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1.
ACS Med Chem Lett ; 15(5): 646-652, 2024 May 09.
Artículo en Inglés | MEDLINE | ID: mdl-38746889

RESUMEN

The potassium (K+) ion channel KCNK13 is specifically expressed in human microglia with elevated expression observed in post-mortem human brain tissue from patients with Alzheimer's disease. Modulation of KCNK13 activity by a small-molecule inhibitor is proposed as a potential treatment for neurodegenerative diseases. Herein, we describe the evolution of a series of KCNK13 inhibitors derived from a high-throughput screening campaign, resulting in CVN293, a potent, selective, and brain permeable clinical candidate molecule. CVN293 demonstrated a concentration-dependent inhibition of the NLRP3-inflammasome mediated production of IL-1ß from LPS-primed murine microglia. Cross-species pharmacokinetic data of CVN293 are also disclosed. These findings support the advancement of CVN293 in clinical trials.

2.
J Neuroinflammation ; 21(1): 58, 2024 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-38409076

RESUMEN

Neuroinflammation is highly influenced by microglia, particularly through activation of the NLRP3 inflammasome and subsequent release of IL-1ß. Extracellular ATP is a strong activator of NLRP3 by inducing K+ efflux as a key signaling event, suggesting that K+-permeable ion channels could have high therapeutic potential. In microglia, these include ATP-gated THIK-1 K+ channels and P2X7 receptors, but their interactions and potential therapeutic role in the human brain are unknown. Using a novel specific inhibitor of THIK-1 in combination with patch-clamp electrophysiology in slices of human neocortex, we found that THIK-1 generated the main tonic K+ conductance in microglia that sets the resting membrane potential. Extracellular ATP stimulated K+ efflux in a concentration-dependent manner only via P2X7 and metabotropic potentiation of THIK-1. We further demonstrated that activation of P2X7 was mandatory for ATP-evoked IL-1ß release, which was strongly suppressed by blocking THIK-1. Surprisingly, THIK-1 contributed only marginally to the total K+ conductance in the presence of ATP, which was dominated by P2X7. This suggests a previously unknown, K+-independent mechanism of THIK-1 for NLRP3 activation. Nuclear sequencing revealed almost selective expression of THIK-1 in human brain microglia, while P2X7 had a much broader expression. Thus, inhibition of THIK-1 could be an effective and, in contrast to P2X7, microglia-specific therapeutic strategy to contain neuroinflammation.


Asunto(s)
Microglía , Proteína con Dominio Pirina 3 de la Familia NLR , Humanos , Microglía/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Enfermedades Neuroinflamatorias , Canales Iónicos/metabolismo , Adenosina Trifosfato/farmacología , Adenosina Trifosfato/metabolismo , Receptores Purinérgicos P2X7/metabolismo
3.
Bioorg Med Chem Lett ; 100: 129629, 2024 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-38295907

RESUMEN

Modulators of orexin receptors are being developed for neurological illnesses such as sleep disorders, addictive behaviours and other psychiatric diseases. We herein describe the discovery of CVN766, a potent orexin 1 receptor antagonist that has greater than 1000-fold selectivity for the orexin 1 receptor over the orexin 2 receptor and demonstrates low off target hits in a diversity screen. In agreement with its in vitro ADME data, CVN766 demonstrated moderate in vivo clearance in rodents and displayed good brain permeability and target occupancy. This drug candidate is currently being investigated in clinical trials for schizophrenia and related psychiatric conditions.


Asunto(s)
Revelación , Trastornos Mentales , Humanos , Orexinas , Antagonistas de los Receptores de Orexina/farmacología , Receptores de Orexina
4.
J Med Chem ; 66(18): 12858-12876, 2023 09 28.
Artículo en Inglés | MEDLINE | ID: mdl-37708305

RESUMEN

From our NETSseq-derived human brain transcriptomics data, we identified GPR55 as a potential molecular target for the treatment of motor symptoms in patients with Parkinson's disease. From a high-throughput screen, we identified and optimized agonists with nanomolar potency against both human and rat GPR55. We discovered compounds with either strong or limited ß-arrestin signaling and receptor desensitization, indicating biased signaling. A compound that showed minimal GPR55 desensitization demonstrated a reduction in firing frequency of medium spiny neurons cultured from rat striatum but did not reverse motor deficits in a rat hypolocomotion model. Further profiling of several desensitizing and non-desensitizing lead compounds showed that they are selective over related cannabinoid receptors CB1 and CB2 and that unbound brain concentrations well above the respective GPR55 EC50 can be readily achieved following oral administration. The novel brain-penetrant GPR55 agonists disclosed can be used to probe the role of this receptor in the brain.


Asunto(s)
Agonistas de Receptores de Cannabinoides , Transducción de Señal , Humanos , Ratas , Animales , Receptores de Cannabinoides , beta-Arrestinas , Cuerpo Estriado/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptor Cannabinoide CB2 , Receptor Cannabinoide CB1
5.
J Med Chem ; 66(17): 11718-11731, 2023 09 14.
Artículo en Inglés | MEDLINE | ID: mdl-37651656

RESUMEN

Nicotinic acetylcholine receptor (nAChR) α6 subunit RNA expression is relatively restricted to midbrain regions and is located presynaptically on dopaminergic neurons projecting to the striatum. This subunit modulates dopamine neurotransmission and may have therapeutic potential in movement disorders. We aimed to develop potent and selective α6-containing nAChR antagonists to explore modulation of dopamine release and regulation of motor function in vivo. High-throughput screening (HTS) identified novel α6-containing nAChR antagonists and led to the development of CVN417. This molecule blocks α6-containing nAChR activity in recombinant cells and reduces firing frequency of noradrenergic neurons in the rodent locus coeruleus. CVN417 modulated phasic dopaminergic neurotransmission in an impulse-dependent manner. In a rodent model of resting tremor, CVN417 attenuated this behavioral phenotype. These data suggest that selective antagonism of α6-containing nAChR, with molecules such as CVN417, may have therapeutic utility in treating the movement dysfunctions observed in conditions such as Parkinson's disease.


Asunto(s)
Dopamina , Receptores Nicotínicos , Encéfalo , Membrana Celular , Cuerpo Estriado , Antagonistas Nicotínicos/farmacología
6.
ACS Med Chem Lett ; 14(4): 442-449, 2023 Apr 13.
Artículo en Inglés | MEDLINE | ID: mdl-37077399

RESUMEN

The low affinity metabotropic glutamate receptor mGluR7 has been implicated in numerous CNS disorders; however, a paucity of potent and selective activators has hampered full delineation of the functional role and therapeutic potential of this receptor. In this work, we present the identification, optimization, and characterization of highly potent, novel mGluR7 agonists. Of particular interest is the chromane CVN636, a potent (EC50 7 nM) allosteric agonist which demonstrates exquisite selectivity for mGluR7 compared to not only other mGluRs, but also a broad range of targets. CVN636 demonstrated CNS penetrance and efficacy in an in vivo rodent model of alcohol use disorder. CVN636 thus has potential to progress as a drug candidate in CNS disorders involving mGluR7 and glutamatergic dysfunction.

7.
Cell Rep Med ; 4(3): 100957, 2023 03 21.
Artículo en Inglés | MEDLINE | ID: mdl-36889319

RESUMEN

Hyperpolarizing GABAAR currents, the unitary events that underlie synaptic inhibition, are dependent upon efficient Cl- extrusion, a process that is facilitated by the neuronal specific K+/Cl- co-transporter KCC2. Its activity is also a determinant of the anticonvulsant efficacy of the canonical GABAAR-positive allosteric: benzodiazepines (BDZs). Compromised KCC2 activity is implicated in the pathophysiology of status epilepticus (SE), a medical emergency that rapidly becomes refractory to BDZ (BDZ-RSE). Here, we have identified small molecules that directly bind to and activate KCC2, which leads to reduced neuronal Cl- accumulation and excitability. KCC2 activation does not induce any overt effects on behavior but prevents the development of and terminates ongoing BDZ-RSE. In addition, KCC2 activation reduces neuronal cell death following BDZ-RSE. Collectively, these findings demonstrate that KCC2 activation is a promising strategy to terminate BDZ-resistant seizures and limit the associated neuronal injury.


Asunto(s)
Estado Epiléptico , Simportadores , Ratones , Animales , Benzodiazepinas/farmacología , Benzodiazepinas/uso terapéutico , Estado Epiléptico/tratamiento farmacológico , Convulsiones/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Simportadores/metabolismo
8.
Neuropharmacology ; 224: 109330, 2023 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-36375694

RESUMEN

Neuroinflammation, specifically the NLRP3 inflammasome cascade, is a common underlying pathological feature of many neurodegenerative diseases. Evidence suggests that NLRP3 activation involves changes in intracellular K+. Nuclear Enriched Transcript Sort Sequencing (NETSseq), which allows for deep sequencing of purified cell types from human post-mortem brain tissue, demonstrated a highly specific expression of the tandem pore domain halothane-inhibited K+ channel 1 (THIK-1) in microglia compared to other glial and neuronal cell types in the human brain. NETSseq also showed a significant increase of THIK-1 in microglia isolated from cortical regions of brains with Alzheimer's disease (AD) relative to control donors. Herein, we report the discovery and pharmacological characterisation of C101248, the first selective small-molecule inhibitor of THIK-1. C101248 showed a concentration-dependent inhibition of both mouse and human THIK-1 (IC50: ∼50 nM) and was inactive against K2P family members TREK-1 and TWIK-2, and Kv2.1. Whole-cell patch-clamp recordings of microglia from mouse hippocampal slices showed that C101248 potently blocked both tonic and ATP-evoked THIK-1 K+ currents. Notably, C101248 had no effect on other constitutively active resting conductance in slices from THIK-1-depleted mice. In isolated microglia, C101248 prevented NLRP3-dependent release of IL-1ß, an effect not seen in THIK-1-depleted microglia. In conclusion, we demonstrated that inhibiting THIK-1 (a microglia specific gene that is upregulated in brains from donors with AD) using a novel selective modulator attenuates the NLRP3-dependent release of IL-1ß from microglia, which suggests that this channel may be a potential therapeutic target for the modulation of neuroinflammation in AD.


Asunto(s)
Enfermedad de Alzheimer , Inflamasomas , Canales de Potasio de Dominio Poro en Tándem , Animales , Humanos , Ratones , Enfermedad de Alzheimer/metabolismo , Encéfalo/metabolismo , Inflamasomas/metabolismo , Microglía , Enfermedades Neuroinflamatorias , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Canales de Potasio de Dominio Poro en Tándem/antagonistas & inhibidores
9.
Nat Commun ; 13(1): 4146, 2022 07 16.
Artículo en Inglés | MEDLINE | ID: mdl-35842429

RESUMEN

Enhancing the removal of aggregate-prone toxic proteins is a rational therapeutic strategy for a number of neurodegenerative diseases, especially Huntington's disease and various spinocerebellar ataxias. Ideally, such approaches should preferentially clear the mutant/misfolded species, while having minimal impact on the stability of wild-type/normally-folded proteins. Furthermore, activation of both ubiquitin-proteasome and autophagy-lysosome routes may be advantageous, as this would allow effective clearance of both monomeric and oligomeric species, the latter which are inaccessible to the proteasome. Here we find that compounds that activate the D1 ATPase activity of VCP/p97 fulfill these requirements. Such effects are seen with small molecule VCP activators like SMER28, which activate autophagosome biogenesis by enhancing interactions of PI3K complex components to increase PI(3)P production, and also accelerate VCP-dependent proteasomal clearance of such substrates. Thus, this mode of VCP activation may be a very attractive target for many neurodegenerative diseases.


Asunto(s)
Adenosina Trifosfatasas , Enfermedades Neurodegenerativas , Proteína que Contiene Valosina , Adenosina Trifosfatasas/metabolismo , Autofagia , Proteínas de Ciclo Celular/metabolismo , Humanos , Enfermedades Neurodegenerativas/genética , Fosfatos de Fosfatidilinositol , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteína que Contiene Valosina/genética , Proteína que Contiene Valosina/metabolismo
10.
Bioorg Med Chem Lett ; 61: 128607, 2022 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-35123006

RESUMEN

We report a significant decrease in transcription of the G protein-coupled receptor GPR39 in striatal neurons of Parkinson's disease patients compared to healthy controls, suggesting that a positive modulator of GPR39 may beneficially impact neuroprotection. To test this notion, we developed various structurally diverse tool molecules. While we elaborated on previously reported starting points, we also performed an in silico screen which led to completely novel pharmacophores. In vitro studies indicated that GPR39 agonism does not have a profound effect on neuroprotection.


Asunto(s)
Pirimidinas/farmacología , Receptores Acoplados a Proteínas G/agonistas , Regulación Alostérica/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Pirimidinas/síntesis química , Pirimidinas/química , Receptores Acoplados a Proteínas G/metabolismo , Relación Estructura-Actividad
11.
Nat Chem Biol ; 17(4): 448-455, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33510452

RESUMEN

Autophagy is an essential cellular process that removes harmful protein species, and autophagy upregulation may be able to protect against neurodegeneration and various pathogens. Here, we have identified the essential protein VCP/p97 (VCP, valosin-containing protein) as a novel regulator of autophagosome biogenesis, where VCP regulates autophagy induction in two ways, both dependent on Beclin-1. Utilizing small-molecule inhibitors of VCP ATPase activity, we show that VCP stabilizes Beclin-1 levels by promoting the deubiquitinase activity of ataxin-3 towards Beclin-1. VCP also regulates the assembly and activity of the Beclin-1-containing phosphatidylinositol-3-kinase (PI3K) complex I, thus regulating the production of PI(3)P, a key signaling lipid responsible for the recruitment of downstream autophagy factors. A decreased level of VCP, or inhibition of its ATPase activity, impairs starvation-induced production of PI(3)P and limits downstream recruitment of WIPI2, ATG16L and LC3, thereby decreasing autophagosome formation, illustrating an important role for VCP in early autophagy initiation.


Asunto(s)
Autofagosomas/metabolismo , Autofagia/fisiología , Proteína que Contiene Valosina/metabolismo , Adenosina Trifosfatasas/metabolismo , Beclina-1/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células HeLa , Humanos , Fosfatos de Fosfatidilinositol/metabolismo , Transducción de Señal , Proteína que Contiene Valosina/fisiología
12.
Cell Rep ; 31(5): 107599, 2020 05 05.
Artículo en Inglés | MEDLINE | ID: mdl-32375049

RESUMEN

Better understanding of the progression of neural stem cells (NSCs) in the developing cerebral cortex is important for modeling neurogenesis and defining the pathogenesis of neuropsychiatric disorders. Here, we use RNA sequencing, cell imaging, and lineage tracing of mouse and human in vitro NSCs and monkey brain sections to model the generation of cortical neuronal fates. We show that conserved signaling mechanisms regulate the acute transition from proliferative NSCs to committed glutamatergic excitatory neurons. As human telencephalic NSCs develop from pluripotency in vitro, they transition through organizer states that spatially pattern the cortex before generating glutamatergic precursor fates. NSCs derived from multiple human pluripotent lines vary in these early patterning states, leading differentially to dorsal or ventral telencephalic fates. This work furthers systematic analyses of the earliest patterning events that generate the major neuronal trajectories of the human telencephalon.


Asunto(s)
Células Madre Embrionarias/metabolismo , Células-Madre Neurales/citología , Neurogénesis/fisiología , Neuronas/metabolismo , Diferenciación Celular/fisiología , Células Cultivadas , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Transducción de Señal/fisiología
13.
Nat Commun ; 11(1): 462, 2020 01 23.
Artículo en Inglés | MEDLINE | ID: mdl-31974374

RESUMEN

Human induced pluripotent stem cells (hiPSCs) are a powerful model of neural differentiation and maturation. We present a hiPSC transcriptomics resource on corticogenesis from 5 iPSC donor and 13 subclonal lines across 9 time points over 5 broad conditions: self-renewal, early neuronal differentiation, neural precursor cells (NPCs), assembled rosettes, and differentiated neuronal cells. We identify widespread changes in the expression of both individual features and global patterns of transcription. We next demonstrate that co-culturing human NPCs with rodent astrocytes results in mutually synergistic maturation, and that cell type-specific expression data can be extracted using only sequencing read alignments without cell sorting. We lastly adapt a previously generated RNA deconvolution approach to single-cell expression data to estimate the relative neuronal maturity of iPSC-derived neuronal cultures and human brain tissue. Using many public datasets, we demonstrate neuronal cultures are maturationally heterogeneous but contain subsets of neurons more mature than previously observed.


Asunto(s)
Diferenciación Celular/genética , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/fisiología , Células-Madre Neurales/fisiología , Transcriptoma , Algoritmos , Animales , Astrocitos/citología , Células Cultivadas , Corteza Cerebral/citología , Técnicas de Cocultivo , Bases de Datos Genéticas , Regulación de la Expresión Génica , Humanos , Modelos Neurológicos , Células-Madre Neurales/citología , Neuronas/citología , Neuronas/fisiología , Ratas
14.
Proc Natl Acad Sci U S A ; 117(1): 677-688, 2020 01 07.
Artículo en Inglés | MEDLINE | ID: mdl-31871190

RESUMEN

A robust body of evidence supports the concept that phosphodiesterase 10A (PDE10A) activity in the basal ganglia orchestrates the control of coordinated movement in human subjects. Although human mutations in the PDE10A gene manifest in hyperkinetic movement disorders that phenocopy many features of early Huntington's disease, characterization of the maladapted molecular mechanisms and aberrant signaling processes that underpin these conditions remains scarce. Recessive mutations in the GAF-A domain have been shown to impair PDE10A function due to the loss of striatal PDE10A protein levels, but here we show that this paucity is caused by irregular intracellular trafficking and increased PDE10A degradation in the cytosolic compartment. In contrast to GAF-A mutants, dominant mutations in the GAF-B domain of PDE10A induce PDE10A misfolding, a common pathological phenotype in many neurodegenerative diseases. These data demonstrate that the function of striatal PDE10A is compromised in disorders where disease-associated mutations trigger a reduction in the fidelity of PDE compartmentalization.


Asunto(s)
Membrana Celular/metabolismo , Enfermedad de Huntington/genética , Neuronas/enzimología , Hidrolasas Diéster Fosfóricas/genética , Dominios Proteicos/genética , Animales , Autofagia/genética , Cuerpo Estriado/citología , Cuerpo Estriado/patología , AMP Cíclico/metabolismo , Embrión de Mamíferos , Células HEK293 , Humanos , Enfermedad de Huntington/patología , Hidrólisis , Isoenzimas/genética , Isoenzimas/metabolismo , Mutación , Neuronas/citología , Técnicas de Placa-Clamp , Hidrolasas Diéster Fosfóricas/metabolismo , Cultivo Primario de Células , Proteolisis , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
15.
Bioorg Med Chem Lett ; 29(15): 1962-1967, 2019 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-31153805

RESUMEN

The TRAF2 and NCK interacting kinase (TNIK) has been proposed to play a role in cytoskeletal organization and synaptic plasticity and has been linked, among others, to neurological disorders. However, target validation efforts for TNIK have been hampered by the limited kinase selectivity of small molecule probes and possible functional compensation in mouse models. Both issues are at least in part due to its close homology to the kinases MINK1 (or MAP4K6) and MAP4K4 (or HGK). As part of our interest in validating TNIK as a therapeutic target for neurological diseases, we set up a panel of biochemical and cellular assays, which are described herein. We then examined the activity of known amino-pyridine-based TNIK inhibitors (1, 3) and prepared structurally very close analogs that lack the ability to inhibit the target. We also developed a structurally orthogonal, naphthyridine-based TNIK inhibitor (9) and an inactive control molecule of the same chemical series. These validated small-molecule probes will enable dissection of the function of TNIK family in the context of human disease biology.


Asunto(s)
Proteínas Serina-Treonina Quinasas/metabolismo , Esquizofrenia/genética , Factor 2 Asociado a Receptor de TNF/metabolismo , Bioensayo , Humanos , Estructura Molecular
16.
Bioorg Med Chem Lett ; 29(1): 83-88, 2019 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-30463802

RESUMEN

We have identified a potent, cell permeable and CNS penetrant class IIa histone deacetylase (HDAC) inhibitor 22, with >500-fold selectivity over class I HDACs (1,2,3) and ∼150-fold selectivity over HDAC8 and the class IIb HDAC6 isoform. Dose escalation pharmacokinetic analysis demonstrated that upon oral administration, compound 22 can reach exposure levels in mouse plasma, muscle and brain in excess of cellular class IIa HDAC IC50 levels for ∼8 h. Given the interest in aberrant class IIa HDAC function for a number of neurodegenerative, neuromuscular, cardiac and oncology indications, compound 22 (also known as CHDI-390576) provides a selective and potent compound to query the role of class IIa HDAC biology, and the impact of class IIa catalytic site occupancy in vitro and in vivo.


Asunto(s)
Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Ácidos Hidroxámicos/farmacología , Animales , Relación Dosis-Respuesta a Droga , Inhibidores de Histona Desacetilasas/síntesis química , Inhibidores de Histona Desacetilasas/química , Humanos , Ácidos Hidroxámicos/síntesis química , Ácidos Hidroxámicos/química , Ratones , Estructura Molecular , Relación Estructura-Actividad
17.
Bioorg Med Chem Lett ; 28(19): 3231-3235, 2018 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-30170942

RESUMEN

Due to increased interest in As(III) S-adenosylmethionine methyltransferase (AS3MT), a search for chemical probes that can help elucidate function was initiated. A homology model was built based on related enzymes, and virtual screening produced 426 potential hits. Evaluation of these compounds in a functional enzymatic assay revealed several modest inhibitors including an O-substituted 2-amino-3-cyano indole scaffold. Two iterations of near neighbor searches revealed compound 5 as a potent inhibitor of AS3MT with good selectivity over representative methyltransferases DOT1L and NSD2 as well as a representative set of diverse receptors. Compound 5 should prove to be a useful tool to investigate the role of AS3MT and a potential starting point for further optimization.


Asunto(s)
Inhibidores Enzimáticos/farmacología , Metiltransferasas/antagonistas & inhibidores , Humanos
18.
J Med Chem ; 61(8): 3491-3502, 2018 04 26.
Artículo en Inglés | MEDLINE | ID: mdl-29617572

RESUMEN

BACE1 is responsible for the first step in APP proteolysis, leading to toxic Aß production, and has been indicated to play a key role in the pathogenesis of Alzheimer's disease. The related isoform BACE2 is thought to be involved in processing of the pigment cell-specific melanocyte protein. To avoid potential effects on pigmentation, we investigated the feasibility for developing isoform-selective BACE1 inhibitors. Cocrystal structures of 47 compounds were analyzed and clustered according to their selectivity profiles. Selective BACE1 inhibitors were found to exhibit two distinct conformational features proximal to the flap and the S3 subpocket. Several new molecules were designed and tested to make use of this observation. The combination of a pyrimidinyl C-ring and a methylcyclohexyl element resulted in lead molecule 28, which exhibited ∼50-fold selectivity. Compared to a nonselective BACE1/2 inhibitor, 28 showed significantly less inhibition of PMEL processing in human melanocytes, indicating good functional selectivity of this inhibitor class.


Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Inhibidores de Proteasas/farmacología , Secretasas de la Proteína Precursora del Amiloide/química , Péptidos beta-Amiloides/metabolismo , Animales , Ácido Aspártico Endopeptidasas/química , Encéfalo/metabolismo , Dominio Catalítico , Perros , Femenino , Humanos , Células de Riñón Canino Madin Darby , Masculino , Ratones Endogámicos C57BL , Estructura Molecular , Oxazoles/síntesis química , Oxazoles/química , Oxazoles/farmacocinética , Oxazoles/farmacología , Fragmentos de Péptidos/metabolismo , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacocinética , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/química , Ratas , Compuestos de Espiro/síntesis química , Compuestos de Espiro/química , Compuestos de Espiro/farmacocinética , Compuestos de Espiro/farmacología , Relación Estructura-Actividad , Antígeno gp100 del Melanoma/metabolismo
19.
J Biol Chem ; 292(52): 21253-21263, 2017 12 29.
Artículo en Inglés | MEDLINE | ID: mdl-29092909

RESUMEN

K+/Cl- cotransporter 2 (KCC2) is selectively expressed in the adult nervous system and allows neurons to maintain low intracellular Cl- levels. Thus, KCC2 activity is an essential prerequisite for fast hyperpolarizing synaptic inhibition mediated by type A γ-aminobutyric acid (GABAA) receptors, which are Cl--permeable, ligand-gated ion channels. Consistent with this, deficits in the activity of KCC2 lead to epilepsy and are also implicated in neurodevelopmental disorders, neuropathic pain, and schizophrenia. Accordingly, there is significant interest in developing activators of KCC2 as therapeutic agents. To provide insights into the cellular processes that determine KCC2 activity, we have investigated the mechanism by which N-ethylmaleimide (NEM) enhances transporter activity using a combination of biochemical and electrophysiological approaches. Our results revealed that, within 15 min, NEM increased cell surface levels of KCC2 and modulated the phosphorylation of key regulatory residues within the large cytoplasmic domain of KCC2 in neurons. More specifically, NEM increased the phosphorylation of serine 940 (Ser-940), whereas it decreased phosphorylation of threonine 1007 (Thr-1007). NEM also reduced with no lysine (WNK) kinase phosphorylation of Ste20-related proline/alanine-rich kinase (SPAK), a kinase that directly phosphorylates KCC2 at residue Thr-1007. Mutational analysis revealed that Thr-1007 dephosphorylation mediated the effects of NEM on KCC2 activity. Collectively, our results suggest that compounds that either increase the surface stability of KCC2 or reduce Thr-1007 phosphorylation may be of use as enhancers of KCC2 activity.


Asunto(s)
Etilmaleimida/metabolismo , Simportadores/metabolismo , Animales , Membrana Celular/metabolismo , Embrión de Mamíferos , Humanos , Moduladores del Transporte de Membrana/metabolismo , Neuronas/metabolismo , Fosforilación/fisiología , Ratas , Ratas Sprague-Dawley , Receptores de GABA/metabolismo , Simportadores/fisiología , Cotransportadores de K Cl
20.
Nat Chem Biol ; 12(6): 444-51, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-27110679

RESUMEN

Usher syndrome type III (USH3), characterized by progressive deafness, variable balance disorder and blindness, is caused by destabilizing mutations in the gene encoding the clarin-1 (CLRN1) protein. Here we report a new strategy to mitigate hearing loss associated with a common USH3 mutation CLRN1(N48K) that involves cell-based high-throughput screening of small molecules capable of stabilizing CLRN1(N48K), followed by a secondary screening to eliminate general proteasome inhibitors, and finally an iterative process to optimize structure-activity relationships. This resulted in the identification of BioFocus 844 (BF844). To test the efficacy of BF844, we developed a mouse model that mimicked the progressive hearing loss associated with USH3. BF844 effectively attenuated progressive hearing loss and prevented deafness in this model. Because the CLRN1(N48K) mutation causes both hearing and vision loss, BF844 could in principle prevent both sensory deficiencies in patients with USH3. Moreover, the strategy described here could help identify drugs for other protein-destabilizing monogenic disorders.


Asunto(s)
Modelos Animales de Enfermedad , Proteínas de la Membrana/antagonistas & inhibidores , Pirazoles/farmacología , Piridazinas/farmacología , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Síndromes de Usher/tratamiento farmacológico , Animales , Ensayos Analíticos de Alto Rendimiento , Humanos , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Estructura Molecular , Pirazoles/síntesis química , Pirazoles/química , Pirazoles/uso terapéutico , Piridazinas/síntesis química , Piridazinas/química , Piridazinas/uso terapéutico , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-Actividad , Síndromes de Usher/genética
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