CYP2C9酶与Warfarin结合模型的立体选择性理论研究

doi:10.7503/cjcu20140713

摘要/Abstract

摘要：

对CYP2C9酶与S-Warfarin复合物的晶体结构进行分子对接、分子动力学模拟、通道分析及结合自由能计算, 发现原晶体结构中的结合模式为“亚稳态”, 提出了CYP2C9与S-Warfarin结合的可催化模式; 比较了CYP2C9与S-和R-Warfarin结合的异同, 确定了在结合过程中起重要作用的锚定氨基酸残基, 尤其是位于活性位点区域的苯丙氨酸簇. 在结合过程中这些残基通过芳香环的移动对稳定底物的结合模式起到至关重要的作用, 阐明了该酶呈现相关底物选择性的原因. 对于CYP2C9与底物对接模式及立体选择性的研究有助于在分子层面上理解特异性底物与酶的结合特点, 为潜在的药物设计提供了合理可信的理论依据.

关键词: 分子对接, 分子动力学模拟, 细胞色素P450, 结合自由能分析, CYP2C9酶和S-Warfarin复合物

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

中图分类号:

O641

TrendMD:

吴云剑, 崔颖璐, 郑清川, 张红星. CYP2C9酶与Warfarin结合模型的立体选择性理论研究. 高等学校化学学报, 2014, 35(12): 2605.

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^†. Chem. J. Chinese Universities, 2014, 35(12): 2605.

图/表 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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