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1.
Sci Rep ; 11(1): 17977, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34504195

RESUMO

Huntington's disease (HD) is caused by a CAG trinucleotide repeat expansion in the first exon of the huntingtin (HTT) gene coding for the huntingtin (HTT) protein. The misfolding and consequential aggregation of CAG-expanded mutant HTT (mHTT) underpin HD pathology. Our interest in the life cycle of HTT led us to consider the development of high-affinity small-molecule binders of HTT oligomerized/amyloid-containing species that could serve as either cellular and in vivo imaging tools or potential therapeutic agents. We recently reported the development of PET tracers CHDI-180 and CHDI-626 as suitable for imaging mHTT aggregates, and here we present an in-depth pharmacological investigation of their binding characteristics. We have implemented an array of in vitro and ex vivo radiometric binding assays using recombinant HTT, brain homogenate-derived HTT aggregates, and brain sections from mouse HD models and humans post-mortem to investigate binding affinities and selectivity against other pathological proteins from indications such as Alzheimer's disease and spinocerebellar ataxia 1. Radioligand binding assays and autoradiography studies using brain homogenates and tissue sections from HD mouse models showed that CHDI-180 and CHDI-626 specifically bind mHTT aggregates that accumulate with age and disease progression. Finally, we characterized CHDI-180 and CHDI-626 regarding their off-target selectivity and binding affinity to beta amyloid plaques in brain sections and homogenates from Alzheimer's disease patients.


Assuntos
Proteína Huntingtina/metabolismo , Doença de Huntington/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Agregados Proteicos/genética , Agregação Patológica de Proteínas/diagnóstico por imagem , Compostos Radiofarmacêuticos/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Autorradiografia/métodos , Encéfalo/metabolismo , Modelos Animais de Doenças , Humanos , Proteína Huntingtina/genética , Doença de Huntington/patologia , Imuno-Histoquímica/métodos , Camundongos , Camundongos Transgênicos , Radioisótopos de Nitrogênio/metabolismo , Traçadores Radioativos , Ensaio Radioligante/métodos , Proteínas Recombinantes/metabolismo
2.
Biomolecules ; 11(8)2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34439801

RESUMO

The potential, multifaceted therapeutic profile of cannabidiol (CBD), a major constituent derived from the Cannabis sativa plant, covers a wide range of neurological and psychiatric disorders, ranging from anxiety to pediatric epilepsy and drug addiction. However, the molecular targets responsible for these effects have been only partially identified. In this view, the involvement of the orexin system, the key regulator in arousal and the sleep/wake cycle, and in motivation and reward processes, including drug addiction, prompted us to explore, using computational and experimental approaches, the possibility that CBD could act as a ligand of orexin receptors, orexin 1 receptor of type 1 (OX1R) and type 2 (OX2R). Ligand-binding assays showed that CBD is a selective ligand of OX1R in the low micromolar range (Ki 1.58 ± 0.2 µM) while in vitro functional assays, carried out by intracellular calcium imaging and mobilization assays, showed that CBD acts as an antagonist at this receptor. Finally, the putative binding mode of CBD has been inferred by molecular docking and molecular dynamics simulations and its selectivity toward the OX1R subtype rationalized at the molecular level. This study provides the first evidence that CBD acts as an OX1R antagonist, supporting its potential use in addictive disorders and/or body weight regulation.


Assuntos
Ansiolíticos/farmacologia , Anticonvulsivantes/farmacologia , Canabidiol/farmacologia , Receptores de Orexina/química , Orexinas/química , Animais , Ansiolíticos/química , Ansiolíticos/metabolismo , Anticonvulsivantes/química , Anticonvulsivantes/metabolismo , Sítios de Ligação , Células CHO , Cálcio/metabolismo , Canabidiol/química , Canabidiol/metabolismo , Cricetulus , Expressão Gênica , Humanos , Cinética , Simulação de Acoplamento Molecular , Imagem Molecular , Antagonistas dos Receptores de Orexina , Receptores de Orexina/genética , Receptores de Orexina/metabolismo , Orexinas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Ensaio Radioligante , Transgenes
3.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360878

RESUMO

Sigma-1 receptor (S1R) is an intracellular, multi-functional, ligand operated protein that also acts as a chaperone. It is considered as a pluripotent drug target in several pathologies. The publication of agonist and antagonist bound receptor structures has paved the way for receptor-based in silico drug design. However, recent studies on this subject payed no attention to the structural differences of agonist and antagonist binding. In this work, we have developed a new ensemble docking-based virtual screening protocol utilizing both agonist and antagonist bound S1R structures. This protocol was used to screen our in-house compound library. The S1R binding affinities of the 40 highest ranked compounds were measured in competitive radioligand binding assays and the sigma-2 receptor (S2R) affinities of the best S1R binders were also determined. This way three novel high affinity S1R ligands were identified and one of them exhibited a notable S1R/S2R selectivity.


Assuntos
Isoxazóis/química , Simulação de Acoplamento Molecular/métodos , Pentazocina/química , Piridinas/química , Receptores sigma/química , Sítios de Ligação , Interações Hidrofóbicas e Hidrofílicas , Isoxazóis/análise , Isoxazóis/farmacologia , Ligantes , Estrutura Molecular , Pentazocina/análise , Pentazocina/farmacologia , Ligação Proteica , Piridinas/análise , Piridinas/farmacologia , Ensaio Radioligante/métodos , Receptores sigma/agonistas , Receptores sigma/análise , Receptores sigma/antagonistas & inibidores
4.
Int J Mol Sci ; 22(9)2021 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-34062920

RESUMO

Radioligand therapy targeting the prostate-specific membrane antigen (PSMA) is rapidly evolving as a promising treatment for metastatic castration-resistant prostate cancer. The PSMA-targeting ligand p-SCN-Bn-TCMC-PSMA (NG001) labelled with 212Pb efficiently targets PSMA-positive cells in vitro and in vivo. The aim of this preclinical study was to evaluate the therapeutic potential of 212Pb-NG001 in multicellular tumour spheroid and mouse models of prostate cancer. The cytotoxic effect of 212Pb-NG001 was tested in human prostate C4-2 spheroids. Biodistribution at various time points and therapeutic effects of different activities of the radioligand were investigated in male athymic nude mice bearing C4-2 tumours, while long-term toxicity was studied in immunocompetent BALB/c mice. The radioligand induced a selective cytotoxic effect in spheroids at activity concentrations of 3-10 kBq/mL. In mice, the radioligand accumulated rapidly in tumours and was retained over 24 h, while it rapidly cleared from nontargeted tissues. Treatment with 0.25, 0.30 or 0.40 MBq of 212Pb-NG001 significantly inhibited tumour growth and improved median survival with therapeutic indexes of 1.5, 2.3 and 2.7, respectively. In BALB/c mice, no signs of long-term radiation toxicity were observed at activities of 0.05 and 0.33 MBq. The obtained results warrant clinical studies to evaluate the biodistribution, therapeutic efficacy and toxicity of 212Pb-NG001.


Assuntos
Neoplasias de Próstata Resistentes à Castração/radioterapia , Ensaio Radioligante , Compostos Radiofarmacêuticos/farmacologia , Esferoides Celulares/efeitos da radiação , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Xenoenxertos , Humanos , Chumbo/farmacologia , Ligantes , Masculino , Camundongos , Metástase Neoplásica , Neoplasias de Próstata Resistentes à Castração/patologia , Radioisótopos/farmacologia , Esferoides Celulares/patologia , Distribuição Tecidual/efeitos da radiação
5.
Anal Bioanal Chem ; 413(17): 4531-4543, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34050775

RESUMO

We adapted a radioligand receptor binding assay for measuring insulin levels in unknown samples. The assay enables rapid and accurate determination of insulin concentrations in experimental samples, such as from insulin-secreting cells. The principle of the method is based on the binding competition of insulin in a measured sample with a radiolabeled insulin for insulin receptor (IR) in IM-9 cells. Both key components, radiolabeled insulin and IM-9 cells, are commercially available. The IR binding assay was used to determine unknown amounts of insulin secreted by MIN6 ß cell line after stimulation with glucose, arginine, ornithine, dopamine, and serotonin. The experimental data obtained by the IR binding assay were compared to the results determined by RIA kits and both methods showed a very good agreement of results. We observed the stimulation of glucose-induced insulin secretion from MIN6 cells by arginine, weaker stimulation by ornithine, but inhibitory effects of dopamine. Serotonin effects were either stimulatory or inhibitory, depending on the concentration of serotonin used. The results will require further investigation. The study also clearly revealed advantages of the IR binding assay that allows the measuring of a higher throughput of measured samples, with a broader range of concentrations than in the case of RIA kits. The IR binding assay can provide an alternative to standard RIA and ELISA assays for the determination of insulin levels in experimental samples and can be especially useful in scientific laboratories studying insulin production and secretion by ß cells and searching for new modulators of insulin secretion.


Assuntos
Secreção de Insulina , Insulina/análise , Insulina/metabolismo , Animais , Arginina/metabolismo , Linhagem Celular , Dopamina/metabolismo , Glucose/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Camundongos , Ornitina/metabolismo , Radioimunoensaio/métodos , Ensaio Radioligante/métodos , Ratos , Ratos Wistar , Serotonina/metabolismo
6.
Front Immunol ; 12: 568959, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34054793

RESUMO

Molecular imaging using PET/CT or PET/MRI has evolved from an experimental imaging modality at its inception in 1972 to an integral component of diagnostic procedures in oncology, and, to lesser extent, in cardiology and neurology, by successfully offering in-vivo imaging and quantitation of key pathophysiological targets or molecular signatures, such as glucose metabolism in cancerous disease. Apart from metabolism probes, novel radiolabeled peptide and antibody PET tracers, including radiolabeled monoclonal antibodies (mAbs) have entered the clinical arena, providing the in-vivo capability to collect target-specific quantitative in-vivo data on cellular and molecular pathomechanisms on a whole-body scale, and eventually, extract imaging biomarkers possibly serving as prognostic indicators. The success of molecular imaging in mapping disease severity on a whole-body scale, and directing targeted therapies in oncology possibly could translate to the management of Coronavirus Disease 2019 (COVID-19), by identifying, localizing, and quantifying involvement of different immune mediated responses to the infection with SARS-COV2 during the course of acute infection and possible, chronic courses with long-term effects on specific organs. The authors summarize current knowledge for medical imaging in COVID-19 in general with a focus on molecular imaging technology and provide a perspective for immunologists interested in molecular imaging research using validated and immediately available molecular probes, as well as possible future targets, highlighting key targets for tailored treatment approaches as brought up by key opinion leaders.


Assuntos
COVID-19/diagnóstico , Imagem Molecular/métodos , RNA Viral/análise , SARS-CoV-2/fisiologia , Animais , Testes Diagnósticos de Rotina , Humanos , Imageamento por Ressonância Magnética , Tomografia por Emissão de Pósitrons , Valor Preditivo dos Testes , Prognóstico , Ensaio Radioligante
7.
Sci Rep ; 11(1): 7700, 2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33833333

RESUMO

GABAA-α5 subunit-containing receptors have been shown to play a key modulatory role in cognition and represent a promising drug target for cognitive dysfunction, as well as other disorders. Here we report on the preclinical and early clinical profile of a novel GABAA-α5 selective negative allosteric modulator (NAM), basmisanil, which progressed into Phase II trials for intellectual disability in Down syndrome and cognitive impairment associated with schizophrenia. Preclinical pharmacology studies showed that basmisanil is the most selective GABAA-α5 receptor NAM described so far. Basmisanil bound to recombinant human GABAA-α5 receptors with 5 nM affinity and more than 90-fold selectivity versus α1, α2, and α3 subunit-containing receptors. Moreover, basmisanil inhibited GABA-induced currents at GABAA-α5 yet had little or no effect at the other receptor subtypes. An in vivo occupancy study in rats showed dose-dependent target engagement and was utilized to establish the plasma exposure to receptor occupancy relationship. At estimated receptor occupancies between 30 and 65% basmisanil attenuated diazepam-induced spatial learning impairment in rats (Morris water maze), improved executive function in non-human primates (object retrieval), without showing anxiogenic or proconvulsant effects in rats. During the Phase I open-label studies, basmisanil showed good safety and tolerability in healthy volunteers at maximum GABAA-α5 receptor occupancy as confirmed by PET analysis with the tracer [11C]-Ro 15-4513. An exploratory EEG study provided evidence for functional activity of basmisanil in human brain. Therefore, these preclinical and early clinical studies show that basmisanil has an ideal profile to investigate potential clinical benefits of GABAA-α5 receptor negative modulation.


Assuntos
Agonistas de Receptores de GABA-A/farmacologia , Receptores de GABA-A/efeitos dos fármacos , Regulação Alostérica , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Feminino , Células HEK293 , Voluntários Saudáveis , Humanos , Aprendizagem/efeitos dos fármacos , Macaca fascicularis , Tomografia por Emissão de Pósitrons , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Receptores de GABA-A/química , Receptores de GABA-A/metabolismo , Xenopus laevis
8.
J Cereb Blood Flow Metab ; 41(10): 2571-2582, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-33853405

RESUMO

Previous work found that [11C]deschloroclozapine ([11C]DCZ) is superior to [11C]clozapine ([11C]CLZ) for imaging Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). This study used PET to quantitatively and separately measure the signal from transfected receptors, endogenous receptors/targets, and non-displaceable binding in other brain regions to better understand this superiority. A genetically-modified muscarinic type-4 human receptor (hM4Di) was injected into the right amygdala of a male rhesus macaque. [11C]DCZ and [11C]CLZ PET scans were conducted 2-24 months later. Uptake was quantified relative to the concentration of parent radioligand in arterial plasma at baseline (n = 3 scans/radioligand) and after receptor blockade (n = 3 scans/radioligand). Both radioligands had greater uptake in the transfected region and displaceable uptake in other brain regions. Displaceable uptake was not uniformly distributed, perhaps representing off-target binding to endogenous receptor(s). After correction, [11C]DCZ signal was 19% of that for [11C]CLZ, and background uptake was 10% of that for [11C]CLZ. Despite stronger [11C]CLZ binding, the signal-to-background ratio for [11C]DCZ was almost two-fold greater than for [11C]CLZ. Both radioligands had comparable DREADD selectivity. All reference tissue models underestimated signal-to-background ratio in the transfected region by 40%-50% for both radioligands. Thus, the greater signal-to-background ratio of [11C]DCZ was due to its lower background uptake.


Assuntos
Clozapina/uso terapêutico , Tomografia por Emissão de Pósitrons/métodos , Ensaio Radioligante/métodos , Animais , Colinérgicos/metabolismo , Clozapina/farmacologia , Macaca mulatta , Masculino , Piperazinas/farmacologia , Transfecção
9.
Circ Heart Fail ; 14(3): e007351, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33663236

RESUMO

BACKGROUND: New heart failure therapies that safely augment cardiac contractility and output are needed. Previous apelin peptide studies have highlighted the potential for APJ (apelin receptor) agonism to enhance cardiac function in heart failure. However, apelin's short half-life limits its therapeutic utility. Here, we describe the preclinical characterization of a novel, orally bioavailable APJ agonist, BMS-986224. METHODS: BMS-986224 pharmacology was compared with (Pyr1) apelin-13 using radio ligand binding and signaling pathway assays downstream of APJ (cAMP, phosphorylated ERK [extracellular signal-regulated kinase], bioluminescence resonance energy transfer-based G-protein assays, ß-arrestin recruitment, and receptor internalization). Acute effects on cardiac function were studied in anesthetized instrumented rats. Chronic effects of BMS-986224 were assessed echocardiographically in the RHR (renal hypertensive rat) model of cardiac hypertrophy and decreased cardiac output. RESULTS: BMS-986224 was a potent (Kd=0.3 nmol/L) and selective APJ agonist, exhibiting similar receptor binding and signaling profile to (Pyr1) apelin-13. G-protein signaling assays in human embryonic kidney 293 cells and human cardiomyocytes confirmed this and demonstrated a lack of signaling bias relative to (Pyr1) apelin-13. In anesthetized instrumented rats, short-term BMS-986224 infusion increased cardiac output (10%-15%) without affecting heart rate, which was similar to (Pyr1) apelin-13 but differentiated from dobutamine. Subcutaneous and oral BMS-986224 administration in the RHR model increased stroke volume and cardiac output to levels seen in healthy animals but without preventing cardiac hypertrophy and fibrosis, effects differentiated from enalapril. CONCLUSIONS: We identify a novel, potent, and orally bioavailable nonpeptidic APJ agonist that closely recapitulates the signaling properties of (Pyr1) apelin-13. We show that oral APJ agonist administration induces a sustained increase in cardiac output in the cardiac disease setting and exhibits a differentiated profile from the renin-angiotensin system inhibitor enalapril, supporting further clinical evaluation of BMS-986224 in heart failure.


Assuntos
Receptores de Apelina/agonistas , Débito Cardíaco/efeitos dos fármacos , Insuficiência Cardíaca/fisiopatologia , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Volume Sistólico/efeitos dos fármacos , Animais , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Células CHO , Cricetulus , Cães , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HEK293 , Haplorrinos , Humanos , Técnicas In Vitro , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fosforilação , Ensaio Radioligante , Ratos , Trítio , Pressão Ventricular/efeitos dos fármacos , beta-Arrestinas/efeitos dos fármacos , beta-Arrestinas/metabolismo
10.
Cell Physiol Biochem ; 55(S3): 1-13, 2021 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-33656308

RESUMO

BACKGROUND/AIMS: The NMDA receptor plays a key role in the pathogenesis of neurodegenerative disorders including Alzheimer's and Huntington's disease, as well as depression and drug or alcohol dependence. Due to its participation in these pathologies, the development of selective modulators for this ion channel is a promising strategy for rational drug therapy. The prototypical negative allosteric modulator ifenprodil inhibits selectively GluN2B subunit containing NMDA receptors. It was conformationally restricted as 2-methyl-3-(4-phenylbutyl)-2,3,4,5-tetrahydro-1H-3-benzazepine-1,7-diol, which showed high GluN2B affinity and inhibitory activity. For a better understanding of the relevance of the functional groups and structural elements, the substituents of this 3-benzazepine were removed successively (deconstruction). Then, additional structural elements were introduced (reconstruction) with the aim to analyze, which additional modifications were tolerated by the GluN2B receptor. METHODS: The GluN2B affinity was recorded in radioligand receptor binding studies with the radioligand [3H]ifenprodil. The activity of the ligands was determined in two-electrode voltage clamp experiments using Xenopus laevis oocytes transfected with cRNA encoding the GluN1-1a and GluN2B subunits of the NMDA receptor. Docking studies showed the crucial interactions with the NMDA receptor protein. RESULTS: The deconstruction approach showed that removal of the methyl moiety and the phenolic OH moiety in 7-positon resulted in almost the same GluN2B affinity as the parent 3-benzazepine. A considerably reduced GluN2B affinity was found for the 3-benzazepine without further substituents. However, removal of one or both OH moieties led to considerably reduced NMDA receptor inhibition. Introduction of a NO2 moiety or bioisosteric replacement of the phenol by a benzoxazolone resulted in comparable GluN2B affinity, but almost complete loss of inhibitory activity. An O-atom, a carbonyl moiety or a F-atom in the tetramethylene spacer led to 6-7-fold reduced ion channel inhibition. CONCLUSION: The results reveal an uncoupling of affinity and activity for the tested 3-benzazepines. Strong inhibition of [3H]ifenprodil binding by a test compound does not necessarily translate into strong inhibition of the ion flux through the NMDA receptor associated ion channel. 3-(4-Phenylbutyl)-2,3,4,5-tetrahydro-1H-3-benzazepine- 1,7-diol (WMS-1410) shows high GluN2B affinity and strong inhibition of the ion channel. Deconstruction by removal of one or both OH moieties reduced the inhibitory activity proving the importance of the OH groups for ion channel blockade. Reconstruction by introduction of various structural elements into the left benzene ring or into the tetramethylene spacer reduced the NMDA receptor inhibition. It can be concluded that these modifications are not able to translate binding into inhibition.


Assuntos
Antagonistas Adrenérgicos alfa/farmacologia , Benzazepinas/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Piperidinas/farmacologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Antagonistas Adrenérgicos alfa/síntese química , Regulação Alostérica , Animais , Benzazepinas/síntese química , Benzoxazóis/química , Sítios de Ligação , Antagonistas de Aminoácidos Excitatórios/síntese química , Humanos , Cinética , Simulação de Acoplamento Molecular , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Piperidinas/síntese química , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Ensaio Radioligante , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Trítio , Xenopus laevis
11.
J Cereb Blood Flow Metab ; 41(9): 2410-2422, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33757319

RESUMO

Colony-stimulating factor 1 receptor (CSF1R) is a specific biomarker for microglia. In this study, we developed a novel PET radioligand for CSF1R, 11C-GW2580, and compared it to a reported CSF1R tracer, 11C-CPPC, in mouse models of acute and chronic neuroinflammation and a rhesus monkey. Dynamic 11C-GW2580- and 11C-CPPC-PET images were quantified by reference tissue-based models and standardized uptake value ratio. Both tracers exhibited increased uptake in the lesioned striata of lipopolysaccharide-injected mice and in the forebrains of AppNL-G-F/NL-G-F-knock-in mice, spatially in agreement with an increased 18-kDa translocator protein radioligand retention. Moreover, 11C-GW2580 captured changes in CSF1R availability more sensitively than 11C-CPPC, with a larger dynamic range and a smaller inter-individual variability, in these model animals. PET imaging of CSF1R in a rhesus monkey displayed moderate-to-high tracer retention in the brain at baseline. Homologous blocker (i. e. unlabeled tracer) treatment reduced the uptake of 11C-GW2580 by ∼30% in all examined brain regions except for centrum semi-ovale white matter, but did not affect the retention of 11C-CPPC. In summary, our results demonstrated that 11C-GW2580-PET captured inflammatory microgliosis in the mouse brain with higher sensitivity than a reported radioligand, and displayed saturable binding in the monkey brain, potentially providing an imaging-based quantitative biomarker for reactive microgliosis.


Assuntos
Inflamação/diagnóstico por imagem , Fator Estimulador de Colônias de Macrófagos/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Ensaio Radioligante/métodos , Doença Aguda , Animais , Modelos Animais de Doenças , Inflamação/patologia , Macaca mulatta , Camundongos
12.
J Nat Prod ; 84(2): 382-394, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33596384

RESUMO

Strychnine is the prototypic antagonist of glycine receptors, a family of pentameric ligand-gated ion channels. Recent high-resolution structures of homomeric glycine receptors have confirmed the presence of five orthosteric binding sites located in the extracellular subunit interfaces of the receptor complex that are targeted by strychnine. Here, we report the synthesis and extensive pharmacological evaluation of bivalent ligands composed of two strychnine pharmacophores connected by appropriate spacers optimized toward simultaneous binding to two adjacent orthosteric sites of homomeric α1 glycine receptors. In all bivalent ligands, the two strychnine units were linked through C-2 by amide spacers of various lengths ranging from 6 to 69 atoms. Characterization of the compounds in two functional assays and in a radioligand binding assay indicated that compound 11a, with a spacer consisting of 57 atoms, may be capable of bridging the homomeric α1 GlyRs by simultaneous occupation of two adjacent strychnine-binding sites. The findings are supported by docking experiments to the crystal structure of the homomeric glycine receptor. Based on its unique binding mode, its relatively high binding affinity and antagonist potency, and its slow binding kinetics, the bivalent strychnine analogue 11a could be a valuable tool to study the functional properties of glycine receptors.


Assuntos
Receptores de Glicina/antagonistas & inibidores , Estricnina/análogos & derivados , Sítios de Ligação , Humanos , Ligantes , Simulação de Acoplamento Molecular , Estrutura Terciária de Proteína , Ensaio Radioligante
13.
Br J Anaesth ; 126(1): 238-244, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33036760

RESUMO

BACKGROUND: The molecular actions underlying the clinical effects of inhaled anaesthetics such as sevoflurane and isoflurane are not fully understood. Unexpected observations in positron emission tomography (PET) studies with [11C]AZD9272, a metabotropic glutamate receptor 5 (mGluR5) radioligand with possible affinity for monoamine oxidase-B (MAO-B), suggest that its binding is sensitive to anaesthesia with sevoflurane. The objective of the present study was to assess the effects of sevoflurane anaesthesia on the binding of [11C]AZD9272 and of [11C]L-deprenyl-D2, a radioligand selective for MAO-B in non-human primates (NHPs). METHODS: Altogether, 12 PET measurements were conducted with a high-resolution research tomograph using the ligands [11C]AZD9272 or [11C]L-deprenyl-D2 in six cynomolgus monkeys anaesthetised with sevoflurane or ketamine/xylazine. RESULTS: The specific binding of [11C]AZD9272 and [11C]L-deprenyl-D2 was markedly reduced during anaesthesia with sevoflurane compared with ketamine/xylazine. The reduction was 80-90% (n=3) for [11C]AZD9272 and 77-80% (n=3) for [11C]L-deprenyl-D2. CONCLUSIONS: Sevoflurane anaesthesia inhibited radioligand binding to MAO-B in the primate brain. The observation of lower MAO-B binding at clinically relevant concentrations of sevoflurane warrants further exploration of the potential role of MAO-B related mechanisms in regulation of systemic blood pressure during anaesthesia.


Assuntos
Anestésicos Inalatórios/farmacologia , Encéfalo/efeitos dos fármacos , Monoaminoxidase/efeitos dos fármacos , Sevoflurano/farmacologia , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Macaca fascicularis , Modelos Animais , Monoaminoxidase/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Ensaio Radioligante/métodos
14.
Neuroimage ; 226: 117523, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33144221

RESUMO

Dopamine D2 receptors (D2-R) in extrastriatal brain regions are of high interest for research in a wide range of psychiatric and neurologic disorders. Pharmacological competition studies and test-retest experiments have shown high validity and reliability of the positron emission tomography (PET) radioligand [11C]FLB 457 for D2-R quantification in extrastriatal brain regions. However, this radioligand is not available at most research centers. Instead, the medium affinity radioligand [11C]raclopride, which has been extensively validated for quantification of D2-R in the high-density region striatum, has been applied also in studies on extrastriatal D2-R. Recently, the validity of this approach has been questioned by observations of low occupancy of [11C]raclopride in extrastriatal regions in a pharmacological competition study with quetiapine. Here, we utilise a data set of 16 healthy control subjects examined with both [11C]raclopride and [11C]FLB 457 to assess the correlation in binding potential (BPND) in extrastriatal brain regions. BPND was quantified using the simplified reference tissue model with cerebellum as reference region. The rank order of mean regional BPND values were similar for both radioligands, and corresponded to previously reported data, both post-mortem and using PET. Nevertheless, weak to moderate within-subject correlations were observed between [11C]raclopride and [11C]FLB 457 BPND extrastriatally (Pearson's R: 0.30-0.56), in contrast to very strong correlations between repeated [11C]FLB 457 measurements (Pearson's R: 0.82-0.98). In comparison, correlations between repeated [11C]raclopride measurements were low to moderate (Pearson's R: 0.28-0.75). These results are likely related to low signal to noise ratio of [11C]raclopride in extrastriatal brain regions, and further strengthen the recommendation that extrastriatal D2-R measures obtained with [11C]raclopride should be interpreted with caution.


Assuntos
Mapeamento Encefálico/métodos , Encéfalo/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos/metabolismo , Receptores de Dopamina D2/análise , Radioisótopos de Carbono/metabolismo , Radioisótopos de Carbono/farmacologia , Antagonistas de Dopamina/metabolismo , Antagonistas de Dopamina/farmacologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pirrolidinas/metabolismo , Pirrolidinas/farmacologia , Racloprida/metabolismo , Racloprida/farmacologia , Ensaio Radioligante/métodos , Compostos Radiofarmacêuticos/farmacologia , Salicilamidas/metabolismo , Salicilamidas/farmacologia
15.
J Med Chem ; 64(1): 458-480, 2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33372800

RESUMO

We present and thoroughly characterize a large collection of 3,4-dihydropyrimidin-2(1H)-ones as A2BAR antagonists, an emerging strategy in cancer (immuno) therapy. Most compounds selectively bind A2BAR, with a number of potent and selective antagonists further confirmed by functional cyclic adenosine monophosphate experiments. The series was analyzed with one of the most exhaustive free energy perturbation studies on a GPCR, obtaining an accurate model of the structure-activity relationship of this chemotype. The stereospecific binding modeled for this scaffold was confirmed by resolving the two most potent ligands [(±)-47, and (±)-38 Ki = 10.20 and 23.6 nM, respectively] into their two enantiomers, isolating the affinity on the corresponding (S)-eutomers (Ki = 6.30 and 11.10 nM, respectively). The assessment of the effect in representative cytochromes (CYP3A4 and CYP2D6) demonstrated insignificant inhibitory activity, while in vitro experiments in three prostate cancer cells demonstrated that this pair of compounds exhibits a pronounced antimetastatic effect.


Assuntos
Antagonistas do Receptor A2 de Adenosina/farmacologia , Pirimidinas/farmacologia , Receptor A2B de Adenosina/efeitos dos fármacos , Antagonistas do Receptor A2 de Adenosina/metabolismo , Animais , Células CHO , Cricetulus , AMP Cíclico/metabolismo , Células HEK293 , Células HeLa , Humanos , Modelos Moleculares , Metástase Neoplásica/prevenção & controle , Pirimidinas/química , Pirimidinas/metabolismo , Ensaio Radioligante , Receptor A2B de Adenosina/metabolismo , Estereoisomerismo , Relação Estrutura-Atividade
16.
Eur J Med Chem ; 209: 112916, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-33328102

RESUMO

Among all of the monoaminergic receptors, the 5-HT6R has the highest number of non-basic ligands (approximately 5% of compounds stored in 25th version of ChEMBL database have the strongest basic pKa below 5, calculated using the Instant JChem calculator plugin). These compounds, when devoid of a basic nitrogen, exhibit high affinity and remarkable selectivity. Despite a decade of research, no clues have been given for explanation of such an intriguing phenomenon. Here, a series of analogs of four known 5-HT6R ligands, has been rationally designed to approach this issue. For each of the synthesized 42 compounds, a binding affinity for 5-HT6R has been measured, together with a selectivity profile against 5-HT1AR, 5-HT2AR, 5-HT7R and D2R. Performed induced fit docking and molecular dynamics experiments revealed that no particular interaction was responsible for the activity of non-basic compounds. In fact, a plain N-phenylsulfonylindole (1e) was found to possess a moderate (5-HT6R, Ki = 159 nM) affinity. No other monoaminergic receptor has as simple and selective ligand as this one. Thus, it is stated that it binds to the receptor solely based on its conformation and as such, possesses a minimum amount of features, required for binding. Also, any functional group able to form an additional interaction with the receptor increase the binding affinity, like in the case of two highly active non-basic compounds 3e and 5g (5-HT6R, Ki = 65 nM and 38 nM, respectively).


Assuntos
Desenho de Fármacos , Indóis/química , Receptores de Serotonina/metabolismo , Células HEK293 , Humanos , Indóis/metabolismo , Indóis/farmacologia , Ligantes , Simulação de Dinâmica Molecular , Ensaio Radioligante , Relação Estrutura-Atividade
17.
Int J Mol Sci ; 21(22)2020 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-33182741

RESUMO

In drug discovery, assays with proximal readout are of great importance to study target-specific effects of potential drug candidates. In the field of G protein-coupled receptors (GPCRs), the determination of GPCR-G protein interactions and G protein activation by means of radiolabeled GTP analogs ([35S]GTPγS, [γ-32P]GTP) has widely been used for this purpose. Since we were repeatedly faced with insufficient quality of radiolabeled nucleotides, there was a requirement to implement a novel proximal functional assay for the routine characterization of putative histamine receptor ligands. We applied the split-NanoLuc to the four histamine receptor subtypes (H1R, H2R, H3R, H4R) and recently engineered minimal G (mini-G) proteins. Using this method, the functional response upon receptor activation was monitored in real-time and the four mini-G sensors were evaluated by investigating selected standard (inverse) agonists and antagonists. All potencies and efficacies of the studied ligands were in concordance with literature data. Further, we demonstrated a significant positive correlation of the signal amplitude and the mini-G protein expression level in the case of the H2R, but not for the H1R or the H3R. The pEC50 values of histamine obtained under different mini-G expression levels were consistent. Moreover, we obtained excellent dynamic ranges (Z' factor) and the signal spans were improved for all receptor subtypes in comparison to the previously performed [35S]GTPγS binding assay.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Receptores Histamínicos/classificação , Receptores Histamínicos/metabolismo , Animais , Descoberta de Drogas , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Células HEK293 , Agonistas dos Receptores Histamínicos/metabolismo , Antagonistas dos Receptores Histamínicos/metabolismo , Humanos , Cinética , Ligantes , Luciferases/metabolismo , Mimetismo Molecular , Conformação Proteica , Ensaio Radioligante , Receptores Acoplados a Proteínas G/metabolismo , Receptores Histamínicos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/classificação , Proteínas Recombinantes/metabolismo
18.
Sci Rep ; 10(1): 20781, 2020 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-33247159

RESUMO

The adenosine A3 receptor (A3R) belongs to a family of four adenosine receptor (AR) subtypes which all play distinct roles throughout the body. A3R antagonists have been described as potential treatments for numerous diseases including asthma. Given the similarity between (adenosine receptors) orthosteric binding sites, obtaining highly selective antagonists is a challenging but critical task. Here we screen 39 potential A3R, antagonists using agonist-induced inhibition of cAMP. Positive hits were assessed for AR subtype selectivity through cAMP accumulation assays. The antagonist affinity was determined using Schild analysis (pA2 values) and fluorescent ligand binding. Structure-activity relationship investigations revealed that loss of the 3-(dichlorophenyl)-isoxazolyl moiety or the aromatic nitrogen heterocycle with nitrogen at α-position to the carbon of carboximidamide group significantly attenuated K18 antagonistic potency. Mutagenic studies supported by molecular dynamic simulations combined with Molecular Mechanics-Poisson Boltzmann Surface Area calculations identified the residues important for binding in the A3R orthosteric site. We demonstrate that K18, which contains a 3-(dichlorophenyl)-isoxazole group connected through carbonyloxycarboximidamide fragment with a 1,3-thiazole ring, is a specific A3R (< 1 µM) competitive antagonist. Finally, we introduce a model that enables estimates of the equilibrium binding affinity for rapidly disassociating compounds from real-time fluorescent ligand-binding studies. These results demonstrate the pharmacological characterisation of a selective competitive A3R antagonist and the description of its orthosteric binding mode. Our findings may provide new insights for drug discovery.


Assuntos
Antagonistas do Receptor A3 de Adenosina/química , Antagonistas do Receptor A3 de Adenosina/farmacologia , Antagonistas do Receptor A3 de Adenosina/farmacocinética , Animais , Sítios de Ligação/genética , Ligação Competitiva , Células CHO , Cricetulus , AMP Cíclico/metabolismo , Avaliação Pré-Clínica de Medicamentos , Humanos , Cinética , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Ensaio Radioligante , Ratos , Receptor A3 de Adenosina/química , Receptor A3 de Adenosina/genética , Receptor A3 de Adenosina/metabolismo , Receptores Purinérgicos P1/química , Receptores Purinérgicos P1/genética , Receptores Purinérgicos P1/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade da Espécie , Relação Estrutura-Atividade
19.
Theranostics ; 10(24): 11178-11196, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33042277

RESUMO

Selective modulation of metabotropic glutamate receptor 2 (mGlu2) represents a novel therapeutic approach for treating brain disorders, including schizophrenia, depression, Parkinson's disease (PD), Alzheimer's disease (AD), drug abuse and addiction. Imaging mGlu2 using positron emission tomography (PET) would allow for in vivo quantification under physiological and pathological conditions and facilitate drug discovery by enabling target engagement studies. In this paper, we aimed to develop a novel specific radioligand derived from negative allosteric modulators (NAMs) for PET imaging of mGlu2. Methods. A focused small molecule library of mGlu2 NAMs with tetrahydro naphthyridine scaffold was synthesized for pharmacology and physicochemical evaluation. GIRK dose-response assays and CNS panel binding selectivity assays were performed to study the affinity and selectivity of mGlu2 NAMs, among which compounds 14a and 14b were selected as PET ligand candidates. Autoradiography in SD rat brain sections was used to confirm the in vitro binding specificity and selectivity of [11C]14a and [11C]14b towards mGlu2. In vivo binding specificity was then studied by PET imaging. Whole body biodistribution study and radiometabolite analysis were conducted to demonstrate the pharmacokinetic properties of [11C]14b as most promising PET mGlu2 PET ligand. Results. mGlu2 NAMs 14a-14g were synthesized in 14%-20% yields in five steps. NAMs 14a and 14b were selected to be the most promising ligands due to their high affinity in GIRK dose-response assays. [11C]14a and [11C]14b displayed similar heterogeneous distribution by autoradiography, consistent with mGlu2 expression in the brain. While PET imaging study showed good brain permeability for both tracers, compound [11C]14b demonstrated superior binding specificity compared to [11C]14a. Further radiometabolite analysis of [11C]14b showed excellent stability in the brain. Conclusions. Compound 14b exhibited high affinity and excellent subtype selectivity, which was then evaluated by in vitro autoradiography and in vivo PET imaging study after labeling with carbon-11. Ligand [11C]14b, which we named [11C]MG2-1904, demonstrated high brain uptake and excellent in vitro/in vivo specific binding towards mGlu2 with high metabolic stability in the brain. As proof-of-concept, our preliminary work demonstrated a successful example of visualizing mGlu2 in vivo derived from NAMs, which represents a promising chemotype for further development and optimization aimed for clinical translation.


Assuntos
Naftiridinas/administração & dosagem , Tomografia por Emissão de Pósitrons/métodos , Ensaio Radioligante/métodos , Compostos Radiofarmacêuticos/administração & dosagem , Receptores de Glutamato Metabotrópico/análise , Regulação Alostérica , Animais , Autorradiografia , Encéfalo/diagnóstico por imagem , Radioisótopos de Carbono , Ligantes , Masculino , Modelos Animais , Naftiridinas/química , Naftiridinas/farmacocinética , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Ratos , Receptores de Glutamato Metabotrópico/metabolismo , Distribuição Tecidual
20.
Brain ; 143(11): 3318-3330, 2020 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-33006604

RESUMO

Overactivation of microglia is associated with most neurodegenerative diseases. In this study we examined whether PET-measurable innate immune cell activation predicts multiple sclerosis disease progression. Activation of microglia/macrophages was measured using the 18-kDa translocator protein (TSPO)-binding radioligand 11C-PK11195 and PET imaging in 69 patients with multiple sclerosis and 18 age- and sex-matched healthy controls. Radioligand binding was evaluated as the distribution volume ratio from dynamic PET images. Conventional MRI and disability measurements using the Expanded Disability Status Scale were performed for patients at baseline and 4.1 ± 1.9 (mean ± standard deviation) years later. Fifty-one (74%) of the patients were free of relapses during the follow-up period. Patients had increased activation of innate immune cells in the normal-appearing white matter and in the thalamus compared to the healthy control group (P = 0.033 and P = 0.003, respectively, Wilcoxon). Forward-type stepwise logistic regression was used to assess the best variables predicting disease progression. Baseline innate immune cell activation in the normal-appearing white matter was a significant predictor of later progression when the entire multiple sclerosis cohort was assessed [odds ratio (OR) = 4.26; P = 0.048]. In the patient subgroup free of relapses there was an association between macrophage/microglia activation in the perilesional normal-appearing white matter and disease progression (OR = 4.57; P = 0.013). None of the conventional MRI parameters measured at baseline associated with later progression. Our results strongly suggest that innate immune cell activation contributes to the diffuse neural damage leading to multiple sclerosis disease progression independent of relapses.


Assuntos
Esclerose Múltipla/diagnóstico por imagem , Esclerose Múltipla/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Receptores de GABA/metabolismo , Adulto , Encéfalo/diagnóstico por imagem , Estudos de Coortes , Avaliação da Deficiência , Progressão da Doença , Feminino , Humanos , Isoquinolinas , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Ensaio Radioligante , Recidiva , Substância Branca/diagnóstico por imagem
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