RESUMO
The development of therapeutic fusion protein drugs is often impeded by the unintended consequences that occur from fusing together domains from independent naturally occurring proteins, consequences such as altered biodistribution, tissue uptake, or rapid clearance and potential immunogenicity. For therapeutic fusion proteins containing globular domains, we hypothesized that aberrant in vivo behavior could be related to low kinetic stability of these domains leading to local unfolding and susceptibility to partial proteolysis and/or salvage and uptake. Herein we describe an assay to measure kinetic stability of therapeutic fusion proteins by way of their sensitivity to the protease thermolysin. The results indicate that in vivo pharmacokinetics of a panel of anti-programmed cell death protein 1 monocolonal antibody:interleukin 21 immunocytokines in both mice and nonhuman primates are highly correlated with their in vitro susceptibility to thermolysin-mediated proteolysis. This assay can be used as a tool to quickly identify in vivo liabilities of globular domains of therapeutic proteins, thus aiding in the optimization and development of new multispecific drug candidates. SIGNIFICANCE STATEMENT: This work describes a novel assay utilizing protein kinetic stability to identify preclinical in vivo pharmacokinetic liabilities of multispecific therapeutic fusion proteins. This provides an efficient, inexpensive method to ascertain inherent protein stability in vitro before conducting in vivo studies, which can rapidly increase the speed of preclinical drug development.
Assuntos
Anticorpos Monoclonais , Interleucinas , Camundongos , Animais , Distribuição Tecidual , Termolisina , Anticorpos Monoclonais/farmacocinéticaRESUMO
As the pharmaceutical industry places greater emphasis on pairing biological pathways with appropriate therapeutic intervention, an increase in the use of biologic drugs has emerged. With increasing complexity of biotherapeutics, absorption, distribution, metabolism, and excretion (ADME) studies have also become increasingly complex. The characterization of ADME properties is critical to tuning the pharmacokinetic profiles of next generation biologics (NGBs). The knowledge of the fate of a drug is essential for the enhancement of the design processes, elongation of exposure at the desired site of action, and achieving efficacy with minimum toxicity. In vivo proteolytic cleavage of biotherapeutics may lead to undesirable in vivo properties, such as rapid clearance, low bioavailability, and loss of pharmacodynamic effect. All of these may affect drug efficacy and/or generate safety concerns through increases in immunogenicity or off-target toxicity. The work herein describes the development of a robust, fully automated immunoaffinity purification (IA)-capillary electrophoresis-mass spectrometry (CE-MS) workflow. The reagents were carefully optimized to maximize isolation yields while minimizing the number of experimental steps to analytical results. The result is the development of a comprehensive integrated platform for the characterization of a wide range of biotherapeutics, including peptibodies, monoclonal antibodies, and bispecific antibodies. Empowered by this automated IA-CE-MS approach, implementing biotransformation studies at an early drug discovery stage can speed up the drug development process.
Assuntos
Produtos Biológicos , Eletroforese Capilar , Anticorpos Monoclonais , Descoberta de Drogas , Espectrometria de MassasRESUMO
Protein engineering is at an all-time high in biopharmaceutics. As a result, absorption, distribution, metabolism and excretion (ADME) of proteins has become more important to understand in the context of engineering strategies to optimize therapeutic properties of potential lead constructs. Immunoaffinity capture coupled with a newly developed capillary electrophoresis - mass spectrometry (CE-MS) system was used to characterize intact protein mass analysis of a wild type Fc-FGF21 construct and a sequence re-engineered Fc-FGF21 construct from an in vivo study. A number of truncated forms were observed and the time courses of the various proteolytic products were identified and compared between the two constructs. The abundances of the intact and truncated forms were used to provide the basis to semi-quantify ADME properties of the two protein forms. The use of this immunoaffinity capture followed by CE-MS based intact mass analysis workflow provided a qualitative and quantitative analysis of the pharmacokinetic profiles of the two proteins. The platform presented here holds great potential in characterization of the ADME properties of proteins.
Assuntos
Eletroforese Capilar/métodos , Espectrometria de Massas , Proteínas Recombinantes de Fusão/química , Animais , Cromatografia de Afinidade , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Meia-Vida , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Camundongos , Estabilidade Proteica , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacocinéticaRESUMO
Characterization of monoclonal antibody (mAb) therapeutics by intact mass analysis provides important information on sequence integrity and post-translational modifications. In order to obtain domain specific information, monoclonal antibodies are reduced to heavy and light chain components or enzymatically digested into smaller portions or peptides. Liquid chromatography (LC) is widely used for separation of the antibody fragments in line with mass spectrometry (MS) for characterization. Capillary electrophoresis (CE) is an analytical technique with high separation efficiency, high sensitivity, and minimal inter-run sample carryover. Combining the resolving power of CE with electrospray ionization (ESI) MS has great potential in regards to accurate mass characterization of protein therapeutics and has been a long sought-after approach. However, the intrinsic technical difficulty in coupling CE to MS has hindered the broad application of CE-MS across the biopharmaceutical industry. Recently, a CE-MS interface has been developed [1] and commercialized. Herein, we report implementation of this technology for coupling CE to an Agilent time-of-flight (TOF) mass spectrometer. CE-MS provides an attractive complement to LC-MS for separation and intact mass determination of mAbs and antibody-based therapeutics.
Assuntos
Anticorpos Monoclonais/análise , Anticorpos Monoclonais/química , Eletroforese Capilar/métodos , Espectrometria de Massas/métodos , Anticorpos Monoclonais/isolamento & purificação , Eletroforese Capilar/instrumentação , Desenho de Equipamento , Glicosilação , Espectrometria de Massas/instrumentaçãoRESUMO
Therapeutic fusion proteins (TFPs) are designed to improve the therapeutic profile of an endogenous protein or protein fragment with a limited dose frequency providing the desired pharmacological activity in vivo. Fusion of a therapeutic protein to a half-life extension or targeting domain can improve the disposition of the molecule or introduce a novel mechanism of action. Prolonged exposure and altered biodistribution of an endogenous protein through fusion technology increases the potential for local protein unfolding during circulation increasing the chance for partial proteolysis of the therapeutic domain. Characterizing the proteolytic liabilities of a TFP can guide engineering efforts to inhibit or hinder partial proteolysis. This review focuses on considerations and techniques for evaluating the stability of a TFP both in vivo and in vitro.
Assuntos
Proteínas/análise , Proteólise , HumanosRESUMO
Bioanalytical support of discovery programs for human monoclonal antibody therapies involves quantitation by immunoassay. Historically, preclinical samples have been analyzed by the traditional Enzyme-Linked Immuno-Sorbent Assay (ELISA). We investigated transferring our generic ELISA for quantitating human IgG constructs in preclinical serum samples to an automated microfluidics immunoassay platform based on nanoscale streptavidin bead columns. Transfer of our immunoassay to the automated platform resulted in not only the anticipated reduction in analysts' time required for manual manipulation (ELISA) but also a substantial increase in the dynamic range of the immunoassay. The generic nature and wide dynamic range of this automated microcolumn immunoassay permit bioanalytical support of novel therapeutic candidates without the need to develop new, specific assay reagents and minimize the chances that sample reassays will be required due to out of range concentration results. Improved process efficiencies and enhanced workflow during the analysis of preclinical PK samples that enable high throughput assessment of a human monoclonal antibody lead in early discovery programs.
Assuntos
Anticorpos Monoclonais/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Imunoglobulina G/sangue , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Especificidade de Anticorpos , Humanos , Fragmentos de Imunoglobulinas/sangue , Fragmentos de Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Macaca fascicularis , Masculino , Técnicas Analíticas Microfluídicas , Ratos , Ratos Sprague-Dawley , Estreptavidina/imunologiaRESUMO
The propensity for cytochrome P450 (P450) enzymes to bioactivate xenobiotics is governed by the inherent chemistry of the xenobiotic itself and the active site architecture of the P450 enzyme(s). Accessible nucleophiles in the active site or egress channels of the P450 enzyme have the potential of sequestering reactive metabolites through covalent modification, thereby limiting their exposure to other proteins. Raloxifene, a drug known to undergo CYP3A-mediated reactive metabolite formation and time-dependent inhibition in vitro, was used to explore the potential for bioactivation and enzyme inactivation of additional P450 enzymes (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A5). Every P450 tested except CYP2E1 was capable of raloxifene bioactivation, based on glutathione adduct formation. However, raloxifene-mediated time-dependent inhibition only occurred in CYP2C8 and CYP3A4. Comparable inactivation kinetics were achieved with K(I) and k(inact) values of 0.26 µM and 0.10 min(-1) and 0.81 µM and 0.20 min(-1) for CYP2C8 and CYP3A4, respectively. Proteolytic digests of CYP2C8 and CYP3A4 Supersomes revealed adducts to Cys225 and Cys239 for CYP2C8 and CYP3A4, respectively. For each P450 enzyme, proposed substrate/metabolite access channels were mapped and active site cysteines were identified, which revealed that only CYP2C8 and CYP3A4 possess accessible cysteine residues near the active site cavities, a result consistent with the observed kinetics. The combined data suggest that the extent of bioactivation across P450 enzymes does not correlate with P450 inactivation. In addition, multiple factors contribute to the ability of reactive metabolites to form apo-adducts with P450 enzymes.
Assuntos
Cisteína/química , Sistema Enzimático do Citocromo P-450/química , Cloridrato de Raloxifeno/química , Domínio Catalítico , Simulação por Computador , Inibidores das Enzimas do Citocromo P-450 , Ativação Enzimática , Cinética , Modelos MolecularesRESUMO
2-(4-(4-(tert-Butylcarbamoyl)-2-(2-chloro-4-cyclopropylphenylsulfonamido)phenoxy)-5-chloro-2-fluorophenyl)acetic acid (AMG 853) is an orally bioavailable and potent dual antagonist of the D-prostanoid and chemoattractant receptor-homologous molecule expressed on T helper 2 cells receptors. The drug interaction potential of AMG 853, both as a victim and a perpetrator, was investigated using in vitro, in silico, and in vivo methodologies. Experiments in human liver microsomes (HLM) and recombinant enzymes identified CYP2C8, CYP2J2, and CYP3A as well as multiple UDP-glucuronosyltransferase isoforms as being responsible for the metabolic clearance of AMG 853. With use of HLM and selective probe substrates, both AMG 853 and its acyl glucuronide metabolite (M1) were shown to be inhibitors of CYP2C8. AMG 853 and M1 did not inhibit any of the other cytochrome P450 isoforms tested, and AMG 853 exhibited minimal enzyme induction properties in human hepatocytes cultures. In light of the in vitro findings, modeling and simulation approaches were used to examine the potential for ketoconazole (a CYP3A inhibitor) to inhibit the metabolism of AMG 853 as well as for AMG 853 to inhibit the metabolism of paclitaxel, rosiglitazone, and montelukast, commonly used substrates of CYP2C8. A weak and clinically insignificant drug interaction (area under the drug concentration-time curve (AUC)(i)/AUC <2) was predicted between ketoconazole and AMG 853. No drug interactions were predicted for AMG 853 and paclitaxel, rosiglitazone, or montelukast. Finally, administration of AMG 853 to healthy human subjects in clinical trials in the presence or absence of ketoconazole confirmed that AMG 853 is unlikely to be involved in clinically significant drug interactions.
Assuntos
Microssomos Hepáticos/metabolismo , Fenilacetatos/farmacologia , Prostaglandinas/metabolismo , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/metabolismo , Receptores de Prostaglandina/antagonistas & inibidores , Receptores de Prostaglandina/metabolismo , Sulfonamidas/farmacologia , Adolescente , Adulto , Sistema Enzimático do Citocromo P-450/metabolismo , Interações Medicamentosas , Feminino , Glucuronosiltransferase/metabolismo , Hepatócitos/metabolismo , Humanos , Cetoconazol/farmacologia , Cinética , Pulmão/metabolismo , Masculino , Microssomos Hepáticos/enzimologia , Pessoa de Meia-Idade , Adulto JovemRESUMO
The in vitro characterization of the inhibition potential of four representative maytansinoid species observed upon hepatic and/or tumor in vivo processing of antibody-maytansine conjugates (AMCs) with cleavable and noncleavable linkers is reported. We investigated the free maytansinoid species N(2')-deacetyl-N(2')-(3-mercapto-1-oxopropyl)-maytansine (DM1), (S)-methyl-DM1, and N(2')-deacetyl-N(2')-(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4) as representative cleavable linker catabolites and Lysine-N(ε)-N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate-DM1 (Lys-MCC-DM1) as the representative noncleavable linker catabolite. Studies with recombinant human cytochromes P450 (P450s) indicate CYP2D6, CYP3A4, and CYP3A5 are the primary isoforms responsible for the oxidative metabolism of DM1, (S)-methyl-DM1, and DM4. Lys-MCC-DM1 was not metabolized by any of the P450 isoforms studied. DM1 was shown to be a reversible inhibitor of CYP2C8 (K(i) = 11 ± 3 µM) and CYP2D6 (K(i) = 14 ± 2 µM). Lys-MCC-DM1 and (S)-methyl-DM1 showed no reversible or time-dependent inactivation of any of the P450s studied. DM1 and DM4 inactivated CYP3A from human liver microsomes with K(i)/k(inact) values of 4.8 ± 0.9 µM/0.035 ± 0.002 min(-1) and 3.3 ± 0.2 µM/0.114 ± 0.002 min(-1), respectively. DM1 and DM4 inactivated recombinant CYP3A4 with K(i)/k(inact) values of 3.4 ± 1.0 µM/0.058 ± 0.005 min(-1) and 1.4 ± 0.3 µM/0.117 ± 0.006 min(-1), respectively. Because of instability in plasma, further characterization of the DM1 and DM4 intramolecular and intermolecular disulfide conjugates observed in vivo is required before an accurate drug-drug interaction (DDI) prediction can be made. AMCs with noncleavable thioether-linked DM1 as the cytotoxic agent are predicted to have no potential for a DDI with any of the major human P450s studied.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Inibidores das Enzimas do Citocromo P-450 , Inibidores Enzimáticos/farmacologia , Imunoconjugados/farmacologia , Maitansina/farmacologia , Anticorpos Monoclonais/metabolismo , Antineoplásicos/metabolismo , Biotransformação , Citocromo P-450 CYP2D6/metabolismo , Inibidores do Citocromo P-450 CYP2D6 , Citocromo P-450 CYP3A/metabolismo , Inibidores do Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/metabolismo , Dextrometorfano/metabolismo , Dextrorfano/metabolismo , Interações Medicamentosas , Inibidores Enzimáticos/metabolismo , Humanos , Imunoconjugados/metabolismo , Cinética , Maitansina/análogos & derivados , Maitansina/metabolismo , Microssomos Hepáticos/enzimologia , Paclitaxel/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Medição de RiscoRESUMO
Mibefradil (Posicor) was developed as a calcium channel blocker for the treatment of chronic hypertension. The compound was withdrawn from the market in 1998 because of the potential for rhabdomyolysis, renal failure, or bradycardia when it was coadministered with other drugs. Mibefradil has previously been shown to be a potent reversible (IC(50) = 0.3-2 µM) and mechanism-based (K(i) = 2.3 µM; k(inact) = 0.4 min(-1)) inhibitor of CYP3A4-catalyzed statin metabolism. At present, the mechanism of CYP3A4 inactivation by mibefradil is not known. Mechanism-based inactivation experiments and spectral studies were used to examine the mechanism of CYP3A4 inactivation by mibefradil and its major metabolite, des-methoxyacetyl mibefradil (Ro 40-5966), in vitro. Both mibefradil and Ro 40-5966 were shown to exhibit type I binding characteristics (K(s) = 0.69 ± 0.06 and 1.39 ± 0.04 µM, respectively) toward CYP3A4. Complete K(i)/k(inact) experiments were performed, revealing a rapid and irreversible decrease in CYP3A4-catalyzed 1'-hydroxymidazolam formation. Approximately 70% of CYP3A4 activity was lost in the first minute of incubation with mibefradil, and inactivation was nonlinear after 2 min. Ro 40-5966 also resulted in time-dependent inhibition of CYP3A4, albeit to a lesser extent than mibefradil. The decrease in CYP3A4 activity in the presence of mibefradil and NADPH was subsequently shown to have a good correlation with the time-dependent loss of CO binding, which, coupled with the lack of stable heme and/or apoprotein adducts, suggests heme destruction as the mechanism of inactivation of CYP3A4 by mibefradil.
Assuntos
Inibidores do Citocromo P-450 CYP3A , Inibidores Enzimáticos/farmacologia , Heme/metabolismo , Mibefradil/farmacologia , Cromatografia Líquida de Alta Pressão , Citocromo P-450 CYP3A , Humanos , Hidrólise , Espectrometria de Massas , Espectrofotometria UltravioletaRESUMO
Catalytically promiscuous enzymes are intermediates in the evolution of new function from an existing pool of protein scaffolds. However, promiscuity will only confer an evolutionary advantage if other useful properties are not compromised or if there is no "negative trade-off" induced by the mutations that yield promiscuity. Therefore, identification and characterization of negative trade-offs incurred during the emergence of promiscuity are required to further develop the evolutionary models and to optimize in vitro evolution. One potential negative trade-off of catalytic promiscuity is increased susceptibility to inhibition, or inhibitory promiscuity. Here we exploit cytochrome P450s (CYPs) as a model protein scaffold that spans a vast range of catalytic promiscuity and apply a quantitative index to determine the relationship between promiscuity of catalysis and promiscuity of inhibition for a series of homologues. The aim of these studies is to begin to identify properties that, in general, correlate with catalytic promiscuity, hypothetically such as inhibitory promiscuity. Interestingly, the data indicate that the potential negative trade-off of inhibitory promiscuity is nearly insignificant because even highly substrate specific CYPs have high inhibitory promiscuity, with little incremental increase in susceptibility to inhibitory interactions as the substrate promiscuity increases across the series of enzymes. In the context of evolution, inhibitory promiscuity is not an obligate negative trade-off for catalytic promiscuity.
Assuntos
Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/metabolismo , Evolução Molecular , Modelos Biológicos , Biocatálise , Fenômenos Químicos , Sistema Enzimático do Citocromo P-450/química , Interações Medicamentosas , Humanos , Fígado/enzimologia , Microssomos/enzimologia , Modelos Moleculares , Proteínas Recombinantes/metabolismo , Estatística como Assunto , Especificidade por SubstratoRESUMO
One goal in drug design is to decrease clearance due to metabolism. It has been suggested that a compound's metabolic stability can be increased by incorporation of a sp(2) nitrogen into an aromatic ring. Nitrogen incorporation is hypothesized to increase metabolic stability by coordination of nitrogen to the heme-iron (termed type II binding). However, questions regarding binding affinity, metabolic stability, and how metabolism of type II binders occurs remain unanswered. Herein, we use pyridinyl quinoline-4-carboxamide analogs to answer these questions. We show that type II binding can have a profound influence on binding affinity for CYP3A4, and the difference in binding affinity can be as high as 1200-fold. We also find that type II binding compounds can be extensively metabolized, which is not consistent with the dead-end complex kinetic model assumed for type II binders. Two alternate kinetic mechanisms are presented to explain the results. The first involves a rapid equilibrium between the type II bound substrate and a metabolically oriented binding mode. The second involves direct reduction of the nitrogen-coordinated heme followed by oxygen binding.
Assuntos
Fenômenos Bioquímicos , Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Heme/metabolismo , Fenômenos Físicos , Heme/química , Humanos , Cinética , Modelos Químicos , Ligação ProteicaRESUMO
Indoleamine 2,3-dioxygenase (IDO) is a heme-containing dioxygenase involved in the degradation of several indoleamine derivatives and has been indicated as an immunosuppressive. IDO is an attractive target for therapeutic intervention in diseases which are known to capitalize on immune suppression, including cancer, HIV, and inflammatory diseases. Conventionally, IDO activity is measured through chemical reduction by the addition of ascorbate and methylene blue. Identification of potential coenzymes involved in the reduction of IDO in vivo should improve in vitro reconstitution systems used to identify potential IDO inhibitors. In this study we show that NADPH-cytochrome P450 reductase (CPR) is capable of supporting IDO activity in vitro and that oxidation of l-Trp follows substrate inhibition kinetics (k(cat) = 0.89 +/- 0.04 s(-1), K(m) = 0.72 +/- 0.15 microM, and K(i) = 9.4 +/- 2.0 microM). Addition of cytochrome b(5) to CPR-supported l-Trp incubations results in modulation from substrate inhibition to sigmoidal kinetics (k(cat) = 1.7 +/- 0.3 s(-1), K(m) = 1.5 +/- 0.9 microM, and K(i) = 1.9 +/- 0.3). CPR-supported d-Trp oxidations (+/-cytochrome b(5)) exhibit Michaelis-Menten kinetics. Addition of methylene blue (minus ascorbate) to CPR-supported reactions resulted in inhibition of d-Trp turnover and modulation of l-Trp kinetics from allosteric to Michaelis-Menten with a concurrent decrease in substrate affinity for IDO. Our data indicate that CPR is capable of supporting IDO activity in vitro and oxidation of tryptophan by IDO displays substrate stereochemistry dependent atypical kinetics which can be modulated by the addition of cytochrome b(5).
Assuntos
Citocromos b/farmacologia , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Azul de Metileno/farmacologia , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Regulação Alostérica/efeitos dos fármacos , Animais , Sítios de Ligação , Células CHO , Cricetinae , Cricetulus , Ativação Enzimática , Indóis/farmacologia , Oxirredução , Proteínas de Saccharomyces cerevisiae/metabolismo , Triptofano/farmacologiaRESUMO
Mangosteen is a xanthone-containing fruit found in Southeast Asia for which health claims include maintaining healthy immune and gastrointestinal systems to slowing the progression of tumor growth and neurodegenerative diseases. Previous studies have identified multiple xanthones in the pericarp of the mangosteen fruit. The aim of the current study was to assess the drug inhibition potential of mangosteen in vitro as well as the cytochrome P450 (P450) enzymes responsible for the metabolism of its individual components. The various xanthone derivatives were found to be both substrates and inhibitors for multiple P450 isoforms. Aqueous extracts of the mangosteen pericarp were analyzed for xanthone content as well as inhibition potency. Finally, in vivo plasma concentrations of alpha-mangostin, the most abundant xanthone derivative found in mangosteen, were predicted using Simcyp and found to be well above their respective in vitro K(i) values for CYP2C8 and CYP2C9.
Assuntos
Inibidores das Enzimas do Citocromo P-450 , Inibidores Enzimáticos/farmacologia , Garcinia mangostana/química , Microssomos Hepáticos/enzimologia , Xantonas/farmacologia , Cromatografia Líquida de Alta Pressão , Inibidores Enzimáticos/química , Humanos , Técnicas In Vitro , Isoenzimas/antagonistas & inibidores , Cinética , Espectrometria de Massas , Microssomos Hepáticos/efeitos dos fármacos , NADP/metabolismo , Preparações Farmacêuticas/metabolismo , Fenótipo , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Xantonas/químicaRESUMO
Cytochrome P450 (P450) reaction phenotyping is a key process toward accurately determining the contribution of different P450s to the metabolism of new chemical entities. The significance of P450s to drug disposition has led to the identification of selective chemical and antibody inhibitors for individual P450 enzymes. Despite these advances, the maximal inhibition attainable is limited by the use of inhibitor concentrations that maintain selectivity for the individual P450s. Thus, most commercially available inhibitors produce a maximal inhibition of approximately 80%. Herein, the combination of chemical plus antibody inhibitors is explored to find whether P450 3A could be selectively and completely (>99%) inhibited by using both inhibitors simultaneously.
Assuntos
Anticorpos Monoclonais/farmacologia , Inibidores do Citocromo P-450 CYP3A , Inibidores Enzimáticos/farmacologia , Microssomos Hepáticos/enzimologia , Anticorpos Monoclonais/imunologia , Área Sob a Curva , Citocromo P-450 CYP3A/imunologia , Citocromo P-450 CYP3A/metabolismo , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/metabolismo , Interações Medicamentosas , Humanos , Hidroxitestosteronas/metabolismo , Cetoconazol/farmacologia , Microssomos Hepáticos/efeitos dos fármacos , Midazolam/análogos & derivados , Midazolam/metabolismo , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Testosterona/análogos & derivados , Testosterona/metabolismoRESUMO
The role of C239 as the active-site residue responsible for forming the covalent linkage with raloxifene during P450 3A4 time-dependent inactivation (TDI) was recently identified. The corresponding residue in CYP3A5 is S239, and when the potential for TDI in P450 3A5 was investigated, only reversible inhibition was observed against midazolam and testosterone, with median inhibitory concentration (IC50) values of 2.4 and 2.9 microM, respectively. In a similar fashion, when C239 was replaced with alanine in P450 3A4, TDI was successfully engineered out, and the reversible inhibition was characterized by IC50 values of 3.7 and 3.5 microM against midazolam and testosterone, respectively. Metabolism studies confirmed that the reactive diquinone methide intermediate required for P450 3A4 inactivation formed in all of the P450 3A enzymes investigated. Furthermore, the absence of TDI in P450 3A5 led to an increase in the formation of GSH-related adducts of raloxifene compared with that for P450 3A4. Consequently, the absence of the nucleophilic cysteine leads to differential TDI and generation of reactive metabolites in the P450 3A enzyme, providing the foundation for pharmacogenetics that contributes to individual differences in susceptibility to adverse drug reactions.
Assuntos
Cisteína/química , Inibidores das Enzimas do Citocromo P-450 , Cloridrato de Raloxifeno/farmacologia , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Clonagem Molecular , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Escherichia coli/genética , Humanos , Modelos Moleculares , Mutação , Cloridrato de Raloxifeno/metabolismo , Moduladores Seletivos de Receptor Estrogênico/metabolismo , Fatores de TempoRESUMO
The heme-containing cytochrome P450s (CYPs) are a major enzymatic determinant of drug clearance and drug-drug interactions. The CYP3A4 isoform is inhibited by antifungal imidazoles or triazoles, which form low-spin heme iron complexes via formation of a nitrogen-ferric iron coordinate bond. However, CYP3A4 also slowly oxidizes the antifungal itraconazole (ITZ) at a site that is approximately 25 A from the triazole nitrogens, suggesting that large antifungal azoles can adopt multiple orientations within the CYP3A4 active site. Here, we report a surface plasmon resonance (SPR) analysis with kinetic resolution of two binding modes of ITZ, and the related drug ketoconazole (KTZ). SPR reveals a very slow off-rate for one binding orientation. Multiphasic binding kinetics are observed, and one of the two binding components resolved by curve fitting exhibits "equilibrium overshoot". Preloading of CYP3A4 with the heme ligand imidazole abolishes this component of the antifungal azole binding trajectories, and it eliminates the conspicuously slow off-rate. The fractional populations of CYP3A4 complexes corresponding to different drug orientations can be manipulated by altering the duration of the pulse of drug exposure. UV-vis difference absorbance titrations yield low-spin spectra and K(D) values that are consistent with the high-affinity complex resolved by SPR. These results demonstrate that ITZ and KTZ bind in multiple orientations, including a catalytically productive mode and a slowly dissociating inhibitory mode. Most importantly, they provide the first example of a SPR-based method for the kinetic characterization of binding of a drug to any human CYP, including mechanistic insight not available from other methods.
Assuntos
Antifúngicos/química , Citocromo P-450 CYP3A/química , Itraconazol/química , Cetoconazol/química , Ressonância de Plasmônio de Superfície/métodos , Antifúngicos/metabolismo , Sítios de Ligação , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Imidazóis/metabolismo , Itraconazol/metabolismo , Cetoconazol/metabolismo , Cinética , Luz , Conformação Proteica/efeitos dos fármacos , Espectrofotometria Ultravioleta , EstereoisomerismoRESUMO
Cytochrome P450's (P450's) catalyze the oxidative metabolism of most drugs and toxins. Although extensive studies have proven that some P450's demonstrate both homotropic and heterotropic cooperativity toward a number of substrates, the mechanistic and molecular details of P450 allostery are still not well-established. Here, we use UV/vis and heteronuclear nuclear magnetic resonance (NMR) spectroscopic techniques to study the mechanism and thermodynamics of the binding of two 9-aminophenanthrene (9-AP) and testosterone (TST) molecules to the erythromycin-metabolizing bacterial P450(eryF). UV/vis absorbance spectra of P450(eryF) demonstrated that binding occurs with apparent negative homotropic cooperativity for TST and positive homotropic cooperativity for 9-AP with Hill-equation-derived dissociation constants of K(S) = 4 and 200 microM, respectively. The broadening and shifting observed in the 2D-{1H,15N}-HSQC-monitored titrations of 15N-Phe-labeled P450(eryF) with 9-AP and TST indicated binding on intermediate and fast chemical exchange time scales, respectively, which was consistent with the Hill-equation-derived K(S) values for these two ligands. Regardless of the type of spectral perturbation observed (broadening for 9-AP and shifting for TST), the 15N-Phe NMR resonances most affected were the same in each titration, suggesting that the two ligands "contact" the same phenylalanines within the active site of P450(eryF). This finding is in agreement with X-ray crystal structures of bound P450(eryF) showing different ligands occupying similar active-site niches. Complex spectral behavior was additionally observed for a small collection of resonances in the TST titration, interpreted as multiple binding modes for the low-affinity TST molecule or multiple TST-bound P450(eryF) conformational substates. A structural and energetic model is presented that combines the energetics and structural aspects of 9-AP and TST binding derived from these observations.
Assuntos
Bactérias/enzimologia , Sistema Enzimático do Citocromo P-450/química , Sítios de Ligação , Cristalografia por Raios X , Sistema Enzimático do Citocromo P-450/metabolismo , Eritromicina/metabolismo , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Fenantrenos/química , Fenantrenos/metabolismo , Fenilalanina/química , Fenilalanina/metabolismo , Espectrofotometria Ultravioleta , Testosterona/química , Testosterona/metabolismo , TermodinâmicaRESUMO
Bacterial glutamine synthetases (GSs) are dodecameric aggregates comprised of two face-to-face hexameric rings, which form a cylindrical aqueous channel. Available crystal structures indicate that each subunit provides a 'central loop' that protrudes into this channel. Residues on either side of this loop contribute directly to substrate or metal ion cofactor binding. Although it has been suggested that this conspicuous structural feature may be functionally important, a systematic structure-function analysis of this loop has not been done. Here, we examine the behavior of a cysteine mutant, E165C, which yields inter-subunit disulfide bonds connecting the central loops. The inter-subunit disulfide bonds are readily detected by electrospray ionization mass spectrometry. Based on molecular models, the disulfide bonds would form only if the engineered cysteines on adjacent subunits moved approximately 5 A. Surprisingly, inter-subunit disulfide bonds between the central loops caused no detectable changes in the KMs for glutamate or ATP, nor the KD for either ATP or the transition state analog (L)-methionine sulfoximine (MSOX). Furthermore, covalent and quantitative adduction of the E165C mutant with iodo-acetamido-pyrene yielded nearly fully active enzyme bearing fluorescent pyrene excimers. The relative contribution of pyrene monomers to excimers in the steady state fluorescence is temperature dependent, suggesting thermal equilibrium between loop conformational states. However, the monomer-excimer ratio is independent of ligands such as MSOX, glutamate, or Mn2+. These results validate the suspected flexibility of the central loop, but raise significant doubt about its direct functional role in GS catalysis via conformational switching, including the proposed regulation of GS via ADP-ribosylation within this loop.
Assuntos
Escherichia coli/enzimologia , Glutamato-Amônia Ligase/química , Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Catálise , Dissulfetos/química , Ácido Glutâmico/química , Cinética , Magnésio/química , Modelos Químicos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Conformação Proteica , Pirenos/química , Ribose/química , Espectrometria de Fluorescência , Espectrometria de Massas por Ionização por Electrospray , TemperaturaRESUMO
Bacterial glutamine synthetases (GSs) are complex dodecameric oligomers that play a critical role in nitrogen metabolism, converting ammonia and glutamate to glutamine. Recently published reports suggest that GS from Mycobacterium tuberculosis (MTb) may be a therapeutic target (Harth, G., and Horwitz, M. A. (2003) Infect. Immun. 71, 456-464). In some bacteria, GS is regulated via adenylylation of some or all of the subunits within the aggregate; catalytic activity is inversely proportional to the extent of adenylylation. The adenylylation and deadenylylation of GS are catalyzed by adenylyl transferase (ATase). Here, we demonstrate via electrospray ionization mass spectrometry that GS from pathogenic M. tuberculosis is adenylylated by the Escherichia coli ATase. The adenylyl group can be hydrolyzed by snake venom phosphodiesterase to afford the unmodified enzyme. The site of adenylylation of MTb GS by the E. coli ATase is Tyr-406, as indicated by the lack of adenylylation of the Y406F mutant, and, as expected, is based on amino acid sequence alignments. Using electrospray ionization mass spectroscopy methodology, we found that GS is not adenylylated when obtained directly from MTb cultures that are not supplemented with glutamine. Under these conditions, the highly related but non-pathogenic Mycobacterium bovis BCG yields partially ( approximately 25%) adenylylated enzyme. Upon the addition of glutamine to the cultures, the MTb GS becomes significantly adenylylated ( approximately 30%), whereas the adenylylation of M. bovis BCG GS does not change. Collectively, the results demonstrate that MTb GS is a substrate for E. coli ATase, but only low adenylylation states are accessible. This parallels the low adenylylation states observed for GS from mycobacteria and suggests the intriguing possibility that adenylylation in the pathogenic versus non-pathogenic mycobacteria is differentially regulated.