RESUMEN
Targeting metalloproteinases has been in the focus of drug design for a long time. However, meprin α and ß emerged as potential drug targets just recently and are linked to several diseases with different pathological background. Nevertheless, the validation of meprins as suitable drug targets still requires highly potent and selective inhibitors as chemical probes to elucidate their role in pathophysiology. Albeit highly selective inhibitors of meprin ß have already been reported, only inhibitors of meprin α with modest activity or selectivity are known. Starting from recently reported heteroaromatic scaffolds, the aim of this study was the optimisation of meprin α and/or meprin ß inhibition while keeping the favourable off-target inhibition profile over other metalloproteases. We report potent pan-meprin inhibitors as well as highly active inhibitors of meprin α with superior selectivity over meprin ß. The latter are suitable to serve as chemical probes and enable further target validation.
Asunto(s)
Metaloendopeptidasas , Metaloproteasas , Relación Estructura-Actividad , Metaloproteasas/metabolismo , Diseño de FármacosRESUMEN
The zinc-dependent metalloprotease meprin α is predominantly expressed in the brush border membrane of proximal tubules in the kidney and enterocytes in the small intestine and colon. In normal tissue homeostasis meprin α performs key roles in inflammation, immunity, and extracellular matrix remodelling. Dysregulated meprin α is associated with acute kidney injury, sepsis, urinary tract infection, metastatic colorectal carcinoma, and inflammatory bowel disease. Accordingly, meprin α is the target of drug discovery programs. In contrast to meprin ß, meprin α is secreted into the extracellular space, whereupon it oligomerises to form giant assemblies and is the largest extracellular protease identified to date (~6 MDa). Here, using cryo-electron microscopy, we determine the high-resolution structure of the zymogen and mature form of meprin α, as well as the structure of the active form in complex with a prototype small molecule inhibitor and human fetuin-B. Our data reveal that meprin α forms a giant, flexible, left-handed helical assembly of roughly 22 nm in diameter. We find that oligomerisation improves proteolytic and thermal stability but does not impact substrate specificity or enzymatic activity. Furthermore, structural comparison with meprin ß reveal unique features of the active site of meprin α, and helical assembly more broadly.
Asunto(s)
Fetuína-B , Metaloendopeptidasas , Humanos , Microscopía por Crioelectrón , Metaloendopeptidasas/metabolismo , Metaloproteasas , Precursores Enzimáticos , ZincRESUMEN
The astacin protease Meprin ß represents an emerging target for drug development due to its potential involvement in disorders such as acute and chronic kidney injury and fibrosis. Here, we elaborate on the structural basis of inhibition by a specific Meprin ß inhibitor. Our analysis of the crystal structure suggests different binding modes of the inhibitor to the active site. This flexibility is caused, at least in part, by movement of the C-terminal region of the protease domain (CTD). The CTD movement narrows the active site cleft upon inhibitor binding. Compared with other astacin proteases, among these the highly homologous isoenzyme Meprin α, differences in the subsites account for the unique selectivity of the inhibitor. Although the inhibitor shows substantial flexibility in orientation within the active site, the structural data as well as binding analyses, including molecular dynamics simulations, support a contribution of electrostatic interactions, presumably by arginine residues, to binding and specificity. Collectively, the results presented here and previously support an induced fit and substantial movement of the CTD upon ligand binding and, possibly, during catalysis. To the best of our knowledge, we here present the first structure of a Meprin ß holoenzyme containing a zinc ion and a specific inhibitor bound to the active site. The structural data will guide rational drug design and the discovery of highly potent Meprin inhibitors.
Asunto(s)
Ácidos Hidroxámicos/química , Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/química , Simulación de Dinámica Molecular , Inhibidores de Proteasas/química , Humanos , Relación Estructura-ActividadRESUMEN
Astacin metalloproteinases, in particular meprins α and ß, as well as ovastacin, are emerging drug targets. Drug-discovery efforts have led to the development of the first potent and selective inhibitors in the last few years. However, the most recent compounds are based on a highly flexible tertiary amine scaffold that could cause metabolic liabilities or decreased potency due to the entropic penalty upon binding to the target. Thus, the aim of this study was to discover novel conformationally constrained scaffolds as starting points for further inhibitor optimization. Shifting from flexible tertiary amines to rigid heteroaromatic cores resulted in a boost in inhibitory activity. Moreover, some compounds already exhibited higher activity against individual astacin proteinases compared to recently reported inhibitors and also a favorable off-target selectivity profile, thus qualifying them as very suitable chemical probes for target validation.
Asunto(s)
Aminas/farmacología , Antineoplásicos/farmacología , Descubrimiento de Drogas , Hidrocarburos Aromáticos/farmacología , Metaloendopeptidasas/antagonistas & inhibidores , Metaloproteasas/antagonistas & inhibidores , Inhibidores de Proteasas/farmacología , Aminas/síntesis química , Aminas/química , Antineoplásicos/síntesis química , Antineoplásicos/química , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Hidrocarburos Aromáticos/síntesis química , Hidrocarburos Aromáticos/química , Metaloendopeptidasas/metabolismo , Metaloproteasas/metabolismo , Estructura Molecular , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/química , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
The formation of amyloid-ß (Aß) peptides is causally involved in the development of Alzheimer's disease (AD). A significant proportion of deposited Aß is N-terminally truncated and modified at the N-terminus by a pGlu-residue (pGlu-Aß). These forms show enhanced neurotoxicity compared to full-length Aß. Although the truncation may occur by aminopeptidases after formation of Aß, recently discovered processing pathways of amyloid-ß protein precursor (AßPP) by proteases such as meprin ß may also be involved. Here, we assessed a role of meprin ß in forming Aß3-40/42, which is the precursor of pGlu-Aß3-40/42 generated by glutaminyl cyclase (QC). Similar to QC, meprin ß mRNA is significantly upregulated in postmortem brain from AD patients. A histochemical analysis supports the presence of meprin ß in neurons and astrocytes in the vicinity of pGlu-Aß containing deposits. Cleavage of AßPP-derived peptides by meprin ß in vitro results in peptides Aß1-x, Aß2-x, and Aß3-x. The formation of N-truncated Aß by meprin ß was also corroborated in cell culture. A subset of the generated peptides was converted into pGlu-Aß3-40 by an addition of glutaminyl cyclase, supporting the preceding formation of Aß3-40. Further analysis of the meprin ß cleavage revealed a yet unknown dipeptidyl-peptidase-like activity specific for the N-terminus of Aß1-x. Thus, our data suggest that meprin ß contributes to the formation of N-truncated Aß by endopeptidase and exopeptidase activity to generate the substrate for QC-catalyzed pGlu-Aß formation.
Asunto(s)
Aminoaciltransferasas/metabolismo , Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Metaloendopeptidasas/metabolismo , Fragmentos de Péptidos/metabolismo , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Aminoaciltransferasas/genética , Péptidos beta-Amiloides/genética , Animales , Encéfalo/patología , Células CHO , Cricetinae , Cricetulus , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/genética , Activación Enzimática/fisiología , Femenino , Células HEK293 , Humanos , Masculino , Metaloendopeptidasas/genética , Fragmentos de Péptidos/genéticaRESUMEN
Common assays for endoprotease activity of meprin α and ß are based on cleavage of internally quenched substrates. Although direct and convenient, for meprins these assays bear disadvantages such as, e.g., significant substrate inhibition or potential fluorescence quenching by compounds applied in inhibitor analysis. Here, we present a novel continuous assay by introducing an auxiliary enzyme, prolyl tripeptidyl aminopeptidase (PtP) and the chromogenic substrate KKGYVADAP-p-nitroanilide. We provide a quick strategy for expression and one-step-purification of the auxiliary enzyme. The enzyme kinetic data for meprin α and ß suggest hyperbolic v/S-characteristics, the kinetic parameters of substrate conversion by meprin ß were Kmâ¯=â¯184⯱â¯32⯵M and kcatâ¯=â¯20⯱â¯4 s-1. We also present conditions for the use of the fluorogenic substrate KKGYVADAP-AMC to assess meprin ß activity. The assays were applied for determination of inhibitory parameters of the natural inhibitor actinonin and two recently published hydroxamates. Hence, we present two novel methods, which can be applied to assess inhibitory mechanism and potency with the attractive current drug targets meprin α and ß. Furthermore, the assay might also provide implications for analysis of other endoproteases as well as their inhibitors.
Asunto(s)
Proteínas Bacterianas/metabolismo , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Metaloendopeptidasas/análisis , Porphyromonas gingivalis/enzimología , Serina Endopeptidasas/metabolismo , Proteínas Bacterianas/química , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/química , Relación Dosis-Respuesta a Droga , Ácidos Hidroxámicos/farmacología , Cinética , Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/metabolismo , Estructura Molecular , Inhibidores de Proteasas/farmacología , Serina Endopeptidasas/química , Relación Estructura-ActividadRESUMEN
Metalloproteinases of the astacin family are drawing ever increasing attention as potential drug targets. However, knowledge regarding inhibitors thereof is limited in most cases. Crucial for the development of metalloprotease inhibitors is high selectivity, to avoid side effects brought about by inhibition of off-target proteases and interference with physiological pathways. In this study we aimed at the design of novel selective inhibitors for the astacin proteinase meprinâ α. Based on a recently identified tertiary amine scaffold, a series of compounds was synthesized and evaluated. The compounds exhibit reasonable inhibitory activity with high selectivity over other metalloproteases. The isoenzyme meprinâ ß is only slightly inhibited. Hence, the present study revealed a novel class of selective meprinâ α inhibitors with improved selectivity over known compounds.
Asunto(s)
Ácidos Hidroxámicos/química , Metaloendopeptidasas/antagonistas & inhibidores , Inhibidores de Proteasas/química , Dominio Catalítico , Diseño de Fármacos , Pruebas de Enzimas , Humanos , Ácidos Hidroxámicos/síntesis química , Ácidos Hidroxámicos/metabolismo , Metaloendopeptidasas/química , Metaloendopeptidasas/metabolismo , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/metabolismo , Unión Proteica , Relación Estructura-ActividadRESUMEN
The metalloproteinase meprin ß emerged as a current drug target for the treatment of a number of disorders, among those fibrosis, inflammatory bowel disease and Morbus Alzheimer. A major obstacle in the development of metalloprotease inhibitors is target selectivity to avoid side effects by blocking related enzymes with physiological functions. Here, we describe the structure-guided design of a novel series of compounds, based on previously reported highly active meprin ß inhibitors. The bioisosteric replacement of the sulfonamide scaffold gave rise to a next generation of meprin inhibitors. Selected compounds based on this novel amine scaffold exhibit high activity against meprin ß and also remarkable selectivity over related metalloproteases, i.e., matrix metalloproteases and A disintegrin and metalloproteinases.
Asunto(s)
Cristalografía por Rayos X , Diseño de Fármacos , Inhibidores de la Metaloproteinasa de la Matriz/química , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Metaloendopeptidasas/antagonistas & inhibidores , Dominio Catalítico , Supervivencia Celular , Células Hep G2 , Humanos , Metaloendopeptidasas/metabolismo , Modelos Moleculares , Estructura Molecular , Conformación Proteica , Relación Estructura-ActividadRESUMEN
Compelling evidence suggests a crucial role of amyloid beta peptides (Aß(1-40/42)) in the etiology of Alzheimer's disease (AD). The N-terminal truncation of Aß(1-40/42) and their modification, e.g. by glutaminyl cyclase (QC), is expected to enhance the amyloid toxicity. In this work, the MALDI-TOF mass spectrometry application proved N-terminal cleavage of Aß(1-40/42) by purified dipeptidyl peptidase IV (DPPIV) in vitro observed earlier. The subsequent transformation of resulted Aß(3-40/42) to pE-Aß(3-40/42) in QC catalyzed glutamate cyclization was manifested. Hence, consecutive conversion of Aß(1-40/42) by DPPIV and QC can be assumed as a potential mechanism of formation of non-degrading pyroglutamated pE-Aß(3-40/42), which might accumulate and contribute to AD progression. The in vitro acceleration of Aß(1-40) aggregation in the simultaneous presence of DPPIV and QC was shown also.
Asunto(s)
Aminoaciltransferasas/metabolismo , Péptidos beta-Amiloides/metabolismo , Dipeptidil Peptidasa 4/metabolismo , Fragmentos de Péptidos/metabolismo , Ácido Pirrolidona Carboxílico/farmacología , Secuencia de Aminoácidos , Péptidos beta-Amiloides/análisis , Péptidos beta-Amiloides/genética , Animales , Bovinos , Humanos , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/genética , Agregado de Proteínas/efectos de los fármacos , Agregado de Proteínas/fisiología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
Recombinant expression and purification of amyloid peptides represents a common basis for investigating the molecular mechanisms of amyloid formation and toxicity. However, the isolation of the recombinant peptides is hampered by inefficient separation from contaminants such as the fusion protein required for efficient expression in E. coli. Here, we present a new approach for the isolation of highly purified Aß(1-42) and pGlu-Aß(3-42), which is based on a separation using preparative SDS-PAGE. The method relies on the purification of the Aß fusion protein by affinity chromatography followed by preparative SDS-PAGE under reducing conditions and subsequent removal of detergents by precipitation. The application of preparative SDS-PAGE represents the key step to isolate highly pure recombinant Aß, which has been applied for characterization of aggregation and toxicity. Thereby, the yield of the purification strategy was >60%. To the best of our knowledge, this is the first description of an electrophoresis-based method for purification of a recombinant Aß peptide. Therefore, the method might be of interest for isolation of other amyloid peptides, which are critical for conventional purification strategies due to their aggregation propensity.
Asunto(s)
Péptidos beta-Amiloides/aislamiento & purificación , Fragmentos de Péptidos/aislamiento & purificación , Proteínas Recombinantes de Fusión/aislamiento & purificación , Péptidos beta-Amiloides/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cromatografía de Afinidad , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Humanos , Espectrometría de Masas , Ratones , Microscopía Electrónica de Transmisión , Neuronas/citología , Fragmentos de Péptidos/farmacología , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
The astacin proteases meprin α and ß are emerging drug targets for treatment of disorders such as kidney failure, fibrosis or inflammatory bowel disease. However, there are only few inhibitors of both proteases reported to date. Starting from NNGH as lead structure, a detailed elaboration of the structure-activity relationship of meprin ß inhibitors was performed, leading to compounds with activities in the lower nanomolar range. Considering the preference of meprin ß for acidic residues in the P1' position, the compounds were optimized. Acidic modifications induced potent inhibition and >100-fold selectivity over other structurally related metalloproteases such as MMP-2 or ADAM10.
Asunto(s)
Ácidos Hidroxámicos/química , Metaloendopeptidasas/antagonistas & inhibidores , Inhibidores de Proteasas/química , Sulfonamidas/química , Ácidos Hidroxámicos/síntesis química , Inhibidores de la Metaloproteinasa de la Matriz/síntesis química , Inhibidores de la Metaloproteinasa de la Matriz/química , Inhibidores de Proteasas/síntesis química , Relación Estructura-Actividad , Sulfonamidas/síntesis químicaRESUMEN
Meprins are zinc-dependent proteases of the metzincin superfamily of metalloproteases. The enzymes are extracellular multi-domain proteins which are stabilized by disulfide bridges, dimerization, and glycosylation. Due to their complex structure, recombinant expression was first established in mammalian and insect cells. However, these methods have several disadvantages such as high costs and the low yields. For this reason, yeast is often considered a preferable expression system. Here, we describe the manipulation and secretory expression of human meprin ß in the methylotrophic yeast P. pastoris. We show that the position of the affinity tag strongly influences the yield of expression, favoring fusion of the affinity tag at the C-terminus.
Asunto(s)
Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismo , Pichia/crecimiento & desarrollo , Glicosilación , Humanos , Metaloendopeptidasas/química , Pichia/genética , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Huntington's disease (HD) is an inherited and fatal polyglutamine neurodegenerative disorder caused by an expansion of the CAG triplet repeat coding region within the HD gene. Progressive dysfunction and loss of striatal GABAergic medium spiny neurons (MSNs) may account for some of the characteristic symptoms in HD patients. Interestingly, in HD, MSNs expressing neuropeptide Y (NPY) are spared and their numbers is even up-regulated in HD patients. Consistent with this, we report here on increased immuno-linked NPY (IL-NPY) levels in human cerebrospinal fluid (hCSF) from HD patients (Control n = 10; early HD n = 9; mid HD n = 11). As this antibody-based detection of NPY may provide false positive differences as a result of the antibody-based detections of only fragments of NPY, the initial finding was validated by investigating the proteolytic stability of NPY in hCSF using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and selective inhibitors. A comparison between resulting NPY-fragments and detailed epitope analysis verified significant differences in IL-NPY1-36/3-36 and NPY1-30 levels between HD patients and control subjects with no significant differences between early vs mid HD cases. Ex vivo degradomics analysis demonstrated that NPY is initially degraded to NPY1-30 by cathepsin D in both HD patients and control subjects. Yet, NPY1-30 is then further differentially hydrolyzed by thimet oligopeptidase (TOP) in HD patients and by neprilysin (NEP) in control subjects. Furthermore, altered hCSF TOP-inhibitor Dynorphin A1-13 (Dyn-A1-13 ) and TOP-substrate Dyn-A1-8 levels indicate an impaired Dyn-A-TOP network in HD patients. Thus, we conclude that elevated IL-NPY-levels in conjunction with TOP-/NEP-activity/protein as well as Dyn-A1-13 -peptide levels may serve as a potential biomarker in human CSF of HD. Huntington's disease (HD) patients' cerebrospinal fluid (CSF) exhibits higher neuropeptide Y (NPY) levels. Further degradomics studies show that CSF-NPY is initially degraded to NPY1-30 by Cathepsin D. The NPY1-30 fragment is then differentially degraded in HD vs control involving Neprilysin (NEP), Thimet Oligopeptidase (TOP), and TOP-Dynorphin-A network. Together, these findings may help in search for HD biomarkers.
Asunto(s)
Enfermedad de Huntington/líquido cefalorraquídeo , Enfermedad de Huntington/diagnóstico , Neuropéptido Y/líquido cefalorraquídeo , Fragmentos de Péptidos/líquido cefalorraquídeo , Proteolisis , Adulto , Anciano , Animales , Biomarcadores/líquido cefalorraquídeo , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Persona de Mediana Edad , RatasRESUMEN
N-terminal truncation and pyroglutamyl (pE) formation are naturally occurring chemical modifications of the Aß peptide in Alzheimer's disease. We show herein that these two modifications significantly reduce the fibril length and the transition midpoint of thermal unfolding of the fibrils, but they do not substantially perturb the fibrillary peptide conformation. This observation implies that the Nâ terminus of the unmodified peptide protects Aß fibrils against mechanical stress and fragmentation and explains the high propensity of pE-modified peptides to form small and particularly toxic aggregates.
Asunto(s)
Péptidos beta-Amiloides/química , Ácido Pirrolidona Carboxílico/química , Secuencia de Aminoácidos , Microscopía Electrónica de Transmisión , Homología de Secuencia de AminoácidoRESUMEN
The bioactivity of neuropeptide Y (NPY) is either N-terminally modulated with respect to receptor selectivity by dipeptidyl peptidase 4 (DP4)-like enzymes or proteolytic degraded by neprilysin or meprins, thereby abrogating signal transduction. However, neither the subcellular nor the compartmental differentiation of these regulatory mechanisms is fully understood. Using mass spectrometry, selective inhibitors and histochemistry, studies across various cell types, body fluids, and tissues revealed that most frequently DP4-like enzymes, aminopeptidases P, secreted meprin-A (Mep-A), and cathepsin D (CTSD) rapidly hydrolyze NPY, depending on the cell type and tissue under study. Novel degradation of NPY by cathepsins B, D, L, G, S, and tissue kallikrein could also be identified. The expression of DP4, CTSD, and Mep-A at the median eminence indicates that the bioactivity of NPY is regulated by peptidases at the interphase between the periphery and the CNS. Detailed ex vivo studies on human sera and CSF samples recognized CTSD as the major NPY-cleaving enzyme in the CSF, whereas an additional C-terminal truncation by angiotensin-converting enzyme could be detected in serum. The latter finding hints to potential drug interaction between antidiabetic DP4 inhibitors and anti-hypertensive angiotensin-converting enzyme inhibitors, while it ablates suspected hypertensive side effects of only antidiabetic DP4-inhibitors application. The bioactivity of neuropeptide Y (NPY) is either N-terminally modulated with respect to receptor selectivity by dipeptidyl peptidase 4 (DP4)-like enzymes or proteolytic degraded by neprilysin or meprins, thereby abrogating signal transduction. However, neither the subcellular nor the compartmental differentiation of these regulatory mechanisms is fully understood. Using mass spectrometry, selective inhibitors and histochemistry, studies across various cell types, body fluids, and tissues revealed that most frequently DP4-like enzymes, aminopeptidases P, secreted meprin-A (Mep-A), and cathepsin D (CTSD) rapidly hydrolyze NPY, depending on the cell type and tissue under study. Novel degradation of NPY by cathepsins B, D, L, G, S, and tissue kallikrein could also be identified. The expression of DP4, CTSD, and Mep-A at the median eminence indicates that the bioactivity of NPY is regulated by peptidases at the interphase between the periphery and the CNS. Detailed ex vivo studies on human sera and CSF samples recognized CTSD as the major NPY-cleaving enzyme in the CSF, whereas an additional C-terminal truncation by angiotensin-converting enzyme could be detected in serum. The latter finding hints to potential drug interaction between antidiabetic DP4 inhibitors and anti-hypertensive angiotensin-converting enzyme inhibitors, while it ablates suspected hypertensive side effects of only antidiabetic DP4-inhibitors application.
Asunto(s)
Sistema Nervioso Central/citología , Dipeptidil Peptidasa 4/metabolismo , Neuroglía/metabolismo , Neuronas/metabolismo , Neuropéptido Y/metabolismo , Sistema Nervioso Periférico/citología , Animales , Proteína C-Reactiva/líquido cefalorraquídeo , Catepsina D/líquido cefalorraquídeo , Células Cultivadas , Dipeptidil Peptidasa 4/genética , Interacciones Farmacológicas , Femenino , Humanos , Hidrólisis/efectos de los fármacos , Masculino , Neuroglía/efectos de los fármacos , Neuronas/efectos de los fármacos , Fragmentos de Péptidos/metabolismo , Proteolisis/efectos de los fármacos , Ratas , Ratas Endogámicas F344 , Ratas TransgénicasRESUMEN
Pyroglutamate (pGlu)-modified amyloid peptides have been identified in sporadic and familial forms of Alzheimer's disease (AD) and the inherited disorders familial British and Danish Dementia (FBD and FDD). In this study, we characterized the aggregation of amyloid-ß protein Aß37, Aß38, Aß40, Aß42 and ADan species in vitro, which were modified by N-terminal pGlu (pGlu-Aß3-x, pGlu-ADan) or possess the intact N-terminus (Aß1-x, ADan). The pGlu-modification confers rapid formation of oligomers and short fibrillar aggregates. In accordance with these observations, the pGlu-modified Aß38, Αß40 and Αß42 species inhibit hippocampal long term potentiation of synaptic response, but pGlu-Aß3-42 showing the highest effect. Among the unmodified Aß peptides, only Aß1-42 exhibites such propensity, which was similar to pGlu-Aß3-38 and pGlu-Aß3-40. Likewise, the amyloidogenic peptide pGlu-ADan impaired synaptic potentiation more pronounced than N-terminal unmodified ADan. The results were validated using conditioned media from cultivated HEK293 cells, which express APP variants favoring the formation of Aß1-x, Aß3-x or N-truncated pGlu-Aß3-x species. Hence, we show that the ability of different amyloid peptides to impair synaptic function apparently correlates to their potential to form oligomers as a common mechanism. The pGlu-modification is apparently mediating a higher surface hydrophobicity, as shown by 1-anilinonaphtalene-8-sulfonate fluorescence, which enforces potential to interfere with neuronal physiology.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Hipocampo/metabolismo , Potenciación a Largo Plazo/fisiología , Ácido Pirrolidona Carboxílico/metabolismo , Péptidos beta-Amiloides/química , Animales , Electroforesis en Gel de Poliacrilamida , Células HEK293 , Hipocampo/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Transmisión , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Ácido Pirrolidona Carboxílico/químicaRESUMEN
Inhibition of acetylcholinesterase (AChE) and therefore prevention of acetylcholine degradation is one of the most accepted therapy opportunities for Alzheimer s disease (AD), today. Due to lack of selectivity of AChE inhibitor drugs on the market, AD-patients suffer from side effects like nausea or vomiting. In the present study the isolation of two alkaloids, infractopicrin (1) and 10-hydroxy-infractopicrin (2), from Cortinarius infractus Berk. (Cortinariaceae) is presented. Both compounds show AChE-inhibiting activity and possess a higher selectivity than galanthamine. Docking studies show that lacking pi-pi-interactions in butyrylcholinesterase (BChE) are responsible for selectivity. Studies on other AD pathology related targets show an inhibitory effect of both compounds on self-aggregation of Abeta-peptides but not on AChE induced Abeta-peptide aggregation. Low cytotoxicity as well as calculated pharmacokinetic data suggest that the natural products could be useful candidates for further drug development.
Asunto(s)
Acetilcolinesterasa/metabolismo , Agaricales/química , Inhibidores de la Colinesterasa/aislamiento & purificación , Inhibidores de la Colinesterasa/farmacología , Cuerpos Fructíferos de los Hongos/química , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Acetilcolinesterasa/química , Animales , Bovinos , Inhibidores de la Colinesterasa/química , Eritrocitos/enzimología , Compuestos Heterocíclicos de 4 o más Anillos/química , Compuestos Heterocíclicos de 4 o más Anillos/aislamiento & purificación , Estructura Molecular , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
N-Terminally truncated and pyroglutamate (pGlu) modified amyloid beta (Abeta) peptides are major constituents of amyloid deposits in sporadic and inherited Alzheimer's disease (AD). Formation of pGlu at the N-terminus confers resistance against cleavage by most aminopeptidases, increases toxicity of the peptides, and may seed Abeta aggregate formation. Similarly, the deposited amyloid peptides ABri and ADan, which cause a very similar histopathology in familial British dementia (FBD) and familial Danish dementia (FDD), are N-terminally blocked by pGlu. Triggered by the coincidence of pGlu-modified amyloid peptides and similar pathology in AD, FBD, and FDD, we investigated the impact of N-terminal pGlu on biochemical and biophysical properties of Abeta, ABri, and ADan. N-Terminal pGlu increases the hydrophobicity and changes the pH-dependent solubility profile, rendering the pGlu-modified peptides less soluble in the basic pH range. The pGlu residue increases the aggregation propensity of all amyloid peptides as evidenced by ThT fluorescence assays and dynamic light scattering. The far-UV CD spectroscopic analysis points toward an enhanced beta-sheet structure of the pGlu-Abeta. Importantly, changes in fibril morphology are clearly caused by the N-terminal pGlu, resulting in the formation of short fibers, which are frequently arranged in bundles. The effect of pGlu on the morphology is virtually indistinguishable between ABri, ADan, and Abeta. The data provide evidence for a comparable influence of the pGlu modification on the aggregation process of structurally different amyloid peptides, thus likely contributing to the molecularly distinct neurodegenerative diseases AD, FBD, and FDD. The main driving force for the aggregation is apparently an increase in the hydrophobicity and thus an accelerated seed formation.
Asunto(s)
Amiloide/metabolismo , Péptidos/metabolismo , Ácido Pirrolidona Carboxílico/metabolismo , Enfermedad de Alzheimer/metabolismo , Secuencia de Aminoácidos , Amiloide/química , Amiloide/ultraestructura , Dicroismo Circular , Humanos , Concentración de Iones de Hidrógeno , Microscopía Electrónica , Datos de Secuencia Molecular , Péptidos/química , Solubilidad , Espectrofotometría UltravioletaRESUMEN
Because of their abundance, resistance to proteolysis, rapid aggregation and neurotoxicity, N-terminally truncated and, in particular, pyroglutamate (pE)-modified Abeta peptides have been suggested as being important in the initiation of pathological cascades resulting in the development of Alzheimer's disease. We found that the N-terminal pE-formation is catalyzed by glutaminyl cyclase in vivo. Glutaminyl cyclase expression was upregulated in the cortices of individuals with Alzheimer's disease and correlated with the appearance of pE-modified Abeta. Oral application of a glutaminyl cyclase inhibitor resulted in reduced Abeta(3(pE)-42) burden in two different transgenic mouse models of Alzheimer's disease and in a new Drosophila model. Treatment of mice was accompanied by reductions in Abeta(x-40/42), diminished plaque formation and gliosis and improved performance in context memory and spatial learning tests. These observations are consistent with the hypothesis that Abeta(3(pE)-42) acts as a seed for Abeta aggregation by self-aggregation and co-aggregation with Abeta(1-40/42). Therefore, Abeta(3(pE)-40/42) peptides seem to represent Abeta forms with exceptional potency for disturbing neuronal function. The reduction of brain pE-Abeta by inhibition of glutaminyl cyclase offers a new therapeutic option for the treatment of Alzheimer's disease and provides implications for other amyloidoses, such as familial Danish dementia.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Aminoaciltransferasas/antagonistas & inhibidores , Péptidos beta-Amiloides/metabolismo , Inhibidores Enzimáticos/uso terapéutico , Ácido Pirrolidona Carboxílico/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Aminoaciltransferasas/fisiología , Animales , Encéfalo/enzimología , Células Cultivadas , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Memoria/efectos de los fármacos , Ratones , Ratones TransgénicosRESUMEN
Incretins, endogenous polypeptide hormones released in response to food intake, potentiate insulin secretion from pancreatic beta cells after oral glucose ingestion (the incretin effect). This response is signaled by the two peptide hormones glucose-dependent insulinotropic polypeptide (GIP) (also known as gastric inhibitory polypeptide) and glucagon-like peptide 1 through binding and activation of their cognate class 2 G protein-coupled receptors (GPCRs). Because the incretin effect is lost or significantly reduced in patients with type 2 diabetes mellitus, glucagon-like peptide 1 and GIP have attracted considerable attention for their potential in antidiabetic therapy. A paucity of structural information precludes a detailed understanding of the processes of hormone binding and receptor activation, hampering efforts to develop novel pharmaceuticals. Here we report the crystal structure of the complex of human GIP receptor extracellular domain (ECD) with its agonist, the incretin GIP(1-42). The hormone binds in an alpha-helical conformation in a surface groove of the ECD largely through hydrophobic interactions. The N-terminal ligand residues would remain free to interact with other parts of the receptor. Thermodynamic data suggest that binding is concomitant with structural organization of the hormone, resulting in a complex mode of receptor-ligand recognition. The presentation of a well structured, alpha-helical ligand by the ECD is expected to be conserved among other hormone receptors of this class.