Synthesis, biological activity and mechanistic insights of 1-substituted cyclopropylamine derivatives: A novel class of irreversible inhibitors of histone demethylase KDM1A

European Journal of Medicinal Chemistry

Volumn 86, Issue , 2014, Pages 352-363

  Vianello, Paola ^a   Botrugno, Oronza A ^a   Cappa, Anna ^a   Ciossani, Giuseppe ^b   Dessanti, Paola ^a   Mai, Antonello ^c   Mattevi, Andrea ^b   Meroni, Giuseppe ^a   Minucci, Saverio ^a,d   Thaler, Florian ^a   Tortorici, Marcello ^b   Trifiró, Paolo ^a   Valente, Sergio ^c   Villa, Manuela ^a   Varasi, Mario ^a   Mercurio, Ciro ^a  

^a EUROPEAN INSTITUTE OF ONCOLOGY   (Italy)

^b UNIVERSITY OF PAVIA   (Italy)

^c SAPIENZA UNIVERSITY OF ROME   (Italy)

^d UNIVERSITY OF MILAN   (Italy)

Author keywords

Antiproliferation; Epigenetics; Histone lysine demethylases; Tranylcypromine; X ray crystallography

Indexed keywords

1 ETHYL 2 PHENYL CYCLOPROPANAMINE HYDROCHLORIDE; 1 ETHYL 2 PHENYL CYCLOPROPANECARBOXYLIC ACID; 1 METHYL 2 PHENYL CYCLOPROPANAMINE HYDROCHLORIDE; 1 METHYL 2 PHENYL CYCLOPROPANECARBOXYLIC ACID; ANTINEOPLASTIC AGENT; ETHYL 1 BENZYL 2 PHENYL CYCLOPROPANECARBOXYLATE; ETHYL 1 BENZYL 2 PHENYL CYCLOPROPANECARBOXYLIC ACID; ETHYL 1 ETHYL 2 PHENYL CYCLOPROPANECARBOXYLATE; ETHYL 1 ETHYL 2 PHENYL CYCLOPROPANECARBOXYLIC ACID; ETHYL 1 METHYL 2 PHENYL CYCLOPROPANECARBOXYLATE; ETHYL 1,2 DIPHENYLCYCLOPROPANECARBOXYLATE; ETHYL TRANS 1 (2 NAPHTHYLMETHYL) 2 PHENYL CYCLOPROPANECARBOXYLATE; ETHYL TRANS 1 (2 PHENYLETHYL) 2 PHENYL CYCLOPROPANECARBOXYLATE; ETHYL TRANS 1 BENZYL 2 PHENYL CYCLOPROPANECARBOXYLATE; ETHYL TRANS 1 ETHYL 2 PHENYL CYCLOPROPANECARBOXYLATE; ETHYL TRANS 1 METHYL 2 PHENYL CYCLOPROPANECARBOXYLATE; ETHYL TRANS 2 PHENYL 1 PROPYL CYCLOPROPANECARBOXYLATE; HISTONE DEMETHYLASE KDM1A INHIBITOR; MONOAMINE OXIDASE A INHIBITOR; MONOAMINE OXIDASE B INHIBITOR; OXYGENASE INHIBITOR; TERT BUTYL N [ 1 METHYL 2 PHENYL CYCLOPROPYL]CARBAMATE; TRANS 1 (2 PHENYLETHYL) 2 PHENYL CYCLOPROPANECARBOXYLIC ACID; TRANS 1 BENZYL 2 PHENYL CYCLOPROPANECARBOXYLIC ACID; TRANS 1 ETHYL 2 (2 NAPHTHYL)CYCLOPROPANECARBOXYLIC ACID; TRANS 1 ETHYL 2 PHENYL CYCLOPROPANECARBOXYLIC ACID; TRANS 1 METHYL 2 PHENYL CYCLOPROPANECARBOXYLIC ACID; TRANS 2 PHENYL 1 PROPYL CYCLOPROPANECARBOXYLIC ACID; TRANYLCYPROMINE DERIVATIVE; UNCLASSIFIED DRUG; UNINDEXED DRUG; 1 (2 PHENYLETHYL) 2 PHENYL CYCLOPROPANECARBOXYLIC ACID; 1 BENZYL 2 PHENYL CYCLOPROPANAMINE HYDROCHLORIDE; 1 BENZYL 2 PHENYL CYCLOPROPANECARBOXYLIC ACID; 1 ETHYL 2 (2 NAPHTHYL)CYCLOPROPANECARBOXYLIC ACID; 1 METHYL 2 PHENYL CYCLOPROPANAMINE 2,2,2- TRIFLUOROACETIC ACID; 2 PHENYL 1 PROPYL CYCLOPROPANECARBOXYLIC ACID; AMINE OXIDASE (FLAVIN CONTAINING); ETHY 1 METHYL 2 PHENYL CYCLOPROPANECARBOXYLATE; ETHYL TRANS 1 PHENYL 1 PROPYL CYCLOPROPANECARBOXYLATE; KDM 1A; SELEGILINE; TERT BUTYL N[1 METHYL 2 PHENYL CYCLOPROPYL]CARBAMATE; TRANYLCYPROMINE; CYCLOPROPANE DERIVATIVE; CYCLOPROPYLAMINE; ENZYME INHIBITOR; HISTONE DEMETHYLASE; KDM1A PROTEIN, HUMAN;

ANTIDEPRESSANT ACTIVITY; ANTIPROLIFERATIVE ACTIVITY; ARTICLE; CELL FUNCTION; CHEMICAL ANALYSIS; CONTROLLED STUDY; COVALENT BOND; DRUG DESIGN; DRUG SYNTHESIS; ENZYME INHIBITION; GENE; GENE EXPRESSION; GENE TARGETING; GFI 1B GENE; HUMAN; HUMAN CELL; IN VITRO STUDY; STEREOCHEMISTRY; STEREOISOMERISM; STRUCTURE ACTIVITY RELATION; TRANS ISOMER; AMINO TERMINAL SEQUENCE; BINDING SITE; BIOLOGICAL ACTIVITY; COLUMN CHROMATOGRAPHY; CRYSTAL STRUCTURE; ENZYME INHIBITION ASSAY; HIGH PERFORMANCE LIQUID CHROMATOGRAPHY; IC50; NONHUMAN; OXIDATION; PROTEIN EXPRESSION; REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION; RNA ANALYSIS; ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY; X RAY CRYSTALLOGRAPHY; ANTAGONISTS AND INHIBITORS; CHEMICAL STRUCTURE; CHEMISTRY; DOSE RESPONSE; METABOLISM; SYNTHESIS;

CRYSTALLOGRAPHY, X-RAY; CYCLOPROPANES; DOSE-RESPONSE RELATIONSHIP, DRUG; ENZYME INHIBITORS; HISTONE DEMETHYLASES; HUMANS; MODELS, MOLECULAR; MOLECULAR STRUCTURE; MONOAMINE OXIDASE; STRUCTURE-ACTIVITY RELATIONSHIP;

EID: 84906839626     PISSN: 02235234     EISSN: 17683254     Source Type: Journal    
DOI: 10.1016/j.ejmech.2014.08.068     Document Type: Article

References (70)

1. T. Jenuwein, and C.D. Allis Translating the histone code Science 293 2001 1074 1080 (Pubitemid 32758077)
2. S.L. Berger Histone modifications in transcriptional regulation Curr. Opin. Genet. Dev. 12 2002 142 148 (Pubitemid 34219485)
3. S.L. Berger The complex language of chromatin regulation during transcription Nature 447 2007 407 412 (Pubitemid 46816746)
4. M.R. Hubner, M.A. Eckersley-Maslin, and D.L. Spector Chromatin organization and transcriptional regulation Curr. Opin. Genet. Dev. 23 2013 89 95
5. C. Biancotto, G. Frige, and S. Minucci Histone modification therapy of cancer Adv. Genet. 70 2010 341 386
6. T. Waldmann, and R. Schneider Targeting histone modifications - epigenetics in cancer Curr. Opin. Cell Biol. 25 2013 184 189
7. P.A. Marks Discovery and development of SAHA as an anticancer agent Oncogene 26 2007 1351 1356 (Pubitemid 46328465)
8. P.A. Marks, and R. Breslow Dimethyl sulfoxide to vorinostat: development of this histone deacetylase inhibitor as an anticancer drug Nat. Biotechnol. 25 2007 84 90 (Pubitemid 46087907)
9. C. Grant, F. Rahman, R. Piekarz, C. Peer, R. Frye, R.W. Robey, E.R. Gardner, W.D. Figg, and S.E. Bates Romidepsin: a new therapy for cutaneous T-cell lymphoma and a potential therapy for solid tumors Expert Rev. Anticancer Ther. 10 2010 997 1008
10. S. Jain, and J. Zain Romidepsin in the treatment of cutaneous T-cell lymphoma J. Blood Med. 2 2011 37 47
11. Y. Zhang, and D. Reinberg Transcription regulation by histone methylation: interplay between different covalent modifications of the core histone tails Genes Dev. 15 2001 2343 2360 (Pubitemid 32899739)
12. A. Spannhoff, A.T. Hauser, R. Heinke, W. Sippl, and M. Jung The emerging therapeutic potential of histone methyltransferase and demethylase inhibitors ChemMedChem 4 2009 1568 1582
13. C. Loenarz, and C.J. Schofield Expanding chemical biology of 2-oxoglutarate oxygenases Nat. Chem. Biol. 4 2008 152 156 (Pubitemid 351264133)
14. S.S. Ng, K.L. Kavanagh, M.A. McDonough, D. Butler, E.S. Pilka, B.M. Lienard, J.E. Bray, P. Savitsky, O. Gileadi, F. von Delft, N.R. Rose, J. Offer, J.C. Scheinost, T. Borowski, M. Sundstrom, C.J. Schofield, and U. Oppermann Crystal structures of histone demethylase JMJD2A reveal basis for substrate specificity Nature 448 2007 87 91 (Pubitemid 47037008)
15. J.C. Culhane, and P.A. Cole LSD1 and the chemistry of histone demethylation Curr. Opin. Chem. Biol. 11 2007 561 568 (Pubitemid 47588999)
16. F. Forneris, C. Binda, E. Battaglioli, and A. Mattevi LSD1: oxidative chemistry for multifaceted functions in chromatin regulation Trends Biochem. Sci. 33 2008 181 189
17. F. Forneris, E. Battaglioli, A. Mattevi, and C. Binda New roles of flavoproteins in molecular cell biology: histone demethylase LSD1 and chromatin FEBS J. 276 2009 4304 4312
18. Y. Shi, F. Lan, C. Matson, P. Mulligan, J.R. Whetstine, P.A. Cole, and R.A. Casero Histone demethylation mediated by the nuclear amine oxidase homolog LSD1 Cell 119 2004 941 953 (Pubitemid 40037608)
19. D.N. Ciccone, H. Su, S. Hevi, F. Gay, H. Lei, J. Bajko, G. Xu, E. Li, and T. Chen KDM1B is a histone H3K4 demethylase required to establish maternal genomic imprints Nature 461 2009 415 418
20. A. Karytinos, F. Forneris, A. Profumo, G. Ciossani, E. Battaglioli, C. Binda, and A. Mattevi A novel mammalian flavin-dependent histone demethylase J. Biol. Chem. 284 2009 17775 17782
21. R. Schneider, A.J. Bannister, F.A. Myers, A.W. Thorne, C. Crane-Robinson, and T. Kouzarides Histone H3 lysine 4 methylation patterns in higher eukaryotic genes Nat. Cell Biol. 6 2004 73 77 (Pubitemid 38425733)
22. G. Liang, J.C. Lin, V. Wei, C. Yoo, J.C. Cheng, C.T. Nguyen, D.J. Weisenberger, G. Egger, D. Takai, F.A. Gonzales, and P.A. Jones Distinct localization of histone H3 acetylation and H3-K4 methylation to the transcription start sites in the human genome Proc. Natl. Acad. Sci. U. S. A. 101 2004 7357 7362 (Pubitemid 38638037)
23. Y. Huang, E. Greene, T. Murray Stewart, A.C. Goodwin, S.B. Baylin, P.M. Woster, and R.A. Casero Jr. Inhibition of lysine-specific demethylase 1 by polyamine analogues results in reexpression of aberrantly silenced genes Proc. Natl. Acad. Sci. U. S. A. 104 2007 8023 8028 (Pubitemid 47185869)
24. E. Metzger, M. Wissmann, N. Yin, J.M. Muller, R. Schneider, A.H. Peters, T. Gunther, R. Buettner, and R. Schule LSD1 demethylates repressive histone marks to promote androgen-receptor-dependent transcription Nature 437 2005 436 439 (Pubitemid 41613508)
25. J. Huang, R. Sengupta, A.B. Espejo, M.G. Lee, J.A. Dorsey, M. Richter, S. Opravil, R. Shiekhattar, M.T. Bedford, T. Jenuwein, and S.L. Berger p53 is regulated by the lysine demethylase LSD1 Nature 449 2007 105 108 (Pubitemid 47373772)
26. J. Wang, S. Hevi, J.K. Kurash, H. Lei, F. Gay, J. Bajko, H. Su, W. Sun, H. Chang, G. Xu, F. Gaudet, E. Li, and T. Chen The lysine demethylase LSD1 (KDM1) is required for maintenance of global DNA methylation Nat. Genet. 41 2009 125 129
27. T.B. Nicholson, and T. Chen LSD1 demethylates histone and non-histone proteins Epigenetics 4 2009 129 132
28. H. Kontaki, and I. Talianidis Lysine methylation regulates E2F1-induced cell death Mol. Cell 39 2010 152 160
29. H.S. Cho, T. Suzuki, N. Dohmae, S. Hayami, M. Unoki, M. Yoshimatsu, G. Toyokawa, M. Takawa, T. Chen, J.K. Kurash, H.I. Field, B.A. Ponder, Y. Nakamura, and R. Hamamoto Demethylation of RB regulator MYPT1 by histone demethylase LSD1 promotes cell cycle progression in cancer cells Cancer Res. 71 2011 655 660
30. J.T. Lynch, W.J. Harris, and T.C. Somervaille LSD1 inhibition: a therapeutic strategy in cancer? Expert Opin. Ther. Targets 16 2012 1239 1249
31. S. Hayami, J.D. Kelly, H.S. Cho, M. Yoshimatsu, M. Unoki, T. Tsunoda, H.I. Field, D.E. Neal, H. Yamaue, B.A. Ponder, Y. Nakamura, and R. Hamamoto Overexpression of LSD1 contributes to human carcinogenesis through chromatin regulation in various cancers Int. J. Cancer 128 2011 574 586
32. I. Hoffmann, M. Roatsch, M.L. Schmitt, L. Carlino, M. Pippel, W. Sippl, and M. Jung The role of histone demethylases in cancer therapy Mol. Oncol. 6 2012 683 703
33. D. Rotili, and A. Mai Targeting histone demethylases: a new avenue for the fight against cancer Genes Cancer 2 2011 663 679
34. T. Schenk, W.C. Chen, S. Gollner, L. Howell, L. Jin, K. Hebestreit, H.U. Klein, A.C. Popescu, A. Burnett, K. Mills, R.A. Casero Jr., L. Marton, P. Woster, M.D. Minden, M. Dugas, J.C. Wang, J.E. Dick, C. Muller-Tidow, K. Petrie, and A. Zelent Inhibition of the LSD1 (KDM1A) demethylase reactivates the all-trans-retinoic acid differentiation pathway in acute myeloid leukemia Nat. Med. 18 2012 605 611
35. F. Crea, L. Sun, A. Mai, Y.T. Chiang, W.L. Farrar, R. Danesi, and C.D. Helgason The emerging role of histone lysine demethylases in prostate cancer Mol. Cancer 11 2012 52
36. S. Lim, A. Janzer, A. Becker, A. Zimmer, R. Schule, R. Buettner, and J. Kirfel Lysine-specific demethylase 1 (LSD1) is highly expressed in ER-negative breast cancers and a biomarker predicting aggressive biology Carcinogenesis 31 2010 512 520
37. M.M. Singh, C.A. Manton, K.P. Bhat, W.W. Tsai, K. Aldape, M.C. Barton, and J. Chandra Inhibition of LSD1 sensitizes glioblastoma cells to histone deacetylase inhibitors Neuro Oncol. 13 2011 894 903
38. J.H. Schulte, S. Lim, A. Schramm, N. Friedrichs, J. Koster, R. Versteeg, I. Ora, K. Pajtler, L. Klein-Hitpass, S. Kuhfittig-Kulle, E. Metzger, R. Schule, A. Eggert, R. Buettner, and J. Kirfel Lysine-specific demethylase 1 is strongly expressed in poorly differentiated neuroblastoma: implications for therapy Cancer Res. 69 2009 2065 2071
39. M.G. Lee, C. Wynder, D.M. Schmidt, D.G. McCafferty, and R. Shiekhattar Histone H3 lysine 4 demethylation is a target of nonselective antidepressive medications Chem. Biol. 13 2006 563 567
40. B. Lohse, J.L. Kristensen, L.H. Kristensen, K. Agger, K. Helin, M. Gajhede, and R.P. Clausen Inhibitors of histone demethylases Bioorg. Med. Chem. 19 2011 3625 3636
41. A. Ortega Munoz, M.C.T. Fyfe, M. Martinell Pedemonte, M. Estiarte Martinez, N. Valls Vidal, G. Kurz, and J.C. Castro Palomino Laria (Hetero)aryl Cyclopropylamine Compounds as LSD1 Inhibitors 2013 WO2013/057320
42. N.W. Johnson, J. Kasparec, W.H. Miller, M.B. Rouse, D. Suarez, and X. Tian Cyclopropylamines as LSD1 Inhibitors 2012 WO2012/135113
43. http://www.clinicaltrials.gov/ct2/show/NCT02034123?term=lsd1&rank=3.
44. www.clinicaltrialsregister.eu/ctr-search/trial/2013-002447-29/ES.
45. R. Neelamegam, E.L. Ricq, M. Malvaez, D. Patnaik, S. Norton, S.M. Carlin, I.T. Hill, M.A. Wood, S.J. Haggarty, and J.M. Hooker Brain-penetrant LSD1 inhibitors can block memory consolidation ACS Chem. Neurosci. 3 2012 120 128
46. R. Ueda, T. Suzuki, K. Mino, H. Tsumoto, H. Nakagawa, M. Hasegawa, R. Sasaki, T. Mizukami, and N. Miyata Identification of cell-active lysine specific demethylase 1-selective inhibitors J. Am. Chem. Soc. 131 2009 17536 17537
47. C. Binda, S. Valente, M. Romanenghi, S. Pilotto, R. Cirilli, A. Karytinos, G. Ciossani, O.A. Botrugno, F. Forneris, M. Tardugno, D.E. Edmondson, S. Minucci, A. Mattevi, and A. Mai Biochemical, structural, and biological evaluation of tranylcypromine derivatives as inhibitors of histone demethylases LSD1 and LSD2 J. Am. Chem. Soc. 132 2010 6827 6833
48. D.M. Schmidt, and D.G. McCafferty trans-2-Phenylcyclopropylamine is a mechanism-based inactivator of the histone demethylase LSD1 Biochemistry 46 2007 4408 4416 (Pubitemid 46559406)
49. M. Yang, J.C. Culhane, L.M. Szewczuk, P. Jalili, H.L. Ball, M. Machius, P.A. Cole, and H. Yu Structural basis for the inhibition of the LSD1 histone demethylase by the antidepressant trans-2-phenylcyclopropylamine Biochemistry 46 2007 8058 8065 (Pubitemid 47051641)
50. D.M. Gooden, D.M. Schmidt, J.A. Pollock, A.M. Kabadi, and D.G. McCafferty Facile synthesis of substituted trans-2-arylcyclopropylamine inhibitors of the human histone demethylase LSD1 and monoamine oxidases A and B Bioorg. Med. Chem. Lett. 18 2008 3047 3051 (Pubitemid 351680392)
51. D.B. Denney, and M.J. Boskin Formation of cyclopropanes from phosphoranes and epoxides J. Am. Chem. Soc. 81 1959 6330 6331
52. W.S.J. Wadsworth, and W.D. Emmons The utility of phosphonate carbanions in olefin synthesis J. Am. Chem. Soc. 83 1961 1733
53. A. Armstrong, and J.N. Scutt Stereocontrolled synthesis of 3-(trans-2-aminocyclopropyl)alanine, a key component of belactosin A Org. Lett. 5 2003 2331 2334 (Pubitemid 37161919)
54. C.D. Bray, and F. Minicone Stereocontrolled synthesis of quaternary cyclopropyl esters Chem. Commun. Camb. 46 2010 5867 5869
55. A.R. Patel 1,2-Diarylcyclopropane derivatives Acta Chem. Scand. 20 1966 1424 1426
56. R.P. Wurz, and A.B. Charette An expedient and practical method for the synthesis of a diverse series of cyclopropane alpha-amino acids and amines J. Org. Chem. 69 2004 1262 1269 (Pubitemid 38233409)
57. R.R. Vunnam, D. Bond, R.A. Schatz, N.S. Radin, and N. Narasimhachari A new class of monoamine oxidase inhibitors J. Neurochem. 34 1980 410 416 (Pubitemid 10172293)
58. A.J. Christmas, C.J. Coulson, D.R. Maxwell, and D. Riddell A comparison of the pharmacological and biochemical properties of substrate-selective monoamine oxidase inhibitors Br. J. Pharmacol. 45 1972 490 503
59. C.L. Zirkle, C. Kaiser, D.H. Tedeschi, R.E. Tedeschi, and A. Burger 2-Substituted cyclopropylamines. Ii. Effect of structure upon monoamine oxidase-inhibitory activity as measured in vivo by potentiation of tryptamine convulsions J. Med. Pharm. Chem. 91 1962 1265 1284
60. A. Burger, C.S. Davis, H. Green, D.H. Tedeschi, and C.L. Zirkle 1-Methyl-2-phenylcyclopropylamine J. Med. Pharm. Chem. 4 1961 571 574
61. M.P. Valley, W. Zhou, E.M. Hawkins, J. Shultz, J.J. Cali, T. Worzella, L. Bernad, T. Good, D. Good, T.L. Riss, D.H. Klaubert, and K.V. Wood A bioluminescent assay for monoamine oxidase activity Anal. Biochem. 359 2006 238 246 (Pubitemid 44821056)
62. L. De Colibus, M. Li, C. Binda, A. Lustig, D.E. Edmondson, and A. Mattevi Three-dimensional structure of human monoamine oxidase A (MAO A): relation to the structures of rat MAO A and human MAO B Proc. Natl. Acad. Sci. U. S. A. 102 2005 12684 12689 (Pubitemid 41279426)
63. S. Saleque, J. Kim, H.M. Rooke, and S.H. Orkin Epigenetic regulation of hematopoietic differentiation by Gfi-1 and Gfi-1b is mediated by the cofactors CoREST and LSD1 Mol. Cell 27 2007 562 572 (Pubitemid 47238633)
64. R.B. Silverman, and P.A. Zieske Mechanism of inactivation of monoamine oxidase by 1-phenylcyclopropylamine Biochemistry 24 1985 2128 2138
65. K. Hackeloer, G. Schnakenburg, and S.R. Waldvogel Oxidative coupling reaction of 1,3-diarylpropene derivatives to dibenzo[a,c]cycloheptene by PIFA Eur. J. Org. Chem. 2011 6314 6319
66. G. Ozer, N. Saracoglu, and M. Balci Synthesis and chemistry of unusual bicyclic endoperoxides containing the pyridazine ring J. Org. Chem. 68 2003 7009 7015 (Pubitemid 37071781)
67. F. Forneris, C. Binda, A. Adamo, E. Battaglioli, and A. Mattevi Structural basis of LSD1-CoREST selectivity in histone H3 recognition J. Biol. Chem. 282 2007 20070 20074 (Pubitemid 47099979)
68. M.D. Winn, C.C. Ballard, K.D. Cowtan, E.J. Dodson, P. Emsley, P.R. Evans, R.M. Keegan, E.B. Krissinel, A.G. Leslie, A. McCoy, S.J. McNicholas, G.N. Murshudov, N.S. Pannu, E.A. Potterton, H.R. Powell, R.J. Read, A. Vagin, and K.S. Wilson Overview of the CCP4 suite and current developments Acta Crystallogr. D Biol. Crystallogr. 67 2011 235 242
69. S. McNicholas, E. Potterton, K.S. Wilson, and M.E. Noble Presenting your structures: the CCP4mg molecular-graphics software Acta Crystallogr. D Biol. Crystallogr. 67 2011 386 394
70. C. Binda, M. Li, F. Hubalek, N. Restelli, D.E. Edmondson, and A. Mattevi Insights into the mode of inhibition of human mitochondrial monoamine oxidase B from high-resolution crystal structures Proc. Natl. Acad. Sci. U. S. A. 100 2003 9750 9755 (Pubitemid 37087013)