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1.
Sci Adv ; 7(16)2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33863724

RESUMO

Several important drug targets, e.g., ion channels and G protein-coupled receptors, are extremely difficult to approach with current antibody technologies. To address these targets classes, we explored kinetically controlled proteases as structural dynamics-sensitive druggability probes in native-state and disease-relevant proteins. By using low-Reynolds number flows, such that a single or a few protease incisions are made, we could identify antibody binding sites (epitopes) that were translated into short-sequence antigens for antibody production. We obtained molecular-level information of the epitope-paratope region and could produce high-affinity antibodies with programmed pharmacological function against difficult-to-drug targets. We demonstrate the first stimulus-selective monoclonal antibodies targeting the transient receptor potential vanilloid 1 (TRPV1) channel, a clinically validated pain target widely considered undruggable with antibodies, and apoptosis-inducing antibodies selectively mediating cytotoxicity in KRAS-mutated cells. It is our hope that this platform will widen the scope of antibody therapeutics for the benefit of patients.


Assuntos
Anticorpos Monoclonais , Antígenos , Anticorpos Monoclonais/química , Sítios de Ligação de Anticorpos , Epitopos , Humanos
2.
Elife ; 72018 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-30417826

RESUMO

Amyloid-ß peptide (Aß) forms plaques in Alzheimer's disease (AD) and is responsible for early cognitive deficits in AD patients. Advancing cognitive decline is accompanied by progressive impairment of cognition-relevant EEG patterns such as gamma oscillations. The endocannabinoid anandamide, a TrpV1-receptor agonist, reverses hippocampal damage and memory impairment in rodents and protects neurons from Aß-induced cytotoxic effects. Here, we investigate a restorative role of TrpV1-receptor activation against Aß-induced degradation of hippocampal neuron function and gamma oscillations. We found that the TrpV1-receptor agonist capsaicin rescues Aß-induced degradation of hippocampal gamma oscillations by reversing both the desynchronization of AP firing in CA3 pyramidal cells and the shift in excitatory/inhibitory current balance. This rescue effect is TrpV1-receptor-dependent since it was absent in TrpV1 knockout mice or in the presence of the TrpV1-receptor antagonist capsazepine. Our findings provide novel insight into the network mechanisms underlying cognitive decline in AD and suggest TrpV1 activation as a novel therapeutic target.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Região CA3 Hipocampal/metabolismo , Capsaicina/farmacologia , Ritmo Gama/efeitos dos fármacos , Células Piramidais/metabolismo , Canais de Cátion TRPV/genética , Potenciais de Ação/fisiologia , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/farmacologia , Animais , Região CA3 Hipocampal/citologia , Região CA3 Hipocampal/efeitos dos fármacos , Capsaicina/análogos & derivados , Capsaicina/antagonistas & inibidores , Cognição/efeitos dos fármacos , Cognição/fisiologia , Eletrodos Implantados , Ritmo Gama/fisiologia , Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microtomia , Modelos Biológicos , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/farmacologia , Células Piramidais/citologia , Células Piramidais/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Canais de Cátion TRPV/deficiência , Técnicas de Cultura de Tecidos
3.
Neuropharmacology ; 118: 13-25, 2017 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-28274820

RESUMO

Histamine is an aminergic neurotransmitter, which regulates wakefulness, arousal and attention in the central nervous system. Histamine receptors have been the target of efforts to develop pro-cognitive drugs to treat disorders such as Alzheimer's disease and schizophrenia. Cognitive functions including attention are closely associated with gamma oscillations, a rhythmical electrical activity pattern in the 30-80 Hz range, which depends on the synchronized activity of excitatory pyramidal cells and inhibitory fast-spiking interneurons. We set out to explore whether histamine has a role in promoting gamma oscillations in the hippocampus. Using in-situ hybridization we demonstrate that histamine receptor subtypes 1, 2 and 3 are expressed in stratum pyramidale of area CA3 in rats. We show that both pyramidal cells and fast-spiking interneurons depolarize and increase action potential firing in response to histamine in vitro. The activation of histamine receptors generates dose-dependent, transient gamma oscillations in area CA3 of the hippocampus - the locus of the gamma rhythm generator. We also demonstrate that this histamine effect is independent of muscarinic receptors. Using specific antagonists we provide evidence that histamine gamma rhythmogenesis specifically depends on the H1 receptor. Histamine also depolarized both pyramidal cells and fast-spiking interneurons and increased membrane resistance in pyramidal cells. The increased membrane resistance is potentially mediated by the inhibition of potassium channels because application of the KCNQ channel opener ICA110381 abolished the oscillations. Taken together our data demonstrate a novel and physiological mechanism for generating gamma oscillations in hippocampus and suggest a role for KCNQ channels in this cognition-relevant brain activity.


Assuntos
Ritmo Gama/efeitos dos fármacos , Hipocampo , Histamínicos/farmacologia , Histamina/farmacologia , Canais de Potássio KCNQ/metabolismo , Receptores Histamínicos/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Benzamidas/farmacologia , Glutamato Descarboxilase/metabolismo , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Técnicas In Vitro , Masculino , Moduladores de Transporte de Membrana/farmacologia , Rede Nervosa/efeitos dos fármacos , Rede Nervosa/fisiologia , Neurônios/efeitos dos fármacos , Neurotransmissores/farmacologia , Piridinas/farmacologia , Ratos , Ratos Sprague-Dawley , Ubiquitina Tiolesterase/metabolismo
4.
J Neurosci Res ; 95(11): 2195-2206, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28150440

RESUMO

Metabolic abnormalities found in epileptogenic tissue provide considerable evidence of brain hypometabolism, while major risk factors for acquired epilepsy all share brain hypometabolism as one common outcome, suggesting that a breakdown of brain energy homeostasis may actually precede epileptogenesis. However, a causal link between deficient brain energy metabolism and epilepsy initiation has not been yet established. To address this issue we developed an in vivo model of chronic energy hypometabolism by daily intracerebroventricular (i.c.v.) injection of the nonmetabolizable glucose analog 2-deoxy-D-glucose (2-DG) and also investigated acute effects of 2-DG on the cellular level. In hippocampal slices, acute glycolysis inhibition by 2-DG (by about 35%) led to contrasting effects on the network: a downregulation of excitatory synaptic transmission together with a depolarization of neuronal resting potential and a decreased drive of inhibitory transmission. Therefore, the potential acute effect of 2-DG on network excitability depends on the balance between these opposing pre- and postsynaptic changes. In vivo, we found that chronic 2-DG i.c.v. application (estimated transient inhibition of brain glycolysis under 14%) for a period of 4 weeks induced epileptiform activity in initially healthy male rats. Our results suggest that chronic inhibition of brain energy metabolism, characteristics of the well-established risk factors of acquired epilepsy, and specifically a reduction in glucose utilization (typically observed in epileptic patients) can initiate epileptogenesis. © 2017 Wiley Periodicals, Inc.


Assuntos
Encéfalo/metabolismo , Encéfalo/fisiopatologia , Metabolismo Energético/fisiologia , Epilepsia/metabolismo , Epilepsia/fisiopatologia , Glicólise/fisiologia , Animais , Encéfalo/efeitos dos fármacos , Desoxiglucose/administração & dosagem , Metabolismo Energético/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Injeções Intraventriculares , Masculino , Camundongos , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-Dawley
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