Metalloprotease Neprilysin Research Articles

Perindopril ameliorates experimental Alzheimer's disease progression: role of amyloid β degradation, central estrogen receptor and hyperlipidemic-lipid raft signaling.

Accumulating evidence indicates that over-stimulation of angiotensin-converting enzyme 1 (ACE1) activity is associated with β-amyloid (Aβ) and phosphorylated tau (p-tau)-induced apoptosis, oxido-nitrosative neuroinflammatory stress and neurodegeneration in Alzheimer's disease (AD). Alternatively, activation of the ACE2, the metalloprotease neprilysin (Neutral Endopeptidase; NEP) and the insulin-degrading enzyme (IDE) could oppose the effects of ACE1 activation. We aim to investigate the relationship between ACE1/ACE2/NEP/IDE and amyloidogenic/hyperlipidemic-lipid raft signaling in hyperlipidemic AD model. Induction of AD was performed in ovariectomized female rats with high-fat high fructose diet (HFFD) feeding after 4weeks following D-galactose injection (150mg/kg). The brain-penetrating ACE1 inhibitor perindopril (0.5mg/kg/day, p.o.) was administered on a daily basis for 30days. Perindopril significantly decreased hippocampal expression of ACE1 and increased expression of ACE2, NEP and IDE. Perindopril markedly decreased Aβ1-42, improved lipid profile and ameliorated the lipid raft protein markers caveolin1 (CAV1) and flotillin 1 (FLOT1). This was accompanied by decreased expression of p-tau and enhancement of cholinergic neurotransmission, coupled with decreased oxido-nitrosative neuroinflammatory stress, enhancement of blood-brain barrier (BBB) functioning and lower expression of the apoptotic markers glial fibrillary acidic protein (GFAP), Bax and β-tubulin. In addition, perindopril ameliorated histopathological damage and improved learning, cognitive and recognition impairment as well as depressive behavior in Morris water maze, Y maze, novel object recognition and forced swimming tests, respectively. Conclusively, perindopril could improve cognitive defects in AD rats, at least through activation of ACE2/NEP/IDE and inhibition of ACE1 and subsequent modulation of amyloidogenic/hyperlipidemic-lipid raft signaling and oxido-nitrosative stress.

N-Terminal Truncated Aβ4-42 Is a Substrate for Neprilysin Degradation in vitro and in vivo.

In sporadic Alzheimer's disease (AD), an imbalance between production and clearance of amyloid-β (Aβ) peptides seems to account for enhanced Aβ accumulation. The metalloprotease neprilysin (NEP) is an important Aβ degrading enzyme as shown by a variety of invitro and in vivo studies. While the degradation of full-length Aβ peptides such as Aβ1-40 and Aβ1-42 is well established, it is less clear whether NEP is also capable of degrading N-terminally truncated Aβ species such as the common variant Aβ4-42. In the present report, we confirmed the degradation of Aβ4-x species by neprilysin using invitro digestion and subsequent analysis using gel-based assays and mass spectrometry. By crossing Tg4-42 mice expressing only Aβ4-42 peptides with homozygous NEP-knock-out mice (NEP-/-), we were able to demonstrate that NEP deficiency increased hippocampal intraneuronal Aβ levels and aggravated neuron loss in the Tg4-42 transgenic mouse model of AD.

Rare Variants in MME, Encoding Metalloprotease Neprilysin, Are Linked to Late-Onset Autosomal-Dominant Axonal Polyneuropathies

Axonal polyneuropathies are a frequent cause of progressive disability in the elderly. Common etiologies comprise diabetes mellitus, paraproteinaemia, and inflammatory disorders, but often the underlying causes remain elusive. Late-onset axonal Charcot-Marie-Tooth neuropathy (CMT2) is an autosomal-dominantly inherited condition that manifests in the second half of life and is genetically largely unexplained. We assumed age-dependent penetrance of mutations in a so far unknown gene causing late-onset CMT2. We screened 51 index case subjects with late-onset CMT2 for mutations by whole-exome (WES) and Sanger sequencing and subsequently queried WES repositories for further case subjects carrying mutations in the identified candidate gene. We studied nerve pathology and tissue levels and function of the abnormal protein in order to explore consequences of the mutations. Altogether, we observed heterozygous rare loss-of-function and missense mutations in MME encoding the metalloprotease neprilysin in 19 index case subjects diagnosed with axonal polyneuropathies or neurodegenerative conditions involving the peripheral nervous system. MME mutations segregated in an autosomal-dominant fashion with age-related incomplete penetrance and some affected individuals were isolated case subjects. We also found that MME mutations resulted in strongly decreased tissue availability of neprilysin and impaired enzymatic activity. Although neprilysin is known to degrade β-amyloid, we observed no increased amyloid deposition or increased incidence of dementia in individuals with MME mutations. Detection of MME mutations is expected to increase the diagnostic yield in late-onset polyneuropathies, and it will be tempting to explore whether substances that can elevate neprilysin activity could be a rational option for treatment.

The Metalloprotease Neprilysin Degrades and Inactivates Apelin Peptides.

The apelinergic system is a mammalian peptide hormone network with key physiological roles. Apelin isoforms and analogues are believed to be promising therapeutics for cardiovascular disease. Despite extensive studies on apelin-13 degradation patterns, only one protease, angiotensin-converting enzyme 2 (ACE2), had been implicated in its physiological regulation. Through use of a peptide-based fluorescent probe, we identified the metalloprotease neprilysin (NEP, a target for Entresto used in treatment of heart failure) as an enzyme that cleaves apelin isoforms. In vitro NEP proteolysis generated fragments that lacked the ability to bind to the apelin receptor, thereby making NEP the first protease to fully inactivate apelin. The involvement of NEP in the apelinergic system contributes to the understanding of its role in cardiovascular physiology.

Neprilysin deficiency alters the neuropathological and behavioral phenotype in the 5XFAD mouse model of Alzheimer's disease.

The deposition of amyloid-β (Aβ) is one of the major neuropathological hallmarks of Alzheimer's disease (AD). In the case of sporadic AD, an imbalance in Aβ in production and clearance seems to be the reason for an enhanced Aβ accumulation. Besides a systematic clearance through the blood-brain barrier, Aβ is cleared from the brain by Aβ-degrading enzymes. The metalloprotease neprilysin (NEP) is an important Aβ-degrading enzyme as shown by numerous in vitro, in vivo and reverse genetics studies. 5XFAD mice represent an early-onset AD mouse model which develops plaque pathology starting with 2 months of age in addition to robust behavioral deficits at later time points. By crossing 5XFAD mice with homozygous NEP-knock-out mice (NEP-/-), we show that hemizygous NEP deficiency aggravates the behavioral and neuropathological phenotype of 5XFAD mice. We found that 5XFAD mice per se showed strongly decreased NEP expression levels compared to wildtype mice, which was aggravated by NEP reduction. 5XFAD/NEP+/- mice demonstrated impairment in spatial working memory and increased astrocytosis in all studied brain areas, in addition to an overall increased level of soluble Aβ42 as well as region-specific increases in extracellular Aβ deposition. Surprisingly, in young mice, a more abundant cortical Aβ plaque pathology was observed in 5XFAD compared to 5XFAD/NEP+/- mice. Additionally, young 5XFAD/NEP+/- as well as hemi- and homozygous NEP knockout mice showed elevated levels of endothelin-converting enzyme 1 (ECE1), suggesting a mutual regulation of ECE1 and NEP at young ages. The present data indicate that NEP mainly degrades soluble Aβ peptides, which confirms previous observations. Increased ECE1 levels correlated well with the strongly reduced extracellular plaque load in young 5XFAD/NEP+/- mice and might suggest a reciprocal effect between ECE and NEP activities in Aβ degradation.

β-Amyloid and Neprilysin Computational Studies Identify Critical Residues Implicated in Binding Specificity

The zinc metalloprotease neprilysin (NEP) promiscuously degrades small bioactive peptides. NEP is among a select group of metalloenzymes that degrade the amyloid beta-peptide (Aβ) in vivo and in situ. Since accumulation of neurotoxic Aβ aggregates in the brain appears to be a causative agent in the pathophysiology of Alzheimer's disease (AD), increased clearance of Aβ resulting from overexpression of NEP exhibits therapeutic potential for AD. However, higher NEP peptidase activity may be harmful without an increased specificity for Aβ over other competing substrates. Crystal structures of NEP-inhibitor complexes and their characterization have highlighted potential amino acid interactions involved in substrate binding and are used as templates to guide our methodology in docking Aβ in NEP. Results from protein-ligand docking calculations predict S2' subsite residues Arg 102 and Arg 110 of NEP participate in specific interactions with Aβ. These interactions provide insight into developing NEP specificity for Aβ.

Optimizing Population Dispersal

Pheromones regulate olfactory plasticity in nematodes.

Copper Downregulates Neprilysin Activity Through Modulation of Neprilysin Degradation

Copper plays a central role in conserved processes such as respiration, and in highly specialized processes, such as protein modification. The metalloprotease neprilysin (NEP) degrades a variety of bioactive peptides and is involved in many physiological processes. However, very little is known about the regulation of NEP activity. In the current study, we focused on the effect of Cu2+ on the enzymatic activity and protein stability of NEP. Using mouse neuroblastoma N2a cells, we found that the enzymatic activity of NEP was decreased by treatment with Cu2+ in a dose- and time-dependent manner. In our investigation of the mechanism by which Cu2+ downregulates NEP enzyme activity, we found that treatment with Cu2+ caused a decrease in the level of NEP as determined by Western blot analysis. Quantitative analysis of NEP mRNA with RT-PCR excluded the possibility that Cu2+ downregulates NEP protein at the gene transcription level. Moreover, specific proteasome inhibitors, MG132 and lactacystin, blocked the turnover of NEP, whereas inhibitors of lysosome had no significant effect, suggesting that Cu2+-induced degradation of NEP is via a proteasome pathway. Taken together, our data suggest that copper downregulates NEP activity through modulation of NEP protein degradation.

Second‐Generation Inhibitors for the Metalloprotease Neprilysin Based on Bicyclic Heteroaromatic Scaffolds: Synthesis, Biological Activity, and X‐Ray Crystal‐Structure Analysis

AbstractA new class of nonpeptidic inhibitors of the ZnII‐dependent metalloprotease neprilysin with IC50 values in the nanomolar activity range (0.034–0.30 μM) 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 π‐π stacking interactions with the imidazole ring of His711. The platform is decorated with a thiol vector to coordinate to the ZnII 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‐Å resolution (Fig. 4 and Table 2). The ligand coordinates with its sulfanyl residue to the ZnII 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 π‐π 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 imidazo‐pyridine 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.

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

AbstractNeprilysin (NEP; neutral endopeptidase EC 3.4.24.11) is a ZnII‐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 ZnII 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 IC50 values in the single‐digit micromolar activity range (2–4 μM, Table), which validates the binding mode postulated by modeling. Useful guidelines for a next lead optimization cycle were obtained in several control runs.

Ontogeny, regional and cellular distribution of the novel metalloprotease neprilysin 2 in the rat: a comparison with neprilysin and endothelin-converting enzyme-1

The localisation of the gene transcripts of a recently discovered peptidase, neprilysin 2 (NEP2), was established by in situ hybridisation in rat tissues during development and adulthood. It was compared with those of neprilysin (NEP), a closely related enzyme in terms of sequence homology or substrate specificity, and of endothelin-converting enzyme 1 (ECE-1) which, like the other two, belongs to the M-13 sub-family of zinc-dependent metallopeptidases. The ontogeny of the three enzymes differed markedly, the expression of NEP2 being restricted to developing and differentiating fields of the CNS, whereas NEP and ECE-1 genes were broadly expressed early on in the CNS and periphery. In contrast to the wide expression of NEP and ECE-1 in peripheral adult tissues and in CNS, NEP2 was almost exclusively expressed in selected neuronal populations of the brain and spinal cord. The only exceptions were the intermediate and anterior lobes of the pituitary as well as the choroid plexuses, where NEP2 was also strongly expressed. These localisations as well as those in the hypothalamic nuclei, together with the previously established pattern of cleaved peptides, suggest the involvement of NEP2 in the metabolism of neurohormones of the hypothalamo-pituitary axis. Complementary distributions of NEP and NEP2 mRNAs were observed in a large number of brain areas with, for instance the former being highly expressed in the striatum in which NEP2 transcripts were almost undetectable. In contrast, NEP2 was highly expressed in numerous thalamic, hypothalamic and brainstem nuclei from which NEP was absent. Since both peptidases are able to cleave the same neuropeptides, this pattern may suggest a complementary role in their peptide inactivation functions in the CNS. Finally, ECE-1 mRNAs were generally observed in neuronal populations known to express the pre-proendothelin-1 gene, confirming the function of the metallopeptidase in endothelin-1 generation.