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1.
Cancer Immunol Res ; 12(7): 921-943, 2024 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-38683145

RESUMO

The prognosis of patients with acute myeloid leukemia (AML) is limited, especially for elderly or unfit patients not eligible for hematopoietic stem cell (HSC) transplantation. The disease is driven by leukemic stem cells (LSCs), which are characterized by clonal heterogeneity and resistance to conventional therapy. These cells are therefore believed to be a major cause of progression and relapse. We designed MP0533, a multispecific CD3-engaging designed ankyrin repeat protein (DARPin) that can simultaneously bind to three antigens on AML cells (CD33, CD123, and CD70), aiming to enable avidity-driven T cell-mediated killing of AML cells coexpressing at least two of the antigens. In vitro, MP0533 induced selective T cell-mediated killing of AML cell lines, as well as patient-derived AML blasts and LSCs, expressing two or more target antigens, while sparing healthy HSCs, blood, and endothelial cells. The higher selectivity also resulted in markedly lower levels of cytokine release in normal human blood compared to single antigen-targeting T-cell engagers. In xenograft AML mice models, MP0533 induced tumor-localized T-cell activation and cytokine release, leading to complete eradication of the tumors while having no systemic adverse effects. These studies show that the multispecific-targeting strategy used with MP0533 holds promise for improved selectivity toward LSCs and efficacy against clonal heterogeneity, potentially bringing a new therapeutic option to this group of patients with a high unmet need. MP0533 is currently being evaluated in a dose-escalation phase 1 study in patients with relapsed or refractory AML (NCT05673057).


Assuntos
Leucemia Mieloide Aguda , Células-Tronco Neoplásicas , Linfócitos T , Humanos , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/patologia , Animais , Camundongos , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Subunidade alfa de Receptor de Interleucina-3/imunologia , Subunidade alfa de Receptor de Interleucina-3/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Complexo CD3/imunologia , Linhagem Celular Tumoral , Citotoxicidade Imunológica
2.
Br J Clin Pharmacol ; 89(7): 2295-2303, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37057679

RESUMO

AIMS: This study aimed to assess safety, tolerability, pharmacokinetic (PK) and pharmacodynamic (PD) effects of ensovibep, a designed ankyrin repeat protein antiviral being evaluated as a COVID-19 treatment, in healthy volunteers in a first-in-human ascending single-dose study. METHODS: Subjects were dosed intravenously, in a randomized double-blinded manner, with either ensovibep at 3, 9 or 20 mg/kg or with placebo, and followed until Day 100. PK and safety were assessed throughout the study duration. Immunogenicity and PD via viral neutralization in serum were also assessed. RESULTS: All adverse events were of mild to moderate severity, and no serious adverse events were observed. One subject who received the 20-mg/kg dose presented with moderate hypersensitivity vasculitis 3 weeks after infusion, which fully resolved using standard procedures. In most subjects ensovibep showed expected mono-exponential decline with a half-life of around 2 weeks. Anti-drug antibodies were detected in 15 of 17 subjects, with the earliest onset detected on Day 29. Viral neutralization assays on subject serum showed effective viral neutralization over the first 3 weeks following dosing with titre values in a dose dependent manner. CONCLUSION: Ensovibep proved safe in this first-in-human safety study and exhibited PK and PD parameters consistent with the expected treatment period required for acute COVID-19 infection.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Antivirais/efeitos adversos , Repetição de Anquirina , Tratamento Farmacológico da COVID-19 , Voluntários Saudáveis , Método Duplo-Cego
3.
Br J Clin Pharmacol ; 89(3): 1105-1114, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36214216

RESUMO

AIM: To assess viral clearance, pharmacokinetics, tolerability and symptom evolution following ensovibep administration in symptomatic COVID-19 outpatients. METHODS: In this open-label, first-in-patient study a single dose of either 225 mg (n = 6) or 600 mg (n = 6) of ensovibep was administered intravenously in outpatients with mild-to-moderate COVID-19 symptoms. Pharmacokinetic profiles were determined (90-day period). Pharmacodynamic assessments consisted of viral load (qPCR and cultures) and symptom questionnaires. Immunogenicity against ensovibep and SARS-CoV-2-neutralizing activity were determined. Safety and tolerability were assessed throughout a 13-week follow-up. RESULTS: Both doses showed similar pharmacokinetics (first-order) with mean half-lives of 14 (SD 5.0) and 13 days (SD 5.7) for the 225- and 600-mg groups, respectively. Pharmacologically relevant serum concentrations were maintained in all subjects for at least 2 weeks postdose, regardless of possible immunogenicity against ensovibep. Viral load changes from baseline at day 15 were 5.1 (SD 0.86) and 5.3 (SD 2.2) log10 copies/mL for the 225- and 600-mg doses, respectively. COVID-19 symptom scores decreased from 10.0 (SD 4.1) and 11.3 (SD 4.0) to 1.6 (SD 3.1) and 3.3 (SD 2.4) in the first week for the 225- and 600-mg groups, respectively. No anti-SARS-CoV-2 neutralizing activity was present predose and all patients had SARS-CoV-2 antibodies at day 91. Adverse events were of mild-to-moderate severity, transient and self-limiting. CONCLUSION: Single-dose intravenous administration of 225 or 600 mg of ensovibep appeared safe and well tolerated in patients with mild-to-moderate COVID-19. Ensovibep showed favourable pharmacokinetics in patients and the pharmacodynamic results warrant further research in a larger phase 2/3 randomized-controlled trail.


Assuntos
COVID-19 , Humanos , SARS-CoV-2 , Proteínas Recombinantes de Fusão , Anticorpos Antivirais , Método Duplo-Cego
5.
Nat Biotechnol ; 40(12): 1845-1854, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-35864170

RESUMO

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with potential resistance to existing drugs emphasizes the need for new therapeutic modalities with broad variant activity. Here we show that ensovibep, a trispecific DARPin (designed ankyrin repeat protein) clinical candidate, can engage the three units of the spike protein trimer of SARS-CoV-2 and inhibit ACE2 binding with high potency, as revealed by cryo-electron microscopy analysis. The cooperative binding together with the complementarity of the three DARPin modules enable ensovibep to inhibit frequent SARS-CoV-2 variants, including Omicron sublineages BA.1 and BA.2. In Roborovski dwarf hamsters infected with SARS-CoV-2, ensovibep reduced fatality similarly to a standard-of-care monoclonal antibody (mAb) cocktail. When used as a single agent in viral passaging experiments in vitro, ensovibep reduced the emergence of escape mutations in a similar fashion to the same mAb cocktail. These results support further clinical evaluation of ensovibep as a broad variant alternative to existing targeted therapies for Coronavirus Disease 2019 (COVID-19).


Assuntos
COVID-19 , SARS-CoV-2 , Animais , Cricetinae , Humanos , SARS-CoV-2/genética , Proteínas de Repetição de Anquirina Projetadas , Microscopia Crioeletrônica , Anticorpos Monoclonais/uso terapêutico , Terapia Combinada de Anticorpos , Anticorpos Neutralizantes
6.
J Clin Oncol ; 39(2): 145-154, 2021 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-33301375

RESUMO

PURPOSE: A first-in-human study was performed with MP0250, a DARPin drug candidate. MP0250 specifically inhibits both vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) with the aim of disrupting the tumor microenvironment. PATIENTS AND METHODS: A multicenter, open-label, repeated-dose, phase I study was conducted to assess the safety, tolerability, and pharmacokinetics of MP0250 in 45 patients with advanced solid tumors. In the dose-escalation part, 24 patients received MP0250 as a 3-hour infusion once every 2 weeks at five different dose levels (0.5-12 mg/kg). Once the maximum tolerated dose (MTD) was established, 21 patients were treated with a 1-hour infusion (n = 13, 8 mg/kg, once every 2 weeks and n = 8, 12 mg/kg, once every 3 weeks) of MP0250 in the dose confirmation cohorts. RESULTS: In the dose-escalation cohort, patients treated with 12 mg/kg MP0250 once every 2 weeks experienced dose-limiting toxicities. Therefore, MTD was 8 mg/kg once every 2 weeks or 12 mg/kg once every 3 weeks. The most common adverse events (AEs) were hypertension (69%), proteinuria (51%), and diarrhea and nausea (both 36%); hypoalbuminemia was reported in 24% of patients. Most AEs were consistent with inhibition of the VEGF and HGF pathways. Exposure was dose-proportional and sustained throughout the dosing period for all patients (up to 15 months). The half-life was about 2 weeks. Signs of single-agent antitumor activity were observed: 1 unconfirmed partial response with a time to progression of 23 weeks and 24 patients with stable disease, with the longest duration of 72 weeks and a median duration of 18 weeks. CONCLUSION: MP0250 is a first-in-class DARPin drug candidate with suitable tolerability and appropriate pharmacokinetic properties for further development in combination with other anticancer therapies.


Assuntos
Fator de Crescimento de Hepatócito/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Proteínas Recombinantes de Fusão/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Adulto , Idoso , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Estudos de Coortes , Relação Dose-Resposta a Droga , Feminino , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Infusões Intravenosas , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Recombinantes de Fusão/efeitos adversos , Proteínas Recombinantes de Fusão/farmacocinética , Microambiente Tumoral , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adulto Jovem
7.
Protein Eng Des Sel ; 30(9): 583-591, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29088432

RESUMO

A long systemic half-life is key for therapeutic proteins. To that end we have generated serum albumin-binding designed ankyrin repeat domains. These domains bind serum albumin of different species with nanomolar affinities, and have significantly improved pharmacokinetic properties both in mouse and cynomolgus monkey compared to non-serum albumin-binding DARPin® domains. In addition, they exhibit high thermal stability and long storage stability, which is an essential feature for their use in drug development. Covalently linking a serum albumin-binding DARPin® domain to domains with other target specificities results in improvements of multiple orders of magnitude in exposure and terminal half-life, both in mouse and cynomolgus monkey. Pharmacokinetic assessment of such constructs revealed terminal half-life values ranging from 27 h to 80 h in mouse, and from 2.6 days to 20 days in cynomolgus monkey. Extrapolation by allometric scaling on these findings suggests terminal half-life values of 5-50 days in human, indicating that pharmacokinetic properties in the range of monoclonal antibodies can be achieved with DARPin® drug candidates. Such serum albumin-binding DARPin® domains are thus valuable tools for the generation of multi-functional drugs with an extended in vivo half-life.


Assuntos
Repetição de Anquirina , Vetores Genéticos/química , Proteínas Recombinantes de Fusão/farmacocinética , Albumina Sérica/genética , Animais , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/metabolismo , Meia-Vida , Humanos , Concentração de Íons de Hidrogênio , Macaca fascicularis , Camundongos , Ligação Proteica , Estabilidade Proteica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Albumina Sérica/metabolismo
8.
MAbs ; 9(8): 1262-1269, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29035637

RESUMO

MP0250 is a multi-domain drug candidate currently being tested in clinical trials for the treatment of cancer. It comprises one anti-vascular endothelial growth factor-A (VEGF-A), one anti-hepatocyte growth factor (HGF), and two anti-human serum albumin (HSA) DARPin® domains within a single polypeptide chain. While there is first clinical validation of a single-domain DARPin® drug candidate, little is known about DARPin® drug candidates comprising multiple domains. Here, we show that MP0250 can be expressed at 15 g/L in soluble form in E. coli high cell-density fermentation, it is stable in soluble/frozen formulation for 2 years as assessed by reverse phase HPLC, it has picomolar potency in inhibiting VEGF-A and HGF in ELISA and cellular assays, and its domains are simultaneously active as shown by surface plasmon resonance. The inclusion of HSA-binding DARPin® domains leads to a favorable pharmacokinetic profile in mouse and cynomolgus monkey, with terminal half-lives of ∼ 30 hours in mouse and ∼ 5 days in cynomolgus monkey. MP0250 is thus a highly potent drug candidate that could be particularly useful in oncology. Beyond MP0250, the properties of MP0250 indicate that multi-domain DARPin® proteins can be valuable next-generation drug candidates.


Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Antineoplásicos/imunologia , Proteínas Recombinantes de Fusão/imunologia , Administração Intravenosa , Animais , Repetição de Anquirina/genética , Repetição de Anquirina/imunologia , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacocinética , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Desenho de Fármacos , Feminino , Meia-Vida , Fator de Crescimento de Hepatócito/antagonistas & inibidores , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/imunologia , Humanos , Infusões Intravenosas , Macaca fascicularis , Masculino , Camundongos Endogâmicos BALB C , Ligação Proteica/imunologia , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacocinética , Albumina Sérica Humana/genética , Albumina Sérica Humana/imunologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/imunologia
9.
Bioorg Med Chem Lett ; 22(24): 7314-21, 2012 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-23147076

RESUMO

SAR studies were performed on a series of 2-arylamido-5,7-dihydro-4H-thieno[2,3-c]pyran-3-carboxamide derivatives as cannabinoid receptor agonists. Starting from a HTS hit both potency and selectivity could be improved. Modifications to the thiophene fusion and C-3 amides were studied. A representative compound 3t produced analgesia when dosed orally in inflammatory pain models of writhing and carrageenan-induced allodynia.


Assuntos
Piranos/farmacologia , Receptores de Canabinoides/metabolismo , Tiofenos/farmacologia , Animais , Humanos , Estrutura Molecular , Piranos/síntese química , Piranos/química , Ratos , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/química
10.
Biochem Pharmacol ; 79(3): 321-9, 2010 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-19737543

RESUMO

Stimulation of the cAMP signaling pathway has been shown to induce apoptosis and augment the effects of glucocorticoids in inducing apoptosis in leukemic cells. We recently reported that in primary B cell chronic lymphocytic leukemic (B-CLL) cells, apoptosis could be induced by stimulating the cAMP signaling pathway with a phosphodiesterase4 (PDE4) inhibitor alone; while in contrast, in the CEM T leukemic cell line, PDE4 inhibitors alone were ineffective, and concurrent stimulation of adenylyl cyclase was required to see effects [Tiwari et al. (2005)]. We report here that in the CEM and Jurkat T leukemic cell lines, the most abundantly expressed PDEs are PDE3B, PDE4A, PDE4D, PDE7A, and PDE8A. Selective inhibition of PDE3, PDE4 or PDE7 alone produces little effect on cell viability, but inhibition of all three of these PDEs together dramatically enhances glucocorticoid-induced apoptosis in CEM cells, and overcomes glucocorticoid resistance in a glucocorticoid-resistant CEM cell line. These studies indicate that for some leukemic cell types, a desired therapeutic effect may be achieved by inhibiting more than one form of PDE.


Assuntos
Apoptose/efeitos dos fármacos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 7/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Glucocorticoides/farmacologia , Leucemia de Células T/enzimologia , Inibidores da Fosfodiesterase 3 , Inibidores da Fosfodiesterase 4 , Apoptose/fisiologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/fisiologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/fisiologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 7/fisiologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Sinergismo Farmacológico , Humanos , Células Jurkat , Leucemia de Células T/tratamento farmacológico , Inibidores de Fosfodiesterase/farmacologia , Células Tumorais Cultivadas
11.
J Immunol ; 178(8): 4820-31, 2007 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-17404263

RESUMO

Type 4 phosphodiesterases (PDE4) are critical regulators in TCR signaling by attenuating the negative constraint of cAMP. In this study, we show that anti-CD3/CD28 stimulation of human primary CD4(+) T cells increases the expression of the PDE4 subtypes PDE4A, PDE4B, and PDE4D in a specific and time-dependent manner. PDE4A and PDE4D mRNAs as well as enzyme activities were up-regulated within 5 days, PDE4B showed a transient up-regulation with highest levels after 24 h. The induction was shown to be independent of different stimulation conditions and was similar in naive and memory T cell subpopulations. To elucidate the functional impact of individual PDE4 subtypes on T cell function, we used PDE4 subtype-specific short-interfering RNAs (siRNAs). Knockdown of either PDE4B or PDE4D inhibited IL-2 release 24 h after stimulation (time point of maximal IL-2 concentrations) to an extent similar to that observed with the panPDE4 inhibitor RP73401 (piclamilast). Substantial amounts of IFN-gamma or IL-5 were measured only at later time points. siRNA targeting PDE4D showed a predominant inhibitory effect on these cytokines measured after 72 h. However, the inhibition of all cytokines was most effective when PDE4 siRNAs were applied in combination. Although the effect of PDE4 inhibition on T cell proliferation is small, the PDE4D-targeting siRNA alone was as effective as the panPDE4 inhibitor, whereas PDE4A or PDE4B siRNAs had hardly an effect. In summary, individual PDE4 subtypes have overall nonredundant, but complementary, time-dependent roles in propagating various T cell functions and PDE4D is the form likely playing a predominant role.


Assuntos
3',5'-AMP Cíclico Fosfodiesterases/classificação , 3',5'-AMP Cíclico Fosfodiesterases/fisiologia , Linfócitos T CD4-Positivos/enzimologia , 3',5'-AMP Cíclico Fosfodiesterases/genética , Antígenos CD28/fisiologia , Complexo CD3/fisiologia , Linfócitos T CD4-Positivos/fisiologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3 , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Regulação da Expressão Gênica , Humanos , Memória Imunológica , RNA Mensageiro/análise , RNA Interferente Pequeno/farmacologia
12.
Blood ; 107(12): 4841-8, 2006 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-16514061

RESUMO

We here investigate the crosstalk of PKC and PKA signaling during primary CD3(+) T-lymphocyte activation using pharmacologic inhibitors and activators in combination with our established panel of PKC isotype-deficient mouse T cells in vitro. PKCtheta and PKA inversely affect the CD3/CD28-induced IL-2 expression, whereas other PKC isotypes are dispensable in this signaling pathway. Gene ablation of PKCtheta selectively results in a profound reduction of IL-2 production; however, complete abrogation of IL-2 production in these PKCtheta(-/-) T cells was achieved only by simultaneous coactivation of the cAMP/PKA pathway in CD3(+) T cells. Conversely, the reduced IL-2 production in PKC inhibitor-treated T cells can be rescued by inhibition of the cAMP/PKA pathway in wild-type but not in PKCtheta(-/-) T cells. Mechanistically, the cAMP/PKA and PKCtheta pathways converge at the level of NF-AT, as shown by DNA binding analysis. The combined increase in PKA and decrease in PKCtheta activity leads to an enhanced inhibition of nuclear NF-AT translocation. This PKCtheta/PKA crosstalk significantly affects neither the NF-kappaB, the AP-1, nor the CREB pathways. Taken together, this opposite effect between the positive PKCtheta and the negative cAMP/PKA signaling pathways appears rate limiting for NF-AT transactivation and IL-2 secretion responses of CD3(+) T lymphocytes.


Assuntos
Complexo CD3/imunologia , Fatores de Transcrição NFATC/imunologia , Proteína Quinase C/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/imunologia , Animais , Antígenos CD28/imunologia , Proteína de Ligação a CREB/imunologia , Núcleo Celular/imunologia , Células Cultivadas , AMP Cíclico/imunologia , Proteínas Quinases Dependentes de AMP Cíclico , Deleção de Genes , Interleucina-2/imunologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , NF-kappa B/imunologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/deficiência , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Linfócitos T/citologia , Fator de Transcrição AP-1/imunologia
13.
J Biol Chem ; 279(13): 12427-37, 2004 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-14718545

RESUMO

Ca2+ entry through store-operated Ca2+release-activated Ca2+ (CRAC) channels is essential for T-cell activation and proliferation. Recently, it has been shown that 3,5-bistrifluoromethyl pyrazole (BTP) derivatives are specific inhibitors of Ca2+-dependent transcriptional activity in T-cells (Trevillyan, J. M., Chiou, X. G., Chen, Y. W., Ballaron, S. J., Sheets, M. P., Smith, M. L., Wiedeman, P. E., Warrior, U., Wilkins, J., Gubbins, E. J., Gagne, G. D., Fagerland, J., Carter, G. W., Luly, J. R., Mollison, K. W., and Djuric, S. W. (2001) J. Biol. Chem. 276, 48118-48126). Whereas inhibition of Ca2+ signals was reported for BTP2 (Ishikawa, J., Ohga, K., Yoshino, T., Takezawa, R., Ichikawa, A., Kubota, H., and Yamada, T. (2003) J. Immunol. 170, 4441-4449), it was not found for BTP3 (Chen, Y., Smith, M. L., Chiou, G. X., Ballaron, S., Sheets, M. P., Gubbins, E., Warrior, U., Wilkins, J., Surowy, C., Nakane, M., Carter, G. W., Trevillyan, J. M., Mollison, K., and Djuric, S. W. (2002) Cell. Immunol. 220, 134-142). We show that BTP2 specifically inhibits CRAC channels in T-cells with an IC(50) of approximately 10 nm. It does not interfere with other mechanisms important for Ca2+ signals in T-cells, including Ca2+ pumps, mitochondrial Ca2+ signaling, endoplasmic reticulum Ca2+ release, and K+ channels. BTP2 inhibits Ca2+ signals in peripheral blood T-lymphocytes (in particular in CD4+ T-cells) and in human Jurkat T-cells. Inhibition of Ca2+ signals is independent of the stimulation method as Ca2+ entry was blocked following stimulation with anti-CD3, which activates the T-cell receptor, and also following stimulation with thapsigargin or inositol 1,4,5-trisphosphate. BTP2 also inhibited Ca2+-dependent gene expression (interleukins 2 and 5 and interferon gamma) and proliferation of T-lymphocytes with similar IC(50) values. BTP2 is the first potent and specific inhibitor of CRAC channels in primary T-lymphocytes. The inhibition of CRAC channels as well as Ca2+-dependent signal transduction with similar IC(50) values in T-lymphocytes emphasizes the importance of CRAC channel activity during T-cell activation. Furthermore, BTP2 could prove to be a tool to finally unmask the molecular identity of CRAC channels.


Assuntos
Anilidas/química , Cálcio/química , Ativação Linfocitária , Pirazóis/farmacologia , Linfócitos T/metabolismo , Tiadiazóis/química , Complexo CD3/biossíntese , Linfócitos T CD4-Positivos/metabolismo , Cálcio/metabolismo , Células Cultivadas , Quelantes/farmacologia , Relação Dose-Resposta a Droga , Ácido Egtázico/farmacologia , Retículo Endoplasmático/metabolismo , Humanos , Concentração Inibidora 50 , Interferon gama/metabolismo , Interleucina-2/biossíntese , Interleucina-5/biossíntese , Células Jurkat , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Canais de Potássio/química , Transdução de Sinais , Linfócitos T/efeitos dos fármacos , Fatores de Tempo , Transcrição Gênica
14.
Biochem J ; 371(Pt 3): 1045-53, 2003 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-12564954

RESUMO

An early key event in the activation of neutrophil granulocytes is Ca(2+) influx. Members of the transient receptor potential (TRP) channel family may be held responsible for this. The aim of the present study is to analyse the expression pattern of TRP mRNA and identify characteristic currents unambiguously attributable to particular TRP channels. mRNA was extracted from human neutrophils, isolated by gradient centrifugation and also by magnetically labelled CD15 antibodies. The presence of mRNA was demonstrated using reverse transcriptase-PCR in neutrophils (controlled to be CD5-negative) as well as in human leukaemic cell line 60 (HL-60) cells, for the following TRP species: the long TRPC2 (LTRPC2), the vanilloid receptor 1, the vanilloid receptor-like protein 1 and epithelial Ca(2+) channels 1 and 2. TRPC6 was specific for neutrophils, whereas only in HL-60 cells were TRPC1, TRPC2, TRPC3, melastatin 1 and melastatin-related 1 found. Patch-clamp measurements in neutrophils revealed non-selective cation currents evoked by intracellular ADP-ribose and by NAD(+). Both these modes of activation have been found to be characteristic of LTRPC2. Furthermore, single-channel activity was resolved in neutrophils and it was indistinguishable from that in LTRPC2-transfected HEK-293 cells. The results provide evidence that LTRPC2 in neutrophil granulocytes forms an entry pathway for Na(+) and Ca(2+), which is regulated by ADP-ribose and the redox state.


Assuntos
Adenosina Difosfato Ribose/farmacologia , Canais de Cálcio/genética , Perfilação da Expressão Gênica , Canais Iônicos , Proteínas de Membrana , NAD/farmacologia , Neutrófilos/efeitos dos fármacos , Sequência de Bases , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/fisiologia , Linhagem Celular , Primers do DNA , Humanos , Neutrófilos/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canais de Cátion TRPC , Canais de Cátion TRPM
15.
FEBS Lett ; 523(1-3): 193-9, 2002 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-12123831

RESUMO

Transient receptor potential proteins (TRP) are supposed to participate in the formation of store-operated Ca(2+) influx channels by co-assembly. However, little is known which domains facilitate the interaction of subunits. Contribution of the N-terminal coiled-coil domain and ankyrin-like repeats and the putative pore region of the mouse TRP1beta (mTRP1beta) variant to the formation of functional cation channels were analyzed following overexpression in HEK293 (human embryonic kidney) cells. MTRP1beta expressing cells exhibited enhanced Ca(2+) influx and enhanced whole-cell membrane currents compared to mTRP1beta deletion mutants. Using a yeast two-hybrid assay only the coiled-coil domain facilitated homodimerization of the N-terminus. These results suggest that the N-terminus of mTRP1beta is required for structural organization thus forming functional channels.


Assuntos
Canais de Cálcio/química , Animais , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Linhagem Celular , Dimerização , Deleção de Genes , Humanos , Potenciais da Membrana/fisiologia , Camundongos , Mutação , Conformação Proteica , Estrutura Terciária de Proteína , Sequências Repetitivas de Aminoácidos , Canais de Cátion TRPC , Transfecção , Técnicas do Sistema de Duplo-Híbrido
16.
Prog Neurobiol ; 66(4): 243-64, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11960680

RESUMO

Stimulation of membrane receptors linked to a phospholipase C and the subsequent production of the second messengers diacylglycerol and inositol-1,4,5-trisphosphate (InsP(3)) is a signaling pathway of fundamental importance in eukaryotic cells. Signaling downstream of these initial steps involves mobilization of Ca(2+) from intracellular stores and Ca(2+) influx through the plasma membrane. For this influx, several contrasting mechanisms may be responsible but particular relevance is attributed to the induction of Ca(2+) influx as consequence of depletion of intracellular calcium stores. This phenomenon (frequently named store-operated calcium entry, SOCE), in turn, may be brought about by various signals, including soluble cytosolic factors, interaction of proteins of the endoplasmic reticulum with ion channels in the plasma membrane, and a secretion-like coupling involving translocation of channels to the plasma membrane. Experimental approaches to analyze these mechanisms have been considerably advanced by the discovery of mammalian homologs of the Drosophila cation channel transient receptor potential (TRP). Some members of the TRP family can be expressed to Ca(2+)-permeable channels that enable SOCE; other members form channels activated independently of stores. TRP proteins may be an essential part of endogenous Ca(2+) entry channels but so far expression of most TRP cDNAs has not resulted in restitution of channels found in any mammalian cells, suggesting the requirement for further unknown subunits. A major exception is CaT1, a TRP channel demonstrated to provide Ca(2+)-selective, store-operated currents identical to those characterized in several cell types. Ongoing and future research on TRP channels will be crucial to understand the molecular basis of receptor-mediated Ca(2+) entry, with respect to the structure of the entry channels as well as to the mechanisms of its activation and regulation.


Assuntos
Canais de Cálcio/fisiologia , Cálcio/metabolismo , Ativação do Canal Iônico/fisiologia , Transdução de Sinais/fisiologia , Animais , Canais de Cátion TRPC
17.
J Biol Chem ; 277(26): 23150-6, 2002 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-11960981

RESUMO

LTRPC2 is a cation channel recently reported to be activated by adenosine diphosphate-ribose (ADP-ribose) and NAD. Since ADP-ribose can be formed from NAD and NAD is elevated during oxidative stress, we studied whole cell currents and increases in the intercellular free calcium concentration ([Ca(2+)](i)) in long transient receptor potential channel 2 (LTRPC2)-transfected HEK 293 cells after stimulation with hydrogen peroxide (H(2)O(2)). Cation currents carried by monovalent cations and Ca(2+) were induced by H(2)O(2) (5 mm in the bath solution) as well as by intracellular ADP-ribose (0.3 mm in the pipette solution) but not by NAD (1 mm). H(2)O(2)-induced currents developed slowly after a characteristic delay of 3-6 min and receded after wash-out of H(2)O(2). [Ca(2+)](i) was rapidly increased by H(2)O(2) in LTRPC2-transfected cells as well as in control cells; however, in LTRPC2-transfected cells, H(2)O(2) evoked a second delayed rise in [Ca(2+)](i). A splice variant of LTRPC2 with a deletion in the C terminus (amino acids 1292-1325) was identified in neutrophil granulocytes. This variant was stimulated by H(2)O(2) as the wild type. However, it did not respond to ADP-ribose. We conclude that activation of LTRPC2 by H(2)O(2) is independent of ADP-ribose and that LTRPC2 may mediate the influx of Na(+) and Ca(2+) during oxidative stress, such as the respiratory burst in granulocytes.


Assuntos
Adenosina Difosfato Ribose/fisiologia , Canais de Cálcio/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Canais Iônicos , Proteínas de Membrana , Adulto , Sequência de Bases , Cálcio/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Clonagem Molecular , Humanos , Dados de Sequência Molecular , NAD/farmacologia , Estresse Oxidativo , Canais de Cátion TRPM
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