RESUMO
In this study, well-defined core-shell ethylenediamine-functional magnetic ferroferric oxide polymers were prepared and were fully characterized by transmission electron microscopy, scanning electron microscopy, FTIR spectroscopy, and vibrating sample magnetometry. Then, it was used as a magnetic solid-phase extraction adsorbent for simultaneous determination of 11 trace quinolone residues in freshwater fish samples coupled to liquid chromatography-tandem mass spectrometry. The obtained results revealed that the adsorbent showed good extraction efficiency and the adsorption mechanisms referred to hydrogen bond and π-π stacking interaction. Moreover, the magnetic solid-phase extraction conditions were also carefully optimized. The limits of quantitation of 11 quinolones were in the range of 0.15-0.36 µg/kg, while spiking recoveries were in the range of 80.2-99.5% for the 11 quinolones in freshwater fish samples at four spiked levels including limits of quantitation, 1.0, 40.0, and 80.0 µg/kg with the relative standard deviations ranging from 0.8 to 9.1%. The proposed method was applied to analyze 45 freshwater fish samples, and enrofloxacin was detected in 91.1% samples with concentrations ranging from 0.659 to 333 µg/kg. It could be concluded that the proposed method is fast, simple, sensitive, and accurate for the routine monitor of freshwater fish.
Assuntos
Cromatografia Líquida/métodos , Resíduos de Drogas/análise , Quinolonas/análise , Extração em Fase Sólida/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Resíduos de Drogas/química , Peixes , Água Doce , Limite de Detecção , Modelos Lineares , Quinolonas/química , Reprodutibilidade dos TestesRESUMO
T-type voltage-gated Ca2+ channels have been implicated in many human disorders, and there has been increasing interest in developing highly selective and potent T-type Ca2+ channel modulators for potential clinical use. However, the unique biophysical properties of T-type Ca2+ channels are not conducive for developing high-throughput screening (HTS) assays to identify modulators, particularly potentiators. To illustrate, T-type Ca2+ channels are largely inactivated and unable to open to allow Ca2+ influx at -25 mV, the typical resting membrane potential of the cell lines commonly used in cellular screening assays. To address this issue, we developed cell lines that express Kir2.3 channels to hyperpolarize the membrane potential to -70 mV, thus allowing T-type channels to return to their resting state where they can be subsequently activated by membrane depolarization in the presence of extracellular KCl. Furthermore, to simplify the HTS assay and to reduce reagent cost, we stably expressed a membrane-tethered genetic calcium sensor, GCaMP6s-CAAX, that displays superior signal to the background compared to the untethered GCaMP6s or the synthetic Ca2+ sensor Fluo-4AM. Here, we describe a novel GCaMP6s-CAAX-based calcium assay utilizing a high-throughput fluorometric imaging plate reader (Molecular Devices, Sunnyvale, CA) format that can identify both activators and inhibitors of T-type Ca2+ channels. Lastly, we demonstrate the utility of this novel fluorescence-based assay to evaluate the activities of two distinct G-protein-coupled receptors, thus expanding the use of GCaMP6s-CAAX to a wide range of applications relevant for developing cellular assays in drug discovery.
RESUMO
Ponatinib (AP24534) was previously identified as a pan-BCR-ABL inhibitor that potently inhibits the T315I gatekeeper mutant, and has advanced into clinical development for the treatment of refractory or resistant CML. In this study, we explored a novel series of five and six membered monocycles as alternate hinge-binding templates to replace the 6,5-fused imidazopyridazine core of ponatinib. Like ponatinib, these monocycles are tethered to pendant toluanilides via an ethynyl linker. Several compounds in this series displayed excellent in vitro potency against both native BCR-ABL and the T315I mutant. Notably, a subset of inhibitors exhibited desirable PK and were orally active in a mouse model of T315I-driven CML.
Assuntos
Alcinos/síntese química , Alcinos/farmacologia , Compostos de Anilina/síntese química , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Tolueno/síntese química , Administração Oral , Alcinos/química , Compostos de Anilina/química , Compostos de Anilina/farmacologia , Animais , Ciclização , Modelos Animais de Doenças , Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Camundongos , Modelos Moleculares , Estrutura Molecular , Mutação , Ratos , Relação Estrutura-Atividade , Tolueno/química , Tolueno/farmacologiaRESUMO
Novel N(9)-arenethenyl purines, optimized potent dual Src/Abl tyrosine kinase inhibitors, are described. The key structural feature is a trans vinyl linkage at N(9) on the purine core which projects hydrophobic substituents into the selectivity pocket at the rear of the ATP site. Their synthesis was achieved through a Horner-Wadsworth-Emmons reaction of N(9)-phosphorylmethylpurines and substituted benzaldehydes or Heck reactions between 9-vinyl purines and aryl halides. Most compounds are potent inhibitors of both Src and Abl kinase, and several possess good oral bioavailability.
Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Purinas/química , Purinas/farmacologia , Quinases da Família src/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Inibidores do Crescimento/química , Inibidores do Crescimento/farmacologia , Humanos , Células K562 , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-abl/fisiologia , RatosRESUMO
Schizophrenia is a severe neuropsychiatric disease that lacks completely effective and safe therapies. As a polygenic disorder, genetic studies have only started to shed light on its complex etiology. To date, the positive symptoms of schizophrenia are well-managed by antipsychotic drugs, which primarily target the dopamine D2 receptor (D2R). However, these antipsychotics are often accompanied by severe side effects, including motoric symptoms. At D2R, antipsychotic drugs antagonize both G-protein dependent (Gαi/o) signaling and G-protein independent (ß-arrestin) signaling. However, the relevant contributions of the distinct D2R signaling pathways to antipsychotic efficacy and on-target side effects (motoric) are still incompletely understood. Recent evidence from mouse genetic and pharmacological studies point to ß-arrestin signaling as the major driver of antipsychotic efficacy and suggest that a ß-arrestin biased D2R antagonist could achieve an additional level of selectivity at D2R, increasing the therapeutic index of next generation antipsychotics. Here, we characterize BRD5814, a highly brain penetrant ß-arrestin biased D2R antagonist. BRD5814 demonstrated good target engagement via PET imaging, achieving efficacy in an amphetamine-induced hyperlocomotion mouse model with strongly reduced motoric side effects in a rotarod performance test. This proof of concept study opens the possibility for the development of a new generation of pathway selective antipsychotics at D2R with reduced side effect profiles for the treatment of schizophrenia.
Assuntos
Antipsicóticos/uso terapêutico , Receptores de Dopamina D2/efeitos dos fármacos , beta-Arrestinas/metabolismo , Animais , Diagnóstico por Imagem/métodos , Proteínas de Ligação ao GTP/antagonistas & inibidores , Humanos , Locomoção/efeitos dos fármacos , Camundongos , Esquizofrenia/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , beta-Arrestinas/antagonistas & inibidoresRESUMO
Brain-derived neurotrophic factor (BDNF) and its receptor, tropomyosin-related kinase B (TrkB), have emerged as key regulators of brain plasticity and represent disease-modifying targets for several brain disorders, including Alzheimer's disease and major depressive disorder. Because of poor pharmacokinetic properties of BDNF, the interest in small-molecule TrkB agonists and modulators is high. Several compounds have been reported to act as TrkB agonists, and their increasing use in various nervous system disorder models creates the perception that these are reliable probes. To examine key pharmacological parameters of these compounds in detail, we have developed and optimized a series of complementary quantitative assays that measure TrkB receptor activation, TrkB-dependent downstream signaling, and gene expression in different cellular contexts. Although BDNF and other neurotrophic factors elicited robust and dose-dependent receptor activation and downstream signaling, we were unable to reproduce these activities using the reported small-molecule TrkB agonists. Our findings indicate that experimental results obtained with these compounds must be carefully interpreted and highlight the challenge of developing reliable pharmacological activators of this key molecular target.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Glicoproteínas de Membrana/agonistas , Neuroblastoma/tratamento farmacológico , Neurônios/efeitos dos fármacos , Receptor trkB/agonistas , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Ensaio de Imunoadsorção Enzimática , Flavanonas/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , Glicoproteínas de Membrana/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Neurônios/citologia , Neurônios/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Receptor trkB/metabolismoRESUMO
In the treatment of echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase positive (ALK+) non-small-cell lung cancer (NSCLC), secondary mutations within the ALK kinase domain have emerged as a major resistance mechanism to both first- and second-generation ALK inhibitors. This report describes the design and synthesis of a series of 2,4-diarylaminopyrimidine-based potent and selective ALK inhibitors culminating in identification of the investigational clinical candidate brigatinib. A unique structural feature of brigatinib is a phosphine oxide, an overlooked but novel hydrogen-bond acceptor that drives potency and selectivity in addition to favorable ADME properties. Brigatinib displayed low nanomolar IC50s against native ALK and all tested clinically relevant ALK mutants in both enzyme-based biochemical and cell-based viability assays and demonstrated efficacy in multiple ALK+ xenografts in mice, including Karpas-299 (anaplastic large-cell lymphomas [ALCL]) and H3122 (NSCLC). Brigatinib represents the most clinically advanced phosphine oxide-containing drug candidate to date and is currently being evaluated in a global phase 2 registration trial.
Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas , Neoplasias Pulmonares/tratamento farmacológico , Compostos Organofosforados/farmacologia , Fosfinas/química , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Administração Oral , Quinase do Linfoma Anaplásico , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Camundongos , Camundongos SCID , Conformação Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Compostos Organofosforados/administração & dosagem , Compostos Organofosforados/química , Fosfinas/farmacologia , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Pirimidinas/administração & dosagem , Pirimidinas/química , Ratos , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Relação Estrutura-AtividadeRESUMO
In this study, mercapto-functionalized nano-Fe(3)O(4) magnetic polymers (SH-Fe(3)O(4)-NMPs) have been prepared and characterized by transmission electron microscopy (TEM), X-ray diffractometer (XRD), vibrating sample magnetometer (VSM), thermogravimetry and differential thermogravimetry analyses (TG-DTA), as well as Fourier-transformed infrared spectroscopy (FTIR). The adsorptive characteristics of the SH-Fe(3)O(4)-NMPs intended for removal of mercury (II) were deeply studied. The results showed that the adsorption efficiency increased with pH increasing and reached a plateau at pH above 3.0. The adsorption data obtained at the optimized condition, i.e., 308 K and pH of 3.0, were well fitted with the Freundlich isotherm. The adsorption of Hg(II) reached equilibrium within 60 min. Thermodynamic parameters such as ΔH(θ), ΔS(θ) and ΔG(θ) suggested that the adsorption processes of Hg(II) onto the SH-Fe(3)O(4)-NMPs were endothermic and entropy favored in nature, with ΔH(θ) at 30.31 kJ mol(-1), ΔS(θ) at 111.41 J mol(-1) K(-1). The effects of mercury salts, i.e., Hg(NO(3))(2), HgSO(4), and different acids, were also deeply investigated and showed that the adsorption capacity of Hg(II) onto the SH-Fe(3)O(4)-NMPs decreased when Cl(-) existed.
RESUMO
The BCR-ABL inhibitor imatinib has revolutionized the treatment of chronic myeloid leukemia. However, drug resistance caused by kinase domain mutations has necessitated the development of new mutation-resistant inhibitors, most recently against the T315I gatekeeper residue mutation. Ponatinib (AP24534) inhibits both native and mutant BCR-ABL, including T315I, acting as a pan-BCR-ABL inhibitor. Here, we undertook a combined crystallographic and structure-activity relationship analysis on ponatinib to understand this unique profile. While the ethynyl linker is a key inhibitor functionality that interacts with the gatekeeper, virtually all other components of ponatinib play an essential role in its T315I inhibitory activity. The extensive network of optimized molecular contacts found in the DFG-out binding mode leads to high potency and renders binding less susceptible to disruption by single point mutations. The inhibitory mechanism exemplified by ponatinib may have broad relevance to designing inhibitors against other kinases with mutated gatekeeper residues.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Imidazóis , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Mutação/efeitos dos fármacos , Piperazinas , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases , Piridazinas , Pirimidinas , Animais , Benzamidas , Linhagem Celular Tumoral , Cristalografia por Raios X , Fluorimunoensaio , Mesilato de Imatinib , Imidazóis/síntese química , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Camundongos , Piperazinas/química , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Piridazinas/síntese química , Piridazinas/farmacologia , Piridazinas/uso terapêutico , Pirimidinas/química , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Relação Estrutura-AtividadeRESUMO
In the treatment of chronic myeloid leukemia (CML) with BCR-ABL kinase inhibitors, the T315I gatekeeper mutant has emerged as resistant to all currently approved agents. This report describes the structure-guided design of a novel series of potent pan-inhibitors of BCR-ABL, including the T315I mutation. A key structural feature is the carbon-carbon triple bond linker which skirts the increased bulk of Ile315 side chain. Extensive SAR studies led to the discovery of development candidate 20g (AP24534), which inhibited the kinase activity of both native BCR-ABL and the T315I mutant with low nM IC(50)s, and potently inhibited proliferation of corresponding Ba/F3-derived cell lines. Daily oral administration of 20g significantly prolonged survival of mice injected intravenously with BCR-ABL(T315I) expressing Ba/F3 cells. These data, coupled with a favorable ADME profile, support the potential of 20g to be an effective treatment for CML, including patients refractory to all currently approved therapies.
Assuntos
Antineoplásicos/síntese química , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Imidazóis/síntese química , Inibidores de Proteínas Quinases/síntese química , Piridazinas/síntese química , Administração Oral , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Barreira Hematoencefálica/metabolismo , Linhagem Celular Tumoral , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Proteínas de Fusão bcr-abl/genética , Imidazóis/farmacocinética , Imidazóis/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/mortalidade , Camundongos , Camundongos SCID , Modelos Moleculares , Mutação , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/farmacologia , Piridazinas/farmacocinética , Piridazinas/farmacologia , RatosRESUMO
A novel series of potent dual Src/Abl kinase inhibitors based on a 9-(arenethenyl)purine core has been identified. Unlike traditional dual Src/Abl inhibitors targeting the active enzyme conformation, these inhibitors bind to the inactive, DFG-out conformation of both kinases. Extensive SAR studies led to the discovery of potent and orally bioavailable inhibitors, some of which demonstrated in vivo efficacy. Once-daily oral administration of inhibitor 9i (AP24226) significantly prolonged the survival of mice injected intravenously with wild type Bcr-Abl expressing Ba/F3 cells at a dose of 10 mg/kg. In a separate model, oral administration of 9i to mice bearing subcutaneous xenografts of Src Y527F expressing NIH 3T3 cells elicited dose-dependent tumor shrinkage with complete tumor regression observed at the highest dose. Notably, several inhibitors (e.g., 14a, AP24163) exhibited modest cellular potency (IC50 = 300-400 nM) against the Bcr-Abl mutant T315I, a variant resistant to all currently marketed therapies for chronic myeloid leukemia.
Assuntos
Desenho de Fármacos , Inibidores de Proteínas Quinases/síntese química , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Purinas/síntese química , Quinases da Família src/antagonistas & inibidores , Animais , Feminino , Humanos , Células K562 , Camundongos , Células NIH 3T3 , Conformação Proteica , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-abl/química , Purinas/farmacologia , Ratos , Relação Estrutura-Atividade , Quinases da Família src/químicaRESUMO
Inhibition of BCR-ABL by imatinib induces durable responses in many patients with chronic myeloid leukemia (CML), but resistance attributable to kinase domain mutations can lead to relapse and a switch to second-line therapy with nilotinib or dasatinib. Despite three approved therapeutic options, the cross-resistant BCR-ABL(T315I) mutation and compound mutants selected on sequential inhibitor therapy remain major clinical challenges. We report design and preclinical evaluation of AP24534, a potent, orally available multitargeted kinase inhibitor active against T315I and other BCR-ABL mutants. AP24534 inhibited all tested BCR-ABL mutants in cellular and biochemical assays, suppressed BCR-ABL(T315I)-driven tumor growth in mice, and completely abrogated resistance in cell-based mutagenesis screens. Our work supports clinical evaluation of AP24534 as a pan-BCR-ABL inhibitor for treatment of CML.
Assuntos
Proteínas de Fusão bcr-abl/antagonistas & inibidores , Imidazóis/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Piridazinas/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cristalografia por Raios X , Proteínas de Fusão bcr-abl/química , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Imidazóis/química , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Camundongos , Camundongos SCID , Modelos Moleculares , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-abl/química , Proteínas Proto-Oncogênicas c-abl/genética , Proteínas Proto-Oncogênicas c-abl/metabolismo , Piridazinas/química , Transdução de Sinais/efeitos dos fármacosRESUMO
Imatinib (Gleevec) is currently the frontline therapy for chronic myeloid leukemia (CML), a disease characterized by the presence of a constitutively activated chimeric tyrosine kinase protein Bcr-AbI. However, drug resistance often occurs at later stages of the disease, principally because of the occurrence of mutations in the kinase domain. Second generation Bcr-AbI inhibitors, such as dasatinib and nilotinib are capable of inhibiting many imatinib-resistant forms of the kinase but not the form in which threonine is mutated to isoleucine at the gatekeeper position (T315I). In this study, we present the crystal structure of the kinase domain of the c-AbI T315I mutant, as well as the wild-type form, in complex with a pyrrolopyridine inhibitor, PPY-A. The side chain of Ile315 is accommodated in the AbI T315I mutant structure without large conformational changes proximal to the site of mutation. In contrast to other inhibitors, such as imatinib and dasatinib, PPY-A does not occupy the hydrophobic pocket behind the gatekeeper residue. This binding mode, coupled with augmented contacts with the glycine-rich loop, appears to be critical for its ability to override the T315I mutation. The data presented here may provide structural guidance for the design of clinically useful inhibitors of Bcr-AbI T315I.
Assuntos
Proteínas Proto-Oncogênicas c-abl/química , Proteínas Proto-Oncogênicas c-abl/metabolismo , Treonina/metabolismo , Animais , Linhagem Celular , Cristalografia por Raios X , Camundongos , Modelos Moleculares , Mutação/genética , Inibidores de Proteínas Quinases/química , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-abl/genética , Piridinas/química , Homologia Estrutural de Proteína , Treonina/genéticaRESUMO
In the course of a high throughput screening, a series of pyrazole compounds were identified with luteinizing hormone receptor (LH-R) agonist activity. A focused pyrazole library was produced by solid-phase synthesis and key pyrazole regioisomers were obtained selectively in solution. Evaluation of those compounds in a cAMP assay in CHO cells transfected with h-LH receptor allowed us to propose a structure-activity relationship model for this series and led to the identification of the first low molecular weight molecule with in vitro activity in a Leydig cells assay (ED(50)=1.31 microM) and in vivo in a model of testosterone induction in rats (significant effect at 32 mpk ip).
Assuntos
Química Farmacêutica/métodos , Pirazóis/síntese química , Pirazóis/farmacologia , Receptores do LH/agonistas , Animais , Células CHO , Cricetinae , Cricetulus , Desenho de Fármacos , Células Intersticiais do Testículo/metabolismo , Masculino , Conformação Molecular , Peso Molecular , Ratos , Relação Estrutura-Atividade , Testosterona/química , Testosterona/metabolismoRESUMO
Uveitis is an important autoimmune disease affecting an estimated 2.3 million Americans. This disease is manifested by inflammation of the retina mediated by the infiltration of T lymphocytes that recognize "S-Antigen" (S-Ag). Current therapies involve the life-long use of immunosuppressive drugs, including steroids. The ability to induce specific tolerance to S-Ag would be desirable and allow patients to be weaned off of steroid therapy. In this study, we determined that S-Ag-Ig retroviral vector was capable of preventing EAU (experimental autoimmune uveoretinitis) in Lewis rats induced by immunization with bovine S-Ag (BoS-Ag). Importantly, B-cell delivered gene therapy with S-Ag-Ig can ameliorate ongoing EAU when the treatment was initiated after rats had been immunized. Furthermore, we have successfully induced tolerance in HLA-DR3 transgenic mice with respect to the T-cell proliferative response. These results demonstrate proof of principle for future efforts to develop this approach for clinical application in patients with uveoretinitis.