RESUMEN
Glucocerebrosidase (GCase) is a lysosomal enzyme encoded by the GBA1 gene, loss of function variants of which cause an autosomal recessive lysosomal storage disorder, Gaucher disease (GD). Heterozygous variants of GBA1 are also known as the strongest common genetic risk factor for Parkinson's disease (PD). Restoration of GCase enzymatic function using a pharmacological chaperone strategy is considered a promising therapeutic approach for PD and GD. We identified compound 4 as a GCase pharmacological chaperone with sub-micromolar activity from a high-throughput screening (HTS) campaign. Compound 4 was further optimised to ER-001230194 (compound 25). ER-001230194 shows improved ADME and physicochemical properties and therefore represents a novel pharmacological chaperone with which to investigate GCase pharmacology further.
Asunto(s)
Enfermedad de Gaucher , Enfermedad de Parkinson , Humanos , Glucosilceramidasa/genética , Mutación , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Gaucher/tratamiento farmacológico , LisosomasRESUMEN
Mitochondrial dysfunction is implicated in Parkinson disease (PD). Mutations in Parkin, an E3 ubiquitin ligase, can cause juvenile-onset Parkinsonism, probably through impairment of mitophagy. Inhibition of the de-ubiquitinating enzyme USP30 may counter this effect to enhance mitophagy. Using different tools and cellular approaches, we wanted to independently confirm this claimed role for USP30. Pharmacological characterisation of additional tool compounds that selectively inhibit USP30 are reported. The consequence of USP30 inhibition by these compounds, siRNA knockdown and overexpression of dominant-negative USP30 on the mitophagy pathway in different disease-relevant cellular models was explored. Knockdown and inhibition of USP30 showed increased p-Ser65-ubiquitin levels and mitophagy in neuronal cell models. Furthermore, patient-derived fibroblasts carrying pathogenic mutations in Parkin showed reduced p-Ser65-ubiquitin levels compared with wild-type cells, levels that could be restored using either USP30 inhibitor or dominant-negative USP30 expression. Our data provide additional support for USP30 inhibition as a regulator of the mitophagy pathway.
Asunto(s)
Proteínas Mitocondriales/metabolismo , Mitofagia , Enfermedad de Parkinson/metabolismo , Proteínas Quinasas/metabolismo , Tioléster Hidrolasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Línea Celular , Fibroblastos , HumanosRESUMEN
Mitochondrial dysfunction and aberrant mitochondrial homeostasis are key aspects of Parkinson's disease (PD) pathophysiology. Mutations in PINK1 and Parkin proteins lead to autosomal recessive PD, suggesting that defective mitochondrial clearance via mitophagy is key in PD etiology. Accelerating the identification and/or removal of dysfunctional mitochondria could therefore provide a disease-modifying approach to treatment. To that end, we performed a high-content phenotypic screen (HCS) of â¼125,000 small molecules to identify compounds that positively modulate mitochondrial accumulation of the PINK1-Parkin-dependent mitophagy initiation marker p-Ser65-Ub in Parkin haploinsufficiency (Parkin +/R275W) human fibroblasts. Following confirmatory counter-screening and orthogonal assays, we selected compounds of interest that enhance mitophagy-related biochemical and functional endpoints in patient-derived fibroblasts. Identification of inhibitors of the ubiquitin-specific peptidase and negative regulator of mitophagy USP30 within our hits further validated our approach. The compounds identified in this work provide a novel starting point for further investigation and optimization.
Asunto(s)
Mitofagia , Enfermedad de Parkinson , Humanos , Mitofagia/genética , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Ubiquitinación/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Mutación , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Tioléster Hidrolasas/genética , Tioléster Hidrolasas/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismoRESUMEN
External innovation initiatives in the pharmaceutical industry have become an integral part of research and development. Collaborations have been built to enhance innovation, mitigate risk, and share cost, especially for neurodegenerative diseases, a therapeutic area that has suffered from high attrition rates. This article outlines the Eisai-University College London (UCL) Drug Discovery and Development Collaboration as a case study of how to implement a productive industry-academic partnership. In the first 10 years, seven projects have been established and the first project, a novel anti-tau antibody for Alzheimer's disease, has entered clinical trials, providing early validation of this collaboration model.
Asunto(s)
Enfermedad de Alzheimer , Descubrimiento de Drogas , Humanos , Universidades , Londres , Enfermedad de Alzheimer/tratamiento farmacológico , Industria FarmacéuticaRESUMEN
Tau deposition in the brain is a pathological hallmark of many neurodegenerative disorders, including Alzheimer's disease (AD). During the course of these tauopathies, tau spreads throughout the brain via synaptically-connected pathways. Such propagation of pathology is thought to be mediated by tau species ("seeds") containing the microtubule binding region (MTBR) composed of either three repeat (3R) or four repeat (4R) isoforms. The tau MTBR also forms the core of the neuropathological filaments identified in AD brain and other tauopathies. Multiple approaches are being taken to limit tau pathology, including immunotherapy with anti-tau antibodies. Given its key structural role within fibrils, specifically targetting the MTBR with a therapeutic antibody to inhibit tau seeding and aggregation may be a promising strategy to provide disease-modifying treatment for AD and other tauopathies. Therefore, a monoclonal antibody generating campaign was initiated with focus on the MTBR. Herein we describe the pre-clinical generation and characterisation of E2814, a humanised, high affinity, IgG1 antibody recognising the tau MTBR. E2814 and its murine precursor, 7G6, as revealed by epitope mapping, are antibodies bi-epitopic for 4R and mono-epitopic for 3R tau isoforms because they bind to sequence motif HVPGG. Functionally, both antibodies inhibited tau aggregation in vitro. They also immunodepleted a variety of MTBR-containing tau protein species. In an in vivo model of tau seeding and transmission, attenuation of deposition of sarkosyl-insoluble tau in brain could also be observed in response to antibody treatment. In AD brain, E2814 bound different types of tau filaments as shown by immunogold labelling and recognised pathological tau structures by immunohistochemical staining. Tau fragments containing HVPGG epitopes were also found to be elevated in AD brain compared to PSP or control. Taken together, the data reported here have led to E2814 being proposed for clinical development.
Asunto(s)
Enfermedad de Alzheimer/inmunología , Enfermedad de Alzheimer/terapia , Anticuerpos Monoclonales/inmunología , Inmunización Pasiva/métodos , Proteínas tau/genética , Proteínas tau/inmunología , Enfermedad de Alzheimer/patología , Animales , Anticuerpos Monoclonales/farmacología , Lóbulo Frontal/metabolismo , Lóbulo Frontal/patología , Inmunoglobulina G/inmunología , Inmunoglobulina G/farmacología , Masculino , Ratones Transgénicos , Agregación Patológica de Proteínas/inmunología , Isoformas de Proteínas/inmunología , Isoformas de Proteínas/farmacologíaRESUMEN
A method to evaluate kinase inhibitor action was reported [L. Morgan, S.J. Neame, H. Child, R. Chung, B. Shah, L. Barden, J.M. Staddon, T.R. Patel, Development of a pentylenetetrazole-induced seizure model to evaluate kinase inhibitor efficacy in the central nervous system, Neurosci. Lett. 395 (2006) 143-148]. In this, acute administration of the GABA antagonist pentylenetetrazole triggers seizures through glutamate-dependent pathways. Under such conditions, activation of the c-Jun N-terminal kinase (JNK) pathway was detected in hippocampal extracts. Phosphorylation of the upstream JNK kinase MKK4 was also revealed through use of a phospho-MKK4-specific antibody. Here, this antibody is shown to also react with a protein of approximately 125 kDa which underwent increased phosphorylation in response to pentylenetetrazole treatment. The present study aimed to identify the approximately 125 kDa protein as it may provide novel insight into signalling, neuronal activity and seizures. Using chromatographic methods and mass spectrometry, the protein was identified as amphiphysin I. This was confirmed by 2D gel analysis and immunoblot with amphiphysin I-specific antibodies. Although the phospho-MKK4 antibody was raised against an MKK4-specific peptide, partial sequence homology between this sequence and a region of amphiphysin was discerned. New antibodies raised against the phospho-threonine 260-amphiphysin-specific sequence detected increased phosphorylation in response to pentylenetetrazole treatment. This particular phosphorylation site does not seem to have been described before, possibly reflecting a novel regulatory aspect of amphiphysin biology. As amphiphysin is involved in the regulation of endocytosis, phosphorylation at this site may play a role in the regulated re-uptake of synaptic vesicles after neurotransmitter release.
Asunto(s)
Antagonistas del GABA , Proteínas del Tejido Nervioso/metabolismo , Pentilenotetrazol , Convulsiones/metabolismo , Treonina/metabolismo , Animales , Hipocampo/metabolismo , MAP Quinasa Quinasa 4/metabolismo , Ratones , Fosforilación , Convulsiones/inducido químicamenteRESUMEN
c-Jun N-terminal kinases (JNKs) are implicated in cell death in neurodegenerative disorders. Therefore, JNK inhibitors could act as neuroprotective agents. To evaluate potential candidates, reproducible and quantitative CNS in vivo models are required. To that end, a pentylenetetrazole-induced seizure model was explored. c-Jun phosphorylation was detected in hippocampal extracts by blotting c-Jun immunoprecipitates with phosphorylation-specific antibodies. Pentylenetetrazole administration induced rapid and reproducible increases in c-Jun phosphorylation. However, special attention had to be paid to the composition of the extraction buffer to ensure stabilization of protein phosphorylation, as demonstrated using internal standards of phosphorylated recombinant c-Jun. As JNK and its upstream activator MKK4 are activated by phosphorylation, these events were also evaluated. In principle, kinase inhibitors could act at the level of JNK or upstream kinases to inhibit c-Jun phosphorylation. MKK4 phosphorylation was dramatically increased in response to pentylenetetrazole but, again, only when appropriate phosphatase inhibitors were in the extraction buffer. In contrast, JNK was found to be constitutively phosphorylated and unaltered upon pentylenetetrazole treatment. The JNK inhibitor SP600125 was shown to inhibit c-Jun phosphorylation without affecting MKK4 phosphorylation. Our procedures enable analysis of JNK pathway signalling in a CNS model and, also, should be applicable to that of other protein phosphorylation events in vivo.
Asunto(s)
Convulsivantes/toxicidad , Modelos Animales de Enfermedad , Hipocampo/metabolismo , MAP Quinasa Quinasa 4/metabolismo , Pentilenotetrazol/toxicidad , Convulsiones/inducido químicamente , Animales , Electroforesis en Gel de Poliacrilamida , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Immunoblotting , Inmunohistoquímica , MAP Quinasa Quinasa 4/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Fosforilación , Inhibidores de Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-jun/efectos de los fármacos , Proteínas Proto-Oncogénicas c-jun/metabolismoRESUMEN
Growing evidence suggests persistent mitochondrial permeability transition pore (mPTP) opening is a key pathophysiological event in cell death underlying a variety of diseases. While it has long been clear the mPTP is a druggable target, current agents are limited by off-target effects and low therapeutic efficacy. Therefore identification and development of novel inhibitors is necessary. To rapidly screen large compound libraries for novel mPTP modulators, a method was exploited to cryopreserve large batches of functionally active mitochondria from cells and tissues. The cryopreserved mitochondria maintained respiratory coupling and ATP synthesis, Ca2+ uptake and transmembrane potential. A high-throughput screen (HTS), using an assay of Ca2+-induced mitochondrial swelling in the cryopreserved mitochondria identified ER-000444793, a potent inhibitor of mPTP opening. Further evaluation using assays of Ca2+-induced membrane depolarisation and Ca2+ retention capacity also indicated that ER-000444793 acted as an inhibitor of the mPTP. ER-000444793 neither affected cyclophilin D (CypD) enzymatic activity, nor displaced of CsA from CypD protein, suggesting a mechanism independent of CypD inhibition. Here we identified a novel, CypD-independent inhibitor of the mPTP. The screening approach and compound described provides a workflow and additional tool to aid the search for novel mPTP modulators and to help understand its molecular nature.
Asunto(s)
Criopreservación , Ciclofilinas/fisiología , Ensayos Analíticos de Alto Rendimiento/métodos , Mitocondrias/efectos de los fármacos , Proteínas de Transporte de Membrana Mitocondrial/antagonistas & inhibidores , Quinolinas/farmacología , Adenosina Trifosfato/biosíntesis , Animales , Peptidil-Prolil Isomerasa F , Metabolismo Energético , Femenino , Células HeLa , Humanos , Mitocondrias/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Ratas , Ratas Sprague-DawleyRESUMEN
In epithelial and endothelial cells, tight junctions limit paracellular flux of ions, proteins and other macromolecules. However, mechanisms regulating tight junction function are not clear. Occludin, a tight junction protein, undergoes phosphorylation changes in several situations but little is known about occludin kinases. A recombinant C-terminal fragment of occludin is a substrate for a kinase in crude extracts of brain. This activity was purified about 10000-fold and identified as CK2 (casein kinase 2) by peptide mass fingerprinting, immunoblotting and mutation of CK2 sites within the occludin sequence. CK2 is therefore a candidate kinase for regulation of occludin phosphorylation in vivo.
Asunto(s)
Encéfalo/enzimología , Extractos Celulares/química , Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión , Quinasa de la Caseína II , Fraccionamiento Celular , Mutagénesis Sitio-Dirigida , Ocludina , Fosforilación , Mutación Puntual , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , PorcinosRESUMEN
Imidazole-based structures of p38 inhibitors served as a starting point for the design of JNK3 inhibitors. Construction of a 6,7-dihydro-5H-pyrrolo[1,2-a]imidazole scaffold led to the synthesis of the (S)-enantiomers, which exhibited p38/JNK3 IC50 ratio of up to 10 and were up to 20 times more potent inhibitors of JNK3 than the relevant (R)-enantiomers. The JNK3 inhibitory potency correlated well with inhibition of c-Jun phosphorylation and neuroprotective properties of the compounds in low K+-induced cell death of rat cerebellar granule neurones.
Asunto(s)
Proteína Quinasa 10 Activada por Mitógenos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/síntesis química , Animales , Muerte Celular/efectos de los fármacos , Cerebelo/citología , Imidazoles , Neuronas/citología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/síntesis química , Fármacos Neuroprotectores/farmacología , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-jun/metabolismo , Ratas , Estereoisomerismo , Relación Estructura-Actividad , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
Members of the Bcl-2 family are critical regulators of apoptosis. Antiapoptotic family proteins such as Bcl-2 and Bcl-x(L) function, at least in part, by binding proapoptotic members such as Bax and Bak and thereby preventing release of apoptotic proteins, including cytochrome c, from the mitochondria. "BH3-only" members of the family disrupt this interaction by binding, via their BH3 domain, to a hydrophobic pocket on the surface of the antiapoptotic members. Disruption of heterodimerizations by small-molecule inhibitors could be used to modulate cell death in both cancer (to increase apoptosis) and degenerative disorders (to decrease apoptosis), and assays are necessary to screen compound libraries. Fluorescence polarization and enzyme-linked immunosorbent assay-based methods to detect Bcl-2 protein interactions have been described. Here, two further methods that are rapid, "mix and read," homogeneous reactions, insensitive to compound autofluorescence, and amenable to high-throughput screening, are described: a scintillation proximity assay and a time-resolved fluorescence resonance energy transfer assay (HTRF). The assays are designed using tags such that different Bcl-2 family members or BH3 domain peptides can be readily applied to either format, as exemplified by the use here of histidine-tagged Bcl-x(L) and biotinylated BH3 peptides.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Conteo por Cintilación/métodos , Secuencia de Aminoácidos , Unión Competitiva , Biotinilación , Quelantes/química , Cobre/química , Proteínas de Unión al ADN/metabolismo , Dimerización , Histidina/genética , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Mapeo de Interacción de Proteínas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Recombinantes/análisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factor de Transcripción STAT3 , Transactivadores/metabolismo , Proteína bcl-XRESUMEN
Death-associated protein kinase (DAP kinase) is a proapoptotic, calcium/calmodulin-dependent serine/threonine kinase. Here, we report that DAP kinase phosphorylates the regulatory light chain of myosin II (MLC) both in vitro and in vivo, and that this phosphorylation occurs preferentially at residue Ser19. In quiescent fibroblasts, DAP kinase stabilizes stress fibers through phosphorylation of MLC, but it is dispensable for the formation of peripheral microfilament bundles. This cytoskeletal effect of DAP kinase occurs before the onset of apoptosis and does not require an intact death domain. In addition, DAP kinase is required for serum-induced stress-fiber formation, which is associated with the upregulation of its catalytic activity. Despite being both sufficient and necessary for the assembly or maintenance of stress fibers, DAP kinase is incapable of stimulating the formation of focal adhesions in quiescent cells. Moreover, it promotes the disassembly of focal adhesions but not stress fibers in cells receiving serum factors. Together, our results identify a novel and unique function of DAP kinase in the uncoupling of stress fibers and focal adhesions. Such uncoupling would lead to a perturbation of the balance between contractile and adhesion forces and subsequent cell detachment, which might contribute to its pro-apoptotic activity.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Adhesiones Focales/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Miosina Tipo II/metabolismo , Fibras de Estrés/metabolismo , Células 3T3 , Actinas/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Baculoviridae , Calmodulina , Células Cultivadas , Clonación Molecular , Proteínas Quinasas Asociadas a Muerte Celular , Humanos , Ratones , Microscopía Fluorescente , Microscopía de Interferencia , Fosforilación , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
Cytotoxic necrotizing factor 1 (CNF-1) is an exotoxin of Escherichia coli that constitutively activates the GTPases Rho, Rac, and CDC42. Stimulation of Rho was shown to enhance myosin light-chain (MLC) phosphorylation via Rho kinase-mediated inhibition of MLC phosphatase in endothelial cells. Here we report that 3 h after CNF stimulation of endothelial cells, RhoA was activated and MLC phosphorylation was increased in a Rho/Rho-kinase-dependent manner, but no decrease in MLC phosphatase activity could be detected. Despite continuous RhoA activation, MLC phosphatase activity was doubled after 24 h of CNF stimulation, and this coincided with decreased MLC phosphorylation and cell spreading. Rac was also activated at 3 to 24 h but did not contribute to MLC phosphorylation, and its amount gradually decreased in the CNF-stimulated cells. CDC42Hs was not activated above control values by CNF. These results suggest that CNF can induce specific decoupling (Rho kinase from MLC phosphatase) and deactivation events in Rho GTPase signaling, potentially reflecting cellular protection mechanisms against permanently active Rho GTPases.
Asunto(s)
Toxinas Bacterianas/toxicidad , Citotoxinas/toxicidad , Proteínas de Escherichia coli , Escherichia coli/patogenicidad , Cadenas Ligeras de Miosina/metabolismo , Actomiosina/metabolismo , Toxinas Bacterianas/genética , Secuencia de Bases , Células Cultivadas , Citotoxinas/genética , ADN Bacteriano/genética , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Escherichia coli/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Cinética , Fosfatasa de Miosina de Cadena Ligera , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/toxicidad , Proteína de Unión al GTP cdc42/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Quinasas Asociadas a rhoRESUMEN
Reactive oxygen species (ROS) increase the contractile response of airway smooth muscle (ASM). Heme oxygenase (HO) catabolizes heme to the powerful antioxidant bilirubin. Because HO is expressed in the airways, we investigated its effects on ASM contractility and ROS production in guinea pig trachea. HO expression was higher in the epithelium than in tracheal smooth muscle. Incubation of tracheal rings (TR) with the HO inhibitor tin protoporphyrin (SnPP IX) or the HO substrate hemin increased and decreased, respectively, ASM contractile response to carbamylcholine. The effect of hemin was reversed by SnPP and mimicked by the antioxidants superoxide dismutase (SOD) and catalase. Hemin significantly reduced the effect of carbamylcholine in rings treated with the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), compared with ODQ-treated rings without hemin incubation, suggesting that the CO-guanosine 3',5'-cyclic monophosphate pathway was not involved in the control of tracheal reactivity. SnPP and hemin increased and decreased ROS production by TR by 18 and 38%, respectively. Bilirubin (100 pM) significantly decreased TR contractility and ROS production. Hemin, bilirubin, and SOD/catalase decreased phosphorylation of the contractile protein myosin light chain, whereas SnPP significantly augmented it. These data suggest that modulation of the redox status by HO and, moreover, by bilirubin modulates ASM contractility by modulating levels of phosphorylated myosin light chain.