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Pavan, Valeria (2016) Design, synthesis and biochemical characterization of Fingolimod analogs for targeting PP2A. [Tesi di dottorato]

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Abstract (inglese)

PP2A (Protein Phosphatase 2A) is a Ser/Thr phosphatase ubiquitously expressed and highly conserved among the species (especially eukariotes). It is involved in delicate signaling pathways, and overall it has the effect to enhance pro-apoptotic and anti-proliferative events; a decrease of its activity makes anti-apoptotic and proliferative signals prevail. Its activity, as the activity of any other phosphatase, is counterbalanced by protein kinases; while the latter have been widely studied through the years, the role of PP2A was taken into account much more recently, and together with that the possibility to consider it as a drug target has been exploited. Indeed, PP2A is demonstrated to be down-regulated in several forms of tumors, in particular leukemias with a peculiar focus on B-CLL (B cell chronic lymphocytic leukemia). To date, the main hypothesis to explain this is that PP2A is inactivated not directly, but because of the over-expression of the protein SET, its endogenous inhibitor. In these cases, the capability of the cell to die is strongly impaired, so trying to restore PP2A activity is nowadays being exploited as a new, possible anti-cancer therapy.
Fingolimod (IUPAC name 2-amino-2-[2-(4-octylphenyl)ethyl]propan-1,3-diol) has been approved in 2010 for the treatment of relapsing-remitting forms of multiple sclerosis. Its activity is due to the fact that, once internalized by the cell, it gets phosphorylated and reaches a structural similarity with sphingosine-1-phosphate (S1P), mimicking it on T cells: by overstimulating S1P receptors, indeed, Fingolimod can lead to their degradation, avoiding the possibility for T cells to egress secondary lymphoid organs and to keep on damaging neural axons. In more recent years, it has been shown that Fingolimod can restore PP2A activity in tumors as a side effect; the mechanism underlying this event is still not clear, but it is probably due to the interaction Fingolimod has with the PP2A/SET complex. Curiously, this activity needs Fingolimod not to be phosphorylated.
This thesis work presents the synthesis and characterization of a library of Fingolimod analogs which are meant to isolate the pro-apoptotic effect from the immunosuppressive one. Keeping in mind that Fingolimod acts on PP2A only if unphosphorylated, this means designing and synthesizing molecules with modulated structural characteristics, but with a peculiar focus on their phosphorylatability. This approach is meant to obtain new potential anti-cancer drug entities that could restore the capability of the involved cells to die for apoptosis. The compounds were assayed in the laboratory of Prof. Brunati (Department of Molecular Medicine, University of Padova) for their capability to restore PP2A activity in B-CLL cells. In this sense, some promising compounds were obtained and the results of the biochemical assays on the most performing compound have been here reported.
In addition to this, the aim of producing PP2A (catalytic subunit) and SET proteins through recombinant DNA technique was pursued in order to obtain more details about the structural features involved in the interactions between the proteins themselves, and also the eventual ones between the proteins and the synthesized compounds; this part of the project was performed at Diamond Light Source (UK). Combining all the pieces of information obtained from these aims will lead both to an enhanced knowledge about the structural interactions between PP2A and SET (which to date is lacking) and to the possibility of rationalize eventual structure-activity relationships between the proteins of interest and anti-cancer compounds; this will help in elucidating the structural characteristics which are needed for the molecules to be active, and as a consequence in designing more, improved, rationalized Fingolimod analogs.

Abstract (italiano)

PP2A (Protein Fosfatasi 2A) è una serin/treonin fosfatasi espressa in maniera ubiquitaria e altamente conservata tra le specie (eucarioti in primis). Essa è coinvolta in delicati pathway di segnalazione cellulare, ed ha come effetto complessivo quello di promuovere eventi pro-apoptotici ed anti-proliferativi; una sua diminuita attività comporta un aumento di segnali anti-apoptotici e proliferativi. La sua attività, come quella di ogni fosfatasi, è controbilanciata dalle protein chinasi; mentre queste ultime sono state ampiamente indagate nel corso degli anni, il ruolo di PP2A è stato preso in considerazione solo molto più di recente, e insieme ad esso è stata esplorata la possibilità di considerare l’enzima un target per farmaci. Infatti, è stato dimostrato che l’attività di PP2A è ridotta in svariate forme tumorali, in particolare leucemie e ancor più nel dettaglio nei casi di B-CLL (leucemia linfatica cronica a cellule B). Al momento, la principale ipotesi per spiegare questo fenomeno è che PP2A sia inattivata non tanto in maniera diretta, quanto piuttosto per aumentata espressione della proteina SET, suo inibitore endogeno. In questi casi, la capacità della cellula di entrare in apoptosi è fortemente compromessa, quindi la possibilità di ripristino dell’attività di PP2A è attualmente presa in considerazione come nuova, possibile terapia anti tumorale.
Fingolimod (nome IUPAC 2-ammino-2-[2-(4-ottilfenil)etil]propan-1,3-diolo) è stato approvato nel 2010 per il trattamento della sclerosi multipla in forma recidivante-remitente. La sua attività è legata al fatto che, una volta internalizzato dalla cellula, esso viene fosforilato e diventa strutturalmente simile alla sfingosina-1-fosfato (S1P), mimandone l’azione sui linfociti T: sovrastimolando i recettori per S1P, infatti, Fingolimod può portare alla loro degradazione, evitando che i linfociti T possano uscire dai linfonodi e perpetrare il danneggiamento degli assoni neuronali. Più di recente, è stato dimostrato che Fingolimod può, come effetto collaterale, ristabilire nei tumori l’attività di PP2A; il meccanismo sottostante a questa attività non è stato ancora del tutto chiarito, ma sembra legato all’interazione tra Fingolimod e il complesso PP2A/SET. Curiosamente, questa funzione richiede che Fingolimod non sia fosforilato.
Questo lavoro di tesi presenta la sintesi e la caratterizzazione di una libreria di analoghi di Fingolimod che vogliono isolare l’effetto pro-apoptotico da quello immunosoppressivo. Tenendo a mente che Fingolimod agisce su PP2A solo se non fosforilato, ciò significa progettare e creare molecole con caratteristiche strutturali modulate rispetto al composto preso a modello, ma con un’attenzione particolare alla loro capacità di subire fosforilazione. Questo approccio ha lo scopo di ottenere nuove, potenziali drug entities anti-cancro che possano ripristinare la capacità delle cellule interessate di subire apoptosi. I composti sono stati testati nel laboratorio della Prof. Brunati (Dipartimento di Medicina Molecolare, Università di Padova) per valutare la loro attività verso PP2A in cellule di B-CLL. In questo senso, sono stati ottenuti alcuni composti meritevoli di interesse e vengono qui riportati i risultati dei saggi biologici sul composto più attivo.
Inoltre, è stato perseguito lo scopo di produrre le proteine PP2A (subunità catalitica) e SET tramite tecnica del DNA ricombinante al fine di ottenere dettagli strutturali sull’interazione tra proteine, e sulla eventuale modalità di legame tra queste e i composti sintetizzati; questa parte del progetto è stata svolta presso Diamond Light Source (UK). Il lavoro svolto getta le basi per un approfondito studio strutturale dell’interazione tra PP2A e SET (che, ad oggi, è piuttosto lacunosa), sia alla razionalizzazione delle relazioni struttura-attività tra le proteine di interesse e composti anti-neoplastici; questo potrà aiutare a chiarire le caratteristiche strutturali di cui necessitano le molecole per essere attive, e di conseguenza a progettare in modo razionalizzato analoghi di Fingolimod nuovi e migliorati.

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Tipo di EPrint:Tesi di dottorato
Relatore:Zagotto, Giuseppe
Dottorato (corsi e scuole):Ciclo 28 > Scuole 28 > SCIENZE MOLECOLARI > SCIENZE FARMACEUTICHE
Data di deposito della tesi:28 Gennaio 2016
Anno di Pubblicazione:28 Gennaio 2016
Parole chiave (italiano / inglese):Protein phosphatase 2A, SET/I2PP2A, Fingolimod, medicinal chemistry, molecular biology
Settori scientifico-disciplinari MIUR:Area 03 - Scienze chimiche > CHIM/08 Chimica farmaceutica
Struttura di riferimento:Dipartimenti > Dipartimento di Scienze Chimiche
Codice ID:9304
Depositato il:21 Ott 2016 15:10
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Le url contenute in alcuni riferimenti sono raggiungibili cliccando sul link alla fine della citazione (Vai!) e tramite Google (Ricerca con Google). Il risultato dipende dalla formattazione della citazione.

1. Bononi, A. et al. Protein Kinases and Phosphatases in the Control of Cell Fate. Enzyme Res. 2011, 1–26 (2011). Cerca con Google

2. Ciccone, M., Calin, G. A. & Perrotti, D. From the biology of PP2A to the PADs for therapy of hematologic malignancies. Front. Oncol. 5, 1–10 (2015). Cerca con Google

3. Hunter, T. Protein Kinases and Phosphatases : The Yin and Yang of Protein Phosphorylation and Signaling. Cell 80, 225–236 (1995). Cerca con Google

4. Virshup, D. M. Protein phosphatase 2A : a panoply of enzymes. Curr. Opin. Cell Biol. 12, 180–185 (2000). Cerca con Google

5. Perrotti, D. & Neviani, P. Protein phosphatase 2A : a target for anticancer therapy. Lancet Oncol. 14, e229–e238 (2013). Cerca con Google

6. Smith, A. M., Roberts, K. G. & Verrills, N. M. in Myeloid Leuk. - Basic Mech. Leukomiogenes. 123–148 (2011). Cerca con Google

7. Agrawal, M., Garg, R. J., Cortes, J. & Quintás-Cardama, A. Tyrosine kinase inhibitors: The first decade. Curr. Hematol. Malig. Rep. 5, 70–80 (2010). Cerca con Google

8. Moorhead, G. B. G., Trinkle-Mulcahy, L. & Ulke-Lemee, A. Emerging roles of nuclear protein phosphatases. Nat. Rev. Mol. Cell Biol. 8, 234–244 (2007). Cerca con Google

9. Marks, F., Klingmuller, U. & Muller-Decker, K. Cellular Signal Processing: An Introduction to the Molecular Mechanisms of Signal Transduction. (Garland Science, Taylor and Francis group, 2009). Cerca con Google

10. Samanta, A. et al. Jak2 inhibition dactivates Lyn kinase through the SET-PP2A-SHP1 pathway, causing apoptosis in drug-resistant cells from chronic myelogenous leukenia patients. Oncogene 28, 1669–1681 (2009). Cerca con Google

11. Zolnierowicz, S. Type 2A Protein Phosphatase , the Complex Regulator of Numerous Signaling Pathways. Biochem. Pharmacol. 60, 1225–1235 (2000). Cerca con Google

12. Eichhorn, P. J. A., Creyghton, M. P. & Bernards, R. Protein phosphatase 2A regulatory subunits and cancer. BBA - Rev. Cancer 1795, 1–15 (2009). Cerca con Google

13. Seshacharyulu, P., Pandey, P., Datta, K. & Batra, S. K. Phosphatase : PP2A structural importance , regulation and its aberrant expression in cancer. Cancer Lett. 335, 9–18 (2013). Cerca con Google

14. Oaks, J. & Ogretmen, B. Regulation of PP2A by sphingolipid metabolism and signaling. Front. Oncol. 4, 1–7 (2015). Cerca con Google

15. Kiely, M. & Kiely, P. PP2A: The Wolf in Sheep’s Clothing? Cancers (Basel). 7, 648–669 (2015). Cerca con Google

16. Ramaswamy, K., Spitzer, B. & Kentsis, A. Therapeutic Re-Activation of Protein Phosphatase 2A in Acute Myeloid Leukemia. Front. Oncol. 5, 1–5 (2015). Cerca con Google

17. Haesen, D., Sents, W., Lemaire, K., Hoorne, Y. & Janssens, V. The Basic Biology of PP2A in Hematologic Cells and Malignancies. Front. Oncol. 4, 1–11 (2014). Cerca con Google

18. Janssens, V. & Goris, J. Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem. J. 353, 417–439 (2001). Cerca con Google

19. Perrotti, D. & Neviani, P. Protein phosphatase 2A (PP2A), a drugable tumor suppressor in Ph1(+) leukemias. Cancer Metastasis Rev. 27, 159–168 (2008). Cerca con Google

20. Groves, M. R. et al. The Structure of the Protein Phosphatase 2A PR65 / A Subunit Reveals the Conformation of Its 15 Tandemly Repeated HEAT Motifs. Cell 96, 99–110 (1999). Cerca con Google

21. Zhou, J., Pham, H., Ruediger, R. & Walter, G. Characterization of the Aalpha and Abeta subunit isoforms of protein phosphatases 2A : differences in expression, subunit interaction, and evolution. Biochem. J. 369, 387–398 (2003). Cerca con Google

22. Janssens, V., Goris, J. & Van Hoof, C. PP2A: The expected tumor suppressor. Curr. Opin. Genet. Dev. 15, 34–41 (2005). Cerca con Google

23. Cooper, G. in Cell A Mol. Approach (Sunderland (MA): Sinauer Associates, 2000). Cerca con Google

24. http://www.bdbiosciences.com. (2015). Vai! Cerca con Google

25. Elmore, S. Apoptosis: a review of programmed cell death. Toxicol. Pathol. 35, 495–516 (2007). Cerca con Google

26. Perrotti, D. & Neviani, P. Protein phosphatase 2A: a target for anticancer therapy. Lancet. Oncol. 14, e229–38 (2013). Cerca con Google

27. Bialojan, C. & Takai, A. Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Biochem. J. 256, 283–290 (1988). Cerca con Google

28. Wera, S. et al. Deregulation of translational control of the 65-kDa regulatory subunit (PR65 alpha) of protein phosphatase 2A leads to multinucleated cells. J. Biol. Chem. 270, 21374–21381 (1995). Cerca con Google

29. Koma, Y., Ito, A., Watabe, K., Kimura, S. H. & Kitamura, Y. A truncated isoform of the PP2A B56 γ regulatory subunit reduces irradiation-induced Mdm2 phosphorylation and could contribute to metastatic melanoma cell radioresistance. Histol. Histopathol. 19, 391–400 (2004). Cerca con Google

30. Ito, A. et al. A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation. EMBO J. 19, 562–571 (2000). Cerca con Google

31. Wang, S. et al. Alterations of the PPP2R1B gene in human lung and colon cancer. Science (80-. ). 282, 284–287 (1998). Cerca con Google

32. Takagi, Y. et al. Alterations of the PPP2R1B gene located at 11q23 in human colorectal cancers. Gut 47, 268–71 (2000). Cerca con Google

33. Chen, W. et al. Identification of specific PP2A complexes involved in human cell transformation. Cancer Cell 5, 127–136 (2004). Cerca con Google

34. Silverstein, A. M., Barrow, C. A., Davis, A. J. & Mumby, M. C. Actions of PP2A on the MAP kinase pathway and apoptosis are mediated by distinct regulatory subunits. Proc. Natl. Acad. Sci. 99, 4221–4226 (2002). Cerca con Google

35. Janssens, V. & Rebollo, A. The Role and Therapeutic Potential of Ser / Thr Phosphatase PP2A in Apoptotic Signalling Networks in Human Cancer Cells. Curr. Mol. Med. 12, 1–19 (2012). Cerca con Google

36. Reilly, P. T., Yu, Y., Hamiche, A. & Wang, L. Cracking the ANP32 whips: important functions, unequal requirement, and hints at disease implications. Bioessays 36, 1062–1071 (2014). Cerca con Google

37. Beresford, P. J. et al. Granzyme A Activates an Endoplasmic Reticulum-associated Caspase-independent Nuclease to Induce Single-stranded DNA Nicks. J. Biol. Chem. 276, 43285–43293 (2001). Cerca con Google

38. Saddoughi, S. A. et al. Sphingosine analogue drug FTY720 targets I2PP2A / SET and mediates lung tumour suppression via activation of PP2A-RIPK1- dependent necroptosis. EMBO Mol. Med. 5, 105–121 (2013). Cerca con Google

39. Bai, X.-L. et al. Inhibition of protein phosphatase 2A enhances cytotoxicity and accessibility of chemotherapeutic drugs to hepatocellular carcinomas. Mol. Cancer Ther. 13, 2062–2072 (2014). Cerca con Google

40. Lu, J. et al. Inhibition of serine/threonine phosphatase PP2A enhances cancer chemotherapy by blocking DNA damage induced defence mechanisms. Proc. Natl. Acad. Sci. 106, 11697–11702 (2009). Cerca con Google

41. Suganuma, M. et al. Calyculin A, an inhibitor of protein phosphatases, a potent tumor promoter on CD-1 mouse skin. Cancer Res. 50, 3521–3525 (1990). Cerca con Google

42. Chatfield, K. & Eastman, A. Inhibitors of protein phosphatases 1 and 2A differentially prevent intrinsic and extrinsic apoptosis pathways. Biochem. Biophys. Res. Commun. 323, 1313–1320 (2004). Cerca con Google

43. Switzer, C. H. et al. Nitric oxide and protein phosphatase 2A provide novel therapeutic opportunities in ER-negative breast cancer. Trends Pharmacol. Sci. 32, 644–651 (2011). Cerca con Google

44. Switzer, C. H. et al. Dithiolethione compounds inhibit Akt signaling in human breast and lung cancer cells by increasing PP2A activity. Oncogene 28, 3837–3846 (2009). Cerca con Google

45. Neviani, P. & Perrotti, D. SETting OP449 into the PP2A-activating drug family. Clin. Cancer Res. 20, 2026–8 (2014). Cerca con Google

46. Vitek, M. P., Ribaudo, G. & Christensen, D. J. ApoE peptide dimers and uses thereof. WO2011/085110A1 (2011). Cerca con Google

47. Cada, D., Levien, T. & Baker, D. Formulary Drug Reviews - Fingolimod. Hosp. Pharm. 46, 122–129 (2011). Cerca con Google

48. Tavazzi, E., Rovaris, M. & La Mantia, L. Drug therapy for multiple sclerosis. Can. Med. Assoc. J. 186, 833–840 (2014). Cerca con Google

49. Brinkmann, V., Billich, A., Baumruker, T. & Heining, P. Fingolimod (FTY720): discovery and development of an oral drug to treat multiple sclerosis. Nat. Publ. Gr. 9, 883–897 (2010). Cerca con Google

50. Miyake, Y., Kozutsumi, Y., Nakamura, S., Fujita, T. & Kawasaki, T. Serine palmitoyltransferase is the primary target of sphingosine-like immunosuppressant, ISP-1/myriocin. Biochem. Biophys. Res. Commun. 211, 396–403 (1995). Cerca con Google

51. Hofrichter, M. in Ind. Appl. (Springer Science & Business Media, 2010). Cerca con Google

52. Sanchez, T. & Hla, T. Structural and functional characteristics of S1P receptors. J. Cell. Biochem. 92, 913–922 (2004). Cerca con Google

53. Chiba, K. & Adachi, K. Sphingosine 1-Phosphate Receptor 1 as a Useful Target for Treatment of Multiple Sclerosis. Pharmaceuticals 1, 514–528 (2012). Cerca con Google

54. Hanson, M. A. & Peach, R. Structural Biology of the S1P 1 Receptor. (2014). doi:10.1007/978-3-319-05879-5 Cerca con Google

55. Neviani, P. et al. FTY720 , a new alternative for treating blast crisis chronic myelogenous leukemia and Philadelphia chromosome – positive acute lymphocytic leukemia. J. Clin. Invest. 117, 2408–2421 (2007). Cerca con Google

56. Leonard, B. Leukemia: a research report. (Diane Pub Co, 1993). Cerca con Google

57. Guenova, M. & Balatzenko, G. Leukemia. (InTech, 2013). doi:10.13140/2.1.3583.4888 Cerca con Google

58. http://www.cancer.gov/research/progress/snapshots/leukemia. Vai! Cerca con Google

59. http://www.cancer.org/cancer/leukemia-chroniclymphocyticcll/detailedguide/leukemia-chronic-lymphocytic-key-statistics. Vai! Cerca con Google

60. Fecteau, J.-F. & Kipps, T. J. Structure and function of the hematopoietic cancer niche: focus on chronic lymphocytic leukemia. Front Biosci (Schol Ed) 4, 61–73 (2012). Cerca con Google

61. Chiorazzi, N., Rai, K. R. & Ferrarini, M. Chronic Lymphocytic Leukemia. N. Engl. J. Med. 352, 804–815 (2005). Cerca con Google

62. Antosz, H. et al. IL-6, IL-10, c-Jun and STAT3 expression in B-CLL. Blood Cells. Mol. Dis. 54, 258–265 (2015). Cerca con Google

63. Herishanu, Y., Katz, B.-Z., Lipsky, A. & Wiestner, A. Biology of chronic lymphocytic leukemia in different microenvironments: clinical and therapeutic implications. Hematol. Oncol. Clin. North Am. 27, 173–206 (2013). Cerca con Google

64. Caligaris-Cappio, F. & Ghia, P. Novel insights in chronic lymphocytic leukemia: Are we getting closer to understanding the pathogenesis of the disease? J. Clin. Oncol. 26, 4497–4503 (2008). Cerca con Google

65. Yang, Y., Huang, Q., Lu, Y., Li, X. & Huang, S. Reactivating PP2A by FTY720 as a novel therapy for AML with c-KIT tyrosine kinase domain mutation. J. Cell. Biochem. 113, 1314–1322 (2012). Cerca con Google

66. Cristòbal, I. et al. SETBP1 overexpression is a novel leukemogenic mechanism that predicts adverse outcome in elderly patients with acute myeloid leukemia. Blood 115, 615–626 (2010). Cerca con Google

67. Neviani, P. et al. PP2A-activating drugs selectively eradicate TKI-resistant chronic myeloid leukemic stem cells. 123, (2013). Cerca con Google

68. Kalla, C. et al. Analysis of 11q22 – q23 deletion target genes in B-cell chronic lymphocytic leukaemia : Evidence for a pathogenic role of NPAT, CUL5, and PPP2R1B. Eur. J. Cancer 3, 1328–1335 (2007). Cerca con Google

69. Christensen, D. J. et al. SET oncoprotein overexpression in B-cell chronic lymphocytic leukemia and non-Hodgkin lymphoma : a predictor of aggressive disease and a new treatment target. Blood 118, 4150–4159 (2011). Cerca con Google

70. Zonta, F. et al. Lyn sustains oncogenic signaling in chronic lymphocytic leukemia by strengthening SET-mediated inhibition of PP2A. Blood 125, 3747–3755 (2015). Cerca con Google

71. Sontag, J.-M. & Sontag, E. Protein phosphatase 2A dysfunction in Alzheimer’s disease. Front. Mol. Neurosci. 7, 16 (2014). Cerca con Google

72. Gamliel, A., Afri, M. & Frimer, A. A. Determining radical penetration of lipid bilayers with new lipophilic spin traps. Free Radic. Biol. Med. 44, 1394–1405 (2008). Cerca con Google

73. Kiuchi, M. et al. Synthesis and Immunosuppressive Activity of 2-Substituted 2-Aminopropane-1,3-diols and 2-Aminoethanols. J. Med. Chem. 43, 2946–2961 (2000). Cerca con Google

74. Fox, M. A. & Whitesell, J. K. Organic Chemistry - Third edition. (Jonas and Barlett, 2004). Cerca con Google

75. Bruckner, R. Organic Mechanisms. Reactions, stereochemistry and synthesis. Eff. Br. mindfulness Interv. acute pain Exp. An Exam. Individ. Differ. 1, (Springer, 2010). Cerca con Google

76. Smith, M. & March, J. March’s advanced organic chemistry: reactions, mechanisms and structures. (Wiley Interscience, 2007). Cerca con Google

77. Clayden, J. & Greeves, N. Organic Chemistry. (Oxford University Press, 2000). Cerca con Google

78. Wnuk, S. F. & Robins, M. J. Antimony(III) chloride exerts potent catalysis of the conversion of sulfoxides to alpha-fluoro thioethers with (diethylamino)sulfur trifluoride. J. Org. Chem. 55, 4757–4760 (1990). Cerca con Google

79. Neuman, R. C. in Org. Chem. (2013). Cerca con Google

80. http://www.cgl.ucsf.edu/chimera/. Vai! Cerca con Google

81. http://avogadro.cc. Vai! Cerca con Google

82. Neuhaus, D. & Williamson, M. The Nuclear Overhauser Effect in structural and conformational analysis. (Wiley-WHC, 2000). Cerca con Google

83. http://autodock.scripps.edu/resources/references. Vai! Cerca con Google

84. Portelli, M. On the synthesis of beta-phenylalanine. Gazz. Chim. Ital. 119, 215–216 (1989). Cerca con Google

85. http://web.expasy.org/compute_pi/. Vai! Cerca con Google

86. Huttunen, K. et al. Novel cyclic phosphate prodrug approach for cytochrome P450-activated drugs containing an alcohol functionality. Pharm. Res. 24, 679–687 (2007). Cerca con Google

87. Bird, L. OPPF-UK Standard Protocols : Cloning and Expression Screening. (2012). Cerca con Google

88. Nettleship, J. OPPF-UK Standard Protocols : Scale-up and Purification. (2015). Cerca con Google

89. Berrow, N. S. et al. A versatile ligation-independent cloning method suitable for high-throughput expression screening applications. Nucleic Acids Res. 35, e45 (2007). Cerca con Google

90. Xing, Y. et al. Structure of Protein Phosphatase 2A Core Enzyme Bound to Tumor-Inducing Toxins. Cell 127, 341–353 (2006). Cerca con Google

91. Ikehara, T., Shinjo, F., Ikehara, S., Imamura, S. & Yasumoto, T. Baculovirus expression , purification , and characterization of human protein phosphatase 2A catalytic subunits alpha and beta. Protein Expr. Purif. 45, 150–156 (2006). Cerca con Google

92. Myles, T., Schmidt, K., Evans, D. R. H., Cron, P. & Hemmings, B. A. Active-site mutations impairing the catalytic function of the catalytic subunit of human protein phosphatase 2A permit baculovirus-mediated overexpression in insect cells. Biochem. J. 232, 225–232 (2001). Cerca con Google

93. Studier, F. W. Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41, 207–234 (2005). Cerca con Google

94. Muto, S. et al. Relationship between the structure of SET/TAF-Ibeta/INHAT and its histone chaperone activity. Proc. Natl. Acad. Sci. U. S. A. 104, 4285–4290 (2007). Cerca con Google

95. Muto, S. et al. Purification, crystallization and preliminary X-ray diffraction analysis of human oncoprotein SET/TAF-1β. Acta Crystallogr. Sect. D - Biol. christallography 60, 712–714 (2004). Cerca con Google

96. CambridgeSoft Corporation. ChemDraw Ultra 8.0. (2003). Cerca con Google

97. Meyer, B. & Peters, T. NMR spectroscopy techniques for screening and identifying ligand binding to protein receptors. Angew. Chemie - Int. Ed. 42, 864–890 (2003). Cerca con Google

98. https://www.oppf.rc-harwell.ac.uk/Opiner/AddTemplate. Vai! Cerca con Google

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