Crystallization And Preliminary X-ray Diffraction Studies Of Isoform α1 Of The Human Thyroid Hormone Receptor Ligand-binding Domain

Thyroid hormone receptors (TR) play critical roles in virtually all tissues. The TR ligand-binding domain (LBD) participates in important activities, such as transcriptional activation and repression, through conformational changes induced by hormone binding. Two crystal forms of isoform α1 of the human thyroid hormone receptor LBD (hTRα1) in complex with the thyroid hormones T3 and Triac were obtained. The hTRα1-T3 complex was crystallized in a previously unobserved crystal form (space group P2 12121, a = 59.98, b = 80.80, c = 102.21 Å), with diffraction patterns extending to 1.90 Å resolution on a rotating-anode X-ray source, and in space group C2 (a = 117.54, b = 80.66, c = 62.55 Å, β = 121.04°), with data extending to 2.32 Å resolution. The hTRα1-Triac complex was also crystallized in the new space group P212121, with unit-cell parameters a = 60.01, b = 80.82, c = 102.39 Å its resolution limit extended to 2.20 Å on a home source. Phasing was carried out by the molecular-replacement method and structural refinement is currently in progress. The refined structures may provide insight into the design of new thyromimetics. © 2004 International Union of Crystallography.601018671870Altschul, S.F., Madden, T., Schffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., (1997) Nucleic Acids Res., 25, pp. 3389-3402(1994) Acta Cryst., D50, pp. 760-763. , Collaborative Computational Project, Number 4Darimont, B.D., Wagner, R.L., Apriletti, J.W., Stallcup, M.R., Kushner, P.J., Baxter, J.D., Fletterick, R.J., Yamamoto, K.R., (1998) Genes Dev., 12, pp. 3343-3356Dow, R.L., (2003) Bioorg. Med. Chem. Lett., 13, pp. 379-382Evans, R.M., (1988) Science, 240, pp. 889-895Forrest, D., Vennström, B., (2000) Thyroid, 10, pp. 41-52Garman, E.F., Schneider, T.R., (1997) J. Appl. Cryst., 30, pp. 211-237Gloss, B., Trost, S., Bluhm, W., Swanson, E., Clark, R., Winkfein, R., Janzen, K., Dillmann, W., (2001) Endocrinology, 142, pp. 544-550Jancarik, J., Kim, S.-H., (1991) J. Appl. Cryst., 24, pp. 409-411Johansson, C., Vennström, B., Thoren, P., (1998) Am. J. Physiol., 275, pp. R640-R646Kleywegt, G., Zou, J., Kjeldgaard, M., Jones, T., (2001) International Tables for Crystallography, F, pp. 353-356. , edited by M. G. Rossmann & E. Arnold, ch. 17.1, Dordrecht: Kluwer Academic PublishersLaudet, V., Hanni, C., Coll, J., Catzeflis, F., Stehelin, D., (1992) EMBO J., 11, pp. 1003-1013Nakai, A., Sakurai, A., Bell, G.I., DeGroot, L.J., (1988) Mol. Endocrinol., 2, pp. 1087-1092Navaza, J., (1994) Acta Cryst., A50, pp. 157-163Ribeiro, R.C., Kushner, P., Baxter, J.D., (1995) Annu. Rev. Med., 46, pp. 443-453Sakurai, A., Nakai, A., DeGroot, L.J., (1990) Mol. Cell. Endocrinol., 71, pp. 83-91Schwartz, H.L., Strait, K.A., Ling, N.C., Oppenheimer, J.H., (1992) J. Biol. Chem., 267, pp. 11794-11799Tsai, M., O'Malley, B.W., (1994) Annu. Rev. Biochem., 63, pp. 451-486Wagner, R.L., Apriletti, J.W., McGrath, M.E., West, B.L., Baxter, J.D., Fletterick, R.J., (1995) Nature (London), 378, pp. 690-697Wagner, R.L., Huber, B.R., Shiau, A.K., Kelly, A., Cunha Lima, S.T., Scanlan, T.S., Apriletti, J.W., Fletterick, R.J., (2001) Mol. Endocrinol., 15, pp. 398-410Weinberger, C., Thompson, C.C., Ong, E.S., Lebo, R., Gruol, D.J., Evans, R.M., (1986) Nature (London), 324, pp. 641-646Wikström, L., Johansson, C., Salto, C., Barlow, C., Campos Barros, A., Bass, F., Forrest, D., Vennström, B., (1998) EMBO J., 17, pp. 455-461Winn, M., Dodson, E.J., Ralph, A., (1997) Methods Enzymol., 277, pp. 620-633Ye, L., Li, Y., Mellstrom, K., Mellin, C., Bladh, L.G., Koehler, K., Garg, N., Malm, J., (2003) J. Med. Chem., 46, pp. 1580-1588Yen, P.M., (2001) Physiol Rev., 81, pp. 1097-114

Determinação de ácido rosmarínico em Cordia verbenacea por cromatografia líquida: aplicabilidade em estudo sazonal

RESUMO Neste estudo, uma técnica de cromatografia líquida de alta resolução em fase reversa (CLAE-FR) para a determinação de ácido rosmarínico em Cordia verbenacea foi desenvolvida e validada. A análise de regressão foi avaliada, com observação de uma boa linearidade (r = 0,9997). Os valores obtidos para a precisão e exatidão estão de acordo com as diretrizes do ICH e com a legislação brasileira. Os valores de repetibilidade e precisão intermediária foram 2,79% e 4,76%, respectivamente. Os limites de detecção e de quantificação de ácido rosmarínico foram de 1,92 µg/mL e 5,81 µg/mL, respectivamente. Os resultados mostraram que o método desenvolvido é uma técnica por CLAE-FR de confiança para a determinação de ácido rosmarínico em tintura de C. verbenacea. Além disso, essa metodologia foi aplicada em estudo sazonal, que revela uma correlação positiva relativamente forte entre o período de chuvas e o teor de ácido rosmarínico

Gq-16, a novel peroxisome proliferator-activated receptor γ (pparγ) ligand, promotes insulin sensitization without weight gain

The recent discovery that peroxisome proliferator-activated receptor γ (PPARγ) targeted anti-diabetic drugs function by inhibiting Cdk5-mediated phosphorylation of the receptor has provided a new viewpoint to evaluate and perhaps develop improved insulin-sensitizing agents. Herein we report the development of a novel thiazolidinedione that retains similar anti-diabetic efficacy as rosiglitazone in mice yet does not elicit weight gain or edema, common side effects associated with full PPARγ activation. Further characterization of this compound shows GQ-16 to be an effective inhibitor of Cdk5-mediated phosphorylation of PPARγ. The structure of GQ-16 bound to PPARγ demonstrates that the compound utilizes a binding mode distinct from other reported PPARγ ligands, although it does share some structural features with other partial agonists, such as MRL-24 and PA-082, that have similarly been reported to dissociate insulin sensitization from weight gain. Hydrogen/deuterium exchange studies reveal that GQ-16 strongly stabilizes the β-sheet region of the receptor, presumably explaining the compound's efficacy in inhibiting Cdk5-mediated phosphorylation of Ser-273. Molecular dynamics simulations suggest that the partial agonist activity of GQ-16 results from the compound's weak ability to stabilize helix 12 in its active conformation. Our results suggest that the emerging model, whereby "ideal" PPARγ-based therapeutics stabilize the β-sheet/Ser-273 region and inhibit Cdk5-mediated phosphorylation while minimally invoking adipogenesis and classical agonism, is indeed a valid framework to develop improved PPARγ modulators that retain antidiabetic actions while minimizing untoward effects. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.The recent discovery that peroxisome proliferator-activated receptor γ (PPARγ) targeted anti-diabetic drugs function by inhibiting Cdk5-mediated phosphorylation of the receptor has provided a new viewpoint to evaluate and perhaps develop improved insulin-sensitizing agents. Herein we report the development of a novel thiazolidinedione that retains similar anti-diabetic efficacy as rosiglitazone in mice yet does not elicit weight gain or edema, common side effects associated with full PPARγ activation. Further characterization of this compound shows GQ-16 to be an effective inhibitor of Cdk5-mediated phosphorylation of PPARγ. The structure of GQ-16 bound to PPARγ demonstrates that the compound utilizes a binding mode distinct from other reported PPARγ ligands, although it does share some structural features with other partial agonists, such as MRL-24 and PA-082, that have similarly been reported to dissociate insulin sensitization from weight gain. Hydrogen/deuterium exchange studies reveal that GQ-16 strongly stabilizes the β-sheet region of the receptor, presumably explaining the compound's efficacy in inhibiting Cdk5-mediated phosphorylation of Ser-273. Molecular dynamics simulations suggest that the partial agonist activity of GQ-16 results from the compound's weak ability to stabilize helix 12 in its active conformation. Our results suggest that the emerging model, whereby "ideal" PPARγ-based therapeutics stabilize the β-sheet/Ser-273 region and inhibit Cdk5-mediated phosphorylation while minimally invoking adipogenesis and classical agonism, is indeed a valid framework to develop improved PPARγ modulators that retain antidiabetic actions while minimizing untoward effects28733281692817