Theoretical Studies on the Substrate Binding Mode and Regioselectivity of Human CYP2C9 with S- and R-Warfarin†

doi:10.7503/cjcu20140713

Abstract

Abstract:

Cytochrome P450(CYP)2C9, a member of the 2C subfamily of CYPs, plays important role in the oxidative metabolism of amount of current clinical drugs. CYP2C9 shows the substrate regioselectivity toward Warfarin. The currently available X-ray structure of CYP2C9-S-Warfarin complex(PDB ID: 1OG5) shows a non-productive orientation of the S-Warfarin bound in the active site of CYP2C9. A series of investigations including automatic docking, molecular dynamics(MD) simulation, combined with tunnel analysis and the MM-GB/SA calculation, identified a 6-, 7-hydroxylation state of the substrate binding mode in the re-dock complex structure, as well as the “metastable state” in the crystal structure. In addition, the comparison of the CYP2C9 binding to S- and R-Warfarin shows the structural features relevant to the substrate regioselectivity of CYP2C9. According to 100 ns MD simulations, the key residues that, in the active binding site, particularly Phe cluster residues, are proposed to play indispensable role in the stabilization of substrates. The investigation of CYP2C9-substrate binding modes provides detailed insights into the structural features of human CYP2C9 toward Warfarin at the atomic level, and will be valuable information for drug development.

Key words: Molecular docking, Molecular dynamics simulation, Cytochrome P450, MM-GB/SA, CYP2C9 and Warfarin complex

CLC Number:

O641

TrendMD:

WU Yunjian, CUI Yinglu, ZHENG Qingchuan, ZHANG Hongxing. Theoretical Studies on the Substrate Binding Mode and Regioselectivity of Human CYP2C9 with S- and R-Warfarin^†[J]. Chem. J. Chinese Universities, 2014, 35(12): 2605.

Figures/Tables 11

Fig.1 Overall structure of CYP2C9, colored from N-terminal to C-terminal(A) and structure of Warfarin(B)

Fig.2 Time-dependent RMSD of CYP2C9 with substrates along the simulation trajectory a. rd-2C9-S-Warfarin; b. cs-2C9-S-Warfarin; c. 2C9-R-Warfarin.

Fig.3 Stereoview of the key residues in the active site of CYP2C9-substrate complexes for cs-2C9-S-Warfarin system(A) and rd-2C9-S-Warfarin system(B)

Table 1 Binding free energies for the ligands in the complexes*

System	ΔE_ele/ (kJ·mol^-1)	ΔE_vdw/ (kJ·mol^-1)	ΔG_GB/ (kJ·mol^-1)	ΔG_SA/ (kJ·mol^-1)	ΔG_MM-GB/SA/ (kJ·mol^-1)	-TΔS/ (kJ·mol^-1)	ΔG_bind/ (kJ·mol^-1)	K_D(expt.)	ΔG_bind-expt./ (kJ·mol^-1)
cs-2C9-S-Warfarin	-112.14	-157.18	191.67	-12.39	-90.04	83.09	-6.95
rd-2C9-S-Warfarin	-100.25	-171.79	170.53	-11.93	-113.44	82.63	-30.81	6	-31.02

Table 2 Decomposition of binding free energy on per-residue basis for cs-2C9-S-Warfarin system

Residue	ΔE_vdw/(kJ·mol^-1)	ΔE_ele/(kJ·mol^-1)	ΔG_GB/(kJ·mol^-1)	ΔG_SASA/(kJ·mol^-1)	Δ $G bind *$ /(kJ·mol^-1)
Leu366	-7.70	-2.26	0.84	-1.09	-10.21
Ala103	-7.12	-6.70	6.03	-1.09	-8.87
Pro367	-7.79	-3.18	3.01	-0.67	-8.62
Phe100	-7.41	-1.30	1.21	-0.50	-7.99
Ile99	-6.07	-5.32	3.77	-0.29	-7.91
Leu102	-3.81	-5.02	4.86	-0.21	-4.19
Phe476	-3.39	0.25	0.63	-0.84	-3.35
Leu208	-3.39	0.08	1.59	-0.88	-2.60
Phe114	-2.55	0.46	-0.04	-0.25	-2.39

Table 2 Decomposition of binding free energy on per-residue basis for cs-2C9-S-Warfarin system

Residue	ΔE_vdw/(kJ·mol^-1)	ΔE_ele/(kJ·mol^-1)	ΔG_GB/(kJ·mol^-1)	ΔG_SASA/(kJ·mol^-1)	Δ $G bind *$ /(kJ·mol^-1)
Leu366	-7.70	-2.26	0.84	-1.09	-10.21
Ala103	-7.12	-6.70	6.03	-1.09	-8.87
Pro367	-7.79	-3.18	3.01	-0.67	-8.62
Phe100	-7.41	-1.30	1.21	-0.50	-7.99
Ile99	-6.07	-5.32	3.77	-0.29	-7.91
Leu102	-3.81	-5.02	4.86	-0.21	-4.19
Phe476	-3.39	0.25	0.63	-0.84	-3.35
Leu208	-3.39	0.08	1.59	-0.88	-2.60
Phe114	-2.55	0.46	-0.04	-0.25	-2.39

Table 3 Decomposition of binding free energy on per-residue basis for rd-2C9-S-Warfarin system

Residue	ΔE_vdw/(kJ·mol^-1)	ΔE_ele/(kJ·mol^-1)	ΔG_GB/(kJ·mol^-1)	ΔG_SASA/(kJ·mol^-1)	ΔG_bind/(kJ·mol^-1)
Glu300	-0.50	-47.63	38.43	-0.96	-10.67
Leu208	-4.56	0.46	-5.19	-1.17	-10.46
Asn204	-0.96	-2.76	-4.10	-0.21	-8.04
Ala297	-6.20	-2.64	2.68	-0.46	-6.61
Leu366	-4.60	-0.75	0.25	-0.71	-5.82
Val113	-3.93	-2.97	1.67	-0.33	-5.57
Phe114	-5.02	-2.26	3.47	-1.09	-4.90
Phe476	-0.38	-0.25	-2.64	-0.04	-3.31
Thr301	-1.88	1.21	-2.60	0.04	-3.22
Leu362	-2.34	0.21	-0.50	-0.46	-3.10

Fig.4 Comparison of the positions of Phe114(A) and Phe476(B) in the representative structures of cs-2C9-S-Warfarin and rd-2C9-S-Warfarin

Fig.5 Access channels identified from the representative structures of CYP2C9-substrate complexes for cs-2C9-S-Warfarin system(A) and rd-2C9-S-Warfarin system(B)

Fig.6 Stereoview of the key residues in the active site of CYP2C9-R-Warfarin complex

Table 4 Binding free energies for 2C9-R-Warfarin complex

ΔE_ele/ (kJ·mol^-1)	ΔE_vdw/ (kJ·mol^-1)	ΔG_GB/ (kJ·mol^-1)	ΔG_SA/ (kJ·mol^-1)	ΔG_MM-GB/SA/ (kJ·mol^-1)	-TΔS/ (kJ·mol^-1)	ΔG_bind/ (kJ·mol^-1)
-85.27	-133.74	145.67	-9.63	-82.96	87.94	4.98

Table 5 Decomposition of binding free energy on per-residue basis for 2C9-R-Warfarin system

Residue	ΔE_vdw/(kJ·mol^-1)	ΔE_ele/(kJ·mol^-1)	ΔG_GB/(kJ·mol^-1)	ΔG_SASA/(kJ·mol^-1)	ΔG_bind/(kJ·mol^-1)
Phe476	-5.48	-0.75	-0.17	-0.54	-6.95
Leu208	-6.95	-0.13	1.55	-1.34	-6.86
Leu366	-6.53	-1.05	1.88	-0.92	-6.61
Phe114	-3.73	-1.26	1.88	-0.88	-3.98
Val113	-3.18	-0.88	0.63	-0.50	-3.93
Phe100	-2.72	-0.21	-0.38	-0.38	-3.68
Leu362	-2.97	0.33	0.08	-0.50	-3.06
Ala103	-2.43	-1.72	2.09	-0.63	-2.68
Ala297	-2.18	-0.08	0.33	-0.42	-2.34
Ile213	-2.05	0.59	-0.33	-0.38	-2.18

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