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1.
Cell ; 187(3): 521-525, 2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38306979

RESUMO

High-quality predicted structures enable structure-based approaches to an expanding number of drug discovery programs. We propose that by utilizing free energy perturbation (FEP), predicted structures can be confidently employed to achieve drug design goals. We use structure-based modeling of hERG inhibition to illustrate this value of FEP.


Assuntos
Desenho de Fármacos , Descoberta de Drogas , Termodinâmica , Entropia
2.
Expert Opin Ther Targets ; 26(9): 811-822, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36424892

RESUMO

INTRODUCTION: The Helping to End Addiction Long-termSM Initiative supports a wide range of programs to develop new or improved prevention and opioid addiction treatment strategies. An essential component of this effort is to accelerate development of non-opioid pain therapeutics. In all fields of medicine, therapeutics development is an arduous process and late-stage translational efforts such as clinical trials to validate targets are particularly complex and costly. While there are plentiful novel targets for pain treatment, successful clinical validation is rare. It is therefore crucial to develop processes whereby therapeutic targets can be reasonably 'de-risked' prior to substantial late-stage validation efforts. Such rigorous validation of novel therapeutic targets in the preclinical space will give potential private sector partners the confidence to pursue clinical validation of promising therapeutic concepts and compounds. AREAS COVERED: In 2020, the National Institutes of Health (NIH) held the Target Validation for Non-Addictive Therapeutics Development for Pain workshop to gather insights from key opinion leaders in academia, industry, and venture-financing. EXPERT OPINION: The result was a roadmap for pain target validation focusing on three modalities: 1) human evidence; 2) assay development in vitro; 3) assay development in vivo.


Assuntos
Transtornos Relacionados ao Uso de Opioides , Dor , Humanos , Dor/tratamento farmacológico , Transtornos Relacionados ao Uso de Opioides/tratamento farmacológico
3.
Drug Discov Today Technol ; 39: 111-117, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34906321

RESUMO

Computational chemistry and structure-based design have traditionally been viewed as a subset of tools that could aid acceleration of the drug discovery process, but were not commonly regarded as a driving force in small molecule drug discovery. In the last decade however, there have been dramatic advances in the field, including (1) development of physics-based computational approaches to accurately predict a broad variety of endpoints from potency to solubility, (2) improvements in artificial intelligence and deep learning methods and (3) dramatic increases in computational power with the advent of GPUs and cloud computing, resulting in the ability to explore and accurately profile vast amounts of drug-like chemical space in silico. There have also been simultaneous advancements in structural biology such as cryogenic electron microscopy (cryo-EM) and computational protein-structure prediction, allowing for access to many more high-resolution 3D structures of novel drug-receptor complexes. The convergence of these breakthroughs has positioned structurally-enabled computational methods to be a driving force behind the discovery of novel small molecule therapeutics. This review will give a broad overview of the synergies in recent advances in the fields of computational chemistry, machine learning and structural biology, in particular in the areas of hit identification, hit-to-lead, and lead optimization.


Assuntos
Inteligência Artificial , Descoberta de Drogas , Desenho Assistido por Computador , Computadores , Desenho de Fármacos , Aprendizado de Máquina , Proteínas
5.
Cell Chem Biol ; 27(1): 32-40.e3, 2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-31653597

RESUMO

Proprotein convertase substilisin-like/kexin type 9 (PCSK9) is a serine protease involved in a protein-protein interaction with the low-density lipoprotein (LDL) receptor that has both human genetic and clinical validation. Blocking this protein-protein interaction prevents LDL receptor degradation and thereby decreases LDL cholesterol levels. Our pursuit of small-molecule direct binders for this difficult to drug PPI target utilized affinity selection/mass spectrometry, which identified one confirmed hit compound. An X-ray crystal structure revealed that this compound was binding in an unprecedented allosteric pocket located between the catalytic and C-terminal domain. Optimization of this initial hit, using two distinct strategies, led to compounds with high binding affinity to PCSK9. Direct target engagement was demonstrated in the cell lysate with a cellular thermal shift assay. Finally, ligand-induced protein degradation was shown with a proteasome recruiting tag attached to the high-affinity allosteric ligand for PCSK9.


Assuntos
Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Pró-Proteína Convertase 9/metabolismo , Proteólise/efeitos dos fármacos , Inibidores de Serino Proteinase/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Humanos , Ligantes , Modelos Moleculares , Estrutura Molecular , Inibidores de Serino Proteinase/química , Bibliotecas de Moléculas Pequenas/química
6.
Cell Metab ; 27(6): 1236-1248.e6, 2018 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-29706567

RESUMO

Diacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step in triglyceride (TG) synthesis and has been shown to play a role in regulating hepatic very-low-density lipoprotein (VLDL) production in rodents. To explore the potential of DGAT2 as a therapeutic target for the treatment of dyslipidemia, we tested the effects of small-molecule inhibitors and gene silencing both in vitro and in vivo. Consistent with prior reports, chronic inhibition of DGAT2 in a murine model of obesity led to correction of multiple lipid parameters. In contrast, experiments in primary human, rhesus, and cynomolgus hepatocytes demonstrated that selective inhibition of DGAT2 has only a modest effect. Acute and chronic inhibition of DGAT2 in rhesus primates recapitulated the in vitro data yielding no significant effects on production of plasma TG or VLDL apolipoprotein B. These results call into question whether selective inhibition of DGAT2 is sufficient for remediation of dyslipidemia.


Assuntos
Diacilglicerol O-Aciltransferase/antagonistas & inibidores , Dislipidemias/metabolismo , Hepatócitos/metabolismo , Obesidade/metabolismo , Triglicerídeos/metabolismo , Animais , Apolipoproteínas B/metabolismo , Células Cultivadas , Diacilglicerol O-Aciltransferase/genética , Modelos Animais de Doenças , Inativação Gênica , Humanos , Lipoproteínas VLDL/metabolismo , Macaca fascicularis , Macaca mulatta , Camundongos , Camundongos Endogâmicos C57BL
7.
J Lipid Res ; 57(12): 2150-2162, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27707816

RESUMO

SREBP cleavage-activating protein (SCAP) is a key protein in the regulation of lipid metabolism and a potential target for treatment of dyslipidemia. SCAP is required for activation of the transcription factors SREBP-1 and -2. SREBPs regulate the expression of genes involved in fatty acid and cholesterol biosynthesis, and LDL-C clearance through the regulation of LDL receptor (LDLR) and PCSK9 expression. To further test the potential of SCAP as a novel target for treatment of dyslipidemia, we used siRNAs to inhibit hepatic SCAP expression and assess the effect on PCSK9, LDLR, and lipids in mice and rhesus monkeys. In mice, robust liver Scap mRNA knockdown (KD) was achieved, accompanied by dose-dependent reduction in SREBP-regulated gene expression, de novo lipogenesis, and plasma PCSK9 and lipids. In rhesus monkeys, over 90% SCAP mRNA KD was achieved resulting in approximately 75, 50, and 50% reduction of plasma PCSK9, TG, and LDL-C, respectively. Inhibition of SCAP function was demonstrated by reduced expression of SREBP-regulated genes and de novo lipogenesis. In conclusion, siRNA-mediated inhibition of SCAP resulted in a significant reduction in circulating PCSK9 and LDL-C in rodent and primate models supporting SCAP as a novel target for the treatment of dyslipidemia.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Lipídeos/sangue , Proteínas de Membrana/genética , Pró-Proteína Convertase 9/genética , RNA Interferente Pequeno/genética , Receptores de LDL/genética , Animais , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Hipolipemiantes/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipogênese , Fígado/enzimologia , Macaca mulatta , Masculino , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Pró-Proteína Convertase 9/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de LDL/metabolismo , Transdução de Sinais , Sinvastatina/farmacologia , Proteínas de Ligação a Elemento Regulador de Esterol/genética , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo
8.
J Lipid Res ; 57(3): 398-409, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26658238

RESUMO

Studies in lipoprotein kinetics almost exclusively rely on steady-state approaches to modeling. Herein, we have used a non-steady-state experimental design to examine the role of cholesteryl ester transfer protein (CETP) in mediating HDL-TG flux in vivo in rhesus macaques, and therefore, we developed an alternative strategy to model the data. Two isotopomers ([(2)H11] and [(13)C18]) of oleic acid were administered (orally and intravenously, respectively) to serve as precursors for labeling TGs in apoB-containing lipoproteins. The flux of a specific TG (52:2) from these donor lipoproteins to HDL was used as the measure of CETP activity; calculations are also presented to estimate total HDL-TG flux. Based on our data, we estimate that the peak total postprandial TG flux to HDL via CETP is ∼ 13 mg · h(-1) · kg(-1) and show that this transfer was inhibited by 97% following anacetrapib treatment. Collectively, these data demonstrate that HDL TG flux can be used as a measure of CETP activity in vivo. The fact that the donor lipoproteins can be labeled in situ using well-established stable isotope tracer techniques suggests ways to measure this activity for native lipoproteins in free-living subjects under any physiological conditions.


Assuntos
Proteínas de Transferência de Ésteres de Colesterol/antagonistas & inibidores , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Oxazolidinonas/farmacologia , Triglicerídeos/metabolismo , Animais , Lipoproteínas HDL/sangue , Macaca mulatta , Masculino , Modelos Biológicos , Triglicerídeos/sangue
9.
J Pharmacol Toxicol Methods ; 71: 137-46, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25304940

RESUMO

INTRODUCTION: In vivo profiles of aldosterone synthase inhibitors (ASIs) have been investigated utilizing various rodent models. Due to lack of CYP17 activity, rodents produce corticosterone rather than cortisol as that of humans, which raised concern to their effectiveness in translational pharmacological characterization of ASI. METHODS: A rhesus monkey model that combines a low sodium diet with adrenocorticotropin (ACTH) treatment was developed. Plasma concentrations of steroid metabolites associated with reactions catalyzed by CYP11B2 and CYP11B1 were measured concurrently by a UPLC/MS method. RESULTS: Plasma concentration of aldosterone in regular diet fed rhesus monkeys was low at 109pg/mL. Aldosterone concentrations were increased to 252pg/mL when animals were maintained on a low sodium diet for 3weeks, and to 300pg/mL with ACTH treatment at 0.3mg/kg. The combination of low sodium diet with ACTH treatment further increased plasma concentration of aldosterone to 730pg/mL and other steroid metabolites at various levels. Intravenous administration of ASI, fadrozole (0.001-1mg/kg) or LCI699 (0.003-3mg/kg), led to dose-dependent reductions in aldosterone and 18-hydroxycorticosterone, increases in 11-deoxycorticosterone and 11-deoxycortisol, and bell-shaped changes in cortisol and corticosterone. In vivo selectivity of CYP11B2/CYP11B1 for fadrazole was 26-fold and LCI-699 was 27-fold, which was consistent with relative selectivity using in vitro values from recombinant cells transfected with rhesus monkey CYP11B2 and CYP11B1. DISCUSSION: This model enables concurrent characterization of pharmacokinetics, pharmacodynamics and selectivity of CYP11B2 over CYP11B1 inhibition in the same animal. It may be used as a translational model for pharmacological characterization of ASI.


Assuntos
Citocromo P-450 CYP11B2/antagonistas & inibidores , Inibidores Enzimáticos/farmacocinética , Modelos Animais , Hormônio Adrenocorticotrópico/administração & dosagem , Hormônio Adrenocorticotrópico/farmacocinética , Animais , Citocromo P-450 CYP11B2/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Macaca mulatta , Masculino , Sódio na Dieta/administração & dosagem , Sódio na Dieta/farmacocinética , Esteroide 11-beta-Hidroxilase/antagonistas & inibidores , Esteroide 11-beta-Hidroxilase/metabolismo , Esteroides/sangue , Esteroides/metabolismo
10.
Rapid Commun Mass Spectrom ; 28(22): 2471-9, 2014 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-25303476

RESUMO

RATIONALE: The ability to quantify rates of formation, regression and/or remodeling of atherosclerotic plaque should facilitate a better understanding of the pathogenesis and management of cardiovascular disease. In the current study, we coupled a stable isotope labeled tracer protocol with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to examine spatial and temporal lipid dynamics in atherosclerotic plaque. METHODS: To promote plaque formation in the aorta region, ApoE KO mice were fed a high cholesterol diet (0.15% cholesterol) and orally dosed with (2,2,3,4,4,6-d(6))-cholesterol over several weeks. Tissue sections of ~10 µm thickness were analyzed by MALDI-MSI using matrix deposition by either chemical sublimation or acoustic droplet ejection. RESULTS: MALDI-MSI yielded distinct spatial distribution information for a variety of lipid classes including specific lysophosphatidylcholines typically associated with atherosclerosis-related tissue damage such as phospholipase 2 (Lp-PLA(2)) that mediate chemotactic responses to inflammation (e.g. LPC 16:0, LPC 18:0 and LPC 18:1) as well as free cholesterol and cholesteryl esters that contribute to atheroma formation. MALDI mass spectra acquired from aorta tissue sections clearly distinguished non-esterified and esterified versions of (2,2,3,4,4,6-d(6))-cholesterol within aortic plaque regions and showed distinct spatial accumulation of the cholesterol tracer. CONCLUSIONS: The ability to couple stable isotope based protocols with MALDI-MSI enables a novel strategy to characterize the effects of therapeutic treatments on atherosclerotic plaque formation, regression and potential remodeling of the complex lipid components with high chemical specificity and spatiotemporal information.

11.
J Lipid Res ; 55(8): 1693-701, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24891332

RESUMO

While genetic determinants strongly influence HDL cholesterol (HDLc) levels, most genetic causes underlying variation in HDLc remain unknown. We aimed to identify novel rare mutations with large effects in candidate genes contributing to extreme HDLc in humans, utilizing family-based Mendelian genetics. We performed next-generation sequencing of 456 candidate HDLc-regulating genes in 200 unrelated probands with extremely low (≤10th percentile) or high (≥90th percentile) HDLc. Probands were excluded if known mutations existed in the established HDLc-regulating genes ABCA1, APOA1, LCAT, cholesteryl ester transfer protein (CETP), endothelial lipase (LIPG), and UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 2 (GALNT2). We identified 93 novel coding or splice-site variants in 72 candidate genes. Each variant was genotyped in the proband's family. Family-based association analyses were performed for variants with sufficient power to detect significance at P < 0.05 with a total of 627 family members being assessed. Mutations in the genes glucokinase regulatory protein (GCKR), RNase L (RNASEL), leukocyte immunoglobulin-like receptor 3 (LILRA3), and dynein axonemal heavy chain 10 (DNAH10) segregated with elevated HDLc levels in families, while no mutations associated with low HDLc. Taken together, we have identified mutations in four novel genes that may play a role in regulating HDLc levels in humans.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Dineínas do Axonema/genética , HDL-Colesterol/sangue , Endorribonucleases/genética , Mutação , Receptores Imunológicos/genética , Transportador 1 de Cassete de Ligação de ATP/genética , Adulto , Idoso , Apolipoproteína A-I/genética , Proteínas de Transferência de Ésteres de Colesterol/genética , HDL-Colesterol/genética , Feminino , Humanos , Lipase/genética , Masculino , Pessoa de Meia-Idade , N-Acetilgalactosaminiltransferases/genética , Fosfatidilcolina-Esterol O-Aciltransferase/genética
12.
Eur J Pharmacol ; 740: 410-6, 2014 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-24769414

RESUMO

Inhibition of cholesteryl ester transfer protein (CETP) has been vigorously pursued as a potential therapy to treat patients who are at an elevated risk for coronary artery disease. Anacetrapib, a novel CETP inhibitor, has been shown clinically to raise HDL cholesterol and reduce LDL cholesterol when provided as monotherapy or when co-administered with a statin. Preclinically, the effects of anacetrapib on the functionality and composition of HDL have been extensively studied. In contrast, the effects of anacetrapib on other parameters related to lipoprotein metabolism and cardiovascular risk have been difficult to explore. The aim of the present investigation was to evaluate the effects of anacetrapib in rhesus macaques and to compare these to effects reported in dyslipidemic humans. Our results from two separate studies show that administration of anacetrapib (150 mg/kg q.d. for 10 days) to rhesus macaques results in alterations in CETP activity (reduced by more than 70%) and HDL cholesterol (increased by more than 110%) which are similar to those reported in dyslipidemic humans. Levels of LDL cholesterol were reduced by more than 60%, an effect slightly greater than what has been observed clinically. Treatment with anacetrapib in this model was also found to lead to statistically significant reductions in plasma PCSK9 and to reduce cholesterol excursion in the combined chylomicron and remnant lipoprotein fraction isolated from plasma by fast protein liquid chromatography. Collectively, these data suggest that rhesus macaques may be a useful translational model to study the mechanistic effects of CETP inhibition.


Assuntos
Anticolesterolemiantes/farmacologia , Proteínas de Transferência de Ésteres de Colesterol/antagonistas & inibidores , Oxazolidinonas/farmacologia , Animais , Apolipoproteínas/sangue , Proteínas de Transferência de Ésteres de Colesterol/sangue , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Macaca mulatta , Masculino , Pró-Proteína Convertases/sangue , Serina Endopeptidases/sangue , Triglicerídeos/sangue
13.
J Lipid Res ; 54(10): 2858-65, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23898048

RESUMO

Cholesteryl ester transfer protein (CETP) transfers cholesteryl ester and triglyceride between HDL and apoB-containing lipoproteins. Anacetrapib (ANA), a reversible inhibitor of CETP, raises HDL cholesterol and lowers LDL cholesterol in dyslipidemic patients. We previously demonstrated that ANA increases macrophage-to-feces reverse cholesterol transport and fecal cholesterol excretion in hamsters, and increased preß HDL-dependent cholesterol efflux via ABCA1 in vitro. However, the effects of ANA on in vivo preß HDL have not been characterized. In vitro, ANA inhibited the formation of preß, however in ANA-treated dyslipidemic hamsters, preß HDL levels (measured by two-dimensional gel electrophoresis) were increased, in contrast to in vitro findings. Because changes in plasma preß HDL have been proposed to potentially affect markers of cholesterol absorption with other CETP inhibitors, a dual stable isotope method was used to directly measure cholesterol absorption in hamsters. ANA treatment of hamsters (on either dyslipidemic or normal diet) had no effect on cholesterol absorption, while dalcetrapib-treated hamsters displayed an increase in cholesterol absorption. Taken together, these data support the notion that ANA promotes preß HDL functionality in vivo, with no effects on cholesterol absorption.


Assuntos
Anticolesterolemiantes/farmacologia , Colesterol/metabolismo , Dislipidemias/tratamento farmacológico , Lipoproteínas de Alta Densidade Pré-beta/sangue , Absorção Intestinal/efeitos dos fármacos , Oxazolidinonas/farmacologia , Amidas , Animais , Anticolesterolemiantes/uso terapêutico , Área Sob a Curva , Azetidinas/farmacologia , Azetidinas/uso terapêutico , Proteínas de Transferência de Ésteres de Colesterol/antagonistas & inibidores , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Cricetinae , Dieta Hiperlipídica/efeitos adversos , Avaliação Pré-Clínica de Medicamentos , Dislipidemias/sangue , Dislipidemias/etiologia , Ésteres , Ezetimiba , Humanos , Masculino , Mesocricetus , Oxazolidinonas/uso terapêutico , Compostos de Sulfidrila/farmacologia , Compostos de Sulfidrila/uso terapêutico
14.
Curr Chem Genomics ; 6: 38-47, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23115612

RESUMO

The function of a particular protein is dependent upon its localization and milieu. The ability to track the "fate" of a protein is a valuable tool to elucidate its function. We present the use of HaloTag technology to study the localization and fate of human Proprotein Convertase Subtilisin-like Kexin type 9 (PCSK9).The role of PCSK9 in the regulation of circulating low density lipoprotein-cholesterol (LDL-c) levels is ascribed to binding of circulating PCSK9 to the LDL receptor (LDLR) and subsequent lysosomal degradation of LDLR. However, hints in the literature indicate that intracellular PCSK9 may act on the LDLR, possibly during processing of newly synthesized protein. To address this question, the source and fate of intracellular PCSK9 requires further investigation.We applied HaloTag technology to distinguish the source of intracellular PCSK9 and showed that newly synthesized intracellular PCSK9 has unique localization from the PCSK9 after re-uptake. This suggests different functions of PCSK9 while interacting with the LDLR.

15.
Respirology ; 11(1): 41-8, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16423200

RESUMO

OBJECTIVE AND BACKGROUND: The course of asthma may be altered during pregnancy with at least one-third of women experiencing a worsening of asthma and 20% having an exacerbation during pregnancy. This study used the novel proteomic technique, surface-enhanced laser desorption ionization-time of flight mass spectrometry to determine if the presence of asthma during pregnancy was associated with alterations in plasma proteins. METHODS: Plasma collected from healthy (n = 23) and asthmatic (n = 27) pregnant women at 18 and 30 weeks gestation was applied to strong anion exchange (SAX2), weak cation exchange (WCX2) and immobilized metal affinity capture (IMAC-Cu(2+)) chips. Mass analysis was conducted using Ciphergen Protein Biology System IIc and significant differences in individual peak intensities between groups determined. RESULTS: At 18 weeks gestation, 91 peaks were significantly different between pregnant women with and without asthma, representing 28% of the total peaks identified. At 30 weeks gestation, 51 peaks were significantly different. There were two peaks that were significantly different between groups at both 18 and 30 weeks gestation and expressed at a similar level at both time points. One was increased in asthmatics (MW = 6444 Da) whereas the other decreased in asthmatics compared with non-asthmatic women (MW = 1846 Da). CONCLUSIONS: This study demonstrated that there are differences in protein patterns between pregnant women with and without asthma. Other techniques are needed to define the molecular species and classify pathophysiological significance. Surface-enhanced laser desorption ionization-time of flight mass spectrometry has potential as a tool to monitor disease progression in situations such as pregnancy.


Assuntos
Asma/sangue , Proteínas Sanguíneas/metabolismo , Complicações na Gravidez/sangue , Análise Serial de Proteínas/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Adulto , Biomarcadores/sangue , Feminino , Humanos , Gravidez , Proteômica
16.
J Soc Gynecol Investig ; 12(5): 349-55, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15979547

RESUMO

OBJECTIVE: We conducted a comparative proteomic analysis of placental and umbilical cord blood proteins using surface-enhanced laser desorption ionization-time of flight mass spectrometry (SELDI-TOF MS) to examine the associations among asthma, fetal gender, and protein profiles. METHODS: Placental tissue and umbilical vein plasma were collected from 10 healthy and 20 asthmatic women. Placental proteins were extracted using phosphate-buffered saline containing protease inhibitors. Samples were applied to the surfaces of strong anion exchange (SAX2), weak cation exchange (WCX2) and immobilized metal affinity capture (IMAC-Cu(2+)) chips. Mass analysis was conducted using a Ciphergen Protein Biology System IIc (Freemont, CA), and differences in individual peak intensities between groups were determined. RESULTS: Fourteen placental peaks were significantly different between asthmatic and non-asthmatic women (seven more highly expressed and seven less highly expressed). Ten umbilical cord blood peak differences were identified, with four peaks more highly expressed and six peaks less highly expressed in asthmatics. Four placental and three umbilical cord blood proteins differed significantly between male and female fetuses. Two placental and five umbilical cord blood peaks were specifically increased in a subgroup of samples collected from asthmatic women who did not use inhaled glucocorticoids and were pregnant with a female fetus, a group previously found to have altered placental function. CONCLUSIONS: This study demonstrates the abilities of the SELDI technique as a tool for protein profiling in tissue or plasma. Further work to positively identify the candidate peptides found in this study may provide a greater understanding of the placental mechanisms leading to alterations in fetal growth in patients with bronchial asthma.


Assuntos
Asma/complicações , Placenta/fisiologia , Complicações na Gravidez , Adulto , Peso ao Nascer , Feminino , Sangue Fetal/química , Desenvolvimento Fetal , Humanos , Recém-Nascido , Masculino , Troca Materno-Fetal , Gravidez , Proteômica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
17.
J Biol Chem ; 280(19): 18853-61, 2005 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-15671030

RESUMO

A new member of a family of proteins characterized by structural similarity to dipeptidyl peptidase (DPP) IV known as DPP10 was recently identified and linked to asthma susceptibility; however, the cellular functions of DPP10 are thus far unknown. DPP10 is highly homologous to subfamily member DPPX, which we previously reported as a modulator of Kv4-mediated A-type potassium channels (Nadal, M. S., Ozaita, A., Amarillo, Y., Vega-Saenz de Miera, E., Ma, Y., Mo, W., Goldberg, E. M., Misumi, Y., Ikehara, Y., Neubert, T. A., and Rudy, B. (2003) Neuron. 37, 449-461). We studied the ability of DPP10 protein to modulate the properties of Kv4.2 channels in heterologous expression systems. We found DPP10 activity to be nearly identical to DPPX activity and significantly different from DPPIV activity. DPPX and DPP10 facilitated Kv4.2 protein trafficking to the cell membrane, increased A-type current magnitude, and modified the voltage dependence and kinetic properties of the current such that they resembled the properties of A-type currents recorded in neurons in the central nervous system. Using in situ hybridization, we found DPP10 to be prominently expressed in brain neuronal populations that also express Kv4 subunits. Furthermore, DPP10 was detected in immunoprecipitated Kv4.2 channel complexes from rat brain membranes, confirming the association of DPP10 proteins with native Kv4.2 channels. These experiments suggest that DPP10 contributes to the molecular composition of A-type currents in the central nervous system. To dissect the structural determinants of these integral accessory proteins, we constructed chimeras of DPPX, DPP10, and DPPIV lacking the extracellular domain. Chimeras of DPPX and DPP10, but not DPPIV, were able to modulate the properties of Kv4.2 channels, highlighting the importance of the intracellular and transmembrane domains in this activity.


Assuntos
Dipeptidil Peptidases e Tripeptidil Peptidases/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Sequência de Aminoácidos , Animais , Biotinilação , Northern Blotting , Encéfalo/metabolismo , Células CHO , Membrana Celular/metabolismo , Sistema Nervoso Central/metabolismo , Cricetinae , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Eletrofisiologia , Humanos , Immunoblotting , Imuno-Histoquímica , Imunoprecipitação , Hibridização In Situ , Cinética , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Neurônios/metabolismo , Peptídeos/química , Canais de Potássio/metabolismo , Canais de Potássio/fisiologia , Conformação Proteica , Estrutura Terciária de Proteína , RNA/química , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/química , Homologia de Sequência de Aminoácidos , Canais de Potássio Shal , Fatores de Tempo , Transfecção
18.
Endocrinology ; 144(5): 1907-19, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12697698

RESUMO

GnRH is the key regulator of the reproductive axis in vertebrates, but little is known about GnRH before the origin of vertebrates. We have identified two genes encoding GnRH in a protochordate, Ciona intestinalis, thought to be related to the ancestral animal that gave rise to vertebrates. Each gene, Ci-gnrh1 and Ci-gnrh2, encodes in tandem three GnRH peptides, each of which is unique compared with known forms. Ci-gnrh1 encodes three peptides and contains no introns, whereas Ci-gnrh2 encodes three more peptides but has two introns. This is the first report in which more than one GnRH peptide is encoded on a single gene. The Ciona genes reveal consensus promoter elements that are conserved compared with human GNRH1. Both tunicate genes are expressed as mRNA early and throughout development, measured at the stages of four-cell, gastrulation, tail release, and tail resorption. In a closely related tunicate species, Ciona savignyi, we used in silico analysis to identify two similar genes encoding six peptides, only one of which is unique compared with C. intestinalis. Immunohistochemistry showed that at least one GnRH peptide was in the nerve net that surrounds the dorsal strand. Synthetic forms of the seven novel tunicate peptides induced release of gametes in adult tunicates. In contrast, the peptides did not activate the human GnRH-I receptor or cause release of LH in a rat pituitary cell assay. These data provide insight into the structural evolution of the GnRH peptides and their genes and show a functional role for GnRH in tunicate spawning.


Assuntos
Ciona intestinalis/genética , Hormônio Liberador de Gonadotropina/genética , Envelhecimento/fisiologia , Sequência de Aminoácidos/genética , Animais , Sítios de Ligação/genética , Ciona intestinalis/crescimento & desenvolvimento , Ciona intestinalis/metabolismo , Reações Cruzadas , Células Germinativas/fisiologia , Hormônio Liberador de Gonadotropina/imunologia , Hormônio Liberador de Gonadotropina/metabolismo , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/fisiologia , RNA Mensageiro/metabolismo , Ratos , Receptores LHRH/metabolismo , Fatores de Transcrição/genética , Sítio de Iniciação de Transcrição , Repetições de Trinucleotídeos
19.
Biochem J ; 373(Pt 1): 179-89, 2003 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-12662155

RESUMO

Two dipeptidyl peptidase IV (DPPIV, DPP4)-related proteins, DPP8 and DPP9, have been identified recently [Abbott, Yu, Woollatt, Sutherland, McCaughan, and Gorrell (2000) Eur. J. Biochem. 267, 6140-6150; Olsen and Wagtmann (2002) Gene 299, 185-193; Qi, Akinsanya, Riviere, and Junien (2002) Patent application WO0231134]. In the present study, we describe the cloning of DPP10, a novel 796-amino-acid protein, with significant sequence identity to DPP4 (32%) and DPP6 (51%) respectively. We propose that DPP10 is a new member of the S9B serine proteases subfamily. The DPP10 gene is located on the long arm of chromosome 2 (2q12.3-2q14.2), close to the DPP4 (2q24.3) and FAP (2q23) genes. The active-site serine residue is replaced by a glycine residue in DPP10, resulting in the loss of DPP activity. The serine residue is also replaced in DPP6, which lacks peptidase activity. DPP8 and DPP9 share an identical active site with DPP4 (Gly-Trp-Ser-Tyr-Gly). In contrast with the previous results suggesting that DPP9 is inactive, we show that DPP9 is a DPP, hydrolysing Ala-Pro-(7-amino-4-methyl-coumarin) with similar pH-specificity and protease-inhibitor-sensitivity to those of DPP4 and DPP8. Northern-blot analysis shows that whereas DPP8 and DPP9 are widely expressed, DPP10 is expressed mainly in the brain and pancreas. DPP6, which has the highest amino acid identity with DPP10, has been shown previously [Nadal, Ozaita, Amarillo, de Miera, Ma, Mo, Goldberg, Misumi, Ikehara, Neubert et al. (2003) Neuron 37, 449-461] to associate with A-type K(+) channel subunits, modulating their transport and function in somatodendritic compartments of neurons. It is possible that DPP10 is involved in similar functions in the brain. Elucidation of the physiological or pathophysiological role of DPP8, DPP9 and DPP10 and characterization of their structure-function relationships will add impetus to the development of inhibitor molecules for pharmacological or therapeutic use.


Assuntos
Cromossomos Humanos Par 2/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Serina Endopeptidases/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Primers do DNA , Dipeptidil Peptidase 4/química , Dipeptidil Peptidase 4/genética , Dipeptidil Peptidase 4/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases/química , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Evolução Molecular , Etiquetas de Sequências Expressas , Humanos , Cinética , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo
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