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1.
Biochemistry ; 55(2): 348-59, 2016 Jan 19.
Artículo en Inglés | MEDLINE | ID: mdl-26704937

RESUMEN

The cyclooxygenase enzymes (COX-1 and COX-2) are the therapeutic targets of nonsteroidal anti-inflammatory drugs (NSAIDs). Neutralization of the carboxylic acid moiety of the NSAID indomethacin to an ester or amide functionality confers COX-2 selectivity, but the molecular basis for this selectivity has not been completely revealed through mutagenesis studies and/or X-ray crystallographic attempts. We expressed and assayed a number of divergent secondary shell COX-2 active site mutants and found that a COX-2 to COX-1 change at position 472 (Leu in COX-2, Met in COX-1) reduced the potency of enzyme inhibition by a series of COX-2-selective indomethacin amides and esters. In contrast, the potencies of indomethacin, arylacetic acid, propionic acid, and COX-2-selective diarylheterocycle inhibitors were either unaffected or only mildly affected by this mutation. Molecular dynamics simulations revealed identical equilibrium enzyme structures around residue 472; however, calculations indicated that the L472M mutation impacted local low-frequency dynamical COX constriction site motions by stabilizing the active site entrance and slowing constriction site dynamics. Kinetic analysis of inhibitor binding is consistent with the computational findings.


Asunto(s)
Amidas/química , Ciclooxigenasa 2/química , Ciclooxigenasa 2/metabolismo , Ésteres/química , Indometacina/farmacología , Biología Computacional , Ciclooxigenasa 2/genética , Activación Enzimática/efectos de los fármacos , Estructura Secundaria de Proteína , Relación Estructura-Actividad
2.
Chem Res Toxicol ; 25(2): 454-61, 2012 Feb 20.
Artículo en Inglés | MEDLINE | ID: mdl-22211372

RESUMEN

Oxidative stress triggers DNA and lipid peroxidation, leading to the formation of electrophiles that react with DNA to form adducts. A product of this pathway, (3-(2'-deoxy-ß-d-erythro-pentofuranosyl)-pyrimido[1,2-α]purine-10(3H)-one), or M(1)dG, is mutagenic in bacterial and mammalian cells and is repaired by the nucleotide excision repair pathway. In vivo, M(1)dG is oxidized to a primary metabolite, (3-(2-deoxy-ß-d-erythro-pentofuranosyl)-pyrimido[1,2-α]purine-6,10(3H,5H)-dione, or 6-oxo-M(1)dG, which is excreted in urine, bile, and feces. We have developed a specific monoclonal antibody against 6-oxo-M(1)dG and have incorporated this antibody into a procedure for the immunoaffinity isolation of 6-oxo-M(1)dG from biological matrices. The purified analyte is quantified by LC-MS/MS using a stable isotope-labeled analogue ([(15)N(5)]-6-oxo-M(1)dG) as an internal standard. Healthy male Sprague-Dawley rats excreted 6-oxo-M(1)dG at a rate of 350-1893 fmol/kg·d in feces. This is the first report of the presence of the major metabolite of M(1)dG in rodents without exogenous introduction of M(1)dG.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Aductos de ADN/análisis , Desoxiguanosina/análisis , Animales , Línea Celular Tumoral , Cromatografía Liquida , Aductos de ADN/inmunología , Aductos de ADN/metabolismo , Desoxiguanosina/análogos & derivados , Desoxiguanosina/inmunología , Desoxiguanosina/metabolismo , Ensayo de Inmunoadsorción Enzimática , Heces/química , Masculino , Ratones , Ratones Endogámicos BALB C , Ratas , Ratas Sprague-Dawley , Espectrometría de Masas en Tándem
3.
Biochemistry ; 48(4): 800-9, 2009 Feb 03.
Artículo en Inglés | MEDLINE | ID: mdl-19132922

RESUMEN

Non-invasive strategies for the analysis of endogenous DNA damage are of interest for the purpose of monitoring genomic exposure to biologically produced chemicals. We have focused our research on the biological processing of DNA adducts and how this may impact the observed products in biological matrixes. Preliminary research has revealed that pyrimidopurinone DNA adducts are subject to enzymatic oxidation in vitro and in vivo and that base adducts are better substrates for oxidation than the corresponding 2'-deoxynucleosides. We tested the possibility that structurally similar exocyclic base adducts may be good candidates for enzymatic oxidation in vitro. We investigated the in vitro oxidation of several endogenously occurring etheno adducts [1,N(2)-epsilon-guanine (1,N(2)-epsilon-Gua), N(2),3-epsilon-Gua, heptanone-1,N(2)-epsilon-Gua, 1,N(6)-epsilon-adenine (1,N(6)-epsilon-Ade), and 3,N(4)-epsilon-cytosine (3,N(4)-epsilon-Cyt)] and their corresponding 2'-deoxynucleosides. Both 1,N(2)-epsilon-Gua and heptanone-1,N(2)-epsilon-Gua were substrates for enzymatic oxidation in rat liver cytosol; heteronuclear NMR experiments revealed that oxidation occurred on the imidazole ring of each substrate. In contrast, the partially or fully saturated pyrimidopurinone analogues [i.e., 5,6-dihydro-M(1)G and 1,N(2)-propanoguanine (PGua)] and their 2'-deoxynucleoside derivatives were not oxidized. The 2'-deoxynucleoside adducts, 1,N(2)-epsilon-dG and 1,N(6)-epsilon-dA, underwent glycolytic cleavage in rat liver cytosol. Together, these data suggest that multiple exocyclic adducts undergo oxidation and glycolytic cleavage in vitro in rat liver cytosol, in some instances in succession. These multiple pathways of biotransformation produce an array of products. Thus, the biotransformation of exocyclic adducts may lead to an additional class of biomarkers suitable for use in animal and human studies.


Asunto(s)
Aductos de ADN/química , Aductos de ADN/metabolismo , Glucólisis/fisiología , Peroxidación de Lípido/fisiología , Nucleósidos de Purina/química , Nucleósidos de Purina/metabolismo , Animales , Bovinos , Reparación del ADN , Guanina/análogos & derivados , Guanina/química , Guanina/metabolismo , Humanos , Estrés Oxidativo/fisiología , Nucleósidos de Pirimidina/química , Nucleósidos de Pirimidina/metabolismo , Ratas
4.
Chem Res Toxicol ; 20(3): 550-7, 2007 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-17311424

RESUMEN

Oxidative damage is considered a major contributing factor to genetic diseases including cancer. Our laboratory is evaluating endogenously formed DNA adducts as genomic biomarkers of oxidative injury. Recent efforts have focused on investigating the metabolic stability of adducts in vitro and in vivo. Here, we demonstrate that the base adduct, M1G, undergoes oxidative metabolism in vitro in rat liver cytosol (RLC, Km = 105 microM and vmax/Km = 0.005 min-1 mg-1) and in vivo when administered intravenously to male Sprague Dawley rats. LC-MS analysis revealed two metabolites containing successive additions of 16 amu. One- and two-dimensional NMR experiments showed that oxidation occurred first at the 6-position of the pyrimido ring, forming 6-oxo-M1G, and then at the 2-position of the imidazole ring, yielding 2,6-dioxo-M1G. Authentic 6-oxo-M1G was chemically synthesized and observed to undergo metabolism to 2,6-dioxo-M1G in RLC (Km = 210 microM and vmax/Km = 0.005 min-1 mg-1). Allopurinol partially inhibited M1G metabolism (75%) and completely inhibited 6-oxo-M1G metabolism in RLC. These inhibition studies suggest that xanthine oxidase is the principal enzyme acting on M1G in RLC and the only enzyme that converts 6-oxo-M1G to 2,6-dioxo-M1G. Both M1G and 6-oxo-M1G are better substrates (5-fold) for oxidative metabolism in RLC than the deoxynucleoside, M1dG. Alternative repair pathways or biological processing of M1dG makes the fate of M1G of interest as a potential marker of oxidative damage in vivo.


Asunto(s)
Aductos de ADN/metabolismo , Purinas/metabolismo , Pirimidinas/metabolismo , Alopurinol/sangre , Animales , Biotransformación , Citosol/metabolismo , Aductos de ADN/farmacocinética , Aductos de ADN/orina , Técnicas In Vitro , Indicadores y Reactivos , Cinética , Hígado/metabolismo , Masculino , Oxidación-Reducción , Purinas/farmacocinética , Purinas/orina , Pirimidinas/farmacocinética , Pirimidinas/orina , Ratas , Ratas Sprague-Dawley
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