RESUMEN
Clathrin-mediated endocytosis (CME) regulates many cell physiological processes such as the internalization of growth factors and receptors, entry of pathogens, and synaptic transmission. Within the endocytic network, clathrin functions as a central organizing platform for coated pit assembly and dissociation via its terminal domain (TD). We report the design and synthesis of two compounds named pitstops that selectively block endocytic ligand association with the clathrin TD as confirmed by X-ray crystallography. Pitstop-induced inhibition of clathrin TD function acutely interferes with receptor-mediated endocytosis, entry of HIV, and synaptic vesicle recycling. Endocytosis inhibition is caused by a dramatic increase in the lifetimes of clathrin coat components, including FCHo, clathrin, and dynamin, suggesting that the clathrin TD regulates coated pit dynamics. Pitstops provide new tools to address clathrin function in cell physiology with potential applications as inhibitors of virus and pathogen entry and as modulators of cell signaling.
Asunto(s)
Clatrina/química , Clatrina/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Técnicas Citológicas/métodos , Bibliotecas de Moléculas Pequeñas , Complejo 2 de Proteína Adaptadora/metabolismo , Animales , Células Cultivadas , Invaginaciones Cubiertas de la Membrana Celular/efectos de los fármacos , Cristalografía por Rayos X , Dinaminas/metabolismo , Endocitosis , Humanos , Ratones , Estructura Terciaria de Proteína , Transducción de Señal , Sinapsis/metabolismo , Sinapsis/ultraestructuraRESUMEN
Hereditary spastic paraplegia is a spastic gait disorder that arises from degeneration of corticospinal axons. The subtype SPG48 is associated with mutations in the zeta subunit of the adaptor protein complex five (AP5). AP5 function and the pathophysiology of SPG48 are only poorly understood. Here, we report an AP5 zeta knockout mouse, which shows an age-dependent degeneration of corticospinal axons. Our analysis of knockout fibroblasts supports a trafficking defect from late endosomes to the transGolgi network and reveals a structural defect of the Golgi. We further show that both autophagic flux and the recycling of lysosomes from autolysosomes were impaired in knockout cells. In vivo, we observe an increase of autophagosomes and autolysosomes and, at later stages, the accumulation of intracellular waste in neurons. Taken together, we propose that loss of AP5 function blocks autophagy and thus leads to the aberrant accumulation of autophagic cargo, which finally results in axon degeneration.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Autofagia/fisiología , Neuronas/metabolismo , Paraplejía Espástica Hereditaria/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Modelos Animales de Enfermedad , Lisosomas/metabolismo , Lisosomas/patología , Ratones , Ratones Noqueados , Degeneración Nerviosa/genética , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Neuronas/patología , Tractos Piramidales/metabolismo , Tractos Piramidales/patología , Paraplejía Espástica Hereditaria/genéticaRESUMEN
Mutations in the CLN7/MFSD8 gene encoding the lysosomal membrane protein CLN7 are causative of CLN7 disease, an inherited neurodegenerative disorder that typically affects children. To gain insight into the pathomechanisms of CLN7 disease, we established an immortalized cell line based on cerebellar (Cb) granule neuron precursors isolated from Cln7-/- mice. Here, we demonstrate that Cln7-deficient neuron-derived Cb cells display an abnormal phenotype that includes increased size and defective outward movement of late endosomes and lysosomes as well as impaired lysosomal exocytosis. Whereas Cln7-/- Cb cells appeared to be autophagy-competent, loss of Cln7 resulted in enhanced cell death under prolonged nutrient deprivation. Furthermore, reduced cell survival of Cln7-deficient cells was accompanied by a significantly impaired protein kinase B/Akt phosphorylation at Ser473 during long-term starvation. In summary, our data demonstrate for the first time that the putative lysosomal transporter CLN7 is relevant for lysosome motility and plays an important role for neuronal cell survival under conditions of starvation.
Asunto(s)
Lisosomas/metabolismo , Proteínas de Transporte de Membrana/genética , Mutación , Lipofuscinosis Ceroideas Neuronales/genética , Neuronas/patología , Animales , Autofagia , Transporte Biológico , Técnicas de Cultivo de Célula , Línea Celular , Supervivencia Celular , Exocitosis , Ratones , Ratones Noqueados , Naftiridinas/farmacología , Neuronas/citología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
The volume-regulated anion channel (VRAC) plays an important role in osmotic cell volume regulation. In addition, it is involved in various physiological processes such as insulin secretion, glia-neuron communication and purinergic signaling. VRAC is formed by hetero-hexamers of members of the LRRC8 protein family, which consists of five members, LRRC8A-E. LRRC8A is an essential subunit for physiological functionality of VRAC. Its obligate heteromerization with at least one of its paralogues, LRRC8B-E, determines the biophysical properties of VRAC. Moreover, the subunit composition is of physiological relevance as it largely influences the activation mechanism and especially the substrate selectivity. However, the endogenous tissue-specific subunit composition of VRAC is unknown. We have now developed and applied a quantitative immunoblot study of the five VRAC LRRC8 subunits in various mouse cell lines and tissues, using recombinant protein for signal calibration. We found tissue-specific expression patterns of the subunits, and generally relative low expression of the essential LRRC8A subunit. Immunoprecipitation of LRRC8A also co-precipitates an excess of the other subunits, suggesting that non-LRRC8A subunits present the majority in hetero-hexamers. With this, we can estimate that in the tested cell lines, the number of VRAC channels per cell is in the order of 10,000, which is in agreement with earlier calculations from the comparison of single-channel and whole-cell currents.
Asunto(s)
Proteínas de la Membrana/metabolismo , Canales Aniónicos Dependientes del Voltaje/metabolismo , Células 3T3 , Animales , Línea Celular , Electroforesis en Gel de Poliacrilamida , Immunoblotting , Inmunoprecipitación , Masculino , Proteínas de la Membrana/genética , Ratones , Canales Aniónicos Dependientes del Voltaje/genéticaRESUMEN
Chlorpromazine is a phenothiazine-derived antipsychotic drug (APD) that inhibits clathrin-mediated endocytosis (CME) in cells by an unknown mechanism. We examined whether its action and that of other APDs might be mediated by the GTPase activity of dynamin. Eight of eight phenothiazine-derived APDs inhibited dynamin I (dynI) in the 2-12 µm range, the most potent being trifluoperazine (IC50 2.6 ± 0.7 µm). They also inhibited dynamin II (dynII) at similar concentrations. Typical and atypical APDs not based on the phenothiazine scaffold were 8- to 10-fold less potent (haloperidol and clozapine) or were inactive (droperidol, olanzapine and risperidone). Kinetic analysis showed that phenothiazine-derived APDs were lipid competitive, while haloperidol was uncompetitive with lipid. Accordingly, phenothiazine-derived APDs inhibited dynI GTPase activity stimulated by lipids but not by various SH3 domains. All dynamin-active APDs also inhibited transferrin (Tfn) CME in cells at related potencies. Structure-activity relationships (SAR) revealed dynamin inhibition to be conferred by a substituent group containing a terminal tertiary amino group at the N2 position. Chlorpromazine was previously proposed to target AP-2 recruitment in the formation of clathrin-coated vesicles (CCV). However, neither chlorpromazine nor thioridazine affected AP-2 interaction with amphiphysin or clathrin. Super-resolution microscopy revealed that chlorpromazine blocks neither clathrin recruitment by AP-2, nor AP-2 recruitment, showing that CME inhibition occurs downstream of CCV formation. Overall, potent dynamin inhibition is a shared characteristic of phenothiazine-derived APDs, but not other typical or atypical APDs, and the data indicate that dynamin is their likely in-cell target in endocytosis.
Asunto(s)
Antipsicóticos/farmacología , Clatrina/metabolismo , Dinaminas/metabolismo , Endocitosis/efectos de los fármacos , Fenotiazinas/farmacología , Línea Celular Tumoral , Vesículas Cubiertas por Clatrina/metabolismo , Humanos , Transferrina/metabolismoRESUMEN
Dynamin is a GTPase that mediates vesicle fission during synaptic vesicle endocytosis. Its long C-terminal proline-rich domain contains 13 PXXP motifs, which orchestrate its interactions with multiple proteins. The SH3 domains of syndapin and endophilin bind the PXXP motifs called Site 2 and 3 (Pro-786-Pro-793) at the N-terminal end of the proline-rich domain, whereas the amphiphysin SH3 binds Site 9 (Pro-833-Pro-836) toward the C-terminal end. In some proteins, SH3/peptide interactions also involve short distance elements, which are 5-15 amino acid extensions flanking the central PXXP motif for high affinity binding. Here we found two previously unrecognized elements in the central and the C-terminal end of the dynamin proline-rich domain that account for a significant increase in syndapin binding affinity compared with a previously reported Site 2 and Site 3 PXXP peptide alone. The first new element (Gly-807-Gly-811) is short distance element on the C-terminal side of Site 2 PXXP, which might contact a groove identified under the RT loop of the SH3 domain. The second element (Arg-838-Pro-844) is located about 50 amino acids downstream of Site 2. These two elements provide additional specificity to the syndapin SH3 domain outside of the well described polyproline-binding groove. Thus, the dynamin/syndapin interaction is mediated via a network of multiple contacts outside the core PXXP motif over a previously unrecognized extended region of the proline-rich domain. To our knowledge this is the first example among known SH3 interactions to involve spatially separated and extended long-range elements that combine to provide a higher affinity interaction.
Asunto(s)
Proteínas Portadoras/química , Dinaminas/química , Neuropéptidos/química , Fosfoproteínas/química , Proteínas Adaptadoras Transductoras de Señales , Secuencias de Aminoácidos , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas del Citoesqueleto , Dinaminas/genética , Dinaminas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Neuropéptidos/genética , Neuropéptidos/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Unión Proteica , Ratas , Dominios Homologos srcRESUMEN
The development of a (Z)-5-((6,8-dichloro-4-oxo-4H-chromen-3-yl)methylene)-2-thioxothiazolidin-4-one (2), rhodanine-based lead that led to the Pitstop® 2 family of clathrin inhibitors is described herein. Head group substitution and bioisosteric replacement of the rhodanine core with a 2-aminothiazol-4(5H)-one scaffold eliminated off target dynamin activity. A series of N-substituents gave first phenylglycine (20, IC50 â¼ 20 µM) then phenyl (25, IC50 â¼ 7.1 µM) and 1-napthyl sulfonamide (26, Pitstop® 2 compound, IC50 â¼ 1.9 µM) analogues with good activity, validating this approach. A final library exploring the head group resulted in three analogues displaying either slight improvements or comparable activity (33, 38, and 29 with IC50 â¼ 1.4, 1.6 and 1.8 µM respectively) and nine others with IC50 < 10 µM. These results were rationalized using in silico docking studies. Docking studies predicted enhanced Pitstop® 2 family binding, not a loss of binding, within the Pistop® groove of the reported clathrin mutant invalidating recent assumptions of poor selectivity for this family of clathrin inhibitors.
Asunto(s)
Clatrina/antagonistas & inhibidores , Sulfonamidas/química , Sulfonamidas/farmacología , Clatrina/química , Clatrina/metabolismo , Diseño de Fármacos , Concentración 50 Inhibidora , Simulación del Acoplamiento Molecular , Conformación Proteica , Relación Estructura-Actividad , Sulfonamidas/metabolismoRESUMEN
Dynamin GTPase activity increases when it oligomerizes either into helices in the presence of lipid templates or into rings in the presence of SH3 domain proteins. Dynasore is a dynamin inhibitor of moderate potency (IC50 ~ 15 µM in vitro). We show that dynasore binds stoichiometrically to detergents used for in vitro drug screening, drastically reducing its potency (IC50 = 479 µM) and research tool utility. We synthesized a focused set of dihydroxyl and trihydroxyl dynasore analogs called the Dyngo™ compounds, five of which had improved potency, reduced detergent binding and reduced cytotoxicity, conferred by changes in the position and/or number of hydroxyl substituents. The Dyngo compound 4a was the most potent compound, exhibiting a 37-fold improvement in potency over dynasore for liposome-stimulated helical dynamin activity. In contrast, while dynasore about equally inhibited dynamin assembled in its helical or ring states, 4a and 6a exhibited >36-fold reduced activity against rings, suggesting that they can discriminate between helical or ring oligomerization states. 4a and 6a inhibited dynamin-dependent endocytosis of transferrin in multiple cell types (IC50 of 5.7 and 5.8 µM, respectively), at least sixfold more potently than dynasore, but had no effect on dynamin-independent endocytosis of cholera toxin. 4a also reduced synaptic vesicle endocytosis and activity-dependent bulk endocytosis in cultured neurons and synaptosomes. Overall, 4a and 6a are improved and versatile helical dynamin and endocytosis inhibitors in terms of potency, non-specific binding and cytotoxicity. The data further suggest that the ring oligomerization state of dynamin is not required for clathrin-mediated endocytosis.
Asunto(s)
Dinaminas/antagonistas & inhibidores , Endocitosis/efectos de los fármacos , Hidrazonas/farmacología , Naftoles/farmacología , Animales , Línea Celular Tumoral , Células Cultivadas , Toxina del Cólera/metabolismo , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas , Dinaminas/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Hidrazonas/síntesis química , Hidrazonas/química , Naftoles/química , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Unión Proteica , Transporte de Proteínas , Ratas , Ratas Sprague-Dawley , Ovinos , Vesículas Sinápticas/efectos de los fármacos , Vesículas Sinápticas/metabolismo , Transferrinas/metabolismoRESUMEN
Intracellular membrane traffic regulates cell physiology at multiple levels ranging from cell growth and development to the function of the nervous and immune systems. Multiple endocytic routes are used by distinct cargoes including ligands bound to their receptors but also viruses and pathogens to gain access to the cell interior. Within the endosomal system, proteins and lipids are sorted for degradation or recycling allowing cells to dynamically respond to environmental signals and to regulate cell shape and morphology. Some receptors or toxins are sorted along the retrograde pathway from endosomes to the Golgi complex, where they intersect with secretory cargo destined for exocytosis. Genetic manipulations of these pathways frequently cause problems with regard to data interpretation as the resulting phenotypes may be indirect consequences resulting from perturbation of multiple steps or trafficking routes. Hence, novel approaches are needed to acutely and reversibly perturb intracellular membrane traffic, e.g., by small molecule inhibitors. Such drugs may also be pharmacologically important as they offer new avenues to fight human diseases. Here, we provide an overview of the small molecules available to interfere with intracellular membrane traffic and outline strategies for future research.
Asunto(s)
Permeabilidad de la Membrana Celular , Proteínas/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas , Animales , Permeabilidad de la Membrana Celular/efectos de los fármacos , Humanos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacocinética , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Structurally related inhibitors of a shared therapeutic target may differ regarding potential toxicity issues that are caused by different off-target bindings. We devised a differential competition capture compound mass spectrometry (dCCMS) strategy to effectively differentiate off-target profiles. Tolcapone and entacapone are potent inhibitors of catechol-O-methyl transferase (COMT) for the treatment of Parkinson's disease. Tolcapone is also known for its hepatotoxic side effects even though it is therapeutically more potent than entacapone. Here, we identified 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) as a possible toxicity-causing off-target of tolcapone, and this protein is not bound by the less toxic COMT inhibitor entacapone. Moreover, two novel compounds from a focused library synthesized in-house, N(2),N(2),N(3),N(3)-tetraethyl-6,7-dihydroxy-5-nitronaphthalene-2,3-dicarboxamide and 5-(3,4-dihydroxy-5-nitrobenzylidene)-3-ethylthiazolidine-2,4-dione, were utilized to gain insight into the structure-activity relationships in binding to COMT and the novel off-target HIBCH. These compounds, especially N(2),N(2),N(3),N(3)-tetraethyl-6,7-dihydroxy-5-nitronaphthalene-2,3-dicarboxamide, could serve as starting point for the development of improved and more specific COMT inhibitors.
Asunto(s)
Inhibidores de Catecol O-Metiltransferasa/farmacología , Catecol O-Metiltransferasa/metabolismo , Inhibidores de Catecol O-Metiltransferasa/síntesis química , Inhibidores de Catecol O-Metiltransferasa/química , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Células Hep G2 , Humanos , Espectrometría de Masas , Modelos Moleculares , Estructura Molecular , Relación Estructura-ActividadRESUMEN
This protocol describes the synthesis of two classes of clathrin inhibitors, Pitstop 1 and Pitstop 2, along with two inactive analogs that can be used as negative controls (Pitstop inactive controls, Pitnot-2 and Pitnot-2-100). Pitstop-induced inhibition of clathrin TD function acutely interferes with clathrin-mediated endocytosis (CME), synaptic vesicle recycling and cellular entry of HIV, whereas clathrin-independent internalization pathways and secretory traffic proceed unperturbed; these reagents can, therefore, be used to investigate clathrin function, and they have potential pharmacological applications. Pitstop 1 is synthesized in two steps: sulfonation of 1,8-naphthalic anhydride and subsequent reaction with 4-amino(methyl)aniline. Pitnot-1 results from the reaction of 4-amino(methyl)aniline with commercially available 4-sulfo-1,8-naphthalic anhydride potassium salt. Reaction of 1-naphthalene sulfonyl chloride with pseudothiohydantoin followed by condensation with 4-bromobenzaldehyde yields Pitstop 2. The synthesis of the inactive control commences with the condensation of 4-bromobenzaldehyde with the rhodanine core. Thioketone methylation and displacement with 1-napthylamine affords the target compound. Although Pitstop 1-series compounds are not cell permeable, they can be used in biochemical assays or be introduced into cells via microinjection. The Pitstop 2-series compounds are cell permeable. The synthesis of these compounds does not require specialist equipment and can be completed in 3-4 d. Microwave irradiation can be used to reduce the synthesis time. The synthesis of the Pitstop 2 family is easily adaptable to enable the synthesis of related compounds such as Pitstop 2-100 and Pitnot-2-100. The procedures are also simple, efficient and amenable to scale-up, enabling cost-effective in-house synthesis for users of these inhibitor classes.
Asunto(s)
Clatrina/antagonistas & inhibidores , Sulfonamidas/síntesis química , Tiazolidinas/síntesis química , Técnicas de Química Sintética , Naftalenos/químicaRESUMEN
We reported the first small molecule inhibitors of the interaction between the clathrin N-terminal domain (TD) and endocyctic accessory proteins (i.e., clathrin inhibition1). Initial screening of a â¼17 000 small molecule ChemBioNet library identified 1. Screening of an existing in-house propriety library identified four substituted 1,8-napthalimides as â¼80-120 µM clathrin inhibitors. Focused library development gave 3-sulfo-N-(4-aminobenzyl)-1,8-naphthalimide, potassium salt (18, IC50 ≈ 18 µM). A second library targeting the 4-aminobenzyl moiety was developed, and four analogues displayed comparable activity (26, 27, 28, 34 with IC50 values of 22, 16, 15, and 15 µM respectively) with a further four (24, 25, 32, 33) more active than 18 with IC50 values of 10, 6.9, 12, and 10 µM, respectively. Docking studies rationalized the structure-activity relationship (SAR) with the biological data. 3-Sulfo-N-benzyl-1,8-naphthalimide, potassium salt (25) with an IC50 ≈ 6.9 µM, is the most potent clathrin terminal domain-amphiphysin inhibitor reported to date.
Asunto(s)
Clatrina/antagonistas & inhibidores , Naftalimidas/síntesis química , Modelos Moleculares , Simulación del Acoplamiento Molecular , Naftalimidas/química , Naftalimidas/farmacología , Relación Estructura-ActividadRESUMEN
Trypanosomatid parasites of the genus Leishmania cause severe human diseases collectively termed leishmaniasis. Parasite ATP-binding proteins have emerged as potent targets for chemotherapeutic intervention. However, many parasite-specific ATP-binding proteins may escape current efforts in drug target identification, validation and deconvolution due to the lack of sequence conservation and functional annotation of these proteins in early branching eukaryotic trypanosomatids. Here, we selectively enriched for ATP-binding proteins from Leishmania donovani axenic promastigote and amastigote total protein extracts utilizing a Capture Compound™ (CC) linked to the ATP-competitive inhibitor staurosporine. As judged by in-gel kinase activity assay and competitive inhibition with free staurosporine, the CC specifically enriched for parasite phosphotransferases. Comparative nanoLC-MS(n) analysis identified 70 captured proteins, including 24 conserved protein kinases, and 32 hypothetical proteins with potential ATP-binding function. We identified conserved signature sequence motifs characteristic for staurosporine-binding protein kinases, and identified the hypothetical proteins LinJ.20.0280 and LinJ.09.1630 as novel ATP-binding proteins. Thus, functional enrichment procedures such as described here, combined with bio-informatics analyses and activity assays, provide powerful tools for the discovery of parasite-specific ATP-binding proteins that escape homology-based identification, which can be subsequently targeted for pharmacological intervention. BIOLOGICAL SIGNIFICANCE: Functional enrichment using a Capture Compound™ linked to the ATP-competitive inhibitor staurosporine provides a powerful new tool for the discovery of parasite-specific ATP-binding proteins that escape homology-based identification, which can be subsequently targeted for pharmacological intervention.
Asunto(s)
Proteínas Portadoras/aislamiento & purificación , Leishmania donovani/metabolismo , Proteínas Quinasas/aislamiento & purificación , Proteínas Protozoarias/aislamiento & purificación , Estaurosporina/química , Adenosina Trifosfato/metabolismo , Cromatografía Liquida/métodos , Ontología de Genes , Leishmania donovani/crecimiento & desarrollo , Proteómica/métodos , Espectrometría de Masas en Tándem/métodosRESUMEN
Six focused rhodanine-based libraries, 60 compounds in total, were synthesized and evaluated as potential dynamin I GTPase inhibitors. Twenty-six were more potent than the lead compound with 13 returning IC50 values ≤10 µM, making the Rhodadyn series among the most active dynamin inhibitors reported. Two analogues were highly effective at blocking receptor-mediated endocytosis: C10 and D10 with IC50(RME) = 7.0 ± 2.2 and 5.9 ± 1.0 µM, respectively. These compounds are equipotent with the best reported in-cell dynamin inhibitors.