目的 研究花椒毒素及其同分異構(gòu)體對(duì)藥物代謝酶細(xì)胞色素P450（CYPs）的抑制差異。方法 采用計(jì)算機(jī)虛擬技術(shù)研究花椒毒素及其同分異構(gòu)體香柑內(nèi)酯、異佛手柑內(nèi)酯與6種CYPs酶（CYP1A2、CYP2D6、CYP3A4、CYP2C19、CYP2C9、CYP2A6）的相互作用；采用Amber（ff99SB）軟件進(jìn)行花椒毒素、香柑內(nèi)酯、異佛手柑內(nèi)酯與CYP2A6的動(dòng)力學(xué)模擬研究；采用“Cocktail”藥物探針?lè)ㄟM(jìn)行驗(yàn)證，分別測(cè)定這3種同分異構(gòu)體對(duì)人肝微粒CYPs酶活性的影響，以及對(duì)CYP2A6與CYP1A2的半數(shù)抑制濃度（IC₅₀）。結(jié)果 計(jì)算機(jī)模擬技術(shù)結(jié)果顯示，花椒毒素、香柑內(nèi)酯、異佛手柑內(nèi)酯對(duì)6種CYPs結(jié)合能力不同，對(duì)CYP2A6的結(jié)合能力差異明顯。分子對(duì)接、分子動(dòng)力學(xué)結(jié)果表明，花椒毒素與CYP2A6形成的氫鍵ASN297：HD22-M：O4是花椒毒素與CYP2A6結(jié)合過(guò)程中最重要的且穩(wěn)定的氫鍵，香柑內(nèi)酯與ASN297產(chǎn)生的分子間氫鍵穩(wěn)定性低于花椒毒素體系，異佛手柑內(nèi)酯在動(dòng)力學(xué)中所產(chǎn)生的氫鍵均不穩(wěn)定；受體活性口袋中疏水性氨基酸與口袋形狀互補(bǔ)是促成配體受體結(jié)合的重要因素。“Cocktail”藥物探針?lè)ū砻?，花椒毒素、香柑?nèi)酯、異佛手柑內(nèi)酯對(duì)CYPs均具有不同程度的抑制作用，其中對(duì)CYP1A2具有顯著抑制作用，相對(duì)剩余酶活力分別為2.67%、4.64%、13.58%，其IC₅₀分別為1.42、2.29、5.12 μmol/L；對(duì)CYP2A6的抑制作用存在差異，相對(duì)剩余酶活力分別為8.10%、38.91%、74.98%，其IC₅₀值分別為3.89、44.03、103.30 μmol/L。結(jié)論 花椒毒素、香柑內(nèi)酯、異佛手柑內(nèi)酯對(duì)CYP1A2具有明顯的抑制作用，對(duì)CYP2A6的抑制效果存在明顯差異。甲氧基的取代位置與呋喃環(huán)駢合的位置對(duì)3種化合物與CYP2A6結(jié)合影響較大，其中CYP2A6活性口袋中ASN297是蛋白受體與配體結(jié)合的關(guān)鍵氨基酸殘基。

Objective To study the inhibitory effects of methoxsalen and its isomers on drug metabolizing enzyme CYP450. Methods The interaction of methoxsalen and its isomers bergapten, isobergapten and six CYPs were studied by computer simulation technology. Amber (ff99SB) software was used to simulate the dynamics of zanthoxylin, bergamot lactone, isobergamot lactone and CYP2A6. The results of computer simulation were verified by "Cocktail" drug probe method, and their half maximal inhibitoryconcentration(IC₅₀) values to CYP2A6 and CYP1A2 were determined. Result Methoxsalen, bergapten, isobergapten had different binding to the six CYPs, among which the binding to CYP2A6 is significantly different. The results of molecular docking and molecular dynamics showed that ASN297:HD22-M:O4 was the most important and stable hydrogen bond between methoxsalen and CYP2A6, The results showed that the stability of intermolecular hydrogen bond between bergapten and ASN297 was lower than that of methoxsalen system, and the hydrogen bond of isobergamot lactone in isobergapten was unstable. The complementary of hydrophobic amino acids and pocket shape in receptor active pocket was an important factor to promote ligand receptor binding. The "Cocktail" drug probe method showed that the three isomers have different degrees of inhibition on CYPs, which had significant inhibition on CYP1A2. The relative remaining enzyme activities were 2.67%, 4.64%, and 13.58%, and their IC 50values were 1.42, 2.29, and 5.12 μmol/L; the inhibitory effect on CYP2A6 was different, the relative remaining enzyme activities were 8.10%, 38.91% and 74.98%, and the IC₅₀ values were 3.89, 44.03, and 103.30 μmol/L. Conclusion Methoxsalen, bergapten, isobergapten have significant inhibitory effects on CYP1A2, and there are significant differences effects on CYP2A6. Molecular docking and molecular dynamics results show that the substitution position of the methoxy group and the position of the furan ring have a greater impact on the binding of the three compounds to CYP2A6. ASN297 in the CYP2A6 active pocket is the key amino acid for the binding of protein receptors and ligands.

教育部創(chuàng)新團(tuán)隊(duì)發(fā)展計(jì)劃資助項(xiàng)目（IRT_14R41）；廣東省自然科學(xué)基金資助項(xiàng)目（2020A1515010156）；廣東省普通高校重點(diǎn)領(lǐng)域?qū)ｍ?xiàng)（2019KZDZX1006）