Structures of cytochrome P450 2B4 complexed with the antiplatelet drugs ticlopidine and clopidogrel .
Prior X-ray crystal structures of rabbit cytochrome P450 2B4 (2B4) in complexes with various imidazoles have demonstrated markedly different enzyme conformations depending on the size of the inhibitor occupying the active site. In this study, structures of 2B4 were determined with the antiplatelet drugs clopidogrel and ticlopidine, which were expected to have greater freedom of movement in the binding pocket. Ticlopidine could be modeled into the electron density maps in two distinct orientations, both of which are consistent with metabolic data gathered with other mammalian P450 enzymes. Results of ligand docking and heme-induced NMR relaxation of drug protons showed that ticlopidine was preferentially oriented with the chlorophenyl group closest to the heme. Because of its stereocenter, clopidogrel was easier to fit in the electron density and exhibited a single orientation, which points the chlorophenyl ring toward the heme. The C(α) traces of both complexes aligned very well with each other and revealed a compact, closed structure that resembles the conformation observed in two previously determined 2B4 structures with the small molecule inhibitors 4-(4-chlorophenyl)imidazole and 1-(4-chlorophenyl)imidazole. The 2B4 active site is able to accommodate small ligands by moving only a small number of side chains, suggesting that ligand reorientation is energetically favored over protein conformational changes for binding of these similarly sized molecules. Adjusting both protein conformation and ligand orientation in the active site gives 2B4 the flexibility to bind to the widest range of molecules, while also being energetically favorable.
Gay
Sean C
SC
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California 92093, USA. scgay@ucsd.edu
Roberts
Arthur G
AG
Maekawa
Keiko
K
Talakad
Jyothi C
JC
Hong
Wen-Xu
WX
Zhang
Qinghai
Q
Stout
C David
CD
Halpert
James R
JR
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D000077144:Clopidogrel; D018360:Crystallography, X-Ray; D000072467:Cytochrome P450 Family 2; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D010975:Platelet Aggregation Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D011485:Protein Binding; D011817:Rabbits; D013988:Ticlopidine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology*
D010975:Platelet Aggregation Inhibitors; D000077144:Clopidogrel; D001189:Aryl Hydrocarbon Hydroxylases; D000072467:Cytochrome P450 Family 2; C465241:cytochrome P-450 CYP2B4 (rabbit); D013988:Ticlopidine
Br J Pharmacol. 2004 Jun;142(3):551-7
15148251
Acta Crystallogr D Biol Crystallogr. 1997 May 1;53(Pt 3):240-55
15299926
Biochemistry. 2005 Nov 1;44(43):14143-51
16245930
Curr Drug Metab. 2007 Jun;8(5):407-47
17584015
Drug Metab Dispos. 2010 Jan;38(1):92-9
19812348
Drug Metab Dispos. 2004 Jan;32(1):49-57
14709620
Biochemistry. 2004 Jun 8;43(22):7207-14
15170358
Methods Enzymol. 1989;177:403-13
2575205
Biochim Biophys Acta. 2007 Mar;1770(3):376-89
17069978
J Comput Chem. 2005 Dec;26(16):1701-18
16211538
Ann Intern Med. 1998 Sep 1;129(5):394-405
9735068
J Clin Pharmacol. 2010 Feb;50(2):126-42
19948947
Eur Biophys J. 2005 Jun;34(4):273-84
15803330
Methods Enzymol. 1997;276:344-58
9048378
Plant Physiol. 2003 Nov;133(3):1198-208
14576280
J Magn Reson. 2000 Jan;142(1):57-63
10617435
Biophys J. 2001 Sep;81(3):1275-84
11509344
J Magn Reson. 2002 Nov;159(1):76-81
12468306
Chem Res Toxicol. 2007 Oct;20(10):1434-41
17894464
Biochemistry. 2009 Jun 9;48(22):4762-71
19397311
Structure. 1997 Mar 15;5(3):325-36
9083112
Drug Metab Dispos. 2009 Mar;37(3):589-93
19047469
Drug Metab Dispos. 2009 Sep;37(9):1904-15
19541827
Clin Pharmacol Ther. 1997 Nov;62(5):572-7
9390115
Chem Rev. 2009 Sep;109(9):4108-39
19522502
Arch Biochem Biophys. 1995 Jul 10;320(2):330-44
7625841
J Med Chem. 2006 Nov 30;49(24):6987-7001
17125252
Drug Metab Pharmacokinet. 2008;23(6):412-20
19122335
Eur J Drug Metab Pharmacokinet. 1981;6(2):91-8
7274310
Angew Chem Int Ed Engl. 2007;46(37):7023-5
17691085
J Pharm Biomed Anal. 1998 Jul;17(3):419-25
9656153
Chem Res Toxicol. 2010 May 17;23(5):909-17
20297803
Mol Pharmacol. 2010 Apr;77(4):529-38
20061448
NMR Biomed. 2001 Jun;14(4):271-7
11410945
Acta Crystallogr D Biol Crystallogr. 1999 Oct;55(Pt 10):1696-702
10531519
Methods Mol Biol. 2008;426:145-59
18542861
NMR Biomed. 1996 May;9(3):93-104
8892395
J Pharmacol Exp Ther. 2004 Jan;308(1):189-97
14563790
Protein Eng. 1990 Dec;4(2):177-84
2075193
Biochim Biophys Acta. 2007 Mar;1770(3):402-12
16935426
Biochemistry. 2007 Jan 9;46(1):106-19
17198380
Acta Crystallogr D Biol Crystallogr. 2010 Apr;66(Pt 4):486-501
20383002
Biophys Chem. 2006 Sep 20;123(2-3):95-101
16701937
Biochemistry. 2007 Oct 16;46(41):11559-67
17887776
Biophys J. 1995 Sep;69(3):810-24
8519982
Biochemistry. 1997 Apr 15;36(15):4461-70
9109653
J Biomol NMR. 1992 Nov;2(6):661-5
1490109
Agents Actions Suppl. 1984;15:68-75
6385655
Nat Struct Mol Biol. 2005 Sep;12(9):822-3
16086027
Drug Metab Dispos. 2004 Jun;32(6):626-31
15155554
Curr Drug Metab. 2001 Jun;2(2):117-37
11469721
J Biol Chem. 2004 Jun 25;279(26):27294-301
15100217
Magn Reson Med. 2000 Oct;44(4):646-9
11025522
J Biol Chem. 2006 Mar 3;281(9):5973-81
16373351
J Biol Chem. 2006 Mar 24;281(12):8051-61
16439365
Biochem Biophys Res Commun. 2005 Dec 9;338(1):331-6
16157296
Biochemistry. 2001 Oct 9;40(40):12112-22
11580286
Proc Natl Acad Sci U S A. 2003 Nov 11;100(23):13196-201
14563924
Biochemistry. 1995 Jul 18;34(28):8982-8
7619797
Acta Crystallogr D Biol Crystallogr. 2003 Jul;59(Pt 7):1124-30
12832754
J Am Chem Soc. 2004 May 12;126(18):5879-96
15125681
J Appl Crystallogr. 2007 Aug 1;40(Pt 4):658-674
19461840
Arch Biochem Biophys. 2000 Dec 1;384(1):47-58
11147835
Biochemistry. 1996 Apr 9;35(14):4540-50
8605204
Acta Crystallogr D Biol Crystallogr. 1994 Mar 1;50(Pt 2):178-85
15299456
Br J Clin Pharmacol. 1999 Apr;47(4):454-7
10233213
Acta Crystallogr A. 2008 Sep;64(Pt 5):519-28
18708715
Magn Reson Med. 1997 May;37(5):651-7
9126938