RESUMO
BACKGROUND: The lipoglycopeptide antibiotic, telavancin, may interfere with some laboratory coagulation tests including prothrombin time (PT) and activated partial thromboplastin time (aPTT). OBJECTIVE: To evaluate the effects of telavancin on PT and aPTT assays in common use. METHODS: Pooled normal human plasma was spiked with telavancin 10, 20, 100 or 200 µg/ml (equivalent to trough, 2 × trough, peak and 2 × peak clinical plasma concentrations, respectively) or diluent control (0.9% sodium chloride). Samples were analysed using 16 PT reagents and seven aPTT reagents. RESULTS: Telavancin 200 µg/ml (corresponding to 2 × peak clinical plasma concentration), produced significant PT prolongation (> 9% difference vs. diluent control) with all the 16 PT reagents (range 12% to > 600%). At lower telavancin concentrations, PT prolongation was dose-dependent and varied among reagents, but appeared greatest with preparations containing recombinant tissue factor. With telavancin 10 µg/ml (equivalent to trough), PT prolongation was 10% with HemosIL(®) PT-Fibrinogen Recombinant, while ranging from 5% to -1% with all other reagents. Significant (> 34% difference vs. baseline) and dose-dependent aPTT prolongation was observed with all the seven reagents in samples spiked with telavancin 100 or 200 µg/ml (range 65-142% at 200 µg/ml). aPTT reagents containing a silica activator appeared to be more sensitive to telavancin interference. Telavancin 10 µg/ml was not associated with increased aPTT with any of the reagents tested. CONCLUSIONS: Telavancin has the potential to prolong both PT and aPTT in vitro. It is recommended that samples for PT or aPTT be obtained just prior to a telavancin dose (trough).
Assuntos
Aminoglicosídeos , Antibacterianos , Coagulação Sanguínea/efeitos dos fármacos , Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Contraindicações , Humanos , Lipoglicopeptídeos , Tempo de Tromboplastina Parcial/normas , Tempo de Protrombina/normas , Valores de ReferênciaRESUMO
BACKGROUND: Involved or implicated in a wide spectrum of diseases, trypsin-like serine proteases comprise well studied drug targets and anti-targets that can be subdivided into two major classes. In one class there is a serine at position 190 at the S1 site, as in urokinase type plasminogen activator (urokinase or uPA) and factor VIIa, and in the other there is an alanine at 190, as in tissue type plasminogen activator (tPA) and factor Xa. A hydrogen bond unique to Ser190 protease-arylamidine complexes between O gamma(Ser190) and the inhibitor amidine confers an intrinsic preference for such inhibitors toward Ser190 proteases over Ala190 counterparts. RESULTS: Based on the structural differences between the S1 sites of Ser190 and Ala190 protease-arylamidine complexes, we amplified the selectivity of amidine inhibitors toward uPA and against tPA, by factors as high as 220-fold, by incorporating a halo group ortho to the amidine of a lead inhibitor scaffold. Comparison of K(i) values of such halo-substituted and parent inhibitors toward a panel of Ser190 and Ala190 proteases demonstrates pronounced selectivity of the halo analogs for Ser190 proteases over Ala190 counterparts. Crystal structures of Ser190 proteases, uPA and trypsin, and of an Ala190 counterpart, thrombin, bound by a set of ortho (halo, amidino) aryl inhibitors and of non-halo parents reveal the structural basis of the exquisite selectivity and validate the design principle. CONCLUSIONS: Remarkable selectivity enhancements of exceptionally small inhibitors are achieved toward the uPA target over the highly similar tPA anti-target through a single atom substitution on an otherwise relatively non-selective scaffold. Overall selectivities for uPA over tPA as high as 980-fold at physiological pH were realized. The increase in selectivity results from the displacement of a single bound water molecule common to the S1 site of both the uPA target and the tPA anti-target because of the ensuing deficit in hydrogen bonding of the arylamidine inhibitor when bound in the Ala190 protease anti-target.
Assuntos
Serina Endopeptidases/química , Inibidores de Serina Proteinase/síntese química , Animais , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Ligação de Hidrogênio , Ligação Proteica , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacologia , Relação Estrutura-Atividade , Ativador de Plasminogênio Tecidual/antagonistas & inibidores , Ativador de Plasminogênio Tecidual/química , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Ativador de Plasminogênio Tipo Uroquinase/química , Água/química , Água/metabolismoRESUMO
A survey of isosteric replacements of the phosphonoalanine side chain coupled with a process of conformational constraint of a bisbenzimidazole-based, Zn(2+)-dependent inhibitor of hepatitis C virus (HCV) NS3 serine protease resulted in the identification of novel series of active compounds with extended side chains. However, Zn(2+)-dependent HCV NS3 inhibition was relatively insensitive to the structural variations examined but dependent on the presence of negatively charged functionality. This result was interpreted in the context of an initial electrostatic interaction between protease and inhibitor that is subsequently consolidated by Zn(2+), with binding facilitated by the featureless active site and proximal regions of the HCV NS3 protein.
Assuntos
Antivirais/química , Antivirais/farmacologia , Benzimidazóis/química , Benzimidazóis/farmacologia , Hepacivirus/enzimologia , Organofosfonatos/química , Organofosfonatos/farmacologia , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Zinco/metabolismo , Antivirais/síntese química , Benzimidazóis/síntese química , Bioquímica/métodos , Avaliação Pré-Clínica de Medicamentos , Concentração Inibidora 50 , Estrutura Molecular , Organofosfonatos/síntese química , Inibidores de Serina Proteinase/síntese química , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/químicaRESUMO
We describe a new serine protease inhibition motif in which binding is mediated by a cluster of very short hydrogen bonds (<2.3 A) at the active site. This protease-inhibitor binding paradigm is observed at high resolution in a large set of crystal structures of trypsin, thrombin, and urokinase-type plasminogen activator (uPA) bound with a series of small molecule inhibitors (2-(2-phenol)indoles and 2-(2-phenol)benzimidazoles). In each complex there are eight enzyme-inhibitor or enzyme-water-inhibitor hydrogen bonds at the active site, three of which are very short. These short hydrogen bonds connect a triangle of oxygen atoms comprising O(gamma)(Ser195), a water molecule co-bound in the oxyanion hole (H(2)O(oxy)), and the phenolate oxygen atom of the inhibitor (O6'). Two of the other hydrogen bonds between the inhibitor and active site of the trypsin and uPA complexes become short in the thrombin counterparts, extending the three-centered short hydrogen-bonding array into a tetrahedral array of atoms (three oxygen and one nitrogen) involved in short hydrogen bonds. In the uPA complexes, the extensive hydrogen-bonding interactions at the active site prevent the inhibitor S1 amidine from forming direct hydrogen bonds with Asp189 because the S1 site is deeper in uPA than in trypsin or thrombin. Ionization equilibria at the active site associated with inhibitor binding are probed through determination and comparison of structures over a wide range of pH (3.5 to 11.4) of thrombin complexes and of trypsin complexes in three different crystal forms. The high-pH trypsin-inhibitor structures suggest that His57 is protonated at pH values as high as 9.5. The pH-dependent inhibition of trypsin, thrombin, uPA and factor Xa by 2-(2-phenol)benzimidazole analogs in which the pK(a) of the phenol group is modulated is shown to be consistent with a binding process involving ionization of both the inhibitor and the enzyme. These data further suggest that the pK(a) of His57 of each protease in the unbound state in solution is about the same, approximately 6.8. By comparing inhibition constants (K(i) values), inhibitor solubilities, inhibitor conformational energies and corresponding structures of short and normal hydrogen bond-mediated complexes, we have estimated the contribution of the short hydrogen bond networks to inhibitor affinity ( approximately 1.7 kcal/mol). The structures and K(i) values associated with the short hydrogen-bonding motif are compared with those corresponding to an alternate, Zn(2+)-mediated inhibition motif at the active site. Structural differences among apo-enzymes, enzyme-inhibitor and enzyme-inhibitor-Zn(2+) complexes are discussed in the context of affinity determinants, selectivity development, and structure-based inhibitor design.
Assuntos
Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/metabolismo , Motivos de Aminoácidos , Ânions , Apoproteínas/química , Apoproteínas/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Fator Xa/química , Fator Xa/metabolismo , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Modelos Moleculares , Fenóis/metabolismo , Conformação Proteica , Solubilidade , Eletricidade Estática , Especificidade por Substrato , Temperatura , Termodinâmica , Trombina/química , Trombina/metabolismo , Tripsina/química , Tripsina/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Água/química , Água/metabolismo , Zinco/metabolismoRESUMO
The efficacy of a series of diazomethyl ketones (DMKs) was measured in rhinovirus-infected cultures and against the HRV14 3C protease. Their specificity and potency were confirmed against purified recombinant enzyme expressed in a yeast secretion system. An internally quenched fluorescent peptide substrate was used to assess the potency against the enzyme, obtaining a 50% inhibitory concentration (IC50) of 1 microM for both Z-L-F-Q-CHN2 and Z-V-L-F-Q-CHN2, while a lower affinity was observed for Z-F-Q-CHN2. The tripeptide Z-L-F-Q-CHN2 blocked viral replication with an IC50 value of 30 microM as judged by the reduction in viral induced cytopathy of HeLa-H1 cells, as well as a marked reduction in viral plaque formation (50% effective concentration=20 microM). Western blot analysis of viral proteins from infected cells indicates that this inhibitor works specifically by blocking viral polyprotein maturation, displaying a reduction of detectable 3C protease and an accumulation of the 3CD polypeptide. These results indicate that DMK inhibitors of the 3C protease have antiviral potency. Furthermore, the pattern of viral protein processing observed suggests that reducing the concentration of mature HRV 3C protease even in the presence of increased 3CD protein is sufficient to block proper viral processing and significantly reduce virus yield.
Assuntos
Antivirais/farmacologia , Diazometano/análogos & derivados , Diazometano/farmacologia , Cetonas/farmacologia , Poliproteínas/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Rhinovirus/efeitos dos fármacos , Rhinovirus/metabolismo , Proteínas Virais/antagonistas & inibidores , Proteases Virais 3C , Antivirais/química , Cisteína Endopeptidases/metabolismo , Diazometano/química , Relação Dose-Resposta a Droga , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Cetonas/química , Estrutura Molecular , Poliproteínas/química , Rhinovirus/enzimologia , Relação Estrutura-Atividade , Ensaio de Placa Viral , Proteínas Virais/metabolismoRESUMO
A statistically exhaustive, 8800 compound tripeptidal amidomethylcoumarin library was synthesized as discreet compounds using solid-phase combinatorial chemistry. A subset of the compounds was purified by HPLC and tested in a high-throughput fluorometric assay against several known serine and cysteine proteases to demonstrate the utility of this library for profiling protease substrate specificity.
Assuntos
Cumarínicos/síntese química , Endopeptidases/metabolismo , Corantes Fluorescentes/síntese química , Cromatografia Líquida de Alta Pressão , Técnicas de Química Combinatória , Cumarínicos/química , Corantes Fluorescentes/química , Sondas Moleculares , Especificidade por SubstratoRESUMO
Active site titration by a reversible tight-binding inhibitor normally depends on prior knowledge of the inhibition constant. Conversely, the determination of tight-binding inhibition constants normally requires prior knowledge of the active enzyme concentration. Often, neither of these quantities is known with sufficient accuracy. This paper describes experimental conditions under which both the enzyme active site concentration and the tight-binding inhibition constant can be determined simultaneously from a single dose-response curve. Representative experimental data are shown for the inhibition of human kallikrein.
Assuntos
Técnicas de Química Combinatória , Enzimas/análise , Enzimas/metabolismo , Calicreínas/análise , Sítios de Ligação , Relação Dose-Resposta a Droga , Humanos , Calicreínas/antagonistas & inibidores , Calicreínas/sangue , Cinética , Modelos Estatísticos , Modelos Teóricos , Método de Monte Carlo , Ligação Proteica , Análise de RegressãoRESUMO
Determination of tight-binding inhibition constants by nonlinear least-squares regression requires sufficiently good initial estimates of the best-fit values. Normally an initial estimate of the inhibition constant must be provided by the investigator. This paper describes an automatic procedure for the estimation of tight-binding inhibition constants directly from dose-response data. Because the procedure does not require human intervention, it was incorporated into an algorithm for high-throughput screening of enzyme inhibitors. A suitable computer program is available electronically (http://www.biokin.com). Representative experimental data are shown for the inhibition of human mast-cell tryptase.
Assuntos
Inibidores Enzimáticos/análise , Serina Endopeptidases/metabolismo , Algoritmos , Automação , Ligação Competitiva , Quimases , Simulação por Computador , Inibidores Enzimáticos/farmacologia , Humanos , Cinética , Método de Monte Carlo , Serina Endopeptidases/efeitos dos fármacos , TriptasesRESUMO
Serine proteases play a role in a variety of disease states and thus are attractive targets for therapeutic intervention. We report the kinetic characterization of a class of serine protease inhibitors whose potencies and selectivities are dramatically enhanced in the presence of Zn(II). The structural basis for Zn(II)-mediated inhibition of trypsin-like proteases has recently been reported [Katz, B. A., Clark, J. M., Finer-Moore, J. S., Jenkins, T. E., Johnson, C. R., Ross, M. J., Luong, C., Moore, W. R., and Stroud, R. M. (1998) Nature 391, 608-612]. A case study of the kinetic behavior of human tryptase inhibitors is provided to illustrate the general phenomenon of Zn(II)-mediated inhibition. Tryptase, Zn(II), and the inhibitor form a ternary complex which exhibits classic tight-binding inhibition. The half-life for release of inhibitor from the tryptase-Zn(II)-inhibitor complex has been measured for a number of inhibitors. Consistent with tight-binding behavior, potent tryptase inhibitors are characterized by extremely slow rates of dissociation from the ternary complex with half-lives on the order of hours. A model of human serum, designed to reproduce physiological levels of Zn(II), has been employed to evaluate the performance of Zn(II)-potentiated tryptase inhibitors under physiological conditions. We demonstrate that Zn(II)-mediated inhibition can be achieved at physiological Zn(II) levels.
Assuntos
Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/farmacologia , Zinco/farmacologia , Cátions Bivalentes/metabolismo , Cátions Bivalentes/farmacologia , Quimases , Sinergismo Farmacológico , Ácido Edético/metabolismo , Ácido Edético/farmacologia , Meia-Vida , Humanos , Cinética , Metais/metabolismo , Metais/farmacologia , Ligação Proteica , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/metabolismo , Albumina Sérica/metabolismo , Especificidade por Substrato , Termodinâmica , Triptases , Zinco/metabolismoRESUMO
Clavaminate synthase (CS), a key enzyme in the clavulanic acid biosynthetic pathway, has been purified to electrophoretic homogeneity from Streptomyces antibioticus (Tü 1718), a species that does not produce clavulanic acid. A comparison of the physical and kinetic properties of clavaminate synthase from S. antibioticus (CS3) and the two isozymes from Streptomyces clavuligerus (CS1 and CS2) has been conducted. In oxidative reactions requiring the co-substrates O2, alpha-ketoglutaric acid, and catalytic Fe2+, both CS1 and CS2 catalyze three distinct transformations, the hydroxylation of deoxyguanidinoproclavaminic acid to guanidinoproclavaminic acid, and the cyclization and desaturation of proclavaminic acid to clavaminic acid. We have demonstrated that CS3 from S. antibioticus also catalyzes these three oxidations. The apparent molecular mass of CS3 from matrix-assisted laser desorption mass spectrometry is 35,839 +/- 36 Da. The enzyme is a monomer in solution as determined by gel filtration chromatography. Analysis of the four possible proclavaminic acid diastereomers confirmed the absolute configuration of the substrate to be 2S,3R. Based upon N-terminal sequence comparisons among the three proteins, CS3 possesses the higher degree of homology with the CS1 isozyme from S. clavuligerus. Although previously associated solely with clavulanic acid biosynthesis, we propose these findings and recent precursor incorporation data support the view that clavaminate synthase plays a critical role in the biosynthesis of the clavam metabolites.
Assuntos
Oxigenases de Função Mista/isolamento & purificação , Oxigenases de Função Mista/metabolismo , Streptomyces antibioticus/enzimologia , Sequência de Aminoácidos , Antibacterianos/biossíntese , Cromatografia em Gel , Cromatografia por Troca Iônica , Ácido Clavulânico , Ácidos Clavulânicos/biossíntese , Ácidos Clavulânicos/química , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , Oxigenases de Função Mista/química , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Streptomyces/enzimologia , Especificidade por SubstratoRESUMO
The reaction catalyzed by phosphoenolpyruvate carboxylase from Zea mays has been studied kinetically. Results of initial velocity patterns and inhibition studies indicate that phosphoenolpyruvate carboxylase has a random sequential mechanism in which there is a high level of synergism in the binding of substrates. The preferred order of addition of reactants is Mg2+, phosphoenolpyruvate, and bicarbonate. The binding of Mg2+ is at equilibrium. Values for the various kinetic parameters are KiMg = 2.3 +/- 0.4 mM, KPEP = 3.6 +/- 0.6 mM, KiPEP = 0.2 +/- 0.07 mM, and Kbicarbonate = 0.18 +/- 0.04 mM. In addition, double inhibition experiments have been performed to examine the nature of the active site interactions with the putative intermediates, carboxy phosphate and the enolate of pyruvate. Highly synergistic inhibition of phosphoenolpyruvate carboxylase was observed in the presence of oxalate and carbamyl phosphate (alpha = 0.0013). However, an antisynergistic relationship exists between oxalate and phosphonoformate (alpha = 2.75).
Assuntos
Fosfoenolpiruvato Carboxilase/metabolismo , Zea mays/enzimologia , Bicarbonatos/metabolismo , Glicolatos/metabolismo , Cinética , Fosfoenolpiruvato Carboxilase/antagonistas & inibidoresRESUMO
The catalytic mechanism of phosphoenolpyruvate (PEP) carboxylase from Zea mays has been studied using (Z)- and (E)-3-fluorophosphoenolpyruvate (F-PEP) as substrates. Both (Z)- and (E)-F-PEP partition between carboxylation to produce 3-fluorooxalacetate and hydrolysis to produce 3-fluoropyruvate. Carboxylation accounts for 3% of the reaction observed with (Z)-F-PEP, resulting in the formation of (R)-3-fluorooxalacetate, and for 86% of the reaction of (E)-F-PEP forming (S)-3-fluorooxalacetate. Carboxylation of F-PEP occurs on the 2-re face, which corresponds to the 2-si face of PEP. The partitioning of F-PEP between carboxylation and hydrolysis is insensitive to pH but varies with metal ion. Use of 18O-labeled bicarbonate produces phosphate that is multiply labeled with 18O; in addition, 18O is also incorporated into residual (Z)- and (E)-F-PEP. The 13(V/K) isotope effect on the carboxylation of F-PEP catalyzed by PEP carboxylase at pH 8.0, 25 degrees C, is 1.049 +/- 0.003 for (Z)-F-PEP and 1.009 +/- 0.006 for (E)-F-PEP. These results are consistent with a mechanism in which carboxylation of PEP occurs via attack of the enolate of pyruvate on CO2 rather than carboxy phosphate. In this mechanism phosphorylation of bicarbonate to give carboxy phosphate and decarboxylation of the latter are reversible steps. An irreversible step, however, precedes partitioning between carboxylation to give oxalacetate and release of CO2, which results in hydrolysis of PEP.
Assuntos
Fosfoenolpiruvato Carboxilase/metabolismo , Fosfoenolpiruvato/análogos & derivados , Fosfoenolpiruvato/metabolismo , Zea mays/enzimologia , Bicarbonatos/metabolismo , Dióxido de Carbono/metabolismo , Cátions Bivalentes , Concentração de Íons de Hidrogênio , Hidrólise , Isomerismo , Cinética , Espectroscopia de Ressonância Magnética , Malato Desidrogenase/metabolismo , Relação Estrutura-AtividadeRESUMO
Formate is an alternate substrate for bicarbonate in the reaction with PEP catalyzed by phosphoenolpyruvate carboxylase from Zea mays, producing formyl phosphate and pyruvate. The Km for formate is 25 +/- 2 mM, and the maximum velocity is 1% of that for bicarbonate at pH 8.0. Use of [18O]formate produces inorganic phosphate containing 1 equiv of 18O, but no label is incorporated into residual phosphoenolpyruvate. PEP carboxylase catalyzes the hydrolysis of phosphoglycolate or L-phospholactate 2000 times more slowly and D-phospholactate 4000 times more slowly than the reaction between bicarbonate and PEP.