Rajendra D. Ghai

Non-peptidic inhibitors of neutral endopeptidase 24.11 1. Discovery and optimization of potency

Abstract Although based on a single α-amino acid residue, N -phosphonomethyl-(S)-(4-phenyl)phenylalanine ( 2 ) was discovered to produce strong inhibition of the zinc metalloprotease, neutral endopeptidase (NEP 24.11). Structural optimization of this new lead culminated with the design of the phosphonic acid tetrazole 17 (CGS 26303), a non-peptidic and extremely potent NEP inhibitor.

Non-peptidic inhibitors of neutral endopeptidase 24.11 2. Design and pharmacology of orally active phosphonate prodrugs

Abstract Prodrugs of 1 (CGS 26303), a novel non-peptidic neutral endopeptidase (NEP 24.11) inhibitor, have been designed. In particular, the diphenylphosphonate 6 (CGS 26393) was found to be a potent, orally bioavailable and long-acting NEP inhibitor. Accordingly, 6 protected atrial natriuretic peptide from enzymatic degradation and displayed a significant antihypertensive activity in the DOCA-salt rat.

Dual inhibition of neutral endopeptidase and angiotensin-converting enzyme by N-phosphonomethyl and N-carboxyalkyl dipeptides

Abstract Structural modifications have been performed on the selective α-amino phosphonic acid NEP inhibitor CGS 24592, to achieve dual ACE/NEP inhibition in vitro. (S)-N-Phosphonomethyl-valyl-(S)-(4-phenyl)phenylalanine (19) is representative of a new type of phosphorus-containing ACE/NEP inhibitors, approaching the in vitro potency of the sulfhydryl ACE inhibitor captopril and the NEP inhibitor thiorphan.

Characterisation of Neutral Endopeptidase 3.4.24.11 (NEP) in the Kidney: Comparison Between Normotensive, Genetically Hypertensive and Experimentally Hypertensive Rats

Neutral endopeptidase 3.4.24.11 (NEP) has been identified as the major atrial natriuretic factor (ANF) degrading enzyme in rat kidney, therefore, suggesting a possible role for this enzyme in blood volume and pressure regulation. Various experimentally induced and genetically hypertensive rat models have been used to test NEP inhibitors. The presence of different isoforms of NEP in the various hypertensive rat models would have relevance when searching for novel NEP inhibitors. Therefore, we compared the properties of NEP in kidney cortex homogenates in order to test for possible differences in the following hypertensive rat models and their appropriate controls: spontaneously hypertensive rats (SHR), Wistar Kyoto strain (WKY), DOCA-salt hypertensive rats, and Sprague Dawley control rats (SD). No relevant differences were found when comparing the following parameters: (1) specific activity (mean: 204 U/mg protein), (2) Michaelis constant (mean: 280 microM), (3) IC50 of thiorphan (mean: 6.5 nM) and phosphoramidon (mean: 54 nM), (4) pH profiles (optimum at pH 8.0), (5) heat inactivation profiles (half-life 20 min at 65 degrees C), (6) immunotitration of kidney cortex homogenates, (7) molecular weight as determined by gel filtration (92,000 Dalton) and (8) affinity chromatography with concanavalin A. Without evidence for the presence of different NEP isoforms, it is unlikely that divergent findings in DOCA-salt rats and SHR using a given NEP inhibitor are due to isoforms of NEP.

Pharmacologic profile of CGS 24128, a potent, long-acting inhibitor of neutral endopeptidase 24.11.

We compared the pharmacologic profiles of thiorphan, a neutral endopeptidase (NEP) inhibitor which is cleared rapidly from the circulation, and CGS 24128, an inhibitor with a much longer half-life (t1/2). Thiorphan and CGS 24128 inhibited NEP in vitro with IC50 values of 5.0 +/- 0.2 and 4.3 +/- 0.2 nM, respectively. After administration at 10 mg/kg intravenously (i.v.), the concentrations of CGS 24128 in the plasma were > 500 nM for 4 h but plasma thiorphan was detectable for only 60 min. Thiorphan 3 mg/kg administered intraarterially (i.a.) increased plasma atrial natriuretic peptide immunoreactivity (ANPir) levels by 58 +/- 12% in rats administered exogenous ANP(99-126). This response lasted < 60 min, whereas the same dose of CGS 24128 produced an average increase of 191 +/- 19% in ANPir concentrations that persisted for 4 h. ANP-induced (1 microgram/kg i.v.) natriuresis was significantly potentiated in anesthetized rats pretreated (60 min) with a bolus of CGS 24128 10 mg/kg i.v. The change in urinary sodium excretion (UNaV) produced by ANP was 28.8 +/- 4.0 and 15.8 +/- 1.8 muEq/kg/min in CGS 24128- and vehicle-treated rats, respectively. ANP-induced natriuresis was also greater during continuous infusion of thiorphan (5 mg/kg bolus + 0.1 mg/kg/min i.v.; delta UNaV = 29.2 +/- 5.8 and 13.8 +/- 3.2 muEq/kg/min in drug- and vehicle-treated rats, respectively) but not when thiorphan was administered as a bolus (10 mg/kg i.v.) 60 min before the ANP challenge.(ABSTRACT TRUNCATED AT 250 WORDS)

Dual inhibition of angiotensin-converting enzyme and neutral endopeptidase by tricyclic benzazepinone thiols §

Abstract Various thioacyl analogs of CGS 28106, a tricyclic dual inhibitor of angiotensin-converting enzyme and neutral endopeptidase, have been synthesized and their inhibitory potencies evaluated in vitro. The structure-activity relationship supports the proposed hypothesis that, despite its conformational constraints, CGS 28106 can inhibit the two distinct metalloproteases by adopting different binding modes. In addition, the structural features of CGS 28106 confer remarkable oral activity to this dual inhibitor, as measured by its ability to block the angiotensin-I pressor response and to potentiate plasma levels of atrial natriuretic peptide.

THE BIOLOGICAL ACTIVITY OF ATRIAL NATRIURETIC FACTOR CLEAVED BY ENDOPROTEASE 3.4.24.11

Ring cleavage of atrial natriuretic peptide (ANF) between Cys7 and Phe8 by endoprotease 3.4.24.11 yields X-ANF. Since endoprotease 3.4.24.11 may contribute to ANF metabolism in vivo, the present study determined if X-ANF exhibits reduced biological activity in comparison to the parent molecule.

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

AbstractHuman 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 Cysl05 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 Km values of 11.7 and 3.1 μM, respectively. The turnover rates (kcat/K...