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1.
J Pharm Sci ; 2024 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-39009346

RESUMO

Past studies have demonstrated higher clearance for monoclonal antibodies possessing increased rates of non-specific endocytosis. However, this metric is oftentimes evaluated indirectly using biophysical techniques or cell surface binding studies that may not provide insight into the specific rates of cellular turnover. Furthermore, few examples evaluating non-specific endocytosis have been reported for a therapeutic antibody that reached clinical assessment. In the current report, we evaluated a therapeutic human immunoglobulin G2 monoclonal antibody targeted against the interleukin-4 receptor alpha chain (IL-4Rα) that exhibited elevated target independent clearance in previous Phase 1 and 2 studies. We confirmed high non-specific clearance of the anti-IL-4Rα antibody as compared to a reference antibody during pharmacokinetic assessments in wild type mice where target-mediated disposition was absent. We then developed a cell-based method capable of measuring cellular protein endocytosis and demonstrated the anti-IL-4Rα antibody exhibited marked non-specific uptake relative to the reference compound. Antibody homology modeling identified the anti-IL-4Rα antibody possessed positive charge patches whose removal via targeted mutations substantially reduced its non-specific endocytosis. We then expanded the scope of the study by evaluating panels of both preclinical and clinically relevant monoclonal antibodies and demonstrate those with the highest rates of non-specific uptake in vitro exhibited elevated target independent clearance, low subcutaneous bioavailability, or both. Our results support the observation that high non-specific endocytosis is a negative attribute in monoclonal antibody development and demonstrate the utility of a generic cell-based screen as a quantitative tool to measure non-specific endocytosis of protein therapeutics at the single-cell level.

2.
J Pharm Sci ; 2024 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-38906252

RESUMO

In vivo clearance mechanisms of therapeutic monoclonal antibodies (mAbs) encompass both target-mediated and target-independent processes. Two distinct determinants of overall mAb clearance largely separate of target-mediated influences are non-specific cellular endocytosis and subsequent pH-dependent mAb recycling mediated by the neonatal Fc receptor (FcRn), where inter-mAb variability in the efficiency of both processes is observed. Here, we implemented a functional cell-based FcRn recycling assay via Madin-Darby canine kidney type II cells stably co-transfected with human FcRn and its light chain ß2-microglobulin. Next, a series of pH-dependent internalization studies using a model antibody demonstrated proper function of the human FcRn complex. We then applied our cellular assays to assess the contribution of both FcRn and non-specific interactions in the cellular turnover for a panel of 8 clinically relevant mAbs exhibiting variable human pharmacokinetic behavior. Our results demonstrate that the interplay of non-specific endocytosis rates, pH-dependent non-specific interactions, and engagement with FcRn all contribute to the overall recycling efficiency of therapeutic monoclonal antibodies. The predictive capacity of our assay approach was highlighted by successful identification of all mAbs within our panel possessing clearance in humans greater than 5 mL/day/kg. These results demonstrate that a combination of cell-based in vitro assays can properly resolve individual mechanisms underlying the overall in vivo recycling efficiency and non-target mediated clearance of therapeutic mAbs.

3.
MAbs ; 15(1): 2263926, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37824334

RESUMO

In this investigation, we tested the hypothesis that a physiologically based pharmacokinetic (PBPK) model incorporating measured in vitro metrics of off-target binding can largely explain the inter-antibody variability in monoclonal antibody (mAb) pharmacokinetics (PK). A diverse panel of 83 mAbs was evaluated for PK in wild-type mice and subjected to 10 in vitro assays to measure major physiochemical attributes. After excluding for target-mediated elimination and immunogenicity, 56 of the remaining mAbs with an eight-fold variability in the area under the curve (AUC0-672h: 1.74 × 106 -1.38 × 107 ng∙h/mL) and 10-fold difference in clearance (2.55-26.4 mL/day/kg) formed the training set for this investigation. Using a PBPK framework, mAb-dependent coefficients F1 and F2 modulating pinocytosis rate and convective transport, respectively, were estimated for each mAb with mostly good precision (coefficient of variation (CV%) <30%). F1 was estimated to be the mean and standard deviation of 0.961 ± 0.593, and F2 was estimated to be 2.13 ± 2.62. Using principal component analysis to correlate the regressed values of F1/F2 versus the multidimensional dataset composed of our panel of in vitro assays, we found that heparin chromatography retention time emerged as the predictive covariate to the mAb-specific F1, whereas F2 variability cannot be well explained by these assays. A sigmoidal relationship between F1 and the identified covariate was incorporated within the PBPK framework. A sensitivity analysis suggested plasma concentrations to be most sensitive to F1 when F1 > 1. The predictive utility of the developed PBPK model was evaluated against a separate panel of 14 mAbs biased toward high clearance, among which area under the curve of PK data of 12 mAbs was predicted within 2.5-fold error, and the positive and negative predictive values for clearance prediction were 85% and 100%, respectively. MAb heparin chromatography assay output allowed a priori identification of mAb candidates with unfavorable PK.


Assuntos
Anticorpos Monoclonais , Modelos Biológicos , Camundongos , Animais , Pinocitose , Bioensaio , Heparina
4.
MAbs ; 15(1): 2256745, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37698932

RESUMO

Biologic drug discovery pipelines are designed to deliver protein therapeutics that have exquisite functional potency and selectivity while also manifesting biophysical characteristics suitable for manufacturing, storage, and convenient administration to patients. The ability to use computational methods to predict biophysical properties from protein sequence, potentially in combination with high throughput assays, could decrease timelines and increase the success rates for therapeutic developability engineering by eliminating lengthy and expensive cycles of recombinant protein production and testing. To support development of high-quality predictive models for antibody developability, we designed a sequence-diverse panel of 83 effector functionless IgG1 antibodies displaying a range of biophysical properties, produced and formulated each protein under standard platform conditions, and collected a comprehensive package of analytical data, including in vitro assays and in vivo mouse pharmacokinetics. We used this robust training data set to build machine learning classifier models that can predict complex protein behavior from these data and features derived from predicted and/or experimental structures. Our models predict with 87% accuracy whether viscosity at 150 mg/mL is above or below a threshold of 15 centipoise (cP) and with 75% accuracy whether the area under the plasma drug concentration-time curve (AUC0-672 h) in normal mouse is above or below a threshold of 3.9 × 106 h x ng/mL.


Assuntos
Anticorpos Monoclonais , Descoberta de Drogas , Animais , Camundongos , Anticorpos Monoclonais/química , Simulação por Computador , Proteínas Recombinantes , Viscosidade
5.
Cancer Immunol Res ; 10(6): 698-712, 2022 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-35413104

RESUMO

Bispecific T-cell engager (BiTE) molecules are biologic T cell-directing immunotherapies. Blinatumomab is approved for treatment of B-cell malignancies, but BiTE molecule development in solid tumors has been more challenging. Here, we employed intravital imaging to characterize exposure and pharmacodynamic response of an anti-muCD3/anti-huEGFRvIII mouse surrogate BiTE molecule in EGFR variant III (EGFRvIII)-positive breast tumors implanted within immunocompetent mice. Our study revealed heterogeneous temporal and spatial dynamics of BiTE molecule extravasation into solid tumors, highlighting physical barriers to BiTE molecule function. We also discovered that high, homogeneous EGFRvIII expression on cancer cells was necessary for a BiTE molecule to efficiently clear tumors. In addition, we found that resident tumor-infiltrating lymphocytes (TIL) were sufficient for optimal tumor killing only at high BiTE molecule dosage, whereas inclusion of peripheral T-cell recruitment was synergistic at moderate to low dosages. We report that deletion of stimulatory conventional type I DCs (cDC1) diminished BiTE molecule-induced T-cell activation and tumor clearance, suggesting that in situ antigen-presenting cell (APC) engagements modulate the extent of BiTE molecule efficacy. In summary, our work identified multiple requirements for optimal BiTE molecule efficacy in solid tumors, providing insights that could be harnessed for solid cancer immunotherapy development.


Assuntos
Anticorpos Biespecíficos , Neoplasias , Animais , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Linfócitos B , Imunoterapia/métodos , Ativação Linfocitária , Camundongos , Neoplasias/patologia , Linfócitos T
6.
Pharmacol Ther ; 212: 107574, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32433985

RESUMO

Therapeutic proteins (TPs) are a diverse drug class that include monoclonal antibodies (mAbs), recombinantly expressed enzymes, hormones and growth factors, cytokines (e.g. chemokines, interleukins, interferons), as well as a wide range of engineered fusion scaffolds containing IgG1 Fc domain for half-life extension. As the pharmaceutical industry advances more potent and selective protein-based medicines through discovery and into the clinical stages of development, it has become widely appreciated that a comprehensive understanding of the mechanisms of TP biodistribution can aid this endeavor. This review aims to highlight the literature that has advanced our understanding of the determinants of TP biodistribution. A particular emphasis is placed on the multi-faceted role of the neonatal Fc receptor (FcRn) in mAb and Fc-fusion protein disposition. In addition, characterization of the TP-target interaction at the cell-level is discussed as an essential strategy to establish pharmacokinetic-pharmacodynamic (PK/PD) relationships that may lead to more informed human dose projections during clinical development. Methods for incorporation of tissue and cell-level parameters defining these characteristics into higher-order mechanistic and semi-mechanistic PK models will also be presented.


Assuntos
Proteínas/farmacocinética , Proteínas/uso terapêutico , Animais , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais/uso terapêutico , Engenharia Genética , Glicosilação , Humanos , Modelos Biológicos , Receptores Fc/fisiologia , Distribuição Tecidual
7.
MAbs ; 12(1): 1746520, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32310023

RESUMO

The decision to pursue a monoclonal antibody (mAb) as a therapeutic for disease intervention requires the assessment of many factors, such as target-biology, including the total target burden and its accessibility at the intended site of action, as well as mAb-specific properties like binding affinity and the pharmacokinetics in serum and tissue. Interleukin-36 receptor (IL-36 R) is a member of the IL-1 family cytokine receptors and an attractive target to treat numerous epithelial-mediated inflammatory conditions, including psoriatic and rheumatoid arthritis, asthma, and chronic obstructive pulmonary disease. However, information concerning the expression profile of IL-36 R at the protein level is minimal, so the feasibility of developing a therapeutic mAb against this target is uncertain. Here, we present a characterization of the properties associated with absorption, distribution, metabolism, and excretion of a high-affinity IL-36 R-targeted surrogate rat (IgG2a) mAb antagonist in preclinical mouse models. The presence of IL-36 R in the periphery was confirmed unequivocally as the driver of non-linear pharmacokinetics in blood/serum, although a predominant site of tissue accumulation was not observed based upon the kinetics of radiotracer. Additionally, the contribution of IL-36 R-mediated catabolism of mAb in kidney was tested in a 5/6 nephrectomized mouse model where minimal effects on serum pharmacokinetics were observed, although analysis of functional mAb in urine suggests that target can influence the amount of mAb excreted. Our data highlight an interesting case of target-mediated drug disposition (TMDD) where low, yet broadly expressed levels of membrane-bound target result in a cumulative effect to drive TMDD behavior typical of a large, saturable target sink. The potential differences between our mouse model and IL-36 R target profile in humans are also presented.


Assuntos
Algoritmos , Anticorpos Monoclonais/farmacocinética , Modelos Biológicos , Receptores de Interleucina/antagonistas & inibidores , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos/imunologia , Feminino , Humanos , Cinética , Masculino , Camundongos Endogâmicos C57BL , Receptores de Interleucina/imunologia , Distribuição Tecidual
8.
J Inorg Biochem ; 205: 110994, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31982812

RESUMO

The Mycobacterium tuberculosis (Mtb) genome encodes 20 different cytochrome P450 enzymes (CYPs), many of which serve essential biosynthetic roles. CYP51B1, the Mtb version of eukaryotic sterol demethylase, remains a potential therapeutic target. The binding of three drug fragments containing nitrogen heterocycles to CYP51B1 is studied here by continuous wave (CW) and pulsed electron paramagnetic resonance (EPR) techniques to determine how each drug fragment binds to the heme active-site. All three drug fragments form a mixture of complexes, some of which retain the axial water ligand from the resting state. Hyperfine sublevel correlation spectroscopy (HYSCORE) and electron-nuclear double resonance spectroscopy (ENDOR) observe protons of the axial water and on the drug fragments that reveal drug binding modes. Binding in CYP51B1 is complicated by the presence of multiple binding modes that coexist in the same solution. These results aid our understanding of CYP-inhibitor interactions and will help guide future inhibitor design.


Assuntos
Antibacterianos/química , Proteínas de Bactérias , Inibidores das Enzimas do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450 , Mycobacterium tuberculosis/enzimologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Domínio Catalítico , Sistema Enzimático do Citocromo P-450/química
9.
Mol Pharm ; 13(7): 2387-96, 2016 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-27248573

RESUMO

Antibody-drug conjugates (ADC) rely on the target-binding specificity of an antibody to selectively deliver potent drugs to cancer cells. IgG antibody half-life is regulated by neonatal Fc receptor (FcRn) binding. Histidine 435 of human IgG was mutated to alanine (H435A) to explore the effect of FcRn binding on the pharmacokinetics, efficacy, and tolerability of two separate maytansine-based ADC pairs with noncleavable linkers, (c-DM1 and c-H435A-DM1) and (7v-Cys-may and 7v-H435A-Cys-may). The in vitro cell-killing potency of each pair of ADCs was similar, demonstrating that H435A showed no measurable impact on ADC bioactivity. The H435A mutant antibodies showed no detectable binding to human or mouse FcRn in vitro, whereas their counterpart wild-type IgG ADCs were found to bind to FcRn at pH = 6.0. In xenograft bearing SCID mice expressing mouse FcRn, the AUC of 7v-Cys-may was 1.6-fold higher than that of 7v-H435A-may, yet the observed efficacy was similar. More severe thrombocytopenia was observed with 7v-H435A-Cys-may as compared to 7v-Cys-may at multiple dose levels. The AUC of c-DM1 was approximately 3-fold higher than that of c-H435A-DM1 in 786-0 xenograft bearing SCID mice, which led to a 3-fold difference in efficacy by dose. Murine FcRn knockout, human FcRn transgenic line 32 SCID animals bearing 786-0 xenografts showed an amplified exposure difference between c-DM1 and c-H435A-DM1 as compared to murine FcRn expressing SCID mice, leading to a 10-fold higher dose required for efficacy despite a 6-fold higher AUC of the c-H435A-DM1. The accelerated clearance observed for the noncleavable maytansine ADCs with the H435A FcRn mutation led to reduced efficacy at equivalent doses and exacerbation of clinical pathology parameters (decreased tolerability) at equivalent doses. The results show that reduced ADC clearance mediated by FcRn modulation can improve therapeutic index.


Assuntos
Anticorpos/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Imunoconjugados/farmacologia , Imunoglobulina G/metabolismo , Receptores Fc/metabolismo , Animais , Anticorpos/genética , Ligante CD27/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Imunoconjugados/química , Maitansina/metabolismo , Camundongos , Camundongos SCID , Receptores Fc/genética
10.
Biochemistry ; 54(5): 1198-207, 2015 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-25591012

RESUMO

Azoles and pyridines are commonly incorporated into small molecule inhibitor scaffolds that target cytochromes P450 (CYPs) as a strategy to increase drug binding affinity, impart isoform-dependent selectivity, and improve metabolic stability. Optical absorbance spectra of the CYP-inhibitor complex are widely used to infer whether these inhibitors are ligated directly to the heme iron as catalytically inert, low-spin (type II) complexes. Here, we show that the low-spin complex between a drug-metabolizing CYP2C9 variant and 4-(3-phenylpropyl)-1H-1,2,3-triazole (PPT) retains an axial water ligand despite exhibiting elements of "classic" type II optical behavior. Hydrogens of the axial water ligand are observed by pulsed electron paramagnetic resonance (EPR) spectroscopy for both inhibitor-free and inhibitor-bound species and show that inhibitor binding does not displace the axial water. A (15)N label incorporated into PPT is 0.444 nm from the heme iron, showing that PPT is also in the active site. The reverse type I inhibitor, LP10, of CYP125A1 from Mycobacterium tuberculosis, known from X-ray crystal structures to form a low-spin water-bridged complex, is found by EPR and by visible and near-infrared magnetic circular dichroism spectroscopy to retain the axial water ligand in the complex in solution.


Assuntos
Aminopiridinas/química , Proteínas de Bactérias/química , Citocromo P-450 CYP2C9/química , Ginsenosídeos/química , Heme/química , Indóis/química , Mycobacterium tuberculosis/química , Sapogeninas/química , Água/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Citocromo P-450 CYP2C9/genética , Citocromo P-450 CYP2C9/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Heme/genética , Heme/metabolismo , Humanos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Água/metabolismo
11.
Drug Metab Dispos ; 42(11): 1906-13, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25209366

RESUMO

The pharmacokinetic (PK) behavior of monoclonal antibodies (mAbs) is influenced by target-mediated drug disposition, off-target effects, antidrug antibody-mediated clearance, and interaction with fragment-crystallizable domain (Fc) receptors such as neonatal Fc receptor. All of these interactions hold the potential to impact mAb biodistribution. Near infrared (NIR) fluorescent probes offer an approach complementary to radionuclides to characterize drug disposition. Notably, the use of FDA-approved IRDye800 (IR800; LI-COR, Lincoln, NE) as a protein-labeling agent in preclinical work holds the potential for quantitative tissue analysis. Here, we tested the utility of the IR800 dye as a quantitative mAb tracer during pharmacokinetic analysis in both plasma and tissues using a model mouse monoclonal IgG1 (8C2) labeled with ≤1.5 molecules of IR800. The plasma PK parameters derived from a mixture of IR800-8C2 and 8C2 dosed intravenously to C57BL/6 mice at 8 mg/kg exhibited a large discrepancy in exposure depending on the method of quantitation [CLplasma = 8.4 ml/d per kilogram (NIR fluorescence detection) versus 2.5 ml/d per kilogram (enzyme-linked immunosorbent assay)]. The disagreement between measurements suggests that the PK of 8C2 is altered by addition of the IR800 dye. Additionally, direct fluorescence analysis of homogenized tissues revealed several large differences in IR800-8C2 tissue uptake when compared with a previously published study using [(125)I]8C2, most notably an over 4-fold increase in liver concentration. Finally, the utility of IR800 in combination with whole body imaging was examined by comparison of IR800-8C2 levels observed in animal sagittal cross-sections to those measured in homogenized tissues. Our results represent the first PK analysis in both mouse plasma and tissues of an IR800-mAb conjugate and suggest that mAb disposition is significantly altered by IR800 conjugation to 8C2.


Assuntos
Anticorpos Monoclonais/farmacocinética , Corantes Fluorescentes/química , Animais , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Espectroscopia de Luz Próxima ao Infravermelho , Distribuição Tecidual
12.
Biochemistry ; 53(9): 1428-34, 2014 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-24576089

RESUMO

The heme-containing cytochrome P450s exhibit isoform-dependent ferric spin equilibria in the resting state and differential substrate-dependent spin equilibria. The basis for these differences is not well understood. Here, magnetic circular dichroism (MCD) reveals significant differences in the resting low spin ligand field of CYPs 3A4, 2E1, 2C9, 125A1, and 51B1, which indicates differences in the strength of axial water ligation to the heme. The near-infrared bands that specifically correspond to charge-transfer porphyrin-to-metal transitions span a range of energies of nearly 2 kcal/mol. In addition, the experimentally determined MCD bands are not entirely in agreement with the expected MCD energies calculated from electron paramagnetic resonance parameters, thus emphasizing the need for the experimental data. MCD marker bands of the high spin heme between 500 and 680 nm were also measured and suggest only a narrow range of energies for this ensemble of high spin Cys(S(-)) → Fe(3+) transitions among these isoforms. The differences in axial ligand energies between CYP isoforms of the low spin states likely contribute to the energetics of substrate-dependent spin state perturbation. However, these ligand field energies do not correlate with the fraction of high spin vs low spin in the resting state enzyme, suggestive of differences in water access to the heme or isoform-dependent differences in the substrate-free high spin states as well.


Assuntos
Sistema Enzimático do Citocromo P-450/química , Água/química , Dicroísmo Circular , Espectroscopia de Ressonância de Spin Eletrônica , Ligadura , Isoformas de Proteínas/química
13.
Biochemistry ; 51(32): 6441-57, 2012 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-22809252

RESUMO

In comparison to imidazole (IMZ) and 1,2,4-triazole (1,2,4-TRZ), the isosteric 1,2,3-triazole (1,2,3-TRZ) is unrepresented among cytochrome P450 (CYP) inhibitors. This is surprising because 1,2,3-TRZs are easily obtained via "click" chemistry. To understand this underrepresentation of 1,2,3-TRZs among CYP inhibitors, thermodynamic and density functional theory computational studies were performed with unsubstituted IMZ, 1,2,4-TRZ, and 1,2,3-TRZ. The results indicate that the lower affinity of 1,2,3-TRZ for the heme iron includes a large unfavorable entropy term likely originating in solvent-1,2,3-TRZ interactions; the difference is not solely due to differences in the enthalpy of heme-ligand interactions. In addition, the 1,2,3-TRZ fragment was incorporated into a well-established CYP3A4 substrate and mechanism-based inactivator, 17-α-ethynylestradiol (17EE), via click chemistry. This derivative, 17-click, yielded optical spectra consistent with low-spin ferric heme iron (type II) in contrast to 17EE, which yields a high-spin complex (type I). Furthermore, the rate of CYP3A4-mediated metabolism of 17-click was comparable to that of 17EE, with a different regioselectivity. Surprisingly, continuous-wave electron paramagnetic resonance (EPR) and HYSCORE EPR spectroscopy indicate that 17-click does not displace water from the sixth axial ligand position of CYP3A4 as expected for a type II ligand. We propose a binding model in which 17-click pendant 1,2,3-TRZ hydrogen bonds with the sixth axial water ligand. The results demonstrate the potential for 1,2,3-TRZ to form metabolically labile water-bridged low-spin heme complexes, consistent with recent evidence that nitrogenous type II ligands of CYPs can be efficiently metabolized. The specific case of [CYP3A4·17-click] highlights the risk of interpreting CYP-ligand complex structure on the basis of optical spectra.


Assuntos
Sistema Enzimático do Citocromo P-450/química , Heme/química , Triazóis/química , Água/química , Química Click , Citocromo P-450 CYP3A/química , Inibidores do Citocromo P-450 CYP3A , Inibidores das Enzimas do Citocromo P-450 , Espectroscopia de Ressonância de Spin Eletrônica , Etinilestradiol/análogos & derivados , Etinilestradiol/síntese química , Etinilestradiol/química , Imidazóis/química , Isomerismo , Cinética , Espectroscopia de Ressonância Magnética , Ligação Proteica , Teoria Quântica , Termodinâmica , Triazóis/síntese química
14.
Arch Biochem Biophys ; 507(1): 56-65, 2011 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-20939998

RESUMO

Cytochrome P450s (CYPs) are heme-containing monooxygenases that contribute to an enormous range of enzymatic function including biosynthetic and detoxification roles. This review summarizes recent studies concerning interactions of CYPs with ligands including substrates, inhibitors, and diatomic heme-ligating molecules. These studies highlight the complexity in the relationship between the heme spin state and active site occupancy, the roles of water in directing protein-ligand and ligand-heme interactions, and the details of interactions between heme and gaseous diatomic CYP ligands. Both kinetic and thermodynamic aspects of ligand binding are considered.


Assuntos
Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Animais , Humanos , Ligantes , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Termodinâmica
15.
Arch Biochem Biophys ; 504(2): 182-9, 2010 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-20836986

RESUMO

4-Hydroxy-2-trans-nonenal (HNE) is a lipid peroxidation product that contributes to the pathophysiology of several diseases with components of oxidative stress. The electrophilic nature of HNE results in covalent adduct formation with proteins, fatty acids and DNA. However, it remains unclear whether enzymes that metabolize HNE avoid inactivation by it. Glutathione transferase A4-4 (GST A4-4) plays a significant role in the elimination of HNE by conjugating it with glutathione (GSH), with catalytic activity toward HNE that is dramatically higher than the homologous GST A1-1 or distantly related GSTs. To determine whether enzymes that metabolize HNE resist its covalent adduction, the rates of adduction of these GST isoforms were compared and the functional effects of adduction on catalytic properties were determined. Although GST A4-4 and GST A1-1 have striking structural similarity, GST A4-4 was insensitive to adduction by HNE under conditions that yield modest adduction of GST A1-1 and extensive adduction of GST P1-1. Furthermore, adduction of GST P1-1 by HNE eliminated its activity toward the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and toward HNE itself. HNE effects on GST A4-4 and A1-1 were less significant. The results indicate that enzymes that metabolize HNE may have evolved structurally to resist covalent adduction by it.


Assuntos
Aldeídos/química , Glutationa Transferase/química , Catálise , Cristalografia por Raios X , Dinitroclorobenzeno/química , Glutationa S-Transferase pi/química , Isoenzimas/química , Cinética , Modelos Moleculares , Conformação Proteica
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