RESUMO
A multidimensional inflammatory response ensues after status epilepticus (SE), driven partly by cyclooxygenase-2-mediated activation of prostaglandin EP2 receptors. The inflammatory response is typified by astrocytosis, microgliosis, erosion of the blood-brain barrier (BBB), formation of inflammatory cytokines, and brain infiltration of blood-borne monocytes. Our previous studies have shown that inhibition of monocyte brain invasion or systemic administration of an EP2 receptor antagonist relieves multiple deleterious consequences of SE. Here we identify those effects of EP2 antagonism that are reproduced by conditional ablation of EP2 receptors in immune myeloid cells and show that systemic EP2 antagonism blocks monocyte brain entry in male mice. The induction of hippocampal IL-6 after pilocarpine SE was nearly abolished in EP2 conditional KO mice. Serum albumin levels in the cortex, a measure of BBB breakdown, were significantly higher after SE in EP2-sufficient mice but not in EP2 conditional KOs. EP2 deficiency in innate immune cells accelerated the recovery from sickness behaviors following SE. Surprisingly, neurodegeneration was not alleviated in myeloid conditional KOs. Systemic EP2 antagonism prevented monocyte brain infiltration and provided broader rescue of SE-induced effects than myeloid EP2 ablation, including neuroprotection and broader suppression of inflammatory mediators. Reporter expression indicated that the cellular target of CD11b-driven Cre was circulating myeloid cells but, unexpectedly, not microglia. These findings indicate that activation of EP2 receptors on immune myeloid cells drives substantial deficits in behavior and disrupts the BBB after SE. The benefits of systemic EP2 antagonism can be attributed, in part, to blocking brain recruitment of blood-borne monocytes.SIGNIFICANCE STATEMENT Unabated seizures reduce quality of life, promote the development of epilepsy, and can be fatal. We previously identified activation of prostaglandin EP2 receptors as a driver of undesirable consequences of seizures. However, the relevant EP2-expressing cell types remain unclear. Here we identify peripheral innate immune cells as a driver of the EP2-related negative consequences of seizures. Removal of EP2 from peripheral immune cells was beneficial, abolishing production of a key inflammatory cytokine, accelerating weight regain, and limiting behavioral deficits. These findings provide evidence that EP2 engagement on peripheral immune and brain endothelia contributes to the deleterious effects of SE, and will assist in the development of beneficial therapies to enhance quality of life in individuals who suffer prolonged seizures.
Assuntos
Imunidade Inata/fisiologia , Células Mieloides/metabolismo , Receptores de Prostaglandina E Subtipo EP2/biossíntese , Estado Epiléptico/metabolismo , Animais , Citometria de Fluxo/métodos , Hipocampo/citologia , Hipocampo/imunologia , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células Mieloides/imunologia , Receptores de Prostaglandina E Subtipo EP2/genética , Receptores de Prostaglandina E Subtipo EP2/imunologia , Estado Epiléptico/genética , Estado Epiléptico/imunologiaRESUMO
BACKGROUND: Alzheimer's disease (AD) causes substantial medical and societal burden with no therapies ameliorating cognitive deficits. Centralized pathologies involving amyloids, neurofibrillary tangles, and neuroinflammatory pathways are being investigated to identify disease-modifying targets for AD. Cyclooxygenase-2 (COX-2) is one of the potential neuroinflammatory agents involved in AD progression. However, chronic use of COX-2 inhibitors in patients produced adverse cardiovascular effects. We asked whether inhibition of EP2 receptors, downstream of the COX-2 signaling pathway, can ameliorate neuroinflammation in AD brains in presence or absence of a secondary inflammatory stimuli. METHODS: We treated 5xFAD mice and their non-transgenic (nTg) littermates in presence or absence of lipopolysaccharide (LPS) with an EP2 antagonist (TG11-77.HCl). In cohort 1, nTg (no-hit) or 5xFAD (single-hit-genetic) mice were treated with vehicle or TG11-77.HCl for 12 weeks. In cohort 2, nTg (single-hit-environmental) and 5xFAD mice (two-hit) were administered LPS (0.5 mg/kg/week) and treated with vehicle or TG11-77.HCl for 8 weeks. RESULTS: Complete blood count analysis showed that LPS induced anemia of inflammation in both groups in cohort 2. There was no adverse effect of LPS or EP2 antagonist on body weight throughout the treatment. In the neocortex isolated from the two-hit cohort of females, but not males, the elevated mRNA levels of proinflammatory mediators (IL-1ß, TNF, IL-6, CCL2, EP2), glial markers (IBA1, GFAP, CD11b, S110B), and glial proteins were significantly reduced by EP2 antagonist treatment. Intriguingly, the EP2 antagonist had no effect on either of the single-hit cohorts. There was a modest increase in amyloid-plaque deposition upon EP2 antagonist treatment in the two-hit female brains, but not in the single-hit genetic female cohort. CONCLUSION: These results reveal a potential neuroinflammatory role for EP2 in the two-hit 5xFAD mouse model. A selective EP2 antagonist reduces inflammation only in female AD mice subjected to a second inflammatory insult.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Doenças Neuroinflamatórias/tratamento farmacológico , Receptores de Prostaglandina E Subtipo EP2/antagonistas & inibidores , Doença de Alzheimer/patologia , Anemia/sangue , Animais , Contagem de Células Sanguíneas , Ciclo-Oxigenase 2/genética , Feminino , Humanos , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Transgênicos , Neuroglia/metabolismo , Doenças Neuroinflamatórias/patologia , Placa Amiloide/metabolismo , Placa Amiloide/patologia , Caracteres Sexuais , Transdução de Sinais/efeitos dos fármacosRESUMO
This review describes an adult rat model of status epilepticus (SE) induced by diisopropyl fluorophosphate (DFP), and the beneficial outcomes of transient inhibition of the prostaglandin-E2 receptor EP2 with a small molecule antagonist, delayed by 2-4â¯h after SE onset. Administration of six doses of the selective EP2 antagonist TG6-10-1 over a 2-3â¯day period accelerates functional recovery, attenuates hippocampal neurodegeneration, neuroinflammation, gliosis and blood-brain barrier leakage, and prevents long-term cognitive deficits without blocking SE itself or altering acute seizure characteristics. This work has provided important information regarding organophosphate-induced seizure related pathologies in adults and revealed the effectiveness of delayed EP2 inhibition to combat these pathologies.
Assuntos
Indóis/farmacologia , Intoxicação por Organofosfatos , Receptores de Prostaglandina E Subtipo EP2/antagonistas & inibidores , Estado Epiléptico/induzido quimicamente , Animais , Inibidores da Colinesterase/toxicidade , Modelos Animais de Doenças , Isoflurofato/toxicidade , RatosRESUMO
Azaindole structural framework is an integral part of several biologically active natural and synthetic organic molecules; and several FDA approved drugs for various diseases. In the last decade, quite a number of literature reports appeared describing the pharmacology, biological activity and therapeutic applications of a variety of azaindole molecules. This prompted the organic and medicinal chemistry community to develop novel synthetic methods for various azaindoles and test them for a bioactivity against a variety of biological targets. Herein, we have summarized the biological activity of therapeutically advanced clinical candidates and several preclinical candidate drugs that contain azaindole structural moiety.
Assuntos
Compostos Aza/química , Indóis/química , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/uso terapêutico , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Humanos , Hipersensibilidade/tratamento farmacológico , Indóis/farmacologia , Indóis/uso terapêutico , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/patologiaRESUMO
The cervix represents a formidable structural barrier for successful induction of labor. Approximately 10% of pregnancies undergo induction of cervical ripening and labor with prostaglandin (PG) E2 or PGE analogs, often requiring many hours of hospitalization and monitoring. On the other hand, preterm cervical ripening in the second trimester predicts preterm birth. The regulatory mechanisms of this paradoxical function of the cervix are unknown. Here, we show that PGE2 uses cell-specific EP2 receptor-mediated increases in Ca2+ to dephosphorylate and translocate histone deacetylase 4 (HDAC4) to the nucleus for repression of 15-hydroxy prostaglandin dehydrogenase (15-PGDH). The crucial role of 15-PGDH in cervical ripening was confirmed in vivo. Although PGE2 or 15-PGDH inhibitor alone did not alter gestational length, treatment with 15-PGDH inhibitor + PGE2 or metabolism-resistant dimethyl-PGE2 resulted in preterm cervical ripening and delivery in mice. The ability of PGE2 to selectively autoamplify its own synthesis in stromal cells by signaling transcriptional repression of 15-PGDH elucidates long sought-after molecular mechanisms that govern PG action in the cervix. This report details unique mechanisms of action in the cervix and serves as a catalyst for (i) the use of 15-PGDH inhibitors to initiate or amplify low-dose PGE2-mediated cervical ripening or (ii) EP2 receptor antagonists, HDAC4 inhibitors, and 15-PGDH activators to prevent preterm cervical ripening and preterm birth.
Assuntos
Maturidade Cervical/metabolismo , Dinoprostona/metabolismo , Histona Desacetilases/metabolismo , Hidroxiprostaglandina Desidrogenases/metabolismo , Nascimento Prematuro/fisiopatologia , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Proteínas Repressoras/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular , Colo do Útero/citologia , Colo do Útero/fisiologia , Ciclo-Oxigenase 2/metabolismo , Regulação para Baixo , Feminino , Histona Desacetilase 2/genética , Histona Desacetilases/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Prostaglandina-E Sintases/antagonistas & inibidores , Prostaglandina-E Sintases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genéticaRESUMO
The prostaglandin E2 receptor, EP2, plays an important role in physiology and in a variety of pathological conditions. Studies indicate that EP2 is pro-inflammatory in chronic peripheral and central nervous system disease and cancer models. Thus, targeting the EP2 receptor with small molecules could be a therapeutic strategy for treating inflammatory diseases and cancer. We recently reported a novel class of competitive antagonists of the EP2 receptor. However, earlier leads displayed low selectivity against the DP1 prostanoid receptor, moderate plasma half-life, and low aqueous solubility, which renders them suboptimal for testing in animal models of disease. We now report a novel compound TG8-69, which has suitable drug-like properties. We present synthesis, lead-optimization studies, pharmacological characterization, and anti-inflammatory properties of this compound that support its use in chronic peripheral inflammatory diseases, including rheumatoid arthritis, endometriosis, and cancer, in which EP2 appears to play a pathogenic role.
Assuntos
Anti-Inflamatórios/farmacologia , Mediadores da Inflamação/metabolismo , Receptores de Prostaglandina E Subtipo EP2/antagonistas & inibidores , Regulação para Cima/efeitos dos fármacos , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/imunologia , Linhagem Celular , Dinoprostona/imunologia , Dinoprostona/metabolismo , Avaliação Pré-Clínica de Medicamentos , Endometriose/tratamento farmacológico , Endometriose/imunologia , Feminino , Meia-Vida , Mediadores da Inflamação/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Ratos , Receptores de Prostaglandina/metabolismo , Receptores de Prostaglandina E Subtipo EP2/imunologia , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Solubilidade , Regulação para Cima/imunologia , Água/químicaRESUMO
Prostaglandin E2 is now widely recognized to play critical roles in brain inflammation and injury, although the responsible prostaglandin receptors have not been fully identified. We developed a potent and selective antagonist for the prostaglandin E2 receptor subtype EP2, TG6-10-1, with a sufficient pharmacokinetic profile to be used in vivo. We found that in the mouse pilocarpine model of status epilepticus (SE), systemic administration of TG6-10-1 completely recapitulates the effects of conditional ablation of cyclooxygenase-2 from principal forebrain neurons, namely reduced delayed mortality, accelerated recovery from weight loss, reduced brain inflammation, prevention of blood-brain barrier opening, and neuroprotection in the hippocampus, without modifying seizures acutely. Prolonged SE in humans causes high mortality and morbidity that are associated with brain inflammation and injury, but currently the only effective treatment is to stop the seizures quickly enough with anticonvulsants to prevent brain damage. Our results suggest that the prostaglandin receptor EP2 is critically involved in neuroinflammation and neurodegeneration, and point to EP2 receptor antagonism as an adjunctive therapeutic strategy to treat SE.
Assuntos
Encéfalo/patologia , Receptores de Prostaglandina E Subtipo EP2/antagonistas & inibidores , Estado Epiléptico/patologia , Animais , Anticonvulsivantes/farmacologia , Anticonvulsivantes/uso terapêutico , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/patologia , Barreira Hematoencefálica/fisiopatologia , Encéfalo/efeitos dos fármacos , Encéfalo/fisiopatologia , Modelos Animais de Doenças , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Hipocampo/fisiopatologia , Humanos , Indóis/farmacologia , Indóis/uso terapêutico , Inflamação/complicações , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Modelos Neurológicos , Degeneração Neural/complicações , Degeneração Neural/tratamento farmacológico , Degeneração Neural/patologia , Degeneração Neural/fisiopatologia , Pilocarpina , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Recuperação de Função Fisiológica/efeitos dos fármacos , Estado Epiléptico/complicações , Estado Epiléptico/tratamento farmacológico , Estado Epiléptico/fisiopatologia , Análise de Sobrevida , Redução de Peso/efeitos dos fármacosRESUMO
The timely resolution of inflammation prevents continued tissue damage after an initial insult. In the brain, the death of activated microglia by apoptosis has been proposed as one mechanism to resolve brain inflammation. How microglial death is regulated after activation is still unclear. We reported that exposure to lipopolysaccharide (LPS) and interleukin (IL)-13 together initially activates and then kills rat microglia in culture by a mechanism dependent on cyclooxygenase-2 (COX-2). We show here that activation of the E prostanoid receptor 2 (EP2, PTGER2) for prostaglandin E2 mediates microglial death induced by LPS/IL-13, and that EP2 activation by agonist alone kills microglia. Both EP2 antagonists and reactive oxygen scavengers block microglial death induced by either LPS/IL-13 or EP2 activation. By contrast, the homeostatic induction of heme oxygenase 1 (Hmox1) by LPS/IL-13 or EP2 activation protects microglia. Both the Hmox1 inducer cobalt protoporphyrin and a compound that releases the Hmox1 product carbon monoxide (CO) attenuated microglial death produced by LPS/IL-13. Whereas CO reduced COX-2 protein expression, EP2 activation increased Hmox1 and COX-2 expression at both the mRNA and protein level. Interestingly, caspase-1 inhibition prevented microglial death induced by either LPS/IL-13 or low (but not high) concentrations of butaprost, suggestive of a predominantly pyroptotic mode of death. Butaprost also caused the expression of activated caspase-3 in microglia, pointing to apoptosis. These results indicate that EP2 activation, which initially promotes microglial activation, later causes delayed death of activated microglia, potentially contributing to the resolution phase of neuroinflammation.
Assuntos
Apoptose , Microglia/patologia , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Transdução de Sinais , Estado Epiléptico/metabolismo , Alprostadil/análogos & derivados , Alprostadil/farmacologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Interleucina-13/imunologia , Lipopolissacarídeos/imunologia , Camundongos , Microglia/metabolismo , Pilocarpina , Gravidez , Ratos , Ratos Sprague-Dawley , Receptores de Prostaglandina E Subtipo EP2/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Estado Epiléptico/induzido quimicamenteRESUMO
Recent progress characterizing the reaction mechanism(s) of fluorescent probes with reactive oxygen species has made it possible to rigorously analyze these reactive species in biological systems. We have developed rapid high throughput-compatible assays for monitoring cellular production of superoxide radical anion and hydrogen peroxide using hydropropidine and coumarin boronic acid probes, respectively. Coupling plate reader-based fluorescence measurements with HPLC-based simultaneous monitoring of superoxide radical anion and hydrogen peroxide provides the basis for the screening protocol for NADPH oxidase (Nox) inhibitors. Using this newly developed approach along with the medium-throughput plate reader-based oximetry and EPR spin trapping as confirmatory assays, it is now eminently feasible to rapidly and reliably identify Nox enzyme inhibitors with a markedly lower rate of false positives. These methodological advances provide an opportunity to discover selective inhibitors of Nox isozymes, through enhanced conceptual understanding of their basic mechanisms of action.
Assuntos
Inibidores Enzimáticos/análise , Ensaios de Triagem em Larga Escala , Peróxido de Hidrogênio/análise , NADPH Oxidases/antagonistas & inibidores , Superóxidos/análise , Cromatografia Líquida de Alta Pressão , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Células HEK293 , Células HL-60 , Humanos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Espectrometria de Massas , NADPH Oxidases/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Marcadores de Spin , Superóxidos/metabolismo , Superóxidos/farmacologiaRESUMO
As a prominent inflammatory effector of cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2) mediates brain inflammation and injury in many chronic central nervous system (CNS) conditions including seizures and epilepsy, largely through its receptor subtype EP2. However, EP2 receptor activation might also be neuroprotective in models of excitotoxicity and ischemia. These seemingly incongruent observations expose the delicacy of immune and inflammatory signaling in the brain; thus the therapeutic window for quelling neuroinflammation might vary with injury type and target molecule. Here, we identify a therapeutic window for EP2 antagonism to reduce delayed mortality and functional morbidity after status epilepticus (SE) in mice. Importantly, treatment must be delayed relative to SE onset to be effective, a finding that could be explained by the time-course of COX-2 induction after SE and compound pharmacokinetics. A large number of inflammatory mediators were upregulated in hippocampus after SE with COX-2 and IL-1ß temporally leading many others. Thus, EP2 antagonism represents a novel anti-inflammatory strategy to treat SE with a tightly-regulated therapeutic window.
Assuntos
Anti-Inflamatórios/uso terapêutico , Ciclo-Oxigenase 2/metabolismo , Indóis/administração & dosagem , Indóis/farmacologia , Indóis/uso terapêutico , Receptores de Prostaglandina E Subtipo EP2/antagonistas & inibidores , Estado Epiléptico/tratamento farmacológico , Estado Epiléptico/metabolismo , Animais , Modelos Animais de Doenças , Encefalite/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Estimativa de Kaplan-Meier , Camundongos , Camundongos Endogâmicos C57BL , Pilocarpina , Transdução de Sinais/efeitos dos fármacos , Estado Epiléptico/mortalidadeRESUMO
With interest waning in the use of cyclooxygenase-2 (COX-2) inhibitors for inflammatory disease, prostaglandin receptors provide alternative targets for the treatment of COX-2-mediated pathological conditions in both the periphery and the central nervous system. Activation of prostaglandin E2 receptor (PGE(2)) subtype EP2 promotes inflammation and is just beginning to be explored as a therapeutic target. To better understand physiological and pathological functions of the prostaglandin EP2 receptor, we developed a suite of small molecules with a 3-aryl-acrylamide scaffold as selective EP2 antagonists. The 12 most potent compounds displayed competitive antagonism of the human EP2 receptor with K(B) 2-20 nM in Schild regression analysis and 268- to 4,730-fold selectivity over the prostaglandin EP4 receptor. A brain-permeant compound completely suppressed the up-regulation of COX-2 mRNA in rat cultured microglia by EP2 activation and significantly reduced neuronal injury in hippocampus when administered in mice beginning 1 h after termination of pilocarpine-induced status epilepticus. The salutary actions of this novel group of antagonists raise the possibility that selective block of EP2 signaling via small molecules can be an innovative therapeutic strategy for inflammation-related brain injury.
Assuntos
Neurônios/efeitos dos fármacos , Neurônios/patologia , Receptores de Prostaglandina E Subtipo EP2/antagonistas & inibidores , Convulsões/metabolismo , Convulsões/patologia , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Ciclo-Oxigenase 2/biossíntese , Indução Enzimática/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/enzimologia , Ratos , Ratos Sprague-Dawley , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Bibliotecas de Moléculas Pequenas/administração & dosagem , Bibliotecas de Moléculas Pequenas/química , Estado Epiléptico/patologia , Relação Estrutura-AtividadeRESUMO
Epilepsy is one of the more prevalent neurologic disorders in the world, affecting approximately 50 million people of different ages and backgrounds. Epileptic seizures propagating through both lobes of the forebrain can have permanent debilitating effects on a patient's cognitive and somatosensory brain functions. Epilepsy, defined by the sporadic occurrence of spontaneous recurrent seizures (SRS), is often accompanied by inflammation of the brain. Pronounced increases in the expression of key inflammatory mediators (e.g., interleukin -1ß [IL-1ß], tumor necrosis factor alpha [TNFα], cyclooxygenase-2 [COX-2], and C-X-C motif chemokine 10 [CXCL10]) after seizures may cause secondary damage in the brain and increase the likelihood of repetitive seizures. The COX-2 enzyme is induced rapidly during seizures. The increased level of COX-2 in specific areas of the epileptic brain can help to identify regions of seizure-induced brain inflammation. A good deal of effort has been expended to determine whether COX-2 inhibition might be neuroprotective and represent an adjunct therapeutic strategy along with antiepileptic drugs used to treat epilepsy. However, the effectiveness of COX-2 inhibitors on epilepsy animal models appears to depend on the timing of administration. With all of the effort placed on making use of COX-2 inhibitors as therapeutic agents for the treatment of epilepsy, inflammation, and neurodegenerative diseases there has yet to be a selective and potent COX-2 inhibitor that has shown a clear therapeutic outcome with acceptable side effects.
Assuntos
Ciclo-Oxigenase 2/fisiologia , Epilepsia/enzimologia , Animais , Anticonvulsivantes/farmacologia , Barreira Hematoencefálica/efeitos dos fármacos , Encéfalo/enzimologia , Inibidores de Ciclo-Oxigenase 2/farmacologia , Epilepsia/tratamento farmacológico , Humanos , Inflamação/enzimologia , Doenças Neurodegenerativas/enzimologia , Convulsões/tratamento farmacológico , Convulsões/enzimologiaRESUMO
The epothilones, including epothilones B and D, are macrocyclic lactones, which have potent cytotoxicities and promote the polymerization of tubulin to mictotubules by binding to and stabilizing the tubulin polymer. They have a very similar mechanism of action to paclitaxel (Taxol®). The determination of the microtubule-binding conformation of the epothilones is an important piece of information in designing improved analogs for possible clinical use, and internuclear distance information that will assist the determination of this conformation can be obtained by rotational echo double resonance (REDOR) NMR studies of microtubule-bound epothilones with appropriate stable isotope labels. Analogs of epothilone B and epothilone D with [(2) H3 ] and [(19) F] labels were prepared from an advanced precursor for potential use in REDOR NMR studies to determine internuclear distances in tubulin-bound ligand.
Assuntos
Deutério/química , Epotilonas/síntese química , Marcação por Isótopo , Moduladores de Tubulina/síntese química , Linhagem Celular Tumoral , Epotilonas/química , Epotilonas/farmacologia , Humanos , Ligação Proteica , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologiaRESUMO
Targeting cancer metabolism to limit cellular energy and metabolite production is an attractive therapeutic approach. Here, we developed analogs of the bisbiguanide, alexidine, to target lung cancer cell metabolism and assess a structure-activity relationship (SAR). The SAR led to the identification of two analogs, AX-4 and AX-7, that limit cell growth via G1/G0 cell-cycle arrest and are tolerated in vivo with favorable pharmacokinetics. Mechanistic evaluation revealed that AX-4 and AX-7 induce potent mitochondrial defects; mitochondrial cristae were deformed and the mitochondrial membrane potential was depolarized. Additionally, cell metabolism was rewired, as indicated by reduced oxygen consumption and mitochondrial ATP production, with an increase in extracellular lactate. Importantly, AX-4 and AX-7 impacted overall cell behavior, as these compounds reduced collective cell invasion. Taken together, our study establishes a class of bisbiguanides as effective mitochondria and cell invasion disrupters, and proposes bisbiguanides as promising approaches to limiting cancer metastasis.
RESUMO
Activation of the Galphas-coupled EP2 receptor for prostaglandin E2 (PGE(2)) promotes cell survival in several models of tissue damage. To advance understanding of EP2 functions, we designed experiments to develop allosteric potentiators of this key prostaglandin receptor. Screens of 292,000 compounds identified 93 that at 20 microM (i) potentiated the cAMP response to a low concentration of PGE(2) by > 50%; (ii) had no effect on EP4 or beta2 adrenergic receptors, the cAMP assay itself, or the parent cell line; and (iii) increased the potency of PGE(2) on EP2 receptors at least 3-fold. In aqueous solution, the active compounds are largely present as nanoparticles that appear to serve as active reservoirs for bioactive monomer. From 94 compounds synthesized or purchased, based on the modification of one hit compound, the most active increased the potency of PGE(2) on EP2 receptors 4- to 5-fold at 10 to 20 microM and showed substantial neuroprotection in an excitotoxicity model. These small molecules represent previously undescribed allosteric modulators of a PGE(2) receptor. Our results strongly reinforce the notion that activation of EP2 receptors by endogenous PGE(2) released in a cell-injury setting is neuroprotective.
Assuntos
Fármacos Neuroprotetores/farmacologia , Receptores de Prostaglandina E/agonistas , Regulação Alostérica , Animais , Técnicas Biossensoriais , AMP Cíclico/metabolismo , Dinoprostona/metabolismo , Avaliação Pré-Clínica de Medicamentos , Transferência Ressonante de Energia de Fluorescência , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Técnicas In Vitro , Nanopartículas , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/química , Ratos , Ratos Endogâmicos SHR , Receptores de Prostaglandina E Subtipo EP2 , Relação Estrutura-AtividadeRESUMO
Cyclooxygenase-1 and -2 (COX1 and COX2) derived endogenous ligand prostaglandin-E2 (PGE2) triggers several physiological and pathological conditions. It mediates signaling through four G-protein coupled receptors, EP1, EP2, EP3, and EP4. Among these, EP2 is expressed throughout the body including the brain and uterus. The functional role of EP2 has been extensively studied using EP2 gene knockout mice, cellular models, and selective small molecule agonists and antagonists for this receptor. The efficacy data from in vitro and in vivo animal models indicate that EP2 receptor is a major proinflammatory mediator with deleterious functions in a variety of diseases suggesting a path forward for EP2 inhibitors as the next generation of selective anti-inflammatory and antiproliferative agents. Interestingly in certain diseases, EP2 action is beneficial; therefore, EP2 agonists seem to be clinically useful. Here, we highlight the strengths, weaknesses, opportunities, and potential threats (SWOT analysis) for targeting EP2 receptor for therapeutic development for a variety of unmet clinical needs.
Assuntos
Dinoprostona , Receptores de Prostaglandina E , Animais , Camundongos , Receptores de Prostaglandina E/agonistas , Receptores de Prostaglandina E/genética , Dinoprostona/farmacologia , Dinoprostona/fisiologia , Ciclo-Oxigenase 2 , Descoberta de Drogas , Receptores de Prostaglandina E Subtipo EP2 , Receptores de Prostaglandina E Subtipo EP4RESUMO
A family of seven NADPH oxidase enzymes (Nox1-5, Duox1-2) has been implicated in a variety of diseases, including inflammatory lung diseases, neurodegenerative diseases, cardiovascular diseases, and cancer. Here, we report the results of our studies aimed at developing novel brain-permeable Nox2 inhibitors with potential application as neuroprotective agents. Using cell-based assays, we identified a novel Nox2 inhibitor, TG15-132, that prevents PMA-stimulated oxygen consumption and reactive oxygen species (superoxide radical anion and hydrogen peroxide) formation upon acute treatment in differentiated HL60 cells. Long-term treatment with TG15-132 attenuates the induction of genes encoding Nox2 subunits, several inflammatory cytokines, and iNOS in differentiated THP-1 cells. Moreover, TG15-132 shows a relatively long plasma half-life (5.6 h) and excellent brain permeability, with a brain-to-plasma ratio (>5-fold) in rodent models. Additionally, TG15-132 does not cause any toxic effects on vital organs or blood biomarkers of toxicity in mice upon chronic dosing for seven days. We propose that TG15-132 may be used as a Nox2 inhibitor and a potential neuroprotective agent, with possible further structural modifications to increase its potency.
RESUMO
SARS-CoV-2-induced impaired antiviral and excessive inflammatory responses cause fatal pneumonia. However, the key pattern recognition receptors that elicit effective antiviral and lethal inflammatory responses in-vivo are not well defined. CoVs possess single-stranded RNA (ssRNA) genome that is abundantly produced during infection and stimulates both antiviral interferon (IFN) and inflammatory cytokine/ chemokine responses. Therefore, in this study, using wild-type control and TLR7 deficient BALB/c mice infected with a mouse-adapted SARS-COV-2 (MA-CoV-2), we evaluated the role of TLR7 signaling in MA-CoV-2-induced antiviral and inflammatory responses and disease outcome. We show that TLR7-deficient mice are more susceptible to MA-CoV-2 infection as compared to infected control mice. Further evaluation of MA-CoV-2 infected lungs showed significantly reduced mRNA levels of antiviral type I (IFNα/ß) and type III (IFNλ) IFNs, IFN stimulated genes (ISGs, ISG15 and CXCL10), and several pro-inflammatory cytokines/chemokines in TLR7 deficient compared to control mice. Reduced lung IFN/ISG levels and increased morbidity/mortality in TLR7 deficient mice correlated with high lung viral titer. Detailed examination of total cells from MA-CoV-2 infected lungs showed high neutrophil count in TLR7 deficient mice compared to control mice. Additionally, blocking TLR7 activity post-MA-CoV-2 infection using a specific inhibitor also enhanced disease severity. In summary, our results conclusively establish that TLR7 signaling is protective during SARS-CoV-2 infection, and despite robust inflammatory response, TLR7-mediated IFN/ISG responses likely protect the host from lethal disease. Given similar outcomes in control and TLR7 deficient humans and mice, these results show that MA-CoV-2 infected mice serve as excellent model to study COVID-19.
RESUMO
Traumatic brain injury (TBI) in patients results in a massive inflammatory reaction, disruption of blood-brain barrier, and oxidative stress in the brain, and these inciting features may culminate in the emergence of post-traumatic epilepsy (PTE). We hypothesize that targeting these pathways with pharmacological agents could be an effective therapeutic strategy to prevent epileptogenesis. To design therapeutic strategies targeting neuroinflammation and oxidative stress, we utilized a fluid percussion injury (FPI) rat model to study the temporal expression of neuroinflammatory and oxidative stress markers from 3 to 24 h following FPI. FPI results in increased mRNA expression of inflammatory mediators including cyclooxygenase-2 (COX-2) and prostanoid receptor EP2, marker of oxidative stress (NOX2), astrogliosis (GFAP), and microgliosis (CD11b) in ipsilateral cortex and hippocampus. The analysis of protein levels indicated a significant increase in the expression of COX-2 in ipsilateral hippocampus and cortex post-FPI. We tested FPI rats with an EP2 antagonist TG8-260 which produced a statistically significant reduction in the distribution of seizure duration post-FPI and trends toward a reduction in seizure incidence, seizure frequency, and duration, hinting a proof of concept that EP2 antagonism must be further optimized for therapeutic applications to prevent epileptogenesis.
RESUMO
Cognitive comorbidities can substantially reduce quality of life in people with epilepsy. Inflammation is a component of all chronic diseases including epilepsy, as well as acute events like status epilepticus (SE). Neuroinflammation is the consequence of several broad signaling cascades including cyclooxygenase-2 (COX-2)-associated pathways. Activation of the EP2 receptor for prostaglandin E2 appears responsible for blood-brain barrier leakage and much of the inflammatory reaction, neuronal injury and cognitive deficit that follows seizure-provoked COX-2 induction in brain. Here we show that brief exposure of mice to TG11-77, a potent, selective, orally available and brain permeant EP2 antagonist, eliminates the profound cognitive deficit in Y-maze performance after SE and reduces delayed mortality and microgliosis, with a minimum effective i.p. dose (as free base) of 8.8 mg/kg. All in vitro studies required to submit an investigational new drug (IND) application for TG11-77 have been completed, and non-GLP dose range-finding toxicology in the rat identified no overt, organ or histopathology signs of toxicity after 7 days of oral administration at 1000 mg/kg/day. Plasma exposure in the rat was dose-linear between 15 and 1000 mg/kg dosing. TG11-77 thus appears poised to continue development towards the initial clinical test of the hypothesis that EP2 receptor modulation after SE can provide the first preventive treatment for one of the chief comorbidities of epilepsy.