The mechanism by which cholesteryl ester transfer protein (CETP) activity affects HDL metabolism was investigated using agents that selectively target CETP (dalcetrapib, torcetrapib, anacetrapib). In contrast with torcetrapib and anacetrapib, dalcetrapib requires cysteine 13 to decrease CETP activity, measured as transfer of cholesteryl ester (CE) from HDL to LDL, and does not affect transfer of CE from HDL3 to HDL2. Only dalcetrapib induced a conformational change in CETP, when added to human plasma in vitro, also observed in vivo and correlated with CETP activity. CETP-induced pre-β-HDL formation in vitro in human plasma was unchanged by dalcetrapib ≤3 µM and increased at 10 µM. A dose-dependent inhibition of pre-β-HDL formation by torcetrapib and anacetrapib (0.1 to 10 µM) suggested that dalcetrapib modulates CETP activity. In hamsters injected with [3H]cholesterol-labeled autologous macrophages, and given dalcetrapib (100 mg twice daily), torcetrapib [30 mg once daily (QD)], or anacetrapib (30 mg QD), only dalcetrapib significantly increased fecal elimination of both [3H]neutral sterols and [3H]bile acids, whereas all compounds increased plasma HDL-[3H]cholesterol. These data suggest that modulation of CETP activity by dalcetrapib does not inhibit CETP-induced pre-β-HDL formation, which may be required to increase reverse cholesterol transport.

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A new class of nonpeptidic inhibitors of the Zn II -dependent metalloprotease neprilysin with IC 50 values in the nanomolar activity range (0.034 ± 0.30 mm) were developed based on structure-based de novo design (Figs. 1 and 2). The inhibitors feature benzimidazole and imidazo [4,5-c]pyridine moieties as central scaffolds to undergo H-bonding to Asn542 and Arg717 and to engage in favorable p-p stacking interactions with the imidazole ring of His711. The platform is decorated with a thiol vector to coordinate to the Zn II ion and an aryl residue to occupy the hydrophobic S1' pocket, but lack a substituent for binding in the S2' pocket, which remains closed by the side chains of Phe106 and Arg110 when not occupied. The enantioselective syntheses of the active compounds ()-1, ()-2, ()-25, and ()-26 were accomplished using Evans auxiliaries (Schemes 2, 4, and 5). The inhibitors ()-2 and ()-26 with an imidazo[4,5-c]pyridine core are ca. 8 times more active than those with a benzimidazole core (()-1 and ()-25) ( Table 1). The predicted binding mode was established by X-ray analysis of the complex of neprilysin with ()-2 at 2.25-resolution (Fig. 4 and Table 2). The ligand coordinates with its sulfanyl residue to the Zn II ion, and the benzyl residue occupies the S1' pocket. The 1H-imidazole moiety of the central scaffold forms the required H-bonds to the side chains of Asn542 and Arg717. The heterobicyclic platform additionally undergoes p-p stacking with the side chain of His711 as well as edge-to-face-type interactions with the side chain of Trp693. According to the X-ray analysis, the substantial advantage in biological activity of the imidazopyridine inhibitors over the benzimidazole ligands arises from favorable interactions of the pyridine N-atom in the former with the side chain of Arg102. Unexpectedly, replacement of the phenyl group pointing into the deep S1' pocket by a biphenyl group does not enhance the binding affinity for this class of inhibitors.1. Introduction. ± In the preceding paper [1], we described a new class of inhibitors of the metalloprotease neprilysin with a central 1H-imidazole platform, featuring IC 50 values (IC 50 : concentration of inhibitor at which 50% V max is observed) in the low micromolar range. The de novo design of these compounds was based on the X-ray crystal structure of NEP complexed with phosphoramidon (Protein Data Bank (PDB) file name 1DMT) [2]. For the design of the second-generation inhibitors, we reverted to an unpublished X-ray crystal structure [3] of NEP complexed with the inhibitor thiorphan [4]. During the analysis, we carefully compared the active sites of the two

Endothelin-1 (ET-1) is mitogenic and/or antiapoptotic in human cancers, and antagonists to ET-1 receptors are under evaluation for cancer treatment. Inhibition of ET-1 activation by the endothelin-converting enzymes 1(a)(-)(d) (ECE-1(a)(-)(d); EC 3.4.24.71) represents another approach to block the ET-1 effect in cancer. To evaluate this potential, we synthesized and characterized a series of low nanomolar nonpeptidic thiol-containing ECE-1 inhibitors, and evaluated their effect, as well as the effect of inhibitors for the related metalloproteases neprilysin (NEP; EC 3.4.24.11) and angiotensin-converting enzyme (ACE; EC 3.4.15.1), on human glioblastoma cell growth. Only ECE-1 inhibitors inhibited DNA synthesis by human glioblastoma cells. Exogenous addition of ET-1 or bigET-1 to glioblastoma cells did not counterbalance the growth inhibition elicited by ECE-1 inhibitors, suggesting that ECE-1 inhibitors block the proliferation of human glioblastoma cells most likely via a mechanism not involving extracellular production of ET-1. This class of molecules may thus represent novel therapeutic agents for the potential treatment of human cancer.

Abstract-We tested the hypothesis that endothelin-converting enzyme (ECE) inhibition ameliorates end-organ damage in rats harboring both human renin and human angiotensinogen genes (dTGR). Hypertension develops in the animals, and they die by age 7 weeks of heart and kidney failure. Three groups were studied: dTGR (nϭ12) receiving vehicle, dTGR receiving ECE inhibitor (RO0687629; 30 mg/kg by gavage; nϭ10), and Sprague-Dawley control rats (SD; nϭ10) receiving vehicle, all after week 4, with euthanasia at week 7. Systolic blood pressure was not reduced by ECE inhibitor compared with dTGR (205Ϯ6 versus 206Ϯ6 mm Hg at week 7, respectively). In contrast, ECE inhibitor treatment significantly reduced mortality rate to 20% (2 of 10), whereas untreated dTGR had a 52% mortality rate (7 of 12). ECE inhibitor treatment ameliorated cardiac damage and reduced left ventricular ECE activity below SD levels. Echocardiography at week 7 showed reduced cardiac hypertrophy (4.8Ϯ0.2 versus 5.7Ϯ0.2 mg/g, PϽ0.01) and increased left ventricular cavity diameter (5.5Ϯ0.3 versus 3.1Ϯ0.1 mm, PϽ0.001) and filling volume (0.42Ϯ0.04 versus 0.16Ϯ0.06 mL, PϽ0.05) after ECE inhibitor compared with untreated dTGR. ECE inhibitor treatment also reduced cardiac fibrosis, tissue factor expression, left ventricular basic fibroblast growth factor mRNA levels, and immunostaining in the vessel wall, independent of high blood pressure. In contrast, the ECE inhibitor treatment showed no renoprotective effect. These data are the first to show that ECE inhibition reduces angiotensin II-induced cardiac damage. Key Words: angiotensin II Ⅲ enzymes Ⅲ fibrosis Ⅲ hypertrophy A ngiotensin (Ang) II-related vascular effects are partially mediated by endothelin-1 (ET-1). Long-term Ang II infusion induces preproendothelin mRNA expression. 1 In rats transgenic for both the human renin and human angiotensinogen genes (dTGR), hypertension as well as severe heart and kidney damage develop, largely independent of blood pressure elevation. The rats die by age 7 weeks. 2 The ET-1 A and B (ETA/B) receptor blocker bosentan inhibits the activation of both nuclear factor-kappa B (NF-B) and transcription factor activator protein (AP)-1 in the kidney and the heart, independent of blood pressure reduction in these rats. 3 Bohlender et al 4 studied the same rat strain and showed that a specific ETA receptor blocker is effective, particularly when combined with an Ang II receptor blocker. ET-1 is a 21-amino acid peptide that was first isolated from porcine endothelial cells. 5 Two structurally related peptides differing by 2 (ET-2) and 6 (ET-3) amino acids were subsequently identified. The endothelin precursors are processed by 2 proteases that create mature active forms, termed preproendothelins. The preproendothelins are cleaved at dibasic sites by furin-like endopeptidases to produce inactive intermediates termed big endothelins. Big endothelins are cleaved to form the final products. A family of membrane-bound zinc metalloproteases from the neprilysin superfamily conducts the last...

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Neprilysin (NEP; neutral endopeptidase EC 3.4.24.11) is a Zn II -dependent, membrane-bound endopeptidase. NEP is widely distributed in the organs, particularly in the kidneys and lungs, and it is involved in the metabolism of a number of smaller regulatory peptides. Inhibition of NEP has been proposed as a potential target for analgesic and antihypertensive therapies. In this study, new nonpeptidic inhibitors of neprilysin ((AE)-1, (AE)-43, (AE)-45, and (AE)-46; Table) were designed, based on the X-ray crystal structure of NEP complexed to phosphoramidon (Fig. 1). They feature an imidazole ring as the central scaffold, acting as a peptide bond isoster to undergo H-bonding with the side chains of Asn542 and Arg717 (Fig. 2). The scaffold is decorated with a thiol group to ligate to the Zn II ion and two aromatic residues to bind into the hydrophobic S1' and S2' pockets. The synthesis of the new inhibitors was approached by two routes (Schemes 1 ± 4 and 5 ± 8), with the second one involving a double directed ortho-metallation of the imidazole platform and a Stille cross-coupling, providing the desired target molecules as hydrochloride salts. In a fluorescence assay, inhibitors (AE)-1, (AE)-43, (AE)-45, and (AE)-46 all exhibit IC 50 values in the single-digit micromolar activity range (2 ± 4 mm, Table), which validates the binding mode postulated by modeling. Useful guidelines for a next lead optimization cycle were obtained in several control runs.

Using X‐ray structure‐based de novo design, a new class of inhibitors of the zinc‐dependent endopeptidase Neprilysin has been developed that feature binding affinities (IC50 values) in the upper nanomolar range. The imidazole moieties of the central benzimidazole or imidazo[4,5‐c]pyridine (see picture) scaffolds act as efficient peptide‐bond isosters.