Thiol-based angiotensin-converting enzyme 2 inhibitors: P1 modifications for the exploration of the S1 subsite

Screening of a metalloprotease library led to the identification of a thiol-based dual ACE/NEP inhibitor as a potent ACE2 inhibitor. Modifications of the P(1) benzyl moiety led to improvements in ACE2 potency as well as to increased selectivity versus ACE and NEP.     

Thiol-based angiotensin-converting enzyme 2 inhibitors: P1' modifications for the exploration of the S1' subsite

Explorations of the S(1') subsite of ACE2 via modifications of the P(1') methylene biphenyl moiety of thiol-based metalloprotease inhibitors led to improvements in ACE2 selectivity versus ACE and NEP, while maintaining potent ACE2 inhibition.     

N-formyl hydroxylamine containing dipeptides: generation of a new class of vasopeptidase inhibitors

Four primary zinc-binding pharmacophores (thiols, carboxylates, phosphorus acids, and hydroxamates) have been utilized in generating inhibitors of zinc metalloproteases such as ACE, NEP, the MMPs, and ECE. Although compounds which inhibit the activity of both ACE and NEP (vasopeptidase inhibitors, VPIs) have been reported which incorporate a thiol, carboxylate, or phosphorus acid pharmacophore, the generation of hydroxamate based vasopeptidase inhibitors has remained elusive. Herein we report the first potent vasopeptidase inhibitors which were generated from the incorporation of conformationally restricted dipeptide mimetics to an N-formyl hydroxylamine zinc-binding group. Compounds such as 13c and 13d are among the most potent in this series, exhibiting in vitro activity comparable to other classes of inhibitors.     

Angiotensin converting enzyme (ACE) and neprilysin hydrolyze neuropeptides: a brief history, the beginning and follow-ups to early studies

Our investigations started when synthetic bradykinin became available and we could characterize two enzymes that cleaved it: kininase I or plasma carboxypeptidase N and kininase II, a peptidyl dipeptide hydrolase that we later found to be identical with the angiotensin I converting enzyme (ACE). When we noticed that ACE can cleave peptides without a free C-terminal carboxyl group (e.g., with a C-terminal nitrobenzylamine), we investigated inactivation of substance P, which has a C-terminal Met(11)-NH(2). The studies were extended to the hydrolysis of the neuropeptide, neurotensin and to compare hydrolysis of the same peptides by neprilysin (neutral endopeptidase 24.11, CD10, NEP). Our publication in 1984 dealt with ACE and NEP purified to homogeneity from human kidney. NEP cleaved substance P (SP) at Gln(6)-Phe(7), Phe(7)[see text]-Phe(8), and Gly(9)-Leu(10) and neurotensin (NT) at Pro(10)-Tyr(11) and Tyr(11)-Ile(12). Purified ACE also rapidly inactivated SP as measured in bioassay. HPLC analysis showed that ACE cleaved SP at Phe(8)-Gly(9) and Gly(9)-Leu(10) to release C-terminal tri- and dipeptide (ratio = 4:1). The hydrolysis was Cl(-) dependent and inhibited by captopril. ACE released only dipeptide from SP free acid. ACE hydrolyzed NT at Tyr(11)-Ile(12) to release Ile(12)-Leu(13). Then peptide substrates were used to inhibit ACE hydrolyzing Fa-Phe-Gly-Gly and NEP cleaving Leu(5)-enkephalin. The K(i) values in microM were as follows: for ACE, bradykinin = 0.4, angiotensin I = 4, SP = 25, SP free acid = 2, NT = 14, and Met(5)-enkephalin = 450, and for NEP, bradykinin = 162, angiotensin I = 36, SP = 190, NT = 39, Met(5)-enkephalin = 22. These studies showed that ACE and NEP, two enzymes widely distributed in the body, are involved in the metabolism of SP and NT. Below we briefly survey how NEP and ACE in two decades have gained the reputation as very important factors in health and disease. This is due to the discovery of more endogenous substrates of the enzymes and to the very broad and beneficial therapeutic applications of ACE inhibitors.     

Bradykinin metabolism in rat hearts with left-ventricular hypertrophy following myocardial infarction

The aim of the present study was to assess the contribution of angiotensin I converting enzyme (ACE)and neutral endopeptidase (NEP) in the coronary degradation of bradykinin (BK) after left-ventricular hypertrophy following myocardial infarction (MI) in rats. Myocardial infarction was induced by left descendant coronary artery ligation, and the contribution of ACE and NEP in the degradation of exogenous BK after a single passage through the coronary bed was assessed at 2, 5, and 36 days post-MI. BK degradation rate (V(max)/Km) was found to be significantly lower in hearts at 36 days (3.30 +/- 0.28 min(-1)) compared with 2 days (4.39 +/- 0.32 min(-1)) for noninfarcted hearts, but this reduction was just above the statistical level of significance for post-MI hearts. In infarcted hearts, V(max)/Km was increased significantly 5 days post-MI (4.91 +/- 0.28 min(-1)) compared with the 2 and 36 day-groups (3.43 +/- 0.20 and 2.78 +/- 0.16 min(-1), respectively). The difference between noninfarcted and MI was significant only 2 days post-MI. Treatment with the vasopeptidase inhibitor, omapatrilat, showed that the relative contribution of ACE and NEP combined increased over time in infarcted hearts and became significantly higher 36 versus 2 days post-MI. Finally, the treatment with an ACE inhibitor (enalaprilat) and a NEP inhibitor (retrothiorphan) in the 36-day infarcted and noninfarcted hearts showed that the relative contribution of ACE in infarcted hearts was comparable with that of noninfarcted hearts, whereas the relative contribution of NEP was increased significantly in infarcted hearts. In conclusion, experimental MI in rats induces complex changes in the metabolism of exogenous BK. The changes resulted in an increased relative contribution of NEP 36 days after infarction.     

Study of bradykinin metabolism by rat lung tissue membranes and rat kidney brush border membranes by HPLC with inductively coupled plasma-mass spectrometry and orthogonal acceleration time-of-flight mass spectrometry.

The coupling of the techniques, high-performance liquid chromatography (HPLC), orthogonal acceleration time-of-flight mass spectrometry (OATOF-MS) and inductively coupled plasma mass spectrometry (ICP-MS) provides a very powerful method for identifying and quantifying the products of bradykinin metabolism. In this study, we were able to identify the major metabolites of bradykinin degradation reported in the literature. In addition, a new bradykinin metabolite corresponding to bradykinin 5,9 fragment (BK-(5,9)-fragment) was identified as a product of neutral endopeptidase (NEP) activity. This finding establishes that NEP cleaves bradykinin simultaneously at the positions 4-5 and 7-8. We also demonstrate the equivalent participation of NEP and angiotensin-converting enzyme (ACE) within the rat lung tissue membranes (RLTM) in bradykinin degradation, suggesting its suitability as a model for the assay of dual ACE/NEP inhibitors. On the contrary, in rat kidney brush border membranes (KBBM), ACE is not significantly involved in bradykinin metabolism, with NEP being the major enzyme.     

Peptidomimetics as receptors agonists or peptidase inhibitors: a structural approach in the field of enkephalins, ANP and CCK.

Stabilization of biologically active conformations of native peptides by cyclization or introduction of hindering residues led to peptidominetics endowed with high affinity and selectivity for one class of receptors and able to cross the blood brain barrier. This is the case of BUBU, Tyr-D-Ser(OtBu)-Gly-Phe-Leu-Thr(OtBu) and BUBUC, Tyr-D-Cys-(OtBu)-Gly-Phe-Leu-Thr(OtBu) for the opioid delta receptors and of BC 254, Boc-gamma-D-Glu-Tyr(SO3H)-Nle-D-Lys-Trp-Nle-Asp-PheNH2 and of BC 264, Boc-Tyr(SO3H)gNle-mGly-Trp-MeNle-Asp-PheNH2 for central CCK-B receptors. Inhibition of metabolizing peptidases such as aminopeptidase N and endopeptidase 24.11 (NEP) for enkephalins and of NEP and ACE for atrial natriuretic peptide and angiotensin I by mixed inhibitors such as kelatorphan and RB 101 or ES14, rationally designed by taking into account the structural differences in the active site of these zinc-metallopeptidases, led to potent analgesics devoid of the major morphine side effects or to new antihypertensives.     

The study of substrate specificity of a new peptide substrate of endothelin-converting enzyme

A new hexapeptide CMC-Ala-Gly-Gly-Trp-Phe-Arg was used as a substrate for assay of endothelin-converting (ECE; EC 3.4.24.71) and angiotensin-converting (ACE; EC 3.4.15.1) enzymes and of neutral endopeptidase (NEP; EC 3.4.24.11). The specific inhibitors lisinopril (for ACE) and thiorphan (for NEP) were used for discrimination between activities of these enzymes.     

Hydrolysis of substance p and neurotensin by converting enzyme and neutral endopeptidase

Angiotensin I converting enzyme (ACE) and neutral endopeptidase ("enkephalinase"; NEP), were purified to homogeneity from human kidney. NEP cleaved substance P (SP) at Gln6-Phe7,-Phe8, and Gly9-Leu10 and neurotensin (NT) at Pro10-Tyr11 and Tyr11-Ile12. NEP hydrolyzed 0.1 mM SP, NT and their C-terminal fragments at the following rates (mumol/min/mg): SP1-11 = 7.8, SP4-11 = 11.7, SP5-11 = 15.4, SP6-11 = 15.6, SP8-11 = 6.7, NT1-13 = 2.9, and NT8-13 = 4.0. Purified ACE rapidly inactivated SP as measured in bioassay. HPLC analysis showed that ACE cleaved SP at Phe8-Gly9 and Gly9-Leu10 to release C-terminal tri- and dipeptide (ratio = 4:1). The hydrolysis was Cl- dependent and inhibited by captopril. ACE released mainly C-terminal tripeptide from SP methyl ester, but only dipeptide from SP free acid. Modification of arginine residues in ACE with cyclohexanedione or butanedione similarly inhibited hydrolysis of SP, bradykinin and Bz-Gly-Phe-Arg (80-93%) indicating an active site arginine is required for hydrolysis of SP. ACE hydrolyzed NT at Tyr11-Ile12 to release Ile12-Leu13. SP, NT and their derivatives (0.1 mM) were cleaved by ACE at the following rates (mumol/min/mg): SP1-11 = 1.2, SP methyl ester = 0.7, SP free acid = 8.5, SP4-11 = 2.4, SP5-11 = 0.9, SP6-11 = 1.4, SP8-11 = 0, NT1-13 = 0.2, and NT8-13 = 1.3. Peptide substrates were used as inhibitors of ACE (substrate = FA-Phe-Gly-Gly) and NEP (substrate = Leu5-enkephalin).(ABSTRACT TRUNCATED AT 250 WORDS)     

Angiotensin converting enzyme has an inhibitory role in CGRP metabolism in human skin

The neutral endopeptidase (NEP) is important for calcitonin gene related peptide (CGRP) degradation, while the role of angiotensin converting enzyme (ACE) remains unclear. By using dermal microdialysis we explored the effect of phosphoramidon (NEP blocker), captopril (ACE blocker) and a mixture of both drugs on the intensity of electrically-induced CGRP-mediated neurogenic flare. The results reveal that phosphoramidon elevated flare intensity, but that this was not further increased by adding captopril. In contrast, neurogenic flare was decreased when the drug mixture was applied in compared to NEP only. Electrically released CGRP levels could be measured directly in perfusates containing phosphoramidon and the mixture. Again, CGRP levels were elevated in phosphoramidon treated sites, and significantly reduced upon adding captopril. These findings suggest that NEP and ACE do not have additive effects regarding neuropeptide degradation. In contrast, inhibition of ACE seems to augment CGRP catabolism.     

Nonpeptidic endothelin-converting enzyme inhibitors and their potential therapeutic applications

Endothelins (ETs) are potent vasoconstrictors, promitogens, and inflammatory mediators. They have been implicated in the pathogenesis of various cardiovascular, renal, pulmonary, and central nervous system diseases. Since the final step of the biosynthesis of ETs is catalyzed by a family of endothelin-converting enzymes (ECEs), inhibitors of these enzymes may represent novel therapeutic agents. Currently, seven isoforms of these metalloproteases have been identified; they all share a significant amino acid sequence identity with neutral endopeptidase 24.11 (NEP), another metalloprotease. Therefore, it is not surprising that the majority of ECE inhibitors also possess potent NEP inhibitory activity. To date, three classes of ECE inhibitors have been synthesized: dual ECE/NEP inhibitors, triple ECE/NEP/ACE inhibitors, and selective ECE inhibitors. Potential clinical applications of these compounds in hypertension, chronic heart failure, restenosis, renal failure, and cerebral vasospasm deduced from studies with relevant animal models are reviewed.     

Dual inhibition of converting enzyme and neutral endopeptidase: a research new way in the field of hypertension]

Two metallopeptidases, angiotensin converting enzyme (ACE) and neutral endopeptidase (NEP) are involved respectively in the release of angiotensin II which is a vasoconstrictor, and in the metabolism of atrial natriuretic peptide which is diuretic and bradykinin which is a vasodilatator. The dual inhibition of these two peptidases represents a new way to regulate the blood pressure in various cardiovascular diseases. Taking into account the mechanism of action of metallopeptidases and the substrate specificity of ACE and NEP, dual inhibitors corresponding to the general formula HS-CH2-CH(R1)CONH-CH(R2)COOH and HS-CH(R1)CONH-CH(R2)CONH-CH(R3)COOH and having inhibitory potencies on each enzyme in the nanomolar range were designed. The most efficient inhibitors have been transformed into lipophilic prodrugs which were found to be active after oral administration. These compounds have been tested on an experimental model of hypertension in rats and, as expected, have been shown to be both diuretic (NEP inhibition) and hypotensive (ACE inhibition).     

Inactivation of a tachykinin-related peptide: identification of four neuropeptide-degrading enzymes in neuronal membranes of insects from four different orders

Tachykinin-related peptides (TRP) are widely distributed in the CNS of insects, where they are likely to function as transmitters/modulators. Metabolic inactivation by membrane ecto-peptidases is one mechanism by which peptide signalling is terminated in the CNS. Using locustatachykinin-1 (LomTK-1, GPSGFYGVRamide) as a substrate and several selective peptidase inhibitors, we have compared the types of membrane associated peptidases present in the CNS of four insects, Locusta migratoria, Leucophaea maderae, Drosophila melanogaster and Lacanobia oleracea. A neprilysin (NEP)-like activity cleaving the G-F peptide bond was the major LomTK-1-degrading peptidase detected in locust brain membranes. NEP activity was also found in Leucophaea brain membranes, but the major peptidase was an angiotensin converting enzyme (ACE), cleaving the G-V peptide bond. Drosophila adult head and larval neuronal membranes cleaved the G-F and G-V peptide bonds. Phosphoramidon inhibited both these cleavages, but with markedly different potencies, indicating the presence in the fly brain of two NEP-like enzymes with different substrate and inhibitor specificity. In Drosophila, membrane ACE did not make a significant contribution to the cleavage of the G-V bond. In contrast, ACE was an important membrane peptidase in Lacanobia brain, whereas very little neuronal NEP could be detected. A dipeptidyl peptidase IV (DPP IV) that removed the GP dipeptide from the N-terminus of LomTK-1 was also found in Lacanobia neuronal membranes. This peptidase was a minor contributor to LomTK-1 metabolism by neuronal membranes from all four insect species. In Lacanobia, LomTK-1 was also a substrate for a deamidase that converted LomTK-1 to the free acid form. However, the deamidase was not an integral membrane protein and could be a lysosomal contaminant. It appears that insects from different orders can have different complements of neuropeptide-degrading enzymes. NEP, ACE and the deamidase are likely to be more efficient than the common DPP IV activity at terminating neuropeptide signalling since they cleave close to the C-terminus of the tachykinin, a region essential for maintaining biological activity.     

N-[2-(Indan-1-yl)-3-mercapto-propionyl] amino acids as highly potent inhibitors of the three vasopeptidases (NEP,ACE, ECE): In vitro and In vivo activities

We have previously reported the design of a lead compound 1a for the joint inhibition of 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), three metallopeptidases which are implicated in the regulation of fluid homeostasis and vascular tone. We report here the synthesis and biological activities of analogues derived from this lead with inhibitory potencies in the nanomolar range for the three enzymes. Compounds 8b and 15c are the most potent triple inhibitors described to date.     

Enhancement of neutral endopeptidase activity in SK-N-SH cells by green tea extract

Green tea extract (EFLA85942) is able to induce specifically the neutral endopeptidase (NEP) activity and to inhibit the proliferation of SK-N-SH cells; the angiotensin-converting enzyme (ACE) activity is not influenced under the same conditions. The treatment of the cells with arabinosylcytosine and green tea extract results in a strong enhancement of cellular NEP activity whereas cellular ACE activity was not changed significantly, indicating a green tea extract-specific regulation of NEP expression. Because of its role in the degradation of amyloid beta peptides this enzyme induction of NEP by long term treatment with green tea extract may have a beneficial effect regarding the prevention of forming amyloid plaques.     

An update on non-peptide angiotensin receptor antagonists and related RAAS modulators

The renin-angiotensin-aldosterone-system (RAAS) is an important regulator of blood pressure and fluid-electrolyte homeostasis. RAAS has been implicated in pathogenesis of hypertension, congestive heart failure, and chronic renal failure. Aliskiren is the first non-peptide orally active renin inhibitor approved by FDA. Angiotensin Converting Enzyme (ACE) Inhibitors are associated with frequent side effects such as cough and angio-oedema. Recently, the role of ACE2 and neutral endopeptidase (NEP) in the formation of an important active metabolite/mediator of RAAS, ang 1-7, has initiated attempts towards development of ACE2 inhibitors and combined ACE/NEP inhibitors. Furukawa and colleagues developed a series of low molecular weight nonpeptide imidazole analogues that possess weak but selective, competitive AT1 receptor blocking property. Till date, many compounds have exhibited promising AT1 blocking activity which cause a more complete RAAS blockade than ACE inhibitors. Many have reached the market for alternative treatment of hypertension, heart failure and diabetic nephropathy in ACE inhibitor intolerant patients and still more are waiting in the queue. But, the hallmark of this area of drug research is marked by a progress in understanding molecular interaction of these blockers at the AT1 receptor and unraveling the enigmatic influence of AT2 receptors on growth/anti-growth, differentiation and the regeneration of neuronal tissue. Different modeling strategies are underway to develop tailor made molecules with the best of properties like Dual Action (Angiotensin And Endothelin) Receptor Antagonists (DARA), ACE/NEP inhibitors, triple inhibitors, AT2 agonists, AT1/TxA2 antagonists, balanced AT1/AT2 antagonists, and nonpeptide renin inhibitors. This abstract gives an overview of these various angiotensin receptor antagonists.     

Differential Cleavage of Urodilatin and Atrial Natriuretic Factor by Thrombin and Protease 3.4.24.11

Abstract

Human urodilatin (residues 95–126) and atrial natriuretic factor (residues 99–126, based on ANF prohor-mone sequence) were incubated separately with three proteases, thrombin, angiotensin converting enzyme (ACE), and neutral endopeptidase 3.4.24.11 (NEP). Thrombin cleaved urodilatin on the carboxyl side of arginine⁹⁸ to yield ANF but under the same conditions did not cleave h-ANF. Neither urodilatin nor ANF was cleaved by ACE. ANF was rapidly degraded by NEP resulting in a major product cleaved between amino acid residues Cys^l05 and Phe¹⁰⁶. Urodilatin was also cleaved by NEP and the amino acid sequencing of the cleaved product revealed the site of cleavage to be the same Cys¹⁰⁵-Phe¹⁰⁶ site as for ANF with a second cleavage site at Gly¹¹⁸-Leu¹¹⁹. However, cleavage of urodilatin by NEP proceeded much more slowly when compared to ANF. A comparison of the affinities of ANF and urodilatin for purified NEP from rabbit kidney revealed K_m values of 11.7 and 3.1 μM, respectively. The turnover rates (k_cat/K_m) for urodilatin and h-ANF with NEP were 4.6 and 37.3 min^?1 μM^?1, respectively. Thus, urodilatin is much less efficiently hydrolyzed by purified NEP than is ANF. The four residue extension at the N-terminus of urodilatin may be important for protection against rapid biological inactivation.     

Direct inhibition of neutral endopeptidase in vasopeptidase inhibitor-mediated amelioration of cardiac remodeling in rats with chronic heart failure

Vasopeptidase inhibitors possess dual inhibitory actions on neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE) and have beneficial effects on cardiac remodeling. However, the contribution of NEP inhibition to their effects is not yet fully understood. To address the role of cardiac NEP inhibition in the anti-remodeling effects of a vasopeptidase inhibitor, we examined the effects of omapatrilat on the development of cardiac remodeling in rats with left coronary artery ligation (CAL) and those on collagen synthesis in cultured fibroblast cells. In vivo treatment with omapatrilat (30 mg/kg/day for 5 weeks) inhibited cardiac NEP activity in rats with CAL, which was associated with a suppression of both cardiac hypertrophy and collagen deposition. In cultured cardiac fibroblasts, omapatrilat (10^–7~10^–5 M) inhibited NEP activity and augmented the ANP-induced decrease in [³H]-proline incorporation. ONO-BB, an active metabolite of the NEP selective inhibitor ONO-9902, also augmented the ANP-induced response, whereas captopril, an ACE inhibitor, did not. The angiotensin I-induced increase in [³H]-proline incorporation was prevented by omapatrilat and captopril, but not by ONO-BB. The results suggest that vasopeptidase inhibitor suppressed cardiac remodeling in the setting of chronic heart failure, possibly acting through the direct inhibition of cardiac NEP. Vasopeptidase inhibitors may have therapeutic advantages over the classical ACE and NEP inhibitors alone with respect to the regression of cardiac fibrosis.     

Degradative enzymes modulate airway responses to intravenous neurokinins A and B

We studied the effects of the neutral endopeptidase (NEP) inhibitor thiorphan (1.7 mg/kg iv) and the angiotensin-converting enzyme (ACE) inhibitor captopril (5.7 mg/kg iv) on airway responses to rapid intravenous infusions of neurokinin A (NKA) and neurokinin B (NKB) in anesthetized, mechanically ventilated guinea pigs. The dose of NKA required to decrease pulmonary conductance to 50% of its base-line value (ED50GL) was fivefold less (P less than 0.0001) in animals treated with thiorphan compared with controls. NKA1-8, a product resulting from cleavage of NKA by NEP, had no bronchoconstrictor activity. Similar results were obtained by using NKB as the bronchoconstricting agent. Captopril had no significant effect on airway responses to NKA or NKB. In contrast, both thiorphan and captopril decrease the ED50GL for substance P (SP). We also compared the relative bronchoconstrictor potency of NKA, NKB, and SP. In control animals, the rank order of ED50GL values was NKA much less than NKB = SP. NKA also caused a more prolonged bronchoconstriction than SP or NKB. Thiorphan had no effect on the rank order of bronchoconstrictor potency, but in animals treated with captopril, the rank order of ED50GL values was altered to NKA less than SP less than NKB. These results suggest that degradation of NKA and NKB by NEP but not by ACE is an important determinant of the bronchoconstriction induced by these peptides. The degradation by ACE of SP but not NKA or NKB influences the observed relative potency of the three tachykinins as bronchoactive agents.