新穎第二型醛脫氫酶促進劑之設計、合成 與離體活性評估

Abstract

第二型粒線體醛脫氫酶 (mitochondrial aldehyde dehydrogenase 2, ALDH2)，為人體進行乙醇代謝的必需氧化酶之一，負責將乙醇經醇脫氫酶 (Alcohol dehydroge-nase, ADH) 氧化所得之乙醛再氧化至低毒性的乙酸。更有研究顯示，此氧化酶除了以乙醛為受質外，亦可以氧化多種內源性與外源性毒性醛類化合物，如：糠醛、丙烯醛與4-羥基壬烯醛，而在人體面對毒性物質的防禦機制上佔有重要的地位。而在臨床研究上，約有5.6億的東亞人種 (佔世界人口比例8%) 具有亞型粒線體醛去氫酶 (ALDH2*2) 之基因，其在氧化乙醛的活性上較野生型粒線體醛去氫酶2減少10倍。近幾年的相關研究更指出，此突變所造成的酵素活性喪失，可能與心血管疾病、慢性神經發炎、消化道癌症等多種病變有關連。 2008年，藉由高通量活性篩選的方式，N-piperonyl-2,6-dichlorobenzamide (Alda-1) 被發現具有相當良好促進ALDH2之活性。本實驗針對Alda-1之骨架，選擇性的修改其結構，製備出一系列類似物，並將其對ALDH2之促進活性與電腦分子模擬進行比對，得到如下之Alda-1類似物結構-活性關係：Alda-1類似物中存在之苄胺結構對ALDH2促進活性非常重要，其可與Phe296及Phe459形成π-π交互作用，並與Asp457形成氫鍵，穩定其與ALDH2之接合構造以防止未反應之受質逸散，以此達到酵素促進作用，上述作用也可能是誘導ALDH2*2結構變形而催化活性增加的主因，而苄胺芳香環上的取代基由於距離靠近Cys302，若為酸性其團，對酵素活性可能造成不良影響。另外，Alda-1類似物之2'或6'位置必須至少存在一個取代基，以防止錯誤的接合模式，並且取代基不可過於親水或是立體障礙過大，其會對ALDH2促進活性造成不良的影響。 本實驗以上述結構-活性關係為基礎設計並合成一系列新骨架ALDH2促進劑，N-(4-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)phenyl)amide (TFOPA)、N-(4-carbamimidoylphenyl)amide (APA)與N-(5-(pyridin-4-yl)thiophen-2-yl)amide (PTHA)類化合物。TFOPA類化合物之水溶解度極低，造成活性評估實驗難以進行；APA類化合物在ALDH2促進實驗中皆展現與Alda-1接近的酵素促進活性，且具有極佳的水溶解度，然而目前尚未能找到活性高於Alda-1之APA骨架化合物；PTHA類化合物在ALDH2促進實驗中，展現出的是抑制活性而非促進活性，未來會針對其相關機轉與修正方式進行研究。

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is an essential enzyme in alcohol metabolism pathway. It converts acetaldehyde, produced from ethanol by alcohol dehydrogenase (ADH), into acetic acid. Past studies indicated that acetaldehyde is not the only one substrate of ALDH2, some endogenous and exogenous toxic aldehyde, e.g.: furaldehyde, acrolein and 4-HNE can also be detoxified by ALDH2. It plays an important role in the defense mechanism against toxic substance of human body. However, about 560 million East Asians (nearly 8% of the world population) carry the ALDH2*2 allel, which is 10 folds less active than wild-type ALDH2. Recent studies also indicated that ALDH2*2 might be relevant to some severe diseases like cardiovascular diseases, chronic neuronal inflammation and cancers. N-Piperonyl-2,6-dichlorobenzamide (Alda-1, 3h) had been found to have ALDH2 -activating activity via high-throughput screening at 2008. In this study, we modified the structure of Alda-1 partially, and synthesized a series of Alda-1 analogs to investigate the structure-activity relationship (SAR) of ALDH2 agonist. After comparing the ALDH2 activating assay with the molecular-docking model, we suggest that the benzylamine moiety might be the pharmacophore of Alda-1 analogs. It can form π-π interaction with Phe296 and Phe459, and form hydrogen bonding with Asp457. These interactions can stabilize the binding conformation of Alda-1 and prevent the substrate from uncatalytic escape, thus, it could activate the activity of ALDH2. Nevertheless, these interactions might also induce the conformational change of ALDH2*2 and enhance its bioactivity. Moreover, the substituted group on the aromatic ring of benzylamine moiety should not be acidic, such substituted group might interfere the catalytic cycle of ALDH2. In addition, at least one substituted group should be located at 2' or 6' position to prevent Alda-1 from incorrect binding, and the substituted group should not be too hydrophilic or bulky. Base on the structure-activity relationship suggested in this study, we designed three scaffolds of novel ALDH2 modulators, N-(4-(5-trifluoromethyl-1,2,4-oxadiazol-3-yl)phenyl)amide (TFOPA), N-(4-carbamimidoylphenyl)amide (APA) and N-(5-(pyridin-4-yl)thiophen-2-yl)amide (PTHA). TFOPA type compounds demonstra-ted very poor water solubility, were unable to run in vitro evaluation. APA type compounds revealed not only the same level of ALDH2-activating activity with Alda-1 but also great water solubility. However, APA type compounds still need more modification to improve the ALDH2-activating activity. And PTHA type compounds inhibited the ALDH2 activity with almost the same activity of isoformononetin (40).