Drug-protein hydrogen bonds govern the inhibition of the ATP hydrolysis of the multidrug transporter P-glycoprotein.
Drug-protein hydrogen bonds govern the inhibition of the ATP hydrolysis of the multidrug transporter P-glycoprotein.
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DOI：
10.1016/j.bcp.2015.12.007
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发表时间：
2016-02-01
影响因子：
5.8
通讯作者：
Ambudkar SV
中科院分区：
文献类型：
--
作者：
Chufan EE;Kapoor K;Ambudkar SV
p -糖蛋白（P-gp）是atp结合盒转运蛋白超家族的一员。这种多药物转运体利用ATP水解产生的能量来排出包括抗癌药物在内的各种疏水和两性化合物。P-gp的大多数底物和调节剂刺激其基础atp酶活性，尽管有些抑制它。在这两种情况下起作用的分子机制是未知的。在本报告中，通过对P-gp的诱变和分子模拟研究，在跨膜区域鉴定了一对苯丙氨酸-酪氨酸结构基序，它们介导某些药物（zosuquidar， elacridar和tariquidar）对ATP水解的抑制，具有高亲和力（IC50 's范围为10至30 nM）。一旦这些残基中的任何一个发生突变，抑制P-gp atp酶活性的药物就会转向刺激其活性。分子模型显示，苯丙氨酸残基F978和F728分别与酪氨酸残基Y953和Y310以边对面构象相互作用，这使得酪氨酸以这样一种方式与抑制剂建立氢键接触。生化研究和完整细胞的转运研究表明，抑制剂在高亲和力位点结合，产生ATP水解和转运功能的抑制。突变后，它们在较低亲和力位点结合，刺激ATP水解，仅对运输有较差的抑制作用。这些结果还表明，筛选能够抑制基础ATP水解的化合物可以作为鉴定P-gp高亲和力调节剂的可靠工具。
P-glycoprotein (P-gp) is a member of the ATP-Binding Cassette transporter superfamily. This multidrug transporter utilizes energy from ATP hydrolysis for the efflux of a variety of hydrophobic and amphipathic compounds including anticancer drugs. Most of the substrates and modulators of P-gp stimulate its basal ATPase activity, although some inhibit it. The molecular mechanisms that are in play in either case are unknown. In this report, mutagenesis and molecular modeling studies on P-gp led to the identification of a pair of phenylalanine-tyrosine structural motifs in the transmembrane region that mediate the inhibition of ATP hydrolysis by certain drugs (zosuquidar, elacridar and tariquidar), with high affinity (IC50’s ranging from 10 to 30 nM). Upon mutation of any of these residues, drugs that inhibit the ATPase activity of P-gp switch to stimulation of the activity. Molecular modeling revealed that the phenylalanine residues F978 and F728 interact with tyrosine residues Y953 and Y310, respectively, in an edge-to-face conformation, which orients the tyrosines in such a way that they establish hydrogen-bond contacts with the inhibitor. Biochemical investigations along with transport studies in intact cells showed that the inhibitors bind at a high affinity site to produce inhibition of ATP hydrolysis and transport function. Upon mutation, they bind at lower affinity sites, stimulating ATP hydrolysis and only poorly inhibiting transport. These results also reveal that screening chemical compounds for their ability to inhibit the basal ATP hydrolysis can be a reliable tool to identify modulators with high affinity for P-gp.