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1.
Artigo em Inglês | MEDLINE | ID: mdl-27816314

RESUMO

Associations between elevated plasma xanthine oxidoreductase (XOR) activity and various pathologies have been widely reported. However, it has been difficult to accurately measure human plasma XOR activity because the XOR activity of humans is lower than that of animals such as mouse. We developed a highly sensitive assay for XOR activity utilizing a combination of [13C2,15N2] xanthine and liquid chromatography/triple quadrupole mass spectrometry. In the present study, we established and validated a novel human plasma XOR activity assay utilizing this technique. The calibration curve of [13C2,15N2]uric acid showed linearity over the range of 4-4000nM (r2>0.995) with a lower limit of quantitation of 4nM which corresponds to an XOR activity of 6.67pmol/h/mL plasma. Intra- and inter-assay coefficients of variation of pooled human plasma XOR activity were 6.5% and 9.1%, respectively. Plasma XOR activities of 20 healthy volunteers ranged from 32.8 to 227pmol/h/mL (mean±SD=89.1±55.1, n=20), which correlated with alanine transaminase (r=0.827), aspartate transaminase (r=0.487), and uric acid (r=0.502). The established assay is expected to be useful for investigating the function of XOR and the effect of its inhibitors in various diseases.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Xantina Desidrogenase/sangue , Ativação Enzimática , Estabilidade Enzimática , Feminino , Humanos , Hipoxantina/metabolismo , Marcação por Isótopo , Isótopos , Masculino , Ácido Úrico/metabolismo , Xantina/metabolismo , Xantina Desidrogenase/metabolismo
2.
J Labelled Comp Radiopharm ; 59(5): 214-20, 2016 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-27006202

RESUMO

In this study, we developed a highly sensitive assay for xanthine oxidoreductase (XOR) activity utilizing a combination of [(13) C2 ,(15) N2 ]xanthine and liquid chromatography (LC)/triple quadrupole mass spectrometry (TQMS). In this assay, the amount of [(13) C2 ,(15) N2 ]uric acid (UA) produced by XOR was determined by using LC/TQMS. For this assay, we synthesized [(13) C2 ,(15) N2 ]xanthine as a substrate, [(13) C2 ,(15) N2 ]UA as an analytical standard, and [(13) C3 ,(15) N3 ]UA as an internal standard. The [(13) C2 ,(15) N2 ]UA calibration curve obtained using LC/TQMS under the selected reaction monitoring mode was evaluated, and the results indicated good linearity (R(2) = 0.998, weighting of 1/x(2) ) in the range of 20 to 4000 nM. As a model reaction of less active samples, the XOR activity of serial-diluted mouse plasma was measured. Thereby, the XOR activity of the 1024-fold-diluted mouse plasma was 4.49 ± 0.44 pmol/100 µL/h (mean ± standard deviation, n = 3). This value is comparable to the predicted XOR activity value of healthy human plasma. Hence, this combination method may be used to obtain high-sensitivity measurements required for XOR activity analysis on various organs or human plasma.


Assuntos
Radioisótopos de Carbono/química , Ensaios Enzimáticos/métodos , Radioisótopos de Nitrogênio/química , Ácido Úrico/química , Ácido Úrico/metabolismo , Xantina Desidrogenase/metabolismo , Animais , Cromatografia Líquida , Humanos , Espectrometria de Massas , Camundongos , Ácido Úrico/farmacologia , Xantina Desidrogenase/sangue
3.
Artigo em Inglês | MEDLINE | ID: mdl-26673227

RESUMO

Studies of pathological mechanisms and XOR inhibitor characterization, such as allopurinol, febuxostat, and topiroxostat, require accurate and sensitive measurements of XOR activity. However, the established assays have some disadvantages such as susceptibility to endogenous substances such as uric acid (UA), xanthine, or hypoxanthine. Here, we aimed to develop a novel XOR activity assay utilizing a combination of high-performance liquid chromatography (LC) and high-resolution mass spectrometry (HRMS) for tissues such as the liver, kidney, and plasma. Stable isotope-labeled [(15)N2]-xanthine was utilized as substrate and the production of [(15)N2]-uric acid was determined. [(15)N2]-UA production by XOR was dependent on the amounts of [(15)N2]-xanthine and enzyme and the time of reaction. Because high concentrations of endogenous xanthine and hypoxanthine affect XOR activities, we employed a multi-component analysis using LC/HRMS to improve the accuracy of XOR activity assay. Quantification of [(15)N2]-UA was validated and showed good linearity, accuracy, and precision. We measured the XOR activities of retired ICR mice using [(15)N2]-xanthine and LC/MS. The XOR activities in plasma, kidney, and liver samples were 38.1±0.7, 158±5, 928±25pmol/min/mg of protein, respectively (mean±SD, n=5). Furthermore, we measured the XOR activities in the same samples using the LC/ultraviolet and LC/fluorescence (FL) methods. The level of [(15)N2]-xanthine oxidation by XOR was equal to that of xanthine oxidation and approximately 7.9-8.9 times higher than that of pterin oxidation. We found a good correlation between XOR activities examined using LC/MS assay with [(15)N2]-xanthine and those examined using LC/FL assay with pterin. This result suggested that although both the LC/MS assay with [(15)N2]-xanthine and the LC/FL assay with pterin were useful, the former provided information regarding XOR activities that more directly reflected the physiological condition than the latter.


Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Xantina Desidrogenase/metabolismo , Animais , Marcação por Isótopo , Camundongos Endogâmicos ICR
4.
J Enzyme Inhib Med Chem ; 30(6): 981-8, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26147347

RESUMO

The single-crystal structure of anagliptin, N-[2-({2-[(2S)-2-cyanopyrrolidin-1-yl]-2-oxoethyl}amino)-2-methylpropyl]-2-methylpyrazolo[1,5-a]pyrimidine-6-carboxamide, was determined. Two independent molecules were held together by intermolecular hydrogen bonds, and the absolute configuration of the 2-cyanopyrrolidine ring delivered from l-prolinamide was confirmed to be S. The interactions of anagliptin with DPP-4 were clarified by the co-crystal structure solved at 2.85 Å resolution. Based on the structure determined by X-ray crystallography, the potency and selectivity of anagliptin were discussed, and an SAR study using anagliptin derivatives was performed.


Assuntos
Dipeptidil Peptidase 4/química , Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/química , Inibidores da Dipeptidil Peptidase IV/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Cristalografia por Raios X , Dipeptidil Peptidase 4/genética , Dipeptidil Peptidase 4/isolamento & purificação , Inibidores da Dipeptidil Peptidase IV/síntese química , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Estrutura Molecular , Pirimidinas/síntese química , Relação Estrutura-Atividade
5.
J Pharm Sci ; 104(8): 2482-8, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26037234

RESUMO

Of organogermanium compounds known to have an immunostimulatory action, propagermanium [PGe; 3-oxygermylpropionic acid polymer, (C3 H5 GeO3.5 )n] is the only one used as a pharmaceutical agent, to treat the hepatitis B virus in Japan. However, because of lack of information about its structure, PGe has been confused with a polymeric solid, repagermanium (RGe, Ge-132, poly-trans-[(2-carboxyethyl) germasesquioxane], (C18 H30 Ge6 O21 )n), which has the same essential formula as PGe. To clarify this issue, the structure of PGe was analyzed using X-ray diffraction (XRD). PGe has a polymeric ladder-shaped structure of a concatenated eight-membered ring composed of Ge-O bonds, which is clearly distinguished from the infinite sheet structure in RGe. Moreover, we observed temperature or moisture-dependent transformations among these compounds using powder XRD. For instance, PGe was easily dissolved in water, and transformed to RGe by exposure to water vapor, but transformed into another straight-chain structure when exposed to aqueous solution. As a result of these findings, PGe was indicated to have labile polymer packing against RGe. These characteristics of PGe may affect pharmaceutical properties such as respective stability and solubility, which indicate its unique impact on physiological activity.


Assuntos
Antineoplásicos/química , Indutores de Interferon/química , Modelos Moleculares , Compostos Organometálicos/química , Precipitação Química , Cristalografia por Raios X , Estabilidade de Medicamentos , Germânio/química , Temperatura Alta , Isomerismo , Conformação Molecular , Estrutura Molecular , Peso Molecular , Polimerização , Difração de Pó , Propionatos , Solubilidade , Água/análise , Água/química
7.
Org Med Chem Lett ; 1(1): 7, 2011 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-22373386

RESUMO

Focused structure-activity relationships of isoindoline class DPP-IV inhibitors have led to the discovery of 4b as a highly selective, potent inhibitor of DPP-IV. In vivo studies in Wistar/ST rats showed that 4b was converted into the strongly active metabolite 4l in high yield, resulting in good in vivo efficacy for antihyperglycemic activity.

8.
J Med Chem ; 48(18): 5659-65, 2005 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-16134934

RESUMO

Structure of the Leu300Pro mutant of human aldose reductase (ALR2) in complex with the inhibitor fidarestat is determined. Comparison with the hALR2-fidarestat complex and the porcine aldehyde reductase (ALR1)-fidarestat complex indicates that the hydrogen bond between the Leu300 amino group of the wild-type and the exocyclic amide group of the inhibitor is the key determinant for the specificity of fidarestat for ALR2 over ALR1. Thermodynamic data also suggest an enthalpic contribution as the predominant difference in the binding energy between the aldose reductase mutant and the wild-type. An additional selectivity-determining feature is the difference in the interaction between the inhibitor and the side chain of Trp219, ordered in the present structure but disordered (corresponding Trp220) in the ALR1-fidarestat complex. Thus, the hydrogen bond ( approximately 7 kJ/mol) corresponds to a 23-fold difference in inhibitor potency while the differences in the interactions between Trp219(ALR2) and fidarestat and between Trp220(ALR1) and fidarestat can account for an additional 10-fold difference in potency.


Assuntos
Aldeído Redutase/antagonistas & inibidores , Aldeído Redutase/química , Imidazolidinas/química , Aldeído Redutase/genética , Sítios de Ligação , Calorimetria , Cristalização , Holoenzimas/química , Holoenzimas/genética , Humanos , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Termodinâmica
9.
J Med Chem ; 48(17): 5536-42, 2005 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-16107153

RESUMO

Structure determination of porcine aldehyde reductase holoenzyme in complex with the potent aldose reductase inhibitor fidarestat was carried out to explain the difference in the potency of the inhibitor for aldose and aldehyde reductases. The hydrogen bonds between the active-site residues Tyr50, His113, and Trp114 and fidarestat are conserved in the two enzymes. In aldose reductase, Leu300 forms a hydrogen bond through its main-chain nitrogen atom with the exocyclic amide group of the inhibitor, which when replaced with a Pro in aldehyde reductase, cannot form a hydrogen bond, thus causing a loss in binding energy. Furthermore, in aldehyde reductase, the side chain of Trp220 occupies a disordered split conformation that is not observed in aldose reductase. Molecular modeling and inhibitory activity measurements suggest that the difference in the interaction between the side chain of Trp220 and fidarestat may contribute to the difference in the binding of the inhibitor to the enzymes.


Assuntos
Oxirredutases do Álcool/antagonistas & inibidores , Oxirredutases do Álcool/química , Aldeído Redutase/antagonistas & inibidores , Aldeído Redutase/química , Imidazolidinas/química , Animais , Sítios de Ligação , Cristalografia por Raios X , Holoenzimas/química , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína , Estereoisomerismo , Relação Estrutura-Atividade , Suínos
10.
J Med Chem ; 47(18): 4530-7, 2004 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-15317464

RESUMO

Structure determinations of human aldose reductase holoenzyme in complex with the 2S4R-,2R4S- and 2R4R-isomers of the potent inhibitor Fidarestat ((2S,4S)-6-fluoro-2',5'-dioxospiro[chroman-4,4'-imidazoline]-2-carboxamide) were carried out in order to elucidate the binding modes responsible for the differences in their inhibitory potencies. In the complex structure with the 2R4S-isomer the cyclic imide moiety formed hydrogen bonds with the side-chains of Trp111, Tyr48 and His110. In the attempt to determine the complex structure with the least potent 2R4R-isomer this ligand was not observed, and instead, the active site was simultaneously occupied by two citrate molecules (occupancies of 60% and 40%). In the case of 2S4R, the active site was occupied by a citrate molecule which anchors the 2S4R-isomer from its carbamoyl group. The structures of the complexes suggest that the differences in the interactions between the cyclic imide rings and carbamoyl groups of the compounds with residues His110, Trp111, Trp219 and Cys298 account for differences in their inhibitory potencies.


Assuntos
Aldeído Redutase/química , Cristalografia por Raios X , Imidazóis/química , Imidazolidinas , Aldeído Redutase/antagonistas & inibidores , Sítios de Ligação , Citratos/química , Inibidores Enzimáticos/química , Humanos , Imidazóis/farmacologia , Estrutura Molecular , Ligação Proteica , Estereoisomerismo , Especificidade por Substrato
11.
Proteins ; 55(4): 805-13, 2004 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-15146479

RESUMO

The X-ray structures of human aldose reductase holoenzyme in complex with the inhibitors Fidarestat (SNK-860) and Minalrestat (WAY-509) were determined at atomic resolutions of 0.92 A and 1.1 A, respectively. The hydantoin and succinimide moieties of the inhibitors interacted with the conserved anion-binding site located between the nicotinamide ring of the coenzyme and active site residues Tyr48, His110, and Trp111. Minalrestat's hydrophobic isoquinoline ring was bound in an adjacent pocket lined by residues Trp20, Phe122, and Trp219, with the bromo-fluorobenzyl group inside the "specificity" pocket. The interactions between Minalrestat's bromo-fluorobenzyl group and the enzyme include the stacking against the side-chain of Trp111 as well as hydrogen bonding distances with residues Leu300 and Thr113. The carbamoyl group in Fidarestat formed a hydrogen bond with the main-chain nitrogen atom of Leu300. The atomic resolution refinement allowed the positioning of hydrogen atoms and accurate determination of bond lengths of the inhibitors, coenzyme NADP+ and active-site residue His110. The 1'-position nitrogen atom in the hydantoin and succinimide moieties of Fidarestat and Minalrestat, respectively, form a hydrogen bond with the Nepsilon2 atom of His 110. For Fidarestat, the electron density indicated two possible positions for the H-atom in this bond. Furthermore, both native and anomalous difference maps indicated the replacement of a water molecule linked to His110 by a Cl-ion. These observations suggest a mechanism in which Fidarestat is bound protonated and becomes negatively charged by donating the proton to His110, which may have important implications on drug design.


Assuntos
Aldeído Redutase/química , Inibidores Enzimáticos/química , Imidazóis/química , Imidazolidinas , Imidas/química , Modelos Moleculares , Quinolonas/química , Aldeído Redutase/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/metabolismo , Holoenzimas/química , Holoenzimas/metabolismo , Imidazóis/metabolismo , Imidas/metabolismo , Substâncias Macromoleculares , Estrutura Molecular , Quinolonas/metabolismo
12.
Science ; 301(5631): 376-9, 2003 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-12869764

RESUMO

We collected and completely sequenced 28,469 full-length complementary DNA clones from Oryza sativa L. ssp. japonica cv. Nipponbare. Through homology searches of publicly available sequence data, we assigned tentative protein functions to 21,596 clones (75.86%). Mapping of the cDNA clones to genomic DNA revealed that there are 19,000 to 20,500 transcription units in the rice genome. Protein informatics analysis against the InterPro database revealed the existence of proteins presented in rice but not in Arabidopsis. Sixty-four percent of our cDNAs are homologous to Arabidopsis proteins.


Assuntos
Genoma de Planta , Oryza/genética , Análise de Sequência de DNA , Processamento Alternativo , Sequência de Aminoácidos , Clonagem Molecular , Biologia Computacional , DNA Complementar , Bases de Dados de Ácidos Nucleicos , Bases de Dados de Proteínas , Genes de Plantas , Dados de Sequência Molecular , Fases de Leitura Aberta , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia , Estrutura Terciária de Proteína , RNA Antissenso/genética , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/química , Fatores de Transcrição/genética , Transcrição Gênica
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