RESUMO
BACKGROUND: Newly proliferated oligodendrocyte precursor cells (OPCs) migrate and surround lesions of patients with multiple sclerosis (MS) and other demyelinating diseases, but fail to differentiate into oligodendrocytes (OLs) and remyelinate remaining viable axons. The abundance of secreted inflammatory factors within and surrounding these lesions likely plays a major inhibitory role, promoting cell death and preventing OL differentiation and axon remyelination. To identify clinical candidate compounds that may protect existing and differentiating OLs in patients, we have developed a high throughput screening (HTS) assay that utilizes purified rat OPCs. RESULTS: Using a fluorescent indicator of cell viability coupled with image quantification, we developed an assay to allow the identification of compounds that promote OL viability and differentiation in the presence of the synergistic inflammatory cytokines, tumor necrosis factor α and interferon-γ. We have utilized this assay to screen the NIH clinical collection library and identify compounds that protect OLs and promote OL differentiation in the presence of these inflammatory cytokines. CONCLUSION: This primary OL-based cytokine protection assay is adaptable for HTS and may be easily modified for profiling of compounds in the presence of other potentially inhibitory molecules found in MS lesions. This assay should be of use to those interested in identifying drugs for the treatment of MS and other demyelinating diseases.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Oligodendroglia/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Inflamação , Interferon gama/metabolismo , Masculino , Esclerose Múltipla/patologia , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Ratos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Multiple sclerosis is caused by an autoimmune response resulting in demyelination and neural degeneration. The adult central nervous system has the capacity to remyelinate axons in part through the generation of new oligodendrocytes (OLs). To identify clinical candidate compounds that may promote remyelination, we have developed a high throughput screening (HTS) assay to identify compounds that promote the differentiation of oligodendrocyte precursor cells (OPCs) into OLs. RESULTS: Using acutely dissociated and purified rat OPCs coupled with immunofluorescent image quantification, we have developed an OL differentiation assay. We have validated this assay with a known promoter of differentiation, thyroid hormone, and subsequently used the assay to screen the NIH clinical collection library. We have identified twenty-seven hit compounds which were validated by dose response analysis and the generation of half maximal effective concentration (EC50) values allowed for the ranking of efficacy. The assay identified novel promoters of OL differentiation which we attribute to (1) the incorporation of an OL toxicity pre-screen to allow lowering the concentrations of toxic compounds and (2) the utilization of freshly purified, non-passaged OPCs. These features set our assay apart from other OL differentiation assays used for drug discovery efforts. CONCLUSIONS: This acute primary OL-based differentiation assay should be of use to those interested in screening large compound libraries for the identification of drugs for the treatment of MS and other demyelinating diseases.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Oligodendroglia/citologia , Oligodendroglia/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Animais , Técnicas In Vitro , Esclerose Múltipla/tratamento farmacológico , Ratos , Células-Tronco/citologiaRESUMO
BACKGROUND: Regeneration of new myelin is impaired in persistent multiple sclerosis (MS) lesions, leaving neurons unable to function properly and subject to further degeneration. Current MS therapies attempt to ameliorate autoimmune-mediated demyelination, but none directly promote the regeneration of lost and damaged myelin of the central nervous system (CNS). Development of new drugs that stimulate remyelination has been hampered by the inability to evaluate axonal myelination in a rapid CNS culture system. RESULTS: We established a high throughput cell-based assay to identify compounds that promote myelination. Culture methods were developed for initiating myelination in vitro using primary embryonic rat cortical cells. We developed an immunofluorescent phenotypic image analysis method to quantify the morphological alignment of myelin characteristic of the initiation of myelination. Using γ-secretase inhibitors as promoters of myelination, the optimal growth, time course and compound treatment conditions were established in a 96 well plate format. We have characterized the cortical myelination assay by evaluating the cellular composition of the cultures and expression of markers of differentiation over the time course of the assay. We have validated the assay scalability and consistency by screening the NIH clinical collection library of 727 compounds and identified ten compounds that promote myelination. Half maximal effective concentration (EC50) values for these compounds were determined to rank them according to potency. CONCLUSIONS: We have designed the first high capacity in vitro assay that assesses myelination of live axons. This assay will be ideal for screening large compound libraries to identify new drugs that stimulate myelination. Identification of agents capable of promoting the myelination of axons will likely lead to the development of new therapeutics for MS patients.
Assuntos
Axônios/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Esclerose Múltipla/tratamento farmacológico , Bainha de Mielina/efeitos dos fármacos , Regeneração Nervosa/efeitos dos fármacos , Secretases da Proteína Precursora do Amiloide/farmacologia , Animais , Axônios/fisiologia , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Córtex Cerebral/fisiologia , Meios de Cultivo Condicionados/farmacologia , Imunofluorescência/métodos , Esclerose Múltipla/fisiopatologia , Bainha de Mielina/fisiologia , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/fisiologia , RatosRESUMO
Oligodendrocyte death contributes to the pathogenesis of the inflammatory demyelinating disease multiple sclerosis (MS). Nevertheless, current MS therapies are mainly immunomodulatory and have demonstrated limited ability to inhibit MS progression. Protection of oligodendrocytes is therefore a desirable strategy for alleviating disease. Here we demonstrate that enhancement of the integrated stress response using the FDA-approved drug guanabenz increases oligodendrocyte survival in culture and prevents hypomyelination in cerebellar explants in the presence of interferon-γ, a pro-inflammatory cytokine implicated in MS pathogenesis. In vivo, guanabenz treatment protects against oligodendrocyte loss caused by CNS-specific expression of interferon-γ. In a mouse model of MS, experimental autoimmune encephalomyelitis, guanabenz alleviates clinical symptoms, which correlates with increased oligodendrocyte survival and diminished CNS CD4+ T cell accumulation. Moreover, guanabenz ameliorates relapse in relapsing-remitting experimental autoimmune encephalomyelitis. Our results provide support for a MS therapy that enhances the integrated stress response to protect oligodendrocytes against the inflammatory CNS environment.
Assuntos
Guanabenzo/farmacologia , Esclerose Múltipla/tratamento farmacológico , Oligodendroglia/citologia , Animais , Linfócitos T CD4-Positivos/citologia , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , Sistema Nervoso Central/metabolismo , Cerebelo/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Encefalomielite Autoimune Experimental/imunologia , Feminino , Inflamação , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Bainha de Mielina/metabolismo , Ratos , Ratos Sprague-Dawley , Células-Tronco/citologiaRESUMO
BACKGROUND: New multiple sclerosis (MS) lesion activity on magnetic resonance imaging (MRI) can test immunomodulatory therapies in proof-of-concept trials. Comparably powerful endpoints to assess tissue protection or repair are lacking. OBJECTIVE: The objective of this paper is to report sample-size calculations for assessment of new lesion recovery. METHODS: In two sets of six active MS cases, new lesions were observed by monthly MRI for approximately 12 months. Averages and quartiles of normalized (proton density/T1/T2 weighted) and quantitative (T1/T2 and mean diffusivity maps for dataset 1, T2 and magnetization transfer ratio maps for dataset 2) measures were used to compare the lesion area before lesion appearance to afterward. A linear mixed-effects model incorporating lesion- and participant-specific random effects estimated average levels and variance components for sample-size calculations. RESULTS: In both datasets, greatest statistical sensitivity was observed for the 25th percentile of normalized proton density-weighted signal. At 3T, using new lesions ⩾15 mm(3), as few as nine participants/arm may be required for a six-month placebo-controlled add-on trial postulating a therapeutic effect size of 20% and statistical power of 90%. CONCLUSION: Lesion recovery is a powerful outcome measure for proof-of-concept clinical trials of tissue protection and repair in MS. The trial design requires active cases and is therefore best implemented near disease onset.
Assuntos
Imageamento por Ressonância Magnética , Esclerose Múltipla/diagnóstico , Esclerose Múltipla/terapia , Substância Branca/patologia , Adolescente , Adulto , Feminino , Humanos , Imunomodulação , Masculino , Tamanho da Amostra , Resultado do TratamentoRESUMO
Based on their structural similarity to previously described compound AMG 009, indole-phenyl acetic acids were proposed to be potent dual inhibitors of chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2 or DP2) and prostanoid D receptor (DP or DP1). This series was equipotent to AMG 009 in binding assays against both receptors but exhibited decreased serum shift. We discovered early in the optimization of these indole-phenylacetic acid compounds that they demonstrated CYP3A4 time-dependent inhibition (TDI). Hypothesizing that the source of TDI was the indole core we modified the 1,2,3-substitution to eventually afford a highly potent modulator of CRTH2 and DP which did not exhibit TDI.
Assuntos
Citocromo P-450 CYP3A/metabolismo , Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Fenilacetatos/farmacologia , Receptores Imunológicos/antagonistas & inibidores , Receptores de Prostaglandina/antagonistas & inibidores , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Indóis/química , Estrutura Molecular , Fenilacetatos/química , Relação Estrutura-Atividade , Fatores de TempoRESUMO
Prostaglandin D2 (PGD2) plays a key role in mediating allergic reactions seen in asthma, allergic rhinitis, and atopic dermatitis. PGD2 exerts its activity through two G protein-coupled receptors (GPCRs), prostanoid D receptor (DP or DP1), and chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2 or DP2). We report the optimization of a series of phenylacetic acid derivatives in an effort to improve the dual activity of AMG 009 against DP and CRTH2. These efforts led to the discovery of AMG 853 (2-(4-(4-(tert-butylcarbamoyl)-2-(2-chloro-4-cyclopropylphenyl sulfonamido)phenoxy)-5-chloro-2-fluorophenyl)acetic acid), which is being evaluated in human clinical trials for asthma.
RESUMO
A general way of improving the potency of CXCR3 antagonists with fused hetero-bicyclic cores was identified. Optimization efforts led to the discovery of a series of imidazo-pyrazine derivatives with improved pharmacokinetic properties in addition to increased potency. The efficacy of the lead compound 21 is evaluated in a mouse lung inflammation model.
Assuntos
Anti-Inflamatórios/química , Benzenoacetamidas/química , Óxidos S-Cíclicos/química , Imidazóis/química , Pirazinas/química , Receptores CXCR3/antagonistas & inibidores , Animais , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/farmacocinética , Benzenoacetamidas/síntese química , Benzenoacetamidas/farmacocinética , Óxidos S-Cíclicos/síntese química , Óxidos S-Cíclicos/farmacocinética , Humanos , Imidazóis/síntese química , Imidazóis/farmacocinética , Camundongos , Conformação Molecular , Pirazinas/síntese química , Pirazinas/farmacocinética , Ratos , Receptores CXCR3/metabolismoRESUMO
Metastasis continues to be the leading cause of mortality for patients with cancer. Several years ago, it became clear that chemokines and their receptors could control the tumor progress. CXCR3 has now been identified in many cancers including osteosarcoma and CXCR3 ligands were expressed by lungs that are the primary sites to which this tumor metastasize. This study tested the hypothesis that disruption of the CXCR3/CXCR3 ligands complexes could lead to a decrease in lungs metastasis. The experimental design involved the use of the CXCR3 antagonist, AMG487 and 2 murine models of osteosarcoma lung metastases. After tail vein injection of osteosarcoma cells, mice that were systematically treated with AMG487 according to preventive or curative protocols had a significant reduction in metastatic disease. Treatment of osteosarcoma cells in vitro with AMG487 led to decreased migration, decreased matrix metalloproteinase activity, decreased proliferation/survival and increased caspase-independent death. Taken together, our results support the hypothesis that CXCR3 and their ligands intervene in the initial dissemination of the osteosarcoma cells to the lungs and stimulate the growth and expansion of the metastatic foci in later stages. Moreover, these studies indicate that targeting CXCR3 may specifically inhibit tumor metastasis without adversely affecting antitumoral host response.
Assuntos
Neoplasias Pulmonares/prevenção & controle , Osteossarcoma/prevenção & controle , Receptores CXCR3/antagonistas & inibidores , Acetamidas/farmacologia , Animais , Apoptose , Western Blotting , Cálcio/metabolismo , Caspases/metabolismo , Movimento Celular , Feminino , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Pirimidinonas/farmacologia , Receptores CXCR3/genética , Receptores CXCR3/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Current immunosuppression regimens are toxic to transplant recipients and, in many cases, acute rejection episodes occur because of escape of donor-reactive lymphocytes from the immunosuppression. T cells are the mediators of acute, cell-mediated graft damage and are hypothesized to use the CXCR3 chemokine axis for migration into the allograft. This study investigates the effect of CXCR3 blockade using a nonpeptide, small molecule inhibitor, AMG1237845, in murine cardiac allograft survival. METHODS: C57BL/6 (H-2) mice received vascularized cardiac allografts from A/J (H-2) donors and were treated with the CXCR3 antagonist. Histologic and flow cytometric analyses were used to measure infiltration of leukocytes, and quantitative reverse-transcriptase polymerase chain reaction and interferon-gamma ELISPOT assays were used to measure donor-specific reactivity. RESULTS: CXCR3 antagonism modestly prolonged allograft survival compared with vehicle treatment, but at time-matched intervals posttransplant, neutrophil, CD8, and CD4 T cell infiltration was indistinguishable. Although proliferation of donor-reactive naïve T cells was unaffected by CXCR3 antagonism, the frequency of interferon-gamma-producing cells in the recipient spleen was significantly reduced by AMG1237845 treatment. CXCR3 blockade for 30 days synergized with short-term, low-dose anti-CD154 monoclonal antibodies to prolong survival past 50 days in 75% of grafts and past 80 days in 25% of the cases. CONCLUSIONS: These results indicate that in synergy with co-stimulation blockade, CXCR3 is a viable therapeutic target to prevent acute graft rejection.
Assuntos
Sobrevivência de Enxerto/imunologia , Transplante de Coração/imunologia , Interferon gama/imunologia , Receptores CXCR3/antagonistas & inibidores , Transplante Homólogo/imunologia , Animais , Rejeição de Enxerto/imunologia , Imunossupressores/uso terapêutico , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transplante HeterotópicoRESUMO
How viral and host factors contribute to the severe pathogenicity of the H5N1 subtype of avian influenza virus infection in humans is poorly understood. We identified three clusters of differentially expressed innate immune response genes in lungs from H5N1 (A/Vietnam/1203/04) influenza virus-infected ferrets by oligonucleotide microarray analysis. Interferon response genes were more strongly expressed in H5N1-infected ferret lungs than in lungs from ferrets infected with the less pathogenic H3N2 subtype. In particular, robust CXCL10 gene expression in H5N1-infected ferrets led us to test the pathogenic role of signaling via CXCL10's cognate receptor, CXCR3, during H5N1 influenza virus infection. Treatment of H5N1-infected ferrets with the drug AMG487, a CXCR3 antagonist, resulted in a reduction of symptom severity and delayed mortality compared to vehicle treatment. We contend that unregulated host interferon responses are at least partially responsible for the severity of H5N1 infection and provide evidence that attenuating the CXCR3 signaling pathway improves the clinical course of H5N1 infection in ferrets.
Assuntos
Perfilação da Expressão Gênica , Virus da Influenza A Subtipo H5N1/imunologia , Virus da Influenza A Subtipo H5N1/fisiologia , Pulmão/imunologia , Pulmão/patologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/patologia , Animais , Quimiocina CXCL10/biossíntese , Furões , Humanos , Vírus da Influenza A Subtipo H3N2/imunologia , Vírus da Influenza A Subtipo H3N2/fisiologia , Pulmão/virologia , Masculino , Infecções por Orthomyxoviridae/virologia , Receptores CXCR3/antagonistas & inibidores , Análise de SobrevidaRESUMO
A series of six-six and six-five fused heterocyclic CXCR3 antagonists has been synthesized and their activities evaluated in an [(125)I]-IP-10 displacement assay and an ITAC mediated in vitro cell migration assay. The pharmacokinetic properties of several top compounds have also been studied. This effort led to the discovery of compounds with increased potency and improved pharmacokinetic properties that could serve as useful tools to study the role of the CXCR3 receptor in vivo.
Assuntos
Compostos Heterocíclicos/farmacologia , Quinazolinonas/farmacologia , Receptores CXCR3/antagonistas & inibidores , Animais , Cromatografia Líquida de Alta Pressão , Humanos , Quinazolinonas/farmacocinética , Ratos , EstereoisomerismoRESUMO
A series of imidazole derivatives have been designed and optimized for CXCR3 antagonism, pharmacokinetic properties, and reduced formation of glutathione conjugates. Our efforts led to the discovery of potent CXCR3 antagonists with good pharmacokinetic properties. These compounds are useful tools for in vivo studies of CXCR3 function.
Assuntos
Imidazóis/química , Imidazóis/farmacologia , Receptores CXCR3/antagonistas & inibidores , Animais , Cromatografia Líquida de Alta Pressão , Desenho de Fármacos , Humanos , Imidazóis/farmacocinética , Camundongos , Camundongos Knockout , Ratos , Receptores CXCR3/genética , Receptores CXCR3/fisiologia , Relação Estrutura-AtividadeRESUMO
Tumor cells aberrantly express chemokines and/or chemokine receptors, and some may promote tumor growth and metastasis. We examined the expression and function of chemokine receptor CXCR3 in a syngeneic murine model of metastatic breast cancer. By flow cytometry, CXCR3 was detected in all murine mammary tumor cell lines examined. All human breast cancer cell lines examined also expressed CXCR3, as did the immortalized but nontumorigenic MCF-10A cell line. Interaction of CXCR3 ligands, CXCL9, CXCL10, and CXCL11, with CXCR3 on the highly malignant murine mammary tumor cell line 66.1 resulted in intracellular calcium mobilization and chemotaxis in vitro. To test the hypothesis that tumor metastasis is facilitated by CXCR3 expressed by tumor cells, we employed a small molecular weight antagonist of CXCR3, AMG487. 66.1 tumor cells were pretreated with AMG487 prior to i.v. injection into immune-competent female mice. Antagonism of CXCR3 on 66.1 tumor cells inhibited experimental lung metastasis, and this antimetastatic activity was compromised in mice depleted of natural killer cells. Systemic administration of AMG487 also inhibited experimental lung metastasis. In contrast to the antimetastatic effect of AMG487, local growth of 66.1 mammary tumors was not affected by receptor antagonism. These studies indicate that murine mammary tumor cells express CXCR3 which facilitates the development of lung metastases. These studies also indicate for the first time that a small molecular weight antagonist of CXCR3 has the potential to inhibit tumor metastasis.
Assuntos
Neoplasias da Mama/patologia , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Receptores de Quimiocinas/antagonistas & inibidores , Adenocarcinoma/imunologia , Adenocarcinoma/prevenção & controle , Adenocarcinoma/secundário , Animais , Neoplasias da Mama/imunologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Neoplasias Pulmonares/imunologia , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos SCID , Receptores CXCR3 , Receptores de Quimiocinas/biossíntese , Receptores de Quimiocinas/imunologiaRESUMO
A series of 2-aminothiazole-derived antagonists of the CCR4 receptor has been synthesized and their affinity for the receptor evaluated using a [(125)I]TARC (CCL17) displacement assay. Optimization of these compounds for potency and pharmacokinetic properties led to the discovery of potent, orally bioavailable antagonists.
Assuntos
Receptores de Quimiocinas/antagonistas & inibidores , Tiazóis/farmacologia , Linhagem Celular , Humanos , Receptores CCR4 , Tiazóis/farmacocinéticaRESUMO
A highly constrained pseudo-tetrapeptide (OC252-324) further defines a new allosteric binding site located near the center of fructose-1,6-bisphosphatase. In a crystal structure, pairs of inhibitory molecules bind to opposite faces of the enzyme tetramer. Each ligand molecule is in contact with three of four subunits of the tetramer, hydrogen bonding with the side chain of Asp187 and the backbone carbonyl of residue 71, and electrostatically interacting with the backbone carbonyl of residue 51. The ligated complex adopts a quaternary structure between the canonical R- and T-states of fructose-1,6-bisphosphatase, and yet a dynamic loop essential for catalysis (residues 52-72) is in a conformation identical to that of the T-state enzyme. Inhibition by the pseudo-tetrapeptide is cooperative (Hill coefficient of 2), synergistic with both AMP and fructose 2,6-bisphosphate, noncompetitive with respect to Mg2+, and uncompetitive with respect to fructose 1,6-bisphosphate. The ligand dramatically lowers the concentration at which substrate inhibition dominates the kinetics of fructose-1,6-bisphosphatase. Elevated substrate concentrations employed in kinetic screens may have facilitated the discovery of this uncompetitive inhibitor. Moreover, the inhibitor could mimic an unknown natural effector of fructose-1,6-bisphosphatase, as it interacts strongly with a conserved residue of undetermined functional significance.