Поиск модуляторов активности шаперона Hsp70 и глицеральдегид-3-фосфат дегидрогеназы при помощи методов молекулярного моделирования тема диссертации и автореферата по ВАК РФ 03.01.04, кандидат химических наук Черноризов, Кирилл Александрович

• Черноризов, Кирилл Александрович
• кандидат химических наук
• 2011

Оглавление диссертации кандидат химических наук Черноризов, Кирилл Александрович

СПИСОК ИСПОЛЬЗОВАННЫХ СОКРАЩЕНИЙ-4

ВВЕДЕНИЕ^

1. ЛИТЕРАТУРНЫЙ ОБЗОР-61.1 ШаперонН5р70 -61.1.1 СТРОЕНИЕ ШАЛЕРОНА НБР70 -81.1.2 ЦИКЛ ФУНКЦИОНИРОВАНИЯ Н5Р70 -Ю

1.1.3 Взаимодействие Нбр70 с факторами обмена нуклеотида -121.1.4 Прочие функции Нбр70 и роль шаперонов в апоптотических процессах -141.1.5 Нйр70 и иммунная система -171.1.6 Модуляция шаперонной активности -19 -1.1.7 Обзор трехмерных структур Н5Р70 -231.2 Глицеральдегид-З-фосфат дегидрогеназа -271.2.1 Строение глицеральдегид-3-фосфат дегидрогеназы и механизм ферментативной реакции - 27 -1.2.2 Функции глицеральдегид-3-фосфат дегидрогеназы -321.2.3 Стратегии модуляции функционирования глицеральдегид-3-фосфат дегидрогеназы - 36

1.2.4 Обзор трехмерных структур глицеральдегид-3-фосфат дегидрогеназы - 41

2. ЭКСПЕРИМЕНТАЛЬНАЯ ЧАСТЬ-452.1 Методики молекулярного моделирования -452.1.1 Симуляция молекулярной динамики -462.1.2 Молекулярный докинг и скрининг -493, РЕЗУЛЬТАТЫ И ОБСУЖДЕНИЕ-50

3.1 Поиск ингибитора взаимодействия н5р70 с фактором нуклеотидного обмена 3.1.1 Моделирование структуры АТФазного домена Нбр70 человека

-50-53

3.1.2 Скрининг баз данных низкомолекулярных соединений -58

3.2 Моделирование полноразмерной трехмерной структуры Нбр70 -65

3.2.1 Модель нНбр70, построенная по гомологии со структурами 1У1)\А/ и Юкг -66

3.2.2 конструирование нНзр70 с аБВО, построенным по шаблонам 11ю0 и 2Р32 -69

3.2.2.1 ИНэрУО с а-субдоменом БВО, построенным на основании структуры 11Ю0 -71

3.2.2.2 ИН5р70 с а-субдоменом 5ВР, построенным на основании структуры 2Р32 -74

3.3 Изучение междоменных взаимодействий н№р70 -78

3.3.1 Зона контакта АТФазного и субстрат-связывающего доменов -78

3.3.2 Симуляция раздвижения субдоменов 1 и II -85

3.4 Моделирование глицеральдегид-3-фосфат дегидрогеназы с ковалентно модицифированным cys149 -87

3.4.1 Алгоритм конструирования ковалентно модифицированной глицеральдегид-3-фосфат дегидрогеназы -87

3.4.2 Проверка алгоритма на модели тиоэфирного интермедиата -90

3.5 Поиск ковалентных ингибиторов глицеральдегид-3-фосфат дегидрогеназы /л/ втсо -93

3.6 Экспериментальная проверка потенциальных 5Н-ингибиторов глицеральдегид-3-фосфат дегидрогеназы -97

3.6.1 Изучение ингибирующего действия GSH и NAC на каталитическую активность глицеральдегид-3фосфат дегидрогеназы -100

3.6.2 Селективное ингибирование глицеральдегид-3-фосфатдегидрогеназы сперматозоидов -103

Заключение диссертации по теме «Биохимия», Черноризов, Кирилл Александрович

4. ОСНОВНЫЕ РЕЗУЛЬТАТЫ И ВЫВОДЫ

1. Предложена стратегия модуляции активности шаперона Нвр70 путем использования низкомолекулярных ингибиторов, способных связываться с АТФазным доменом и предотвращать взаимодействия комплекса Нзр70:АДФ:Субстрат с фактором нуклеотидного обмена;

2. Предложены низкомолекулярные ингибиторы, способные стабилизировать комплекс Н5р70:АДФ:Пептид;

3. Разработана модель полноразмерной трехмерной структуры Нзр70 человека;

4. Проведено молекулярное моделирование и описан процесс взаимодействия АТФазного и субстрат-связывающего доменов Нзр70 человека, ключевую роль в котором играют солевые мостики Аг§171 :Ст1и516 и Аг§416:С1и218;

5. Проведено моделирование структур тиоэфирного интермедиата в механизме функционирования ГАФД и коваленгных фермент-ингибиторных комплексов с модифицированным аминокислотным остатком Суя 149 в активном центре фермента;

6. Найдены новые ковалентные ингибиторы ГАФД, в том числе селективные ингибиторы сперматозоидной формы фермента.

Список литературы диссертационного исследования кандидат химических наук Черноризов, Кирилл Александрович, 2011 год

1. Финкельштейн A.B., Птицын O.E. Физика белка. КДУ, Москва, 2005

2. Ritossa P. Problems of prophylactic vaccinations of infants. Riv 1st Sicroter Ital. 1962;37:79-108.

3. Young JC, Agashe VR, Siegers K, Hartl FU. Pathways of chaperone-mediated protein folding in the cytosol. Nat Rev Mol Cell Biol. 2004;5(10):781-91.

4. Noonan EJ, Place RF, Giardina C, Hightower LE. Hsp70B' regulation and function. Cell Stress Chaperones. 2007;12(4):393-402.

5. Song J, Takeda M, Morimoto RI. Bagl-Hsp70 mediates a physiological stress signalling pathway that regulates Raf-l/ERK and cell growth. Nat Cell Biol. 2001;3(3):276-82.

6. Morimoto RT. Cells in stress: transcriptional activation of heat shock genes. Science. 1993 ;259(5100): 1409-1410.

7. Callahan MK, Chaillot D, Jacquin C, Clark PR, Menoret A. Differential acquisition of antigenic peptides by Hsp70 and Hsc70 under oxidative conditions. J Biol Chem. 2002;277(37):33604-9.

8. Brown IR. Heat shock proteins and protection of the nervous system. Ann N Y Acad Sei. 2007;1113:147-58.

9. Multhoff G, Pfister K, Gehrmann M, Hantschel M, Gross C, Hafner M, Hiddemann W. A 14-mer Hsp70 peptide stimulates natural killer (NK) cell activity. Cell Stress Chaperones. 2001;6(4):337-44.

10. Schmitt E, Gehrmann M, Brunei M, Multhoff G, Garrido C. Intracellular and extracellular functions of heat shock proteins: repercussions in cancer therapy. J Leukoc Biol. 2007;81(1): 15-27.

11. Vogel M, Bukau B, Mayer MP. Allosteric regulation of Hsp70 chaperones by a proline switch. Mol Cell. 2006;21 (3):359-67.

12. Nollen EA, Kabakov AE, Brunsting JF, Kanon В, Höhfeld J, Kampinga HH. Modulation of in vivo HSP70 chaperone activity by Hip and Bag-1. J Biol Chem. 2001;276(7):4677-82.

13. O'Brien MC, Flaherty KM, McKay DB. Lysine 71 of the chaperone protein Hsc70 Is essential for ATP hydrolysis. J Biol Chem. 1996;271(27):15874-S.

14. Hennessy F, Nicoll WS, Zimmermann R, Cheetham ME, Blatch GL. Not all J domains arc created equal: implications for the specificity of IIsp40-Hsp70 interactions. Protein Sei. 2005; 14(7): 1697-1709. ч

15. Гужова И, Новоселов С, Маргулис Б. Шаперон Hsp70 и перспективы его использования в противоопухолевой терапии. Цитология. 2005;47(3): 187-199.

16. Osipiuk J, Walsh MA, Freeman ВС, Morimoto RI, Joachimiak A. Stmcture of a new crystal form of human Hsp70 ATPase domain. Acta Crystallogr. D Biol. Crystallogr. 1999;55(Pt 5): 11051107.

17. Mayer MP, Bukau B. Hsp70 chaperones: cellular functions and molecular mechanism. Cell. Mol. Life Sci. 2005;62(6):670-684.

18. Chang Y, Sun Y, Wang C, Hsiao C. Crystal structures of the 70-kDa heat shock proteins in domain disjoining conformation. J. Biol. Chem. 2008;283(22): 15502-15511.

19. Freeman ВС, Myers MP, Schumacher R, Morimoto RI. Identification of a regulatory motif in Hsp70 that affects ATPase activity, substrate binding and interaction with HDJ-1. EMBO J. 1995; 14( 10):2281 -92.

20. Qian X, Hou W, Zhengang L, Sha B. Direct interactions between molecular chaperones heat-shock protein (lisp) 70 and Hsp40: yeast Hsp70 Ssal binds the extreme C-terminal region of yeast Hsp40 Sisl. Biochcm J. 2002;361(Pt 1У.27-34.

21. Sondermann H, Scheufler C, Schneider C, Hohfeld J, Haiti FU, Moarefi I. Structure of a Bag/Hsc70 complex: convergent functional evolution of Hsp70 nucleotide exchange factors. Science. 2001 ;291(5508): 1553-7.

22. Brehmcr D, Rudiger S, Gassier CS, Klostermeier D, Packschies L, Reinstein J, Mayer MP, Bukau B. Tuning of chaperone activity of Hsp70 proteins by.modulation of nucleotide exchange. Nat Struct Biol. 2001;8(5):427-32.

23. Stuart JK, Myszka DG, Joss L, Mitchell RS, McDonald SM, Xie Z, Takayama S, Reed JC, Ely KR. Characterization of interactions between the anti-apoptotic protein BAG-1 and Hsc70 molecular chaperones. J Biol Chem. 1998;273(35):22506-14.

24. Mitra A, Shevde LA, Samant RS. Multi-faceted role ofHSP40 in cancer. Clin. Exp. Metastasis. 2009;26(6):559-567.

25. Chou C, Forouhar F, Yeh Y, Shr H, Wang C, Hsiao C. Crystal structure of the C-terminal 10-kDa subdomain of Hsc70. J. Biol. Chem. 2003;278(32):30311-30316.

26. Jiang J, Maes EG, Taylor AB, Wang L, Hinck AP, Lafer EM, Sousa R. Structural basis of J cochaperone binding and regulation of Hsp70. Mol Cell. 2007;28(3):422-33.

27. Li J, Wu Y, Qian X, Sha B. Crystal structure of yeast Sisl peptide-binding fragment and Hsp70 Ssal C-terminal complex. Biochem. J. 2006;398(3):353-360.

28. Wang HG, Takayama S, Rapp UR, Reed JC. Bcl-2 interacting protein, BAG-1, binds to and activates the kinase Raf-1. Proc. Natl. Acad. Sci. U.S.A 1996;93(14):7063-7068.

29. McLellan С A, Raynes DA, Guerriero V. IIspBPl, an Hsp70 cochaperone, has two structuraldomains and is capable of altering the conformation of the Hsp70 ATPase domain. J. Biol. Chem. 2003;278(21): 19017-19022.

30. Polier S, Dragovic Z, Hartl FU, Bracher A. Structural basis for the cooperation of Hsp70 and Hspl 10 chapcrones in protein folding. Cell. 2008;133(6):1068-79.

31. Schuermann JP, Jiang J, Cuellar J, Llorca O, Wang L, Gimenez LE, Jin S, Taylor AB, Demeler B, Morano KA, Hart PJ, Valpuesta JM, Lafer EM, Sousa R. Structure of the Hspll0:Hsc70 nucleotide exchange machine. Mol Cell. 2008;31(2):232-43.

32. Odunuga OO, Longshaw VM, Blatch GL. Hop: more than an Hsp70/Hsp90 adaptor protein. Bioessays. 2004;26( 10): 1058-68.

33. Hartl FU, Hayer-IIartl M. Molecular Chaperones in the.CytosoI: from Nascent Chain to Folded Protein. Science. 2002;295(5561): 1852-1858.

34. Esser C, Alberti S, Hohfeld J. Cooperation of molecular chaperones with the ubiquitin/proteasome system. Biochim Biophys Acta. 2004; 1695(1-3): 171-88.

35. Bronk P, Wenniger JJ, Dawson-Scully K, Guo X, Hong S, Atwood IIL, Zinsmaier KE. Drosophila Hsc70-4 is critical for neurotransmitter exocytosis in vivo. Neuron. 200l;30(2):475-88.

36. Nicholson DW. Caspase structure, proteolytic substrates, and function during apoptotic cell death. Cell Death Differ. 1999;6(11): 1028-1042.

37. Gehrmann M, Brunner M, Pfister K, Reichle A, Kremmer E, Multhoff G. Differential up-regulation of cytosolic and membrane-bound heat shock protein 70 in tumor cells by antiinflammatory drugs. Clin Cancer Res. 2004;10(10):3354-64.

38. Ito A, Honda H, Kobayashi T. Cancer immunotherapy based on intracellular hyperthermia using magnetite nanoparticles: a novel concept of "heat-controlled necrosis" with heat shock protein expression. Cancer Immunol Immunother. 2006;55(3):320-8.

39. Suck G. Novel approaches using natural killer cells in cancer therapy. Semin. Cancer Biol. 2006; 16(5):412-8.

40. Browning MJ, Bodmer WF. MHC antigens and cancer: implications for T-cell surveillance. Curr. Opin. Immunol. 1992;4(5):613-618.

41. Bausero MA, Gastpar R, Multhoff G, Asea A. Alternative mechanism by which IFN-gamma enhances tumor recognition: active release of heat! shock protein 72. J Immunol. 2005;175(5):2900-12.

42. Wang R, Kovalchin JT, Muhlenkamp P, Chandawarkar RY. Exogenous heat shock protein 70 binds macrophage lipid raft microdomain and stimulates phagocytosis, processing, and. MHC-II presentation of antigens. Blood. 2006;107(4): 1636-42.

43. Arispe N, Doh M, Simakova O, Kurganov B, De Maio A. Hsc70 and Hsp70 internet with phosphatidylserine on the surface of PC 12 cells resulting in a decrease of viability. FASEB J.2004; 18(14): 1636-1645.

44. Broquet AH, Thomas G, Masliah J, Trugnan G, Bachelet M. Expression of the molecular chaperone Hsp70 in detergent-resistant microdomains correlates with its membrane delivery and release. J. Biol. Chem. 2003;278(24):21601-21606.

45. Nylandsted J, Brand K, Jaattela M. Heat shock protein 70 is required for the survival of cancer cells. Ann. N. Y. Acad. Sci. 2000;926:122-125.

46. O'Brien MC, McKay DB. Threonine 204 of the chaperone protein Hsc70 influences the structure of the active site, but is not essential for ATP hydrolysis. J. Biol. Chem. 1993;268(32):24323-24329.

47. Wilbanks SM, McKay DB. How potassium affects the activity of the molecular chaperone Hse70. II. Potassium binds specifically in the ATPase active site. J. Biol. Chem. 1995;270(5):2251-2257.

48. Jiang J, Prasad K, Lafer EM, Sousa R. Structural basis of interdomain communication in the Hsc70 chaperone. Mol Cell. 2005;20(4):513-24.

49. Sousa MC, McKay DB. The hydroxyl of threonine 13 of the bovine 70-kDa heat shock cognate protein is essential for transducing the ATP-induced conformational change. Biochemistry. 1998;37(44): 15392-15399.

50. Flaherty KM, DeLuca-Flaherty C, McKay DB. Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein. Nature. 1990;346(6285):623-628.

51. Harrison CJ, Hayer-Hartl M, Di Liberto M, Hartl F, Kuriyan J. Crystal structure of the nucleotide exchange factor GrpE bound to the ATPase domain of the molecular chaperone DnaK. Science. 1997;276(5311):431 -435.

52. Zhu X, Zhao X, Burkholder WF, Gragerov 'A, Ogata CM, Gottesman ME, Hendrickson WA. Structural analysis of substrate binding by the molecular chaperone DnaK. Science. 1996;272(5268): 1606-1614.

53. Shomura Y, Dragovic Z, Chang H, Tzvetkov N, Young JC, Brodsky JL, Gucrriero V, Hartl FU, Bracher A. Regulation of Hsp70 function by HspBPl: structural analysis reveals an alternate mechanism for Hsp70 nucleotide exchange. Mol. Cell. 2005;17(3):367-379.

54. Liu Q, Hendrickson WA. Insights into Hsp70 chaperone activity from a crystal structure of theyeast Hsp 110 Sse 1. Cell. 2007; 131 (1): 106-120.

55. Worrall LJ, Walkinshaw MD. Crystal structure of the C-terminal three-helix bundle subdomain of C. elegans Hsp70. Biochem. Biophys. Res. Commun. 2007;357(1): 105-110.

56. Cupp-Vickery JR, Peterson JC, Ta DT, Vickery LE. Crystal structure of the molecular chapcrone HscA substrate binding domain- complexed with the IscU recognition peptide ELPPVKIHC. J. Mol. Biol. 2004;342(4): 1265-1278.

57. Arakawa A, Handa N, Ohsawa N, Shida M, Kigawa T, Hayashi F, Shirouzu M, Yokoyama S. The C-terminal BAG domain of BAG5 induces conformational changes of the IIsp70 nucleotide -binding domain for A DP-ATP exchange. Structure. 2010;18(3):309-319.

58. Wisniewska M, Karlberg T, Lehtiö L, Johansson I, Kotcnyova T, Moche M, Schüler II. Crystal structures of the ATPase domains of four human Hsp70 isoforms: HSPAlL/Hsp70-hom, HSPA2/Hsp70-2, HSPA6/Hsp70B', andHSPA5/BiP/GRP78. PLoS ONE. 2010;5(l):e8625.

59. Xu Z, Page RC, Gomes MM, Kohli E, Nix JC, Herr AB, Patterson C, Misra S. Structural basis of nucleotide exchange and client binding by the Hsp70 cochaperone Bag2. Nat. Struct. Mol. Biol. 2008; 15(12): 1309-1317.

60. Liebscher M, Roujeinikova A. Allosteric coupling between the lid and interdomain linker in DnaK revealed by inhibitor binding studies. J. Bacteriol. 2009; 191(5): 1456-1462.

61. Jenkins JL, Tanner JJ. High-resolution structure of human D-glyceraldchyde-3-phosphate dehydrogenase. Acta Crystallogr D Biol Crystallogr. 2006;62(Pt 3):290-301.

62. Chuang D, Hough C, Senatorov VV. Glyceraldehyde-3-phosphate dehydrogenase, apoptosis, and neurodegenerative diseases. Annu Rev Pharmacol Toxicol. 2005;45:269-90.

63. Foucault G, Nakano M, Pudles J. Role of lysine-183 in D-glyceraldehyde-3-phosphate dehydrogenases. Properties of the N-acetylated yeast, sturgeon muscle and rabbit muscle enzymes. Eur J Biochem. 1978;83(1):113-23.

64. Sirover MA. New nuclear functions of the glycolytic protein, glyceraldehyde-3-phosphate dehydrogenase, in mammalian cells. J Cell Biochem. 2005;95(l):45-52.

65. Yun M, Park CG, Kim JY, Park HW. Structural analysis of glyceraldehyde 3-phosphate dehydrogenase from Escherichia coli: direct evidence of substrate binding and cofactor-induced conformational changes. Biochemistry. 2000;39(35): 10702-10.

66. Kliman HJ, Steck TL. Association of glyceraldehyde-3-phosphate dehydrogenase with the human red cell membrane. A kinetic analysis. J Biol Chem. 1980;255(13):6314-21.

67. Orru S, Ruoppolo M, Francesc S, Vitagliano L, Marino G, Esposito C. Identification of tissue transglutaminase-reactive lysine residues in glyceraldehyde-3-phosphate dehydrogenase. Protein Sci. 2002; 1 l(l):137-46.

68. Arrasate M, Mitra S, Schweitzer ES, Segal MR, Finkbeiner S. Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death. Nature. 2004;431(7010):805-10.

69. Steffan JS, Agrawal N, Pallos J, Rockabrand E, Trotman LC, Slepko N, llles K, Lukacsovich T, Zhu Y, Cattanco E, Pandolfi PP, Thompson LM, Marsh JL. SUMO modification of I luntingtin and Huntington's disease pathology. Science. 2004;304(5667): 100-104.

70. Suresh S, Bressi JC, Kennedy KJ, Verlinde CL, Gelb MH, Hoi WG. Conformational changes in Leishmania mexicana glyceraldehyde-3-phosphate dehydrogenase induced by designed inhibitors. J Mol Biol. 2001 ;309(2):423-35.

71. Nyasse B, Nono J, Nganso Y, Ngantehou I, Schneider B. Uapaca genus (Euphorbiaceae), a good, source of betulinic acid. Fitoterapia. 2008;80(l):32-24.

72. Gregus Z, Nemeti B. The glycolytic enzyme glyceraidehydc-3-phosphate dehydrogenase works as an arsenate reductase in human red blood cells and rat liver cytosol. Toxicol Sei. 2005;85(2):859-69:

73. Sakai K, Hasumi K, Endo A. Two glyceraldehyde-3-phosphate dehydrogenase isozymes from the koningic acid (heptelidic acid) producer Trichoderma koningii. Eur J Bioehem. 1990; 193(1): 195-202.

74. Fujita SC, Oshima T, Imahori K. Purification and properties of D-glyceraldehyde-3-phosphate dehydrogenase from an extreme thermophile, Thermus thermophilus strain HB 8. Eur J Bioehem. 1976;64(l):57-68.

75. Kim H, Hol WG. Crystal structure of Leishmania mexicana glycosomal glyceraldehyde-3-phosphate dehydrogenase in a new crystal form confirms the putative physiological active site structure. J. Mol. Biol. 1998;278(I):5-11.

76. Isupov MN, Fleming TM, Dalby AR, Crowhurst GS, Bourne PC, Littlechild JA. Crystal structure of the glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaeon Sulfolobus solfataricus. J. Mol. Biol. 1999;291(3):651-660.

77. Tanner JJ, Hecht RM, Krause KL. Determinants of enzyme thermostability observed in the molecular structure of Thermus aquaticus D-glyceraldehyde-3-phosphate dehydrogenase at 25 Angstroms Resolution. Biochemistry. 1996;35(8):2597-2609.

78. Shen YQ, Li J, Song SY, Lin ZJ. Structure of apo-glyceraldehyde-3-phosphate dehydrogenase from Palinurus versicolor. J. Struct. Biol. 2000; 130(1): 1-9.

79. Song SY, Xu YB, Lin ZJ, Tsou CL. Structure of active site carboxymethylated D-gIyceraldehyde-3-phosphate dehydrogenase from Palinurus versicolor. J. Mol. Biol. 1999;287(4):719-725.

80. Shen YQ, Song SY, Lin ZJ. Structures of D-glyceraldehyde-3-phosphate dehydrogenase complexed with coenzyme analogues. Acta Crystallogr. D Biol. Crystallogr. 2002;58(Pt 8): 12871297.

81. Cowan-Jacob SW, Kauftnann M, Anselmo AN, Stark W, Grütter MG. Structure of rabbit-muscle glyceraldehyde-3-phosphate dehydrogenase. Acta Crystallogr. D Biol. Crystallogr. 2003;59(Pt 12):2218-2227.

82. Antonyuk SV, Eady RR, Strange RW, Hasnain SS. The structure of glyceraldehyde 3-phosphate dehydrogenase from Alcaligenes xylosoxidans at 1.7 A resolution. Acta Crystallogr. D Biol. Crystallogr. 2003;59(Pt 5):835-842.

83. Shin, D.H., Thor, J., Yokota, H., Kim, R., Kim, S.H. Crystal structure of MES buffer bound form of glyceraldehyde 3-phosphate dehydrogenase from Escherichia coli. http://www.pdb.org/pdb/explore/explore.do?structureld= 1S7C.

84. Song SY, Gao YG, Zhou JM, Tsou CL. Preliminary crystallographic studies of lobster D-glyceraldehyde-3-phosphate dehydrogenase and the modified enzyme carrying the fluorescent derivative. J. Mol. Biol. 1983; 171 (2):225-228.

85. Satchell JF, Malby RL, Luo CS, Adisa A, Alpyurek AE, Klonis N, Smith BJ, Tilley L, Colman PM. Structure of gIyceraldehyde-3-phosphate dehydrogenase from Plasmodium falciparum. Acta Crystallogr. D Biol. Crystallogr. 2005;61(Pt 9): 1213-1221.

86. Ismail, S.A., Park, H.W. Crystal Structure Analysis of Human liver GAPDH. http://www.pdb. org/pdb/explore/explore.do?structureId= 1ZNQ.

87. I to, K., Arai, R., Kamo-Uchikubo, T., Shirouzu, M., Yokoyama, S. Crystal structure of glyceraldehyde-3-phosphate dehydrogenase from Pyrococcus horikoshii OT3. http://www.pdb.org/pdb/explore/explore.do? structureId=2CZC.

88. Asada," Y., Kunishima, N. Stnictural study of Project ID aq1065 from Aquifex aeolicus VF5. http://www.pdb.org/pdb/explore/explore.do7structurekh12EP7.

89. Jenkins, J.L., Buencamino, R., Tanner, J.J. High Resolution Structures of Thermus.aquaticus Glyceraldehyde-3-Phosphate ■ Dehydrogenase: Role of 220's Loop Motion in- Catalysis. http://www.pdb.org/pdb/explore/explore.do?structureId=2G82.

90. Nikolaev V.K., Leontovich A.M., Drachev V.A., Brodsky L.I. Building multiple alignment using iterative analyzing biopolymers structure dynamic improvement of the initial motif alignment. Biochemistry. 1997;62(6):578-582.

91. Kiefer F, Arnold K, Kunzli M, Bordoli L, Sclnvede T. The SWISS-MODEL Repository and associated resources. Nucleic Acids Res. 2009;37(Database issue):D387-392.

92. Arnold K, Bordoli L, Kopp J, Schwede T. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics. 2006;22(2): 195-201.

93. Van Der Spoel D, Lindahl E, Hess B, Groenhof G, Mark AE, Bercndsen HJC. GROMACS:fast, flexible, and free. J Comput Chem. 2005;26( 16): 1701 -1718.i

94. Stroganov OV, Novikov FN, Stroylov VS, Kulkov V, Chilov GG. Lead finder: an approach to improve accuracy of protein-ligand docking, binding energy estimation, and virtual screening. J Chem Inf Model. 2008;48(12):2371-2385.

95. Guex, N and Peitsch, M.C. Swiss-PdbViewer: A Fast and Easy-to-use PDB Viewer for Macintosh and PC. Protein Data Bank Quaterly Newsletter. 1996;77:7.125. http://opcnbabel.org/wiki/MainPagc.

96. Pavelites JJ, Gao J, Bash PA, Jr ADM. A molecular mechanics force field for NAD+ NADH, and the pyrophosphate groups of nucleotides. Journal of Computational Chemistry. 1997;18(2):221-239.

97. Walker RC, de Souza MM, Mercer IP, Gould IR, Klug DR. Large and Fast Relaxations inside a Protein: Calculation and Measurement of Reorganization Energies in Alcohol Dehydrogenase. The Journal ofPhysical Chemistry B. 2002; 106(44): 11658-11665.

98. Wishart DS, Knox C, Guo AC, Shrivastava S, Hassanali M, Stothard P, Chang Z, Woolsey J. DrugBank: a comprehensive resource for in silico drug discovery and exploration. Nucleic Acids Res. 2006;34(Database issue):D668-72.

99. Wishart DS, Knox C, Guo AC, Cheng D, Shrivastava S, Tzur D, Gautam B, Hassanali M. DrugBank: a knowledgebase for drugs, drug actions and drug targets. Nucleic Acids Res. 2008;36(Database issue):D901-6.134. http://mi.caspur.it/PMDB/main.php.

100. Castrignanö T, De Meo PD, Cozzetto D, Talamo IG, Tramontano A. The PMDB Protein Model

101. Database. Nucleic Acids Res. 2006;34(Database issue):D306-309.136. de Vijldcr JJ, Boers W, Slater EC. Binding and properties of NAD+ in glyccraldehydephosphate dehydrogenase from lobster-tail muscle. Biochim. Biophys. Acta. 1969; 191(2):214-220.