)-nitro-L-arginine methyl ester, L-NAME, 10 mg kg(-1)), a kininase I inhibitor (DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, MGTA, 10 mg kg(-1)+20 mg kg(-1) 20 min(-1) infusion) and (2) comparing the effects in STZ-induced diabetic (STZ-BN) and control Brown-Norway kininogen-deficient (C-BN) rats. 2. In C-SDs, CAP and CAP+RT increased FVC similarly. In STZ-SDs, FVC and FBF were decreased compared to C-SDs. CAP+RT increased them more effectively than CAP alone. 3. In both C-SDs and STZ-SDs, the femoral bed vasodilatation elicited by CAP was inhibited by Hoe-140 and L-NAME. The FVC increase elicited by CAP+RT was not significantly reduced by Hoe-140 but was inhibited by L-NAME and Hoe-140+MGTA. 4. In C-BNs, the vasodilatator responses to CAP and CAP+RT were abolished and highly reduced, respectively. In STZ-BNs, these responses were abolished. 5. These results show that in STZ-SDs, CAP+RT improve FBF and FVC more effectively than CAP alone. These effects are linked to an increased activation of the kinin-NO pathway. BK could lead to NO production by BK B2 receptor activation and another pathway in which kininase I may be involved.

Exploration of the S(')(1) subsite of neprilysin: a joined molecular modeling and site-directed mutagenesis study.

Based on the recently described three-dimensional model of the 507-749 region of neprilysin, which contains the catalytic site of the enzyme, experiments were performed to improve the proposed topology of its large and hydrophobic S(')(1) subsite. Docking studies, site-directed mutagenesis, and biochemical studies were combined. The mutations of various residues proposed to be part of the S(')(1) subsite (F563A, F564A, M579A, F716A, and I718A) did not induce major structural reorganization of the active site as demonstrated by the slight modification of the enzyme activity. The mutations were also analyzed by measuring the inhibitory potencies of thiol inhibitors containing P(')(1) moieties of increasing sizes. These results combined with molecular modeling studies support the proposed topology of the S(')(1) subsite. This, and the critical role of F563 and M579 in inhibitor binding, could facilitate the synthesis of new potent and selective inhibitors.

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.

Phosphinic derivatives as new dual enkephalin-degrading enzyme inhibitors: synthesis, biological properties, and antinociceptive activities.

The development of dual inhibitors of the two zinc metallopeptidases, neprilysin (neutral endopeptidase) and aminopeptidase N involved in the inactivation of the opioid peptides, enkephalins, represents an attractive physiological approach in the search for new analgesics devoid of the major drawbacks of morphine. Phosphinic compounds, corresponding to the general formula H(3)N(+)-CH(R(1))-P(O)(OH)-CH(2)-CH(R(2))-CONH-CH(R(3))-COO(-), able to act as transition-state analogues and to fit the S(1), S(1)', and S(2)' subsites of both enzymes were designed. Selection of the R(1), R(2), and R(3) residues for optimal recognition of these enzymes led to the first dual competitive inhibitors with K(i) values in the nanomolar range for neprilysin and aminopeptidase N. These compounds induce potent analgesic responses after intracerebroventricular or intravenous administrations in mice (hot plate test), and several of them were shown to be, at least, 10 times more potent than the previously described dual inhibitors.

Investigation of subsite preferences in aminopeptidase A (EC 3.4.11.7) led to the design of the first highly potent and selective inhibitors of this enzyme.

The study of the physiological roles of the membrane-bound zinc-aminopeptidase A (glutamyl aminopeptidase, EC 3.4.11.7) needs the design of efficient and selective inhibitors of this enzyme. An acute exploration of aminopeptidase A active site was performed by a combinatorial approach using (3-amino-2-mercapto-acyl)dipeptides able to fit its S(1), S(1)', and S(2)' subsites. This analysis confirmed that the S(1) subsite is optimally blocked by a glutamate or isosteric residues and demonstrated that the S(1)' subsite is hydrophobic whereas the S(2)' subsite recognizes preferentially negatively charged residues derived from aspartic acid. The optimization of these structural parameters led to the synthesis of nanomolar and subnanomolar inhibitors of aminopeptidase A such as H(3)N(+)CH(CH(2)CH(2)SO(3)(-))CH(SH)CO-Ile-(3-COOH)Pro that exhibits a K(i) of 0.87 nM. The best compounds were synthesized by a stereochemically controlled route. These first described highly potent inhibitors could allow studies about the role of physiological substrates of APA such as angiotensin II and cholecystokinin CCK(8) in the central nervous system.

Aminopeptidase A inhibitors as potential central antihypertensive agents.

Overactivity of the brain renin-angiotensin system (RAS) has been implicated in the development and maintenance of hypertension in several experimental models, such as spontaneously hypertensive rats and transgenic mice expressing both human renin and human angiotensinogen transgenes. We recently reported that, in the murine brain, angiotensin II (AngII) is converted to angiotensin III (AngIII) by aminopeptidase A (APA), whereas AngIII is inactivated by aminopeptidase N (APN). If injected into cerebral ventricles (ICV), AngII and AngIII cause similar pressor responses. Because AngII is metabolized in vivo into AngIII, the exact nature of the active peptide is not precisely determined. Here we report that, in rats, ICV injection of the selective APA inhibitor EC33 [(S)-3-amino-4-mercaptobutyl sulfonic acid] blocked the pressor response of exogenous AngII, suggesting that the conversion of AngII to AngIII is required to increase blood pressure (BP). Furthermore, ICV injection, but not i.v. injection, of EC33 alone caused a dose-dependent decrease in BP by blocking the formation of brain but not systemic AngIII. This is corroborated by the fact that the selective APN inhibitor, PC18 (2-amino-4-methylsulfonyl butane thiol), administered alone via the ICV route, increases BP. This pressor response was blocked by prior treatment with the angiotensin type 1 (AT(1)) receptor antagonist, losartan, showing that blocking the action of APN on AngIII metabolism leads to an increase in endogenous AngIII levels, resulting in BP increase, through interaction with AT(1) receptors. These data demonstrate that AngIII is a major effector peptide of the brain RAS, exerting tonic stimulatory control over BP. Thus, APA, the enzyme responsible for the formation of brain AngIII, represents a potential central therapeutic target that justifies the development of APA inhibitors as central antihypertensive agents.

Crystal structures of alpha-mercaptoacyldipeptides in the thermolysin active site: structural parameters for a Zn monodentation or bidentation in metalloendopeptidases.

Three alpha-mercaptoacyldipeptides differing essentially in the size of their C-terminal residues have been crystallized in the thermolysin active site. A new mode of binding was observed for 3 [HS-CH(CH(2)Ph)CO-Phe-Tyr] and 4 [HS-CH((CH(2))(4)CH(3))CO-Phe-Ala], in which the mercaptoacyl moieties act as bidentates with Zn-S and Zn-O distances of 2.3 and 2.4 A, respectively, the side chains fitting the S(1), S(1)', and S(2)' pockets. Moreover, a distance of 3.1 A between the sulfur atom and the OE1 of Glu(143) suggests that they are H-bonded and that one of these atoms is protonated. This H-bond network involving Glu(143), the mercaptoacyl group of the inhibitor, and the Zn ion could be considered a "modified" transition state mimic of the peptide bond hydrolysis. Due to the presence of the hindering (5-phenyl)proline, the inhibitor HS-CH(CH(2)Ph)CO-Gly-(5-Ph)Pro (2) interacts through the usual Zn monodentation via the thiol group and occupancy of S(1)' and S(2)' subsites by the aromatic moieties, the proline ring being outside the active site. The inhibitory potencies are consistent with these structural data, with higher affinities for 3 (4.2 x 10(-)(8) M) and 4 (4.8 x 10(-)(8) M) than for 2 (1.2 x 10(-)(6) M). The extension of the results, obtained with thermolysin being considered as the model of physiological zinc metallopeptidases, allows inhibitor-recognition modes for other peptidases, such as angiotensin converting enzyme and neutral endopeptidase, to be proposed and opens interesting possibilities for the design of new classes of inhibitors.