A New Class of Inhibitors for the Metalloprotease Neprilysin Based on a Central Imidazole Scaffold

Abstract

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.

A fluorine scan of non-peptidic inhibitors of neprilysin: Fluorophobic and fluorophilic regions in an enzyme active site

“…This pocket has been shown to accommodate large substituents, such as biphenyl residues [6,7,37,38]; however, changing phenyl (in (+)-1a/(+)-1b) to biphenyl (in (+)-2a/(+)-2b) does not generate additional binding potency [29]. The imidazole ring of the central platform acts as peptide isoster [27] and forms H-bonds to the side chains of Arg717 and Asp542, while the annulated benzene/ pyridine ring undergoes aromatic interactions with the side chains of His711 and Trp693. The crystal structure provides a good explanation for the measured enhanced binding affinity of the imidazo [4,5-c]pyridine-based ligands as compared to the benzimidazole derivatives (Table 1).…”

“…After workup, the crude thiol was immediately purified by reverse-phase (RP)-HPLC and isolated as its trifluoroacetate salt, which did not oxidize even after several weeks being exposed to air. The in vitro activity of this series of inhibitors towards NEP was determined in a fluorimetric assay (Table 1, for details see [27,40]). In addition, the previously reported inhibitors (+)-1a and (+)-1b (R = H) were retested [29].…”

Bare Histidine–Serine Models: Implication and Impact of Hydrogen Bonding on Nucleophilicity

“…Compounds 1 – 6 were prepared in a few steps and good overall yield (≈40 %) (Scheme ; see the Supporting Information for details). The 2‐hydroxymethyl derivative 1 was obtained by direct reduction of the 2‐formyl precursor12a according to reported procedures 12b–e. Selective lithiation of the C2 position of N2‐sulfamoyl imidazoles, followed by ethylene oxide ring opening,13 yielded after deprotection the hydroxyethyl derivative 2 ,14a,b as previously described.…”

Endogenous Opioids and the Treatment of Multiple Sclerosis

“…Enkephalins and enkephalin proteases also may have an important role in the etiology of MS [22][23][24][25][26][27][28][29]. The neprilysin (NEP) family of zinc metallopeptidases, including neprilysin and endothelin-converting enzyme-2 (ECE-2), are expressed at the cell surface [26]. Enzymatically, neprilysin functions both as an endopeptidase and as a dipeptidylcarboxypeptidase to inactivate neuropeptides.…”

“…Expression of CD10 is an indicator of certain lymphomas and leukemias such as T cell lymphoma and Burkitt's lymphoma. Neprilysin actively degrades a number of signaling neuropeptides including enkephalins, substance P, and atrial natriuretic factor [26][27][28][29], thus implicating a role of aberrant levels of endogenous opioids and/or their enkephalinases in disease.…”