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.
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 1a-d (ECE-1a-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.
We have isolated cDNAs coding for two novel human pancreatic lipase (hPL)-related human proteins, referred to as hPL-related proteins 1 and 2 (hPLRP1 and hPLRP2) and for hPL. The two novel proteins show an amino acid sequence identity to hPL of 68 and 65% for hPLRP1 and 2, respectively. All three proteins are secreted into the medium after transfection of COS cells with the corresponding cDNAs. The size of the three expressed proteins is similar and ranges between 45 and 50 kDa. The expressed hPLRP2 shows a lipolytic activity that is, however, in contrast to that of hPL only marginally dependent on the presence of colipase, whereas hPLRP1 shows no activity in this assay. A Northern analysis of normal human pancreas mRNA shows that the expression levels of hPLRP1 and hPLRP2 are about 4-fold and 24-fold lower, respectively, than that of hPL. hPLRP2 is, additionally, most closely related to a lipase reported to be expressed in mouse T-cells. A comparison of the sequences of the three proteins with sequences described as pancreatic lipases of other animal species shows three subfamilies of closer kinship. This suggests that the two novel proteins also exist in other species and that some of the sequences reported to be pancreatic lipase might more likely be the orthologues of hPLRP1 or hPLRP2 in those species.
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 ((±)‐ 1 , (±)‐ 43 , (±)‐ 45 , and (±)‐ 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 (±)‐ 1 , (±)‐ 43 , (±)‐ 45 , and (±)‐ 46 all exhibit IC 50 values in the single‐digit micromolar activity range (2–4 μ M , Table ), which validates the binding mode postulated by modeling. Useful guidelines for a next lead optimization cycle were obtained in several control runs.
