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1.
ACS Chem Neurosci ; 10(9): 4076-4101, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31441641

RESUMO

Here, we present a series of dual-target phosphodiesterase 9 (PDE9) and histone deacetylase (HDAC) inhibitors devised as pharmacological tool compounds for assessing the implications of these two targets in Alzheimer's disease (AD). These novel inhibitors were designed taking into account the key pharmacophoric features of known selective PDE9 inhibitors as well as privileged chemical structures, bearing zinc binding groups (hydroxamic acids and ortho-amino anilides) that hit HDAC targets. These substituents were selected according to rational criteria and previous knowledge from our group to explore diverse HDAC selectivity profiles (pan-HDAC, HDAC6 selective, and class I selective) that were confirmed in biochemical screens. Their functional response in inducing acetylation of histone and tubulin and phosphorylation of cAMP response element binding (CREB) was measured as a requisite for further progression into complete in vitro absorption, distribution, metabolism and excretion (ADME) and in vivo brain penetration profiling. Compound 31b, a selective HDAC6 inhibitor with acceptable brain permeability, was chosen for assessing in vivo efficacy of these first-in-class inhibitors, as well as studying their mode of action (MoA).

2.
Front Aging Neurosci ; 11: 149, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31281249

RESUMO

The discouraging results with therapies for Alzheimer's disease (AD) in clinical trials, highlights the urgent need to adopt new approaches. Like other complex diseases, it is becoming clear that AD therapies should focus on the simultaneous modulation of several targets implicated in the disease. Recently, using reference compounds and the first-in class CM-414, we demonstrated that the simultaneous inhibition of histone deacetylases [class I histone deacetylases (HDACs) and HDAC6] and phosphodiesterase 5 (PDE5) has a synergistic therapeutic effect in AD models. To identify the best inhibitory balance of HDAC isoforms and PDEs that provides a safe and efficient therapy to combat AD, we tested the compound CM-695 in the Tg2576 mouse model of this disease. CM-695 selectively inhibits HDAC6 over class I HDAC isoforms, which largely overcomes the toxicity associated with HDAC class 1 inhibition. Furthermore, CM-695 inhibits PDE9, which is expressed strongly in the brain and has been proposed as a therapeutic target for AD. Chronic treatment of aged Tg2576 mice with CM-695 ameliorates memory impairment and diminishes brain Aß, although its therapeutic effect was no longer apparent 4 weeks after the treatment was interrupted. An increase in the presence of 78-KDa glucose regulated protein (GRP78) and heat shock protein 70 (Hsp70) chaperones may underlie the therapeutic effect of CM-695. In summary, chronic treatment with CM-695 appears to reverse the AD phenotype in a safe and effective manner. Taking into account that AD is a multifactorial disorder, the multimodal action of these compounds and the different events they affect may open new avenues to combat AD.

3.
Mol Neurobiol ; 56(8): 5900-5910, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30687889

RESUMO

Cannabinoid CB1 receptors (CB1R) and the GPR55 receptor are expressed in striatum and are potential targets in the therapy of Parkinson's disease (PD), one of the most prevalent neurodegenerative diseases in developed countries. The aim of this paper was to address the potential of ligands acting on those receptors to prevent the action of a neurotoxic agent, MPP+, that specifically affects neurons of the substantia nigra due to uptake via the dopamine DAT transporter. The SH-SY5Y cell line model was used as it expresses DAT and, therefore, is able to uptake MPP+ that inhibits complex I of the respiratory mitochondrial chain and leads to cell death. Cells were transfected with cDNAs coding for either or both receptors. Receptors in cotransfected cells formed heteromers as indicated by the in situ proximity ligation assays. Cell viability was assayed by oxygen rate consumption and by the bromide-based MTT method. Assays of neuroprotection using two concentrations of MPP+ showed that cells expressing receptor heteromers were more resistant to the toxic effect. After correction by effects on cell proliferation, the CB1R antagonist, SR141716, afforded an almost full neuroprotection in CB1R-expressing cells even when a selective agonist, ACEA, was present. In contrast, SR141716 was not effective in cells expressing CB1/GPR55 heteromeric complexes. In addition, an agonist of GPR55, CID1792197, did not enhance neuroprotection in GPR55-expressing cells. These results show that neurons expressing heteromers are more resistant to cell death but question the real usefulness of CB1R, GPR55, and their heteromers as targets to afford PD-related neuroprotection.


Assuntos
Terapia de Alvo Molecular , Fármacos Neuroprotetores/uso terapêutico , Doença de Parkinson/tratamento farmacológico , Receptor CB1 de Canabinoide/antagonistas & inibidores , Linhagem Celular Tumoral , Humanos , Ligantes , Modelos Biológicos , Neuroproteção/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Consumo de Oxigênio/efeitos dos fármacos , Receptor CB1 de Canabinoide/metabolismo , Receptores de Canabinoides/metabolismo
4.
ACS Chem Neurosci ; 10(3): 1765-1782, 2019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30525452

RESUMO

In order to determine the contributions of histone deacetylase (HDAC) isoforms to the beneficial effects of dual phosphodiesterase 5 (PDE5) and pan-HDAC inhibitors on in vivo models of Alzheimer's disease (AD), we have designed, synthesized, and tested novel chemical probes with the desired target compound profile of PDE5 and class I HDAC selective inhibitors. Compared to previous hydroxamate-based series, these molecules exhibit longer residence times on HDACs. In this scenario, shorter or longer preincubation times may have a significant impact on the IC50 values of these compounds and therefore on their corresponding selectivity profiles on the different HDAC isoforms. On the other hand, different chemical series have been explored and, as expected, some pairwise comparisons show a clear impact of the scaffold on biological responses (e.g., 35a vs 40a). The lead identification process led to compound 29a, which shows an adequate ADME-Tox profile and in vivo target engagement (histone acetylation and cAMP/cGMP response element-binding (CREB) phosphorylation) in the central nervous system (CNS), suggesting that this compound represents an optimized chemical probe; thus, 29a has been assayed in a mouse model of AD (Tg2576).

5.
Front Neuroanat ; 12: 52, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30002620

RESUMO

Gaucher's disease (GD) is the most prevalent lysosomal storage disorder. GD is caused by homozygous mutations of the GBA1 gene, which codes for beta-glucocerebrosidase (GCase). Although GD primarily affects peripheral tissues, the presence of neurological symptoms has been reported in several GD subtypes. GBA1 mutations have recently deserved increased attention upon the demonstration that both homo- and heterozygous GBA1 mutations represent the most important genetic risk factor for the appearance of synucleinopathies like Parkinson's disease (PD) and dementia with Lewy bodies (LBD). Although reduced GCase activity leads to alpha-synuclein aggregation, the mechanisms sustaining a role for GCase in alpha-synuclein homeostasis still remain largely unknown. Furthermore, the role to be played by impairment in the physiological function of endoplasmic reticulum, mitochondria and other subcellular membranous components is currently under investigation. Here we focus on the impact of GCase loss-of-function that impact on the levels of sterylglucosides, molecules that are known to trigger a PD-related synucleinopathy upon administration in rats. Moreover, the concurrence of another gene also coding for an enzyme with GCase activity (GBA2 gene) should also be taken into consideration, bearing in mind that in addition to a hydrolytic function, both GCases also share transglycosylation as a second catalytic activity. Accordingly, sterylglycoside levels should also be considered to further assess their impact on the neurodegenerative process. In this regard-and besides GBA1 genotyping-we suggest that screening for GBA2 mutations should be considered, together with analytical measurements of cholesterol glycosides in body fluids, as biomarkers for both PD risk and disease progression.

6.
ACS Med Chem Lett ; 9(5): 428-433, 2018 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-29795754

RESUMO

In an effort to find novel chemical series as antifibrinolytic agents, we explore α-phenylsulfonyl-α-spiropiperidines bearing different zinc-binding groups (ZBGs) to target those metalloproteinases involved in the fibrinolytic process: MMP3 and MMP10. Surprisingly, all these new chemical series were inactive against these metalloproteinases; however, several new molecules retained the antifibrinolytic activity in a phenotypic functional assay using thromboelastometry and human whole blood. Further optimization led to compound 38 as a potent antifibrinolytic agent in vivo, three times more efficacious than the current standard-of-care (tranexamic acid, TXA) at 300 times lower dose. Finally, in order to decipher the underlying mode-of-action leading to this phenotypic response, an affinity-based probe 39 was successfully designed to identify the target involved in this response: a potentially unknown mechanism-of-action in the fibrinolytic process.

7.
Eur J Med Chem ; 150: 506-524, 2018 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-29549837

RESUMO

We have identified chemical probes that act as dual phosphodiesterase 5 (PDE5) and histone deacetylase 6 (HDAC6)-selective inhibitors (>1 log unit difference versus class I HDACs) to decipher the contribution of HDAC isoforms to the positive impact of dual-acting PDE5 and HDAC inhibitors on mouse models of Alzheimer's disease (AD) and fine-tune this systems therapeutics approach. Structure- and knowledge-based approaches led to the design of first-in-class molecules with the desired target compound profile: dual PDE5 and HDAC6-selective inhibitors. Compound 44b, which fulfilled the biochemical, functional and ADME-Tox profiling requirements and exhibited adequate pharmacokinetic properties, was selected as pharmacological tool compound and tested in a mouse model of AD (Tg2576) in vivo.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Desenho de Drogas , Desacetilase 6 de Histona/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Inibidores da Fosfodiesterase 5/farmacologia , Doença de Alzheimer/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Desacetilase 6 de Histona/metabolismo , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/química , Humanos , Estrutura Molecular , Neuroglia/efeitos dos fármacos , Inibidores da Fosfodiesterase 5/síntese química , Inibidores da Fosfodiesterase 5/química , Relação Estrutura-Atividade
8.
J Am Heart Assoc ; 6(6)2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28572282

RESUMO

BACKGROUND: Intracerebral hemorrhage (ICH) is an acute neurological disorder with high mortality and no effective treatment. In addition to the initial bleeding event, rebleeding and hematoma expansion are associated with poor outcome in these patients. We studied the effectiveness of the new antifibrinolytic agent CM352, a short-half-life matrix metalloproteinase inhibitor, for achieving early hemostasis and improving functional recovery in a rat model of collagenase-induced ICH. METHODS AND RESULTS: ICH was induced by striatal injection of collagenase, and 1 hour later, rats received an intravenous injection of saline (n=6) or CM352 (1 mg/kg, n=6). Hematoma (basal and after 3 and 24 hours) and lesion (14 days) volumes were quantified on T2-weighted (T2) magnetic resonance images. Neurological and functional recovery was evaluated by using Bederson score and a cylinder test (basal, 24 hours, and 14 days). Early treatment (1 hour) with CM352 was efficient reducing hematoma expansion at 3 hours (P<0.01) and, more markedly, at 24 hours (P<0.01). Decreased bleeding after antifibrinolytic treatment was accompanied by reduced interleukin-6 levels at 3 hours (P<0.05) and smaller lesion volume at 14 days (P<0.01). CM352 drastically reduced sensorimotor impairment (cylinder test) after ICH in rats at 24 hours (P<0.01) and 14 days (P<0.01). Similarly, it also attenuated neurological deficit (Bederson scale) at 24 hours (P<0.01) and 14 days (P<0.01). Interestingly, late (3 hours) CM352 administration also resulted in reduced lesion size and better functional outcome. CONCLUSIONS: CM352, a new antifibrinolytic agent and matrix metalloproteinase inhibitor, effectively prevented hematoma growth and reduced lesion size in ICH in association with improved functional and neurological recovery.


Assuntos
Antifibrinolíticos/administração & dosagem , Benzamidas/administração & dosagem , Encéfalo/efeitos dos fármacos , Hemorragia Cerebral/tratamento farmacológico , Hematoma/tratamento farmacológico , Ácidos Hidroxâmicos/administração & dosagem , Inibidores de Metaloproteinases de Matriz/administração & dosagem , Acidente Vascular Cerebral/tratamento farmacológico , Animais , Antifibrinolíticos/farmacocinética , Benzamidas/farmacocinética , Encéfalo/enzimologia , Encéfalo/patologia , Encéfalo/fisiopatologia , Morte Celular/efeitos dos fármacos , Hemorragia Cerebral/enzimologia , Hemorragia Cerebral/patologia , Hemorragia Cerebral/fisiopatologia , Modelos Animais de Doenças , Hematoma/enzimologia , Hematoma/patologia , Hematoma/fisiopatologia , Ácidos Hidroxâmicos/farmacocinética , Injeções Intravenosas , Imagem por Ressonância Magnética , Masculino , Inibidores de Metaloproteinases de Matriz/farmacocinética , Metaloproteinases da Matriz/metabolismo , Atividade Motora/efeitos dos fármacos , Ratos Sprague-Dawley , Acidente Vascular Cerebral/enzimologia , Acidente Vascular Cerebral/patologia , Acidente Vascular Cerebral/fisiopatologia
9.
Nat Commun ; 8: 15424, 2017 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-28548080

RESUMO

The indisputable role of epigenetics in cancer and the fact that epigenetic alterations can be reversed have favoured development of epigenetic drugs. In this study, we design and synthesize potent novel, selective and reversible chemical probes that simultaneously inhibit the G9a and DNMTs methyltransferase activity. In vitro treatment of haematological neoplasia (acute myeloid leukaemia-AML, acute lymphoblastic leukaemia-ALL and diffuse large B-cell lymphoma-DLBCL) with the lead compound CM-272, inhibits cell proliferation and promotes apoptosis, inducing interferon-stimulated genes and immunogenic cell death. CM-272 significantly prolongs survival of AML, ALL and DLBCL xenogeneic models. Our results represent the discovery of first-in-class dual inhibitors of G9a/DNMTs and establish this chemical series as a promising therapeutic tool for unmet needs in haematological tumours.


Assuntos
Antineoplásicos/farmacologia , Metilases de Modificação do DNA/antagonistas & inibidores , Desenho de Drogas , Inibidores Enzimáticos/farmacologia , Neoplasias Hematológicas/tratamento farmacológico , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Apoptose/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Cristalografia por Raios X , Metilases de Modificação do DNA/química , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/metabolismo , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Epigênese Genética/efeitos dos fármacos , Feminino , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/imunologia , Neoplasias Hematológicas/mortalidade , Antígenos de Histocompatibilidade/química , Antígenos de Histocompatibilidade/genética , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Interferons/imunologia , Interferons/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Microssomos Hepáticos , Simulação de Acoplamento Molecular , Análise de Sobrevida , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
10.
ACS Chem Neurosci ; 8(3): 638-661, 2017 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-27936591

RESUMO

A novel systems therapeutics approach, involving simultaneous inhibition of phosphodiesterase 5 (PDE5) and histone deacetylase (HDAC), has been validated as a potentially novel therapeutic strategy for the treatment of Alzheimer's disease (AD). First-in-class dual inhibitors bearing a sildenafil core have been very recently reported, and the lead molecule 7 has proven this strategy in AD animal models. Because scaffolds may play a critical role in primary activities and ADME-Tox profiling as well as on intellectual property, we have explored alternative scaffolds (vardenafil- and tadalafil-based cores) and evaluated their impact on critical parameters such as primary activities, permeability, toxicity, and in vivo (pharmacokinetics and functional response in hippocampus) to identify a potential alternative lead molecule bearing a different chemotype for in vivo testing.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Inibidores de Histona Desacetilases/uso terapêutico , Inibidores da Fosfodiesterase 5/uso terapêutico , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Trifosfato de Adenosina/metabolismo , Doença de Alzheimer/patologia , Animais , Linhagem Celular Transformada , GMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/farmacologia , Humanos , Leucócitos Mononucleares , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Modelos Moleculares , Neuroglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Inibidores da Fosfodiesterase 5/química , Inibidores da Fosfodiesterase 5/farmacologia
11.
Neuropsychopharmacology ; 42(2): 524-539, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27550730

RESUMO

The targeting of two independent but synergistic enzymatic activities, histone deacetylases (HDACs, class I and HDAC6) and phosphodiesterase 5 (PDE5), has recently been validated as a potentially novel therapeutic approach for Alzheimer's disease (AD). Here we report the discovery of a new first-in-class small-molecule (CM-414) that acts as a dual inhibitor of PDE5 and HDACs. We have used this compound as a chemical probe to validate this systems therapeutics strategy, where an increase in the activation of cAMP/cGMP-responsive element-binding protein (CREB) induced by PDE5 inhibition, combined with moderate HDAC class I inhibition, leads to efficient histone acetylation. This molecule rescued the impaired long-term potentiation evident in hippocampal slices from APP/PS1 mice. Chronic treatment of Tg2576 mice with CM-414 diminished brain Aß and tau phosphorylation (pTau) levels, increased the inactive form of GSK3ß, reverted the decrease in dendritic spine density on hippocampal neurons, and reversed their cognitive deficits, at least in part by inducing the expression of genes related to synaptic transmission. Thus, CM-414 may serve as the starting point to discover balanced dual inhibitors with an optimal efficacy and safety profile for clinical testing on AD patients.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Hipocampo/efeitos dos fármacos , Inibidores de Histona Desacetilases/administração & dosagem , Plasticidade Neuronal/efeitos dos fármacos , Inibidores da Fosfodiesterase 5/administração & dosagem , Pirazóis/uso terapêutico , Pirimidinonas/uso terapêutico , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/prevenção & controle , Animais , Comportamento Animal/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Hipocampo/fisiopatologia , Inibidores de Histona Desacetilases/farmacologia , Camundongos , Camundongos Transgênicos , Atividade Motora/efeitos dos fármacos , Inibidores da Fosfodiesterase 5/farmacologia , Cultura Primária de Células , Pirazóis/administração & dosagem , Pirazóis/farmacologia , Pirimidinonas/administração & dosagem , Pirimidinonas/farmacologia
12.
J Med Chem ; 59(19): 8967-9004, 2016 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-27606546

RESUMO

Simultaneous inhibition of phosphodiesterase 5 (PDE5) and histone deacetylases (HDAC) has recently been validated as a potentially novel therapeutic approach for Alzheimer's disease (AD). To further extend this concept, we designed and synthesized the first chemical series of dual acting PDE5 and HDAC inhibitors, and we validated this systems therapeutics approach. Following the implementation of structure- and knowledge-based approaches, initial hits were designed and were shown to validate our hypothesis of dual in vitro inhibition. Then, an optimization strategy was pursued to obtain a proper tool compound for in vivo testing in AD models. Initial hits were translated into molecules with adequate cellular functional responses (histone acetylation and cAMP/cGMP response element-binding (CREB) phosphorylation in the nanomolar range), an acceptable therapeutic window (>1 log unit), and the ability to cross the blood-brain barrier, leading to the identification of 7 as a candidate for in vivo proof-of-concept testing ( Cuadrado-Tejedor, M.; Garcia-Barroso, C.; Sánchez-Arias, J. A.; Rabal, O.; Mederos, S.; Ugarte, A.; Franco, R.; Segura, V.; Perea, G.; Oyarzabal, J.; Garcia-Osta, A. Neuropsychopharmacology 2016 , in press, doi: 10.1038/npp.2016.163 ).


Assuntos
Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/farmacologia , Inibidores da Fosfodiesterase 5/química , Inibidores da Fosfodiesterase 5/farmacologia , Acetilação/efeitos dos fármacos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Desenho de Drogas , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/farmacocinética , Histona Desacetilases/metabolismo , Humanos , Camundongos , Modelos Moleculares , Inibidores da Fosfodiesterase 5/síntese química , Inibidores da Fosfodiesterase 5/farmacocinética
13.
J Pharmacol Exp Ther ; 358(3): 580-7, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27358483

RESUMO

Endocannabinoids act on G protein-coupled receptors that are considered potential targets for a variety of diseases. There are two different cannabinoid receptor types: ligands for cannabinoid type 2 receptors (CB2Rs) show more promise than those for cannabinoid type 1 receptors (CB1Rs) because they lack psychotropic actions. However, the complex pharmacology of these receptors, coupled with the lipophilic nature of ligands, is delaying the translational success of medications targeting the endocannabinoid system. We here report the discovery and synthesis of a fluorophore-conjugated CB2R-selective compound, CM-157 (3-[[4-[2-tert-butyl-1-(tetrahydropyran-4-ylmethyl)benzimidazol-5-yl]sulfonyl-2-pyridyl]oxy]propan-1-amine), which was useful for pharmacological characterization of CB2R by using a time-resolved fluorescence resonance energy transfer assay. This methodology does not require radiolabeled compounds and may be undertaken in homogeneous conditions and in living cells (i.e., without the need to isolate receptor-containing membranes). The affinity of the labeled compound was similar to that of the unlabeled molecule. Time-resolved fluorescence resonance energy transfer assays disclosed a previously unreported second affinity site and showed conformational changes in CB2R forming receptor heteromers with G protein-coupled receptor GPR55, a receptor for l-α-lysophosphatidylinositol. The populations displaying subnanomolar and nanomolar affinities were undisclosed in competitive assays using a well known cannabinoid receptor ligand, AM630 (1-[2-(morpholin-4-yl)ethyl]-2-methyl-3-(4-methoxybenzoyl)-6-iodoindole), and TH-chrysenediol, not previously tested on binding to cannabinoid receptors. Variations in binding parameters upon formation of dimers with GPR55 may reflect decreases in binding sites or alterations of the quaternary structure of the macromolecular G protein-coupled receptor complexes. In summary, the homogeneous binding assay described here may serve to better characterize agonist binding to CB2R and to identify specific properties of CB2R on living cells.


Assuntos
Bioensaio , Receptor CB2 de Canabinoide/metabolismo , Sítios de Ligação , Crisenos/metabolismo , Corantes Fluorescentes/química , Células HEK293 , Humanos , Indóis/metabolismo , Ligantes , Simulação de Acoplamento Molecular , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Receptor CB2 de Canabinoide/química , Receptores Acoplados a Proteínas-G/química , Relação Estrutura-Atividade
14.
Mol Neurobiol ; 53(4): 2312-9, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-25976369

RESUMO

Growing evidence suggests that the endocannabinoid system plays a role in neuroprotection in Parkinson's disease. Recently, we have shown the neuroprotective effect of monoacylglycerol lipase (MAGL) inhibition with JZL184 in the chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model. However, further investigation is needed to determine the neuroprotective mechanisms of the endocannabinoid system on the nigrostriatal pathway. The aim of this work was to investigate whether the neuroprotective effect of JZL184 in mice could be extended to an in vitro cellular model to further understand the mechanism of action of the drug. The SH-SY5Y cell line was selected based on its dopaminergic-like phenotype and its susceptibility to 1-methyl-4-phenylpyridinium iodide (MPP(+)) toxicity. Furthermore, SH-SY5Y cells express both cannabinoid receptors, CB1 and CB2. The present study describes the neuroprotective effect of MAGL inhibition with JZL184 in SH-SY5Y cells treated with MPP(+). The effect of JZL184 in cell survival was blocked by AM630, a CB2 receptor antagonist, and it was mimicked with JWH133, a CB2 receptor agonist. Rimonabant, a CB1 receptor antagonist, did not affect JZL184-induced cell survival. These results demonstrate that the neuroprotective effect of MAGL inhibition with JZL184 described in animal models of Parkinson's disease could be extended to in vitro models such as SH-SY5Y cells treated with MPP(+). This represents a useful tool to study mechanisms of neuroprotection mediated by MAGL inhibition, and we provide evidence for the possible involvement of CB2 receptors in the improvement of cell survival.


Assuntos
1-Metil-4-fenilpiridínio/toxicidade , Benzodioxóis/farmacologia , Fármacos Neuroprotetores/farmacologia , Piperidinas/farmacologia , Receptor CB2 de Canabinoide/metabolismo , Amidoidrolases/metabolismo , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Humanos , Monoacilglicerol Lipases/antagonistas & inibidores , Monoacilglicerol Lipases/metabolismo , Neurotoxinas/toxicidade , Receptor CB1 de Canabinoide/metabolismo
15.
J Med Chem ; 58(7): 2941-57, 2015 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-25686022

RESUMO

Discovery of potent and safe therapeutics that improve upon currently available antifibrinolytics, e.g., tranexamic acid (TXA, 1) and aprotinin, has been challenging. Matrix metalloproteinases (MMPs) participate in thrombus dissolution. Then we designed a novel series of optimized MMP inhibitors that went through phenotypic screening consisting of thromboelastometry and mouse tail bleeding. Our optimized lead compound, CM-352 (2), inhibited fibrinolysis in human whole blood functional assays and was more effective than the current standard of care, 1, in the tail-bleeding model using a 30 000 times lower dose. Moreover, 2 reduced blood loss during liver hepatectomy, while 1 and aprotinin had no effect. Molecule 2 displayed optimal pharmacokinetic and safety profiles with no evidence of thrombosis or coagulation impairment. This novel mechanism of action, targeting MMP, defines a new class of antihemorrhagic agents without interfering with normal hemostatic function. Furthermore, 2 represents a preclinical candidate for the acute treatment of bleeding.


Assuntos
Benzamidas/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Hemorragia/prevenção & controle , Hemostáticos/química , Hemostáticos/farmacologia , Ácidos Hidroxâmicos/farmacologia , Animais , Antifibrinolíticos/química , Antifibrinolíticos/farmacologia , Benzamidas/química , Células CACO-2/efeitos dos fármacos , Descoberta de Drogas/métodos , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Hemorragia/tratamento farmacológico , Hemorragia/metabolismo , Humanos , Ácidos Hidroxâmicos/química , Metaloproteinase 10 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/química , Inibidores de Metaloproteinases de Matriz/farmacologia , Camundongos Endogâmicos C57BL , Estrutura Molecular , Terapia de Alvo Molecular/métodos
16.
J Med Chem ; 58(5): 2465-88, 2015 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-25686153

RESUMO

Growing evidence suggests that matrix metalloproteinases (MMP) are involved in thrombus dissolution; then, considering that new therapeutic strategies are required for controlling hemorrhage, we hypothesized that MMP inhibition may reduce bleeding by delaying fibrinolysis. Thus, we designed and synthesized a novel series of MMP inhibitors to identify potential candidates for acute treatment of bleeding. Structure-based and knowledge-based strategies were utilized to design this novel chemical series, α-spiropiperidine hydroxamates, of potent and soluble (>75 µg/mL) pan-MMP inhibitors. The initial hit, 12, was progressed to an optimal lead 19d. Racemic 19d showed a remarkable in vitro phenotypic response and outstanding in vivo efficacy; in fact, the mouse bleeding time at 1 mg/kg was 0.85 min compared to 29.28 min using saline. In addition, 19d displayed an optimal ADME and safety profile (e.g., no thrombus formation). Its corresponding enantiomers were separated, leading to the preclinical candidate 5 (described in Drug Annotations series, J. Med. Chem. 2015, ).


Assuntos
Benzamidas/síntese química , Benzamidas/farmacologia , Desenho de Drogas , Hemorragia/tratamento farmacológico , Ácidos Hidroxâmicos/química , Inibidores de Metaloproteinases de Matriz/química , Inibidores de Metaloproteinases de Matriz/farmacologia , Metaloproteinases da Matriz/química , Animais , Permeabilidade da Membrana Celular/efeitos dos fármacos , Humanos , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/farmacologia , Masculino , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade
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