一、七種人類細胞色素P450 3A4受質抑制土震素代謝反應之研究; 二、以RAF值探討細胞色素P450 3A於大鼠及人體肝臟微粒體中 土震素代謝的角色

1.Inhibition Study of Territrem 4β-C hydroxylation and O-demethylation by Seven Human Cytochrome P450 3A4 substrates; 2.Application of the Relative Activity Factor to Approach the Role of Cytochrome P450 3A to the Metabolism of Territrems by Wistar Rats and Human Liver Microsomes

林侑璇(Yu-Hsuan Lin)

指導教授 ： 彭福佐

國立臺灣大學/醫學院/毒理學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.01284

摘要

一、七種人類細胞色素P450 3A4受質抑制土震素代謝反應之研究
土震素為黴菌Aspergillus terreus 23-1產生的震顫性毒素。過去研究顯示土震素A (TRA)可經4β-C hydroxylation產生MA1，TRB可進行4β-C hydroxylation生成MB2及O-demethylation生成MB4，以上反應在人體肝臟均主要由細胞色素P450 3A4催化。CYP3A4構造內具有相當大的催化活性區(active site)，可同時有兩種以上受質協同結合，且受質間存在交互作用，使CYP3A4對其受質之代謝動力學常為非典型(atypical)，另外，活性區內特定胺基酸對於受質之結合扮演相當重要的角色。
為了瞭解TRA、TRB代謝過程中在CYP3A4上結合位置的關係，本實驗使用可表現人類CYP3A4之V79MZh3A4細胞株，同時給予TRA及TRB進行代謝，以HPLC分析兩者代謝物產量，繪製雙倒數圖形以了解兩者間抑制作用的類型。結果顯示：TRA生成MA1及TRB生成MB2過程中，TRB和TRA互為混合型抑制作用；TRB生成MB4過程中，則和TRA具有未競爭型抑制作用。
為了進一步探求TRB在CYP3A4上結合的位置，選定七種和TRB進行類似代謝反應之CYP3A4受質，分別和TRB同時進行代謝，以抑制類型間接推測TRB在CYP3A4上結合的位置。結果顯示：在TRB代謝至MB2過程中，和testosterone具有競爭型抑制作用、和budesonide具有未競爭型抑制作用；而在TRB代謝至MB4過程中，和testosterone、etoposide具有未競爭型抑制作用；和phenacetin、erythromycin具有混合型抑制作用。
綜合以上結果推測：TRA和TRB在CYP3A4上的結合位置不同，但其中之ㄧ和CYP3A4結合後會改變另一結合位置的結構，使CYP 3A4對另一受質結合的親合力下降，另外，推測TRB和testosterone在CYP 3A4活性區中會競爭同一結合位置，即可與CYP 3A4活性區中serine-119形成氫鍵進行4β-C hydroxylation，但和上述其餘抑制劑均不在同一結合位置，僅會互相牽制，至於TRB和受質間交互作用的模式仍未知，另外，本實驗亦觀察到數種CYP3A4的非典型動力學的特性，證實了CYP3A4的龐雜性。

二、以RAF值探討細胞色素P450 3A於大鼠及人體肝臟微粒體中
土震素代謝的角色
細胞色素P450是人體內最重要之氧化酵素系統，負責代謝超過90%之內生性及外生性物質，因為其廣泛的受質特異性，易有受質間交互作用，且兩種或以上的CYP會同時催化同一受質進行代謝反應，因此研究各種CYP 在藥物代謝中貢獻的程度是相當重要的課題。RAF (relative activity factor，相關活性因素) 值是Crespi於1995年首度提出的概念，其可以整合來自以基因重組技術表現單一P450 (recombinant P450, rP450s)及人類肝臟微粒體 (human liver microsome, HLM)之代謝數據，並定量比較兩者之間的差異，另外，計算各種CYP之CLint (intrinsic clearance) 值，以了解各種CYP P450對於整體代謝之貢獻百分比。
本實驗室過去以人類肝臟微粒體及rP450s的研究指出：在土震素B (TRB)代謝至MB2過程中，以CYP3A4占97.1%為主要參與酵素；代謝至MB4過程中，以CYP3A5占64.3%為主要參與酵素。
為了更進一步釐清各種酵素在TRA及TRB代謝過程中的貢獻程度，我們使用基因重組之rP450s、兩週和八週大之雌、雄大鼠肝臟微粒體及八份HLM，以適量的濃度進行酵素動力學反應，求得反應Vmax 及Km値，並計算各酵素之RAF及CLint值。結果顯示：在大鼠及人體肝臟微粒體代謝TRA過程中，分別以CYP3A2占84.7%及CYP3A4占85.9%為主要參與酵素；在大鼠肝臟微粒體代謝TRB至MB2過程中，以CYP3A1占92%為主要參與酵素、代謝至MB4過程中，以CYP3A2占65.2%為主要參與酵素。另外，觀察到大鼠代謝過程具有年齡及性別差異性，普遍以八週大公鼠最好，而人體代謝過程中個體差異大，但和年齡及性別無明顯相關。許多因素可能導致以上代謝之差異性，包括TRA、TRB相異之化學結構、不同年齡及性別之大鼠肝臟中CYP3A1/2 mRNA及蛋白質表現量之差異、不同人體肝臟中CYP3A4/5表現量及活性亦會因許多因素而異、CYP3A1/2 及CYP3A4/5間胺基酸序列同質性及酵素結合位置的差異等，均是未來必須要進一步探討之課題。

關鍵字

土震素 ； 細胞色素P450 ； 受質結合位置 ； 相關活性因素

並列摘要

1. Inhibition Study of Territrem 4β-C hydroxylation and O-demethylation by Seven Human Cytochrome P450 3A4 substrates
Territrems are the structure related tremogenic mycotoxins, which are produced by Aspergillus terreus 23-1. Previous research indicated that MA1 can be produced from TRA hydroxylation, and TRB can produce MB2 and MB4 by 4β-C hydroxylation and O-demethylation. These reactions are mainly catalyzed by P450 3A4 in liver.
Structure of CYP3A4 contains great active site, and also capable of binding of more than two substrates. Interactions between substrates cause the kinetics of CYP3A4 become atypical. In addition, some amino acids in CYP3A4 active site plays an important role in substrate binding.
To understand the relations of the location of TRA and TRB metabolism, this experience uses cell line V79MZh3A4 which expressing human CYP3A4. We provide TRA and TRB in time to metabolize and analyze the metabolite by HPLC. Double reciprocal plot facilitates understanding of types of inhibition.
Results shows that in the process of TRA producing MA1 and TRB producing MB2, TRB and TRA are mutually mixed-type inhibition. In the process of TRB producing MB4, TRB and TRA are un-competitive inhibition.
To delve more into the combinational location of TRB on CYP3A4, select seven kinds of CYP3A4 substrates to metabolize with TRB at the same time, in order to indirectly infer the location of TRB on CYP3A4. The results presents that in the metabolizing process from TRB to MB2, inhibition occurs with testosterone and un-inhibition occurs with budesonide. Also, in the metabolizing process of TRB to MB4, un-competitive inhibition occurs with testosterone, etoposide and mixed-type inhibition occurs with budesonide.
Inferring from above, combination of TRA and TRB on CYP3A4 have different location, but one combination of another might change the structural location, causing declines affinity of CYP3A4.
Additionally, by predicting the competitive locations of TRB and testosterone, we suggest that TRB can form a hydrogen bond with serine-119 in CYP 3A4 active site. But above inhibitors locate differently, and they only interact with each other. About the modes of TRB and substrate, it still remains unknown. Moreover, our experiment observed many atypical characteristics of CYP3A4, proving the complexity of CYP3A4.

2.Application of the Relative Activity Factor to Approach the Role of Cytochrome P450 3A to the Metabolism of Territrems by Wistar Rats and Human Liver Microsomes
Cytochrome P450 is the most crucial oxidative enzyme system responsible for metabolizing over 90% endogenous and xenobiotics in human. Its prevalent characteristics of substrate specificity often result in interactions between substrates and, moreover, two or more CYP enzymes often contribute to the metabolism of a single drug. Due to above reasons, it’s important to determine the relative contribution of each CYP to net metabolism of the drug..
The concept of RAF (relative activity factor) was first proposed by Crespi at 1995. The RAF approach has been applied successfully to estimate CYP isoforms contribution to drug metabolism.
According to the previous research in our Laboratory, during metabolism of TRB to MB2, CYP3A4 was major involving enzyme, containing 97.1%: during metabolism of TRB to MB4, CYP3A4 was the major, containing 64.3%.
To clarify further the relative contributions of each CYP isoforms to the metabolism of TRA and TRB, we use rP450s and different ages and genders of human, and Wistar rats, and calculate the RAF.
Experiment results indicated that during metabolism of TRA to MA1 in rat and human liver microsomes, CYP3A2 (84.7%) and CYP3A4 (85.9%) are major involving enzyme. During metabolism of TRB to MB2 and MB4 in rat liver microsomes, CYP3A1 (92%) and CYP3A2 (65.2%) are major involving enzyme.
Furthermore, the results indicated that comparing the sex and age differences, 8-week-old male rats presented best results in the process of metabolism. But for human bodies, there is no obvious correlation between age and sex. Many factors might explain this, including the chemical structure of TRA and TRB, the mRNA and protein expression level of CYP3A1/2 in rats, the expression level and activity of CYP3A4/5 in different human bodies, amino acid sequence homology between CYP3A1/2 and CYP3A4/5, and the substrate binding sites in CYPs.

並列關鍵字

Territrem ； CYP P450 ； substrate binding site ； Relative Activity Factor

參考文獻

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