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1.
Science ; 383(6682): eadi5798, 2024 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-38301010

RESUMO

Increasing use of covalent and noncovalent inhibitors of Bruton's tyrosine kinase (BTK) has elucidated a series of acquired drug-resistant BTK mutations in patients with B cell malignancies. Here we identify inhibitor resistance mutations in BTK with distinct enzymatic activities, including some that impair BTK enzymatic activity while imparting novel protein-protein interactions that sustain B cell receptor (BCR) signaling. Furthermore, we describe a clinical-stage BTK and IKZF1/3 degrader, NX-2127, that can bind and proteasomally degrade each mutant BTK proteoform, resulting in potent blockade of BCR signaling. Treatment of chronic lymphocytic leukemia with NX-2127 achieves >80% degradation of BTK in patients and demonstrates proof-of-concept therapeutic benefit. These data reveal an oncogenic scaffold function of mutant BTK that confers resistance across clinically approved BTK inhibitors but is overcome by BTK degradation in patients.


Assuntos
Tirosina Quinase da Agamaglobulinemia , Resistencia a Medicamentos Antineoplásicos , Fator de Transcrição Ikaros , Leucemia Linfocítica Crônica de Células B , Inibidores de Proteínas Quinases , Proteólise , Humanos , Tirosina Quinase da Agamaglobulinemia/genética , Tirosina Quinase da Agamaglobulinemia/metabolismo , Fator de Transcrição Ikaros/metabolismo , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Mutação , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de Sinais , Proteólise/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos
2.
J Allergy Clin Immunol ; 152(5): 1131-1140.e6, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37474025

RESUMO

BACKGROUND: The emerging role of sphingosine-1-phosphate (S1P) in regulating smooth muscle functions has led to the exploration of the possibility that this sphingolipid could represent a potential therapeutic target in asthma and other lung diseases. Several studies in animal surrogates have suggested a role for S1P-mediated signaling in the regulation of airway smooth muscle (ASM) contraction, airway hyperresponsiveness, and airway remodeling, but evidence from human studies is lacking. OBJECTIVE: We sought to compare the responsiveness of the airways to S1P in healthy and asthmatic individuals in vivo, in isolated human airways ex vivo, and in murine airways dissected from healthy and house dust mite (HDM)-sensitized animals. METHODS: Airway responsiveness was measured by spirometry during inhalation challenges and by wire myography in airways isolated from human and mouse lungs. Thymidine incorporation and calcium mobilization assays were used to study human ASM cell responses. RESULTS: S1P did not induce contraction of airways isolated from healthy and HDM-exposed mice, nor in human airways. Similarly, there was no airway constriction observed in healthy and asthmatic subjects in response to increasing concentrations of inhaled S1P. However, a 30-minute exposure to S1P induced a significant concentration-dependent enhancement of airway reactivity to methacholine and to histamine in murine and human airways, respectively. HDM-sensitized mice demonstrated a significant increase in methacholine responsiveness, which was not further enhanced by S1P treatment. S1P also concentration-dependently enhanced proliferation of human ASM cells, an effect mediated through S1P receptor type 2, as shown by selective antagonism and S1P receptor type 2 small-interfering RNA knockdown. CONCLUSIONS: Our data suggest that S1P released locally into the airways may be involved in the regulation of ASM hyperresponsiveness and hyperplasia, defining a novel target for future therapies.


Assuntos
Asma , Humanos , Camundongos , Animais , Receptores de Esfingosina-1-Fosfato/metabolismo , Cloreto de Metacolina , Asma/metabolismo , Músculo Liso/metabolismo , Proliferação de Células
3.
World Neurosurg ; 174: 213-220.e2, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-36958719

RESUMO

BACKGROUND: Long-standing overt ventriculomegaly in adults (LOVA) is a heterogeneous term describing forms of adult hydrocephalus. LOVA incidence is increasing, yet the optimal treatment strategy for symptomatic cases remains unclear. We compared success rates and complication rates between endoscopic third ventriculostomy (ETV) and ventriculoperitoneal shunt (VPS) as first-line treatment for LOVA. METHODS: A systematic review and meta-analysis was conducted in accordance with PRISMA guidelines. Three databases were searched, and articles published from 2000 to October 2022 were included (last search date October 24, 2022). Success rates and complications of both ETV and VPS were compared using random-effects models. RESULTS: Of 895 articles identified, 22 studies were included in the analysis (556 patients: 346 in ETV group, 210 in VPS group). Mean age was 44.8 years. The most common presenting symptoms were gait disturbance (n = 178), headache (n = 156), and cognitive decline (n = 134). Combined success rates were 81.8% (n = 283/346) in the ETV group and 86.7% (n = 182/210) in the VPS group (median follow-up 41 months). There was no difference in success rates between ETV and VPS groups (odds ratio 0.94, 95% confidence interval 0.86-1.03, I2 = 0%). Combined complication rates were 4.6% (n = 16/346) in the ETV group and 27.1% (n = 57/210) in the VPS group. ETV had a lower rate of postoperative complications (odds ratio 0.22, 95% confidence interval 0.11-0.33, I2 = 0%). CONCLUSIONS: Symptomatic LOVA can be successfully managed with surgical intervention. ETV and VPS have similar success rates when used as first-line treatment. VPS has a higher complication rate.


Assuntos
Hidrocefalia , Neuroendoscopia , Terceiro Ventrículo , Humanos , Adulto , Ventriculostomia/efeitos adversos , Derivação Ventriculoperitoneal/efeitos adversos , Terceiro Ventrículo/cirurgia , Hidrocefalia/etiologia , Próteses e Implantes/efeitos adversos , Resultado do Tratamento , Neuroendoscopia/efeitos adversos , Estudos Retrospectivos
4.
Blood ; 141(13): 1584-1596, 2023 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-36375120

RESUMO

Bruton tyrosine kinase (BTK) is essential for B-cell receptor (BCR) signaling, a driver of chronic lymphocytic leukemia (CLL). Covalent inhibitors bind C481 in the active site of BTK and have become a preferred CLL therapy. Disease progression on covalent BTK inhibitors is commonly associated with C481 mutations. Here, we investigated a targeted protein degrader, NRX-0492, that links a noncovalent BTK-binding domain to cereblon, an adaptor protein of the E3 ubiquitin ligase complex. NRX-0492 selectively catalyzes ubiquitylation and proteasomal degradation of BTK. In primary CLL cells, NRX-0492 induced rapid and sustained degradation of both wild-type and C481 mutant BTK at half maximal degradation concentration (DC50) of ≤0.2 nM and DC90 of ≤0.5 nM, respectively. Sustained degrader activity was maintained for at least 24 hours after washout and was equally observed in high-risk (deletion 17p) and standard-risk (deletion 13q only) CLL subtypes. In in vitro testing against treatment-naïve CLL samples, NRX-0492 was as effective as ibrutinib at inhibiting BCR-mediated signaling, transcriptional programs, and chemokine secretion. In patient-derived xenografts, orally administered NRX-0492 induced BTK degradation and inhibited activation and proliferation of CLL cells in blood and spleen and remained efficacious against primary C481S mutant CLL cells collected from a patient progressing on ibrutinib. Oral bioavailability, >90% degradation of BTK at subnanomolar concentrations, and sustained pharmacodynamic effects after drug clearance make this class of targeted protein degraders uniquely suitable for clinical translation, in particular as a strategy to overcome BTK inhibitor resistance. Clinical studies testing this approach have been initiated (NCT04830137, NCT05131022).


Assuntos
Leucemia Linfocítica Crônica de Células B , Humanos , Tirosina Quinase da Agamaglobulinemia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Xenoenxertos , Resistencia a Medicamentos Antineoplásicos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico
5.
J Hematol ; 10(1): 22-24, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33643506

RESUMO

A 60-year-old Caucasian man had a 55-year history of recurrent severe epistaxis and later presented with multiple gastrointestinal (GI) bleeding from hereditary hemorrhagic telangiectasia (HHT). Bleeding was exacerbated due to coexistent mild hemophilia A. Despite repeated conventional surgical interventions, tranexamic acid and recombinant factor VIII (FVIII) prophylaxis, bleeding episodes worsened in frequency and severity, resulting in the patient becoming transfusion dependent. The introduction of tamoxifen therapy resulted in reduced transfusion requirement.

6.
Nat Commun ; 10(1): 1402, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30926793

RESUMO

Protein-protein interactions (PPIs) governing the recognition of substrates by E3 ubiquitin ligases are critical to cellular function. There is significant therapeutic potential in the development of small molecules that modulate these interactions; however, rational design of small molecule enhancers of PPIs remains elusive. Herein, we report the prospective identification and rational design of potent small molecules that enhance the interaction between an oncogenic transcription factor, ß-Catenin, and its cognate E3 ligase, SCFß-TrCP. These enhancers potentiate the ubiquitylation of mutant ß-Catenin by ß-TrCP in vitro and induce the degradation of an engineered mutant ß-Catenin in a cellular system. Distinct from PROTACs, these drug-like small molecules insert into a naturally occurring PPI interface, with contacts optimized for both the substrate and ligase within the same small molecule entity. The prospective discovery of 'molecular glue' presented here provides a paradigm for the development of small molecule degraders targeting hard-to-drug proteins.


Assuntos
Bibliotecas de Moléculas Pequenas/análise , Bibliotecas de Moléculas Pequenas/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Células HEK293 , Humanos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Especificidade por Substrato/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos , beta Catenina/metabolismo , Proteínas Contendo Repetições de beta-Transducina/metabolismo
8.
FASEB J ; 28(7): 2790-803, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24671708

RESUMO

Controlled mechanical ventilation (CMV) is associated with the development of diaphragm atrophy and contractile dysfunction, and respiratory muscle weakness is thought to contribute significantly to delayed weaning of patients. Therefore, therapeutic strategies for preventing these processes may have clinical benefit. The aim of the current study was to investigate the role of the Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in CMV-mediated diaphragm wasting and weakness in rats. CMV-induced diaphragm atrophy and contractile dysfunction coincided with marked increases in STAT3 phosphorylation on both tyrosine 705 (Tyr705) and serine 727 (Ser727). STAT3 activation was accompanied by its translocation into mitochondria within diaphragm muscle and mitochondrial dysfunction. Inhibition of JAK signaling during CMV prevented phosphorylation of both target sites on STAT3, eliminated the accumulation of phosphorylated STAT3 within the mitochondria, and reversed the pathologic alterations in mitochondrial function, reduced oxidative stress in the diaphragm, and maintained normal diaphragm contractility. In addition, JAK inhibition during CMV blunted the activation of key proteolytic pathways in the diaphragm, as well as diaphragm atrophy. These findings implicate JAK/STAT3 signaling in the development of diaphragm muscle atrophy and dysfunction during CMV and suggest that the delayed extubation times associated with CMV can be prevented by inhibition of Janus kinase signaling.-Smith, I. J., Godinez, G. L., Singh, B. K., McCaughey, K. M., Alcantara, R. R., Gururaja, T., Ho, M. S., Nguyen, H. N., Friera, A. M., White, K. A., McLaughlin, J. R., Hansen, D., Romero, J. M., Baltgalvis, K. A., Claypool, M. D., Li, W., Lang, W., Yam, G. C., Gelman, M. S., Ding, R., Yung, S. L., Creger, D. P., Chen, Y., Singh, R., Smuder, A. J., Wiggs, M. P., Kwon, O.-S., Sollanek, K. J., Powers, S. K., Masuda, E. S., Taylor, V. C., Payan, D. G., Kinoshita, T., Kinsella, T. M. Inhibition of Janus kinase signaling during controlled mechanical ventilation prevents ventilation-induced diaphragm dysfunction.


Assuntos
Diafragma/metabolismo , Janus Quinases/metabolismo , Respiração Artificial/efeitos adversos , Transdução de Sinais/fisiologia , Animais , Interleucina-6/metabolismo , Masculino , Mitocôndrias/metabolismo , Debilidade Muscular/metabolismo , Atrofia Muscular/metabolismo , Estresse Oxidativo/fisiologia , Fosforilação/fisiologia , Proteólise , Ratos , Ratos Sprague-Dawley , Fator de Transcrição STAT3/metabolismo , Serina/metabolismo , Tirosina/metabolismo
9.
PLoS One ; 8(12): e81870, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24339975

RESUMO

Modulation of mitochondrial function through inhibiting respiratory complex I activates a key sensor of cellular energy status, the 5'-AMP-activated protein kinase (AMPK). Activation of AMPK results in the mobilization of nutrient uptake and catabolism for mitochondrial ATP generation to restore energy homeostasis. How these nutrient pathways are affected in the presence of a potent modulator of mitochondrial function and the role of AMPK activation in these effects remain unclear. We have identified a molecule, named R419, that activates AMPK in vitro via complex I inhibition at much lower concentrations than metformin (IC50 100 nM vs 27 mM, respectively). R419 potently increased myocyte glucose uptake that was dependent on AMPK activation, while its ability to suppress hepatic glucose production in vitro was not. In addition, R419 treatment of mouse primary hepatocytes increased fatty acid oxidation and inhibited lipogenesis in an AMPK-dependent fashion. We have performed an extensive metabolic characterization of its effects in the db/db mouse diabetes model. In vivo metabolite profiling of R419-treated db/db mice showed a clear upregulation of fatty acid oxidation and catabolism of branched chain amino acids. Additionally, analyses performed using both (13)C-palmitate and (13)C-glucose tracers revealed that R419 induces complete oxidation of both glucose and palmitate to CO2 in skeletal muscle, liver, and adipose tissue, confirming that the compound increases mitochondrial function in vivo. Taken together, our results show that R419 is a potent inhibitor of complex I and modulates mitochondrial function in vitro and in diabetic animals in vivo. R419 may serve as a valuable molecular tool for investigating the impact of modulating mitochondrial function on nutrient metabolism in multiple tissues and on glucose and lipid homeostasis in diabetic animal models.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Diabetes Mellitus Experimental/metabolismo , Mitocôndrias Hepáticas/metabolismo , Células Musculares/metabolismo , Aminoácidos de Cadeia Ramificada/metabolismo , Animais , Diabetes Mellitus Experimental/patologia , Ativação Enzimática/efeitos dos fármacos , Ácidos Graxos/metabolismo , Glucose/metabolismo , Células Hep G2 , Humanos , Hipoglicemiantes/farmacologia , Metformina/farmacologia , Camundongos , Mitocôndrias Hepáticas/patologia , Células Musculares/patologia , Oxirredução/efeitos dos fármacos , Palmitatos/farmacologia , Inibidores de Proteínas Quinases/farmacologia
10.
Chem Biol ; 14(10): 1105-18, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17961823

RESUMO

Small-molecule library screening to find compounds that inhibit TNFalpha-induced, but not interleukin 1beta (IL-1beta)-induced, intercellular adhesion molecule 1 (ICAM-1) expression in lung epithelial cells identified a class of triazoloquinoxalines. These compounds not only inhibited the TNFalpha-induced nuclear factor kappaB (NFkappaB) survival pathway but also blocked death-pathway activation. Such dual activity makes them unique against other known NFkappaB-pathway inhibitors that inhibit only a subset of TNFalpha signals leading to increased TNFalpha-induced cytotoxicity. Interestingly, these compounds inhibited association of TNFalpha receptor (TNFalphaR) I with TNFalphaR-associated death domain protein (TRADD) and receptor interacting protein 1 (RIP1), the initial intracellular signaling event following TNFalpha stimulation. Further study showed that they blocked ligand-dependent internalization of the TNFalpha-TNFalphaR complex, thereby inhibiting most of the TNFalpha-induced cellular responses. Thus, compounds with a triazoloquinoxaline scaffold could be a valuable tool to investigate small molecule-based anti-TNFalpha therapies.


Assuntos
Apoptose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Molécula 1 de Adesão Intercelular/metabolismo , Quinoxalinas/farmacologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Proteína de Domínio de Morte Associada a Receptor de TNF/metabolismo , Triazóis/farmacologia , Fator de Necrose Tumoral alfa , Apoptose/fisiologia , Membrana Celular/química , Membrana Celular/metabolismo , Células Epiteliais/citologia , Células Epiteliais/patologia , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Molécula 1 de Adesão Intercelular/genética , Pulmão/citologia , Pulmão/patologia , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Bibliotecas de Moléculas Pequenas , Proteína de Domínio de Morte Associada a Receptor de TNF/genética
11.
J Pharmacol Exp Ther ; 320(2): 900-6, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17110523

RESUMO

A previously described VPAC2-selective agonist, BAY 55-9837 (peptide HSDAVFTDNYTRLRKQVAAKKYLQSIKNKRY), had several limitations with respect to its potential as an insulin secretagogue for the treatment of type 2 diabetes. These limitations were primarily poor stability in aqueous buffer and short duration of action in vivo. In this report, we describe a series of novel analogs of BAY 55-9837 that were designed around the likely degradation mechanisms and structure-activity relationship of this peptide with a view to overcoming its limitations. These analogs were tested for improved liquid stability and retention of VPAC2-selective binding and activation, as well as prolonged activity in vivo. Although several degradation mechanisms were possible based on the degradation pattern, it was determined that deamidation at the two asparagines (N9 and N28) was the major instability determinant. Changing these two asparagines to glutamines did not negatively affect VPAC2-selective binding and activation. The double glutamine mutein analog, BAY(Q9Q28), retained full VPAC2 activity and selectivity while displaying no significant degradation when stored at 40 degrees C for 4 weeks. This is in contrast to BAY 55-9837, which showed greater than 80% degradation when stored at 40 degrees C for 2 weeks. A cysteine was added to the C terminus of BAY(Q9Q28), followed by site-specific cysteine conjugation with a 22- or 43-kDa polyethylene glycol (PEG) to yield BAY(Q9Q28C32)PEG22 or BAY(Q9Q28C32)PEG43, respectively. These PEGylated peptides retain the ability to selectively bind and activate the VPAC2 receptor and have prolonged glucose-lowering activity in vivo.


Assuntos
Hipoglicemiantes/farmacologia , Fragmentos de Peptídeos/farmacologia , Receptores Tipo II de Peptídeo Intestinal Vasoativo/agonistas , Sequência de Aminoácidos , Animais , Glicemia/análise , Células CHO , Cricetinae , Cricetulus , Estabilidade de Medicamentos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Polietilenoglicóis , Ratos , Ratos Wistar , Peptídeo Intestinal Vasoativo/farmacologia
12.
Clin Cancer Res ; 12(12): 3831-42, 2006 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-16778111

RESUMO

PURPOSE: The design and development of synthetic small molecules to disrupt microtubule dynamics is an attractive therapeutic strategy for anticancer drug discovery research. Loss of clinical efficacy of many useful drugs due to drug resistance in tumor cells seems to be a major hurdle in this endeavor. Thus, a search for new chemical entities that bind tubulin, but neither are a substrate of efflux pump, P-glycoprotein 170/MDR1, nor cause undesired side effects, would potentially increase the therapeutic index in certain cancer treatments. EXPERIMENTAL DESIGN: A high-content cell-based screen of a compound library led to the identification of a new class of compounds belonging to a thienopyrimidine series, which exhibited significant antitumor activities. On structure-activity relationship analysis, R-253 [N-cyclopropyl-2-(6-(3,5-dimethylphenyl)thieno[3,2-d]pyrimidin-4-yl)hydrazine carbothioamide] emerged as a potent antiproliferative agent (average EC(50), 20 nmol/L) when examined in a spectrum of tumor cell lines. RESULTS: R-253 is structurally unique and destabilizes microtubules both in vivo and in vitro. Standard fluorescence-activated cell sorting and Western analyses revealed that the effect of R-253 on cell growth was associated with cell cycle arrest in mitosis, increased select G(2)-M checkpoint proteins, and apoptosis. On-target activity of R-253 on microtubules was further substantiated by immunofluorescence studies and selected counter assays. R-253 competed with fluorescent-labeled colchicine for binding to tubulin, indicating that its binding site on tubulin could be similar to that of colchicine. R-253 neither is a substrate of P-glycoprotein 170/MDR1 nor is cytotoxic to nondividing human hepatocytes. CONCLUSION: Both biochemical and cellular mechanistic studies indicate that R-253 could become a promising new tubulin-binding drug candidate for treating various malignancies.


Assuntos
Linhagem Celular Tumoral/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Microtúbulos/efeitos da radiação , Tiofenos/farmacologia , Tioureia/análogos & derivados , Adenocarcinoma , Antineoplásicos/farmacologia , Apoptose/efeitos da radiação , Neoplasias Ósseas , Ciclo Celular/efeitos da radiação , Neoplasias do Colo , Citometria de Fluxo , Células HeLa , Humanos , Neoplasias Pulmonares , Peso Molecular , Osteossarcoma , Pirimidinas/química , Tioureia/farmacologia
13.
J Biol Chem ; 281(18): 12506-15, 2006 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-16505481

RESUMO

The closely related peptides glucagon-like peptide (GLP-1) and glucagon have opposing effects on blood glucose. GLP-1 induces glucose-dependent insulin secretion in the pancreas, whereas glucagon stimulates gluconeogenesis and glycogenolysis in the liver. The identification of a hybrid peptide acting as both a GLP-1 agonist and a glucagon antagonist would provide a novel approach for the treatment of type 2 diabetes. Toward this end a series of hybrid peptides made up of glucagon and either GLP-1 or exendin-4, a GLP-1 agonist, was engineered. Several peptides that bind to both the GLP-1 and glucagon receptors were identified. The presence of glucagon sequence at the N terminus removed the dipeptidylpeptidase IV cleavage site and increased plasma stability compared with GLP-1. Targeted mutations were incorporated into the optimal dual-receptor binding peptide to identify a peptide with the highly novel property of functioning as both a GLP-1 receptor agonist and a glucagon receptor antagonist. To overcome the short half-life of this mutant peptide in vivo, while retaining dual GLP-1 agonist and glucagon antagonist activities, site-specific attachment of long chained polyethylene glycol (PEGylation) was pursued. PEGylation at the C terminus retained the in vitro activities of the peptide while dramatically prolonging the duration of action in vivo. Thus, we have generated a novel dual-acting peptide with potential for development as a therapeutic for type 2 diabetes.


Assuntos
Peptídeos/química , Receptores de Glucagon/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Desenho de Fármacos , Receptor do Peptídeo Semelhante ao Glucagon 1 , Humanos , Masculino , Dados de Sequência Molecular , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Receptores de Glucagon/agonistas , Homologia de Sequência de Aminoácidos
14.
J Biol Chem ; 278(12): 10273-81, 2003 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-12525492

RESUMO

Pituitary adenylate cyclase-activating peptide (PACAP) has a specific receptor PAC1 and shares two receptors VPAC1 and VPAC2 with vasoactive intestinal peptide (VIP). VPAC2 activation enhances glucose-induced insulin release while VPAC1 activation elevates glucose output. To generate a large pool of VPAC2 selective agonists for the treatment of type 2 diabetes, structure-activity relationship studies were performed on PACAP, VIP, and a VPAC2 selective VIP analog. Chemical modifications on this analog that prevent recombinant expression were sequentially removed to show that a recombinant peptide would retain VPAC2 selectivity. An efficient recombinant expression system was then developed to produce and screen hundreds of mutant peptides. The 11 mutations found on the VIP analog were systematically replaced with VIP or PACAP sequences. Three of these mutations, V19A, L27K, and N28K, were sufficient to provide most of the VPAC2 selectivity. C-terminal extension with the KRY sequence from PACAP38 led to potent VPAC2 agonists with improved selectivity (100-1000-fold). Saturation mutagenesis at positions 19, 27, 29, and 30 of VIP and charge-scanning mutagenesis of PACAP27 generated additional VPAC2 selective agonists. We have generated the first set of recombinant VPAC2 selective agonists described, which exhibit activity profiles that suggest therapeutic utility in the treatment of diabetes.


Assuntos
Neuropeptídeos/farmacologia , Receptores de Peptídeo Intestinal Vasoativo/agonistas , Peptídeo Intestinal Vasoativo/farmacologia , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Neuropeptídeos/química , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Receptores Tipo II de Peptídeo Intestinal Vasoativo , Proteínas Recombinantes/farmacologia , Relação Estrutura-Atividade , Peptídeo Intestinal Vasoativo/química
15.
Diabetes ; 51(5): 1453-60, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-11978642

RESUMO

Pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP) activate two shared receptors, VPAC1 and VPAC2. Activation of VPAC1 has been implicated in elevating glucose output, whereas activation of VPAC2 may be involved in insulin secretion. A hypothesis that a VPAC2-selective agonist would enhance glucose disposal by stimulating insulin secretion without causing increased hepatic glucose production was tested using a novel selective agonist of VPAC2. This agonist, BAY 55-9837, was generated through site-directed mutagenesis based on sequence alignments of PACAP, VIP, and related analogs. The peptide bound to VPAC2 with a dissociation constant (K(d)) of 0.65 nmol/l and displayed >100-fold selectivity over VPAC1. BAY 55-9837 stimulated glucose-dependent insulin secretion in isolated rat and human pancreatic islets, increased insulin synthesis in purified rat islets, and caused a dose-dependent increase in plasma insulin levels in fasted rats, with a half-maximal stimulatory concentration of 3 pmol/kg. Continuous intravenous or subcutaneous infusion of the peptide reduced the glucose area under the curve following an intraperitoneal glucose tolerance test. The peptide had effects on intestinal water retention and mean arterial blood pressure in rats, but only at much higher doses. BAY 55-9837 may be a useful therapy for the treatment of type 2 diabetes.


Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Fragmentos de Peptídeos/farmacologia , Receptores de Peptídeo Intestinal Vasoativo/agonistas , Peptídeo Intestinal Vasoativo/farmacologia , Sequência de Aminoácidos , Animais , Pressão Sanguínea/efeitos dos fármacos , Células Cultivadas , Diarreia/tratamento farmacológico , Diarreia/metabolismo , Glucose/farmacologia , Frequência Cardíaca/efeitos dos fármacos , Hormônios/sangue , Humanos , Injeções Intravenosas , Injeções Subcutâneas , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ratos , Ratos Wistar , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Receptores do Hormônio Hipofisário/metabolismo , Receptores de Peptídeo Intestinal Vasoativo/metabolismo , Receptores Tipo II de Peptídeo Intestinal Vasoativo , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo , Peptídeo Intestinal Vasoativo/análogos & derivados , Peptídeo Intestinal Vasoativo/química , Peptídeo Intestinal Vasoativo/metabolismo
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