N,N-diethylaminobenzaldehyde (DEAB) as a substrate and mechanism-based inhibitor for human ALDH isoenzymes.
N,N-diethylaminobenzaldehyde (DEAB) as a substrate and mechanism-based inhibitor for human ALDH isoenzymes.
复制标题
DOI：
10.1016/j.cbi.2014.12.008
复制
发表时间：
2015-06-05
影响因子：
5.1
通讯作者：
Hurley, Thomas D.
中科院分区：
文献类型：
--
作者：
Morgan, Cynthia A.;Parajuli, Bibek;Buchman, Cameron D.;Dria, Karl;Hurley, Thomas D.
N，N-二乙氨基苯甲醛(DEAB)是肿瘤干细胞生物学中常用的乙醛脱氢酶同工酶的选择性抑制剂，因为它在广泛使用的Aldeflor试验中被作为阴性对照化合物。最近越来越多的证据表明，相对于ALDH1、ALDH2和ALDH3家族成员，Deab在体外测试时不是一种选择性抑制药。我们试图测定Deab对ALDH1A1、ALDH1A2、ALDH1A3、ALDH1B1、ALDH1L1、ALDH2、ALDH3A1、ALDH4A1和ALDH5A1同工酶的选择性，并确定Deab发挥抑制作用的机制。我们发现DEAB是ALDH3A1的优良底物，表现出超过其常用底物苯甲醛的Vmax/Km。DEAB也是ALDH1A1的底物，尽管这是一种异常缓慢的底物(周转速度~0.03min−1)。相反，当与ALDH1A2、ALDH1A3、ALDH1B1、ALDH2或ALDH5A1孵育时，几乎没有观察到Deab的周转。Deab既不是ALDH1L1或ALDH4A1的底物，也不是ALDH4A1的抑制剂。酶动力学和QTOF-MS分析表明，DEAB是ALDH1A2和ALDH2的不可逆抑制剂，表观双分子速率常数分别为2900和86000 M−1 S−1。失活机制与氢化物转移后形成的类奎宁共振态是一致的，这种共振态是由几种ALDH同工酶中存在的局部结构特征稳定的。
N,N-diethylaminobenzaldehyde (DEAB) is a commonly used “selective” inhibitor of aldehyde dehydrogenase isoenzymes in cancer stem cell biology due to its inclusion as a negative control compound in the widely utilized Aldefluor assay. Recent evidence has accumulated that DEAB is not a selective inhibitory agent when assayed in vitro versus ALDH1, ALDH2 and ALDH3 family members. We sought to determine the selectivity of DEAB toward ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1, ALDH1L1, ALDH2, ALDH3A1, ALDH4A1 and ALDH5A1 isoenzymes and determine the mechanism by which DEAB exerts its inhibitory action. We found that DEAB is an excellent substrate for ALDH3A1, exhibiting a Vmax/KM that exceeds that of its commonly used substrate, benzaldehyde. DEAB is also a substrate for ALDH1A1, albeit an exceptionally slow one (turnover rate ~0.03 min−1). In contrast, little if any turnover of DEAB was observed when incubated with ALDH1A2, ALDH1A3, ALDH1B1, ALDH2 or ALDH5A1. DEAB was neither a substrate nor an inhibitor for ALDH1L1 or ALDH4A1. Analysis by enzyme kinetics and QTOF mass spectrometry demonstrates that DEAB is an irreversible inhibitor of ALDH1A2 and ALDH2 with apparent bimolecular rate constants of 2900 and 86,000 M−1 s−1, respectively. The mechanism of inactivation is consistent with the formation of quinoid-like resonance state following hydride transfer that is stabilized by local structural features that exist in several of the ALDH isoenzymes.
登录
查看更多内容
影响因子：
5.1
作者：
Moreb, Jan S.;Ucar, Deniz;Han, Shuhong;Amory, John K.;Goldstein, Alex S.;Ostmark, Blanca;Chang, Lung-Ji
通讯作者：
Chang, Lung-Ji
影响因子：
8
作者：
Mann, CJ;Weiner, H
通讯作者：
Weiner, H
DOI：
10.1073/pnas.96.16.9118
发表时间：
1999-08-03
影响因子：
11.1
作者：
Storms, RW;Trujillo, AP;Smith, C
通讯作者：
Smith, C
影响因子：
3.1
作者：
Vasiliou, V;Pappa, A
通讯作者：
Pappa, A
影响因子：
2.9
作者：
FARRES, J;WANG, TTY;WEINER, H
通讯作者：
WEINER, H