Patel P J, Messer W S, Hudson R A
Department of Medicinal and Biological Chemistry, College of Pharmacy, University of Toledo, Ohio 43606.
J Med Chem. 1993 Jun 25;36(13):1893-901. doi: 10.1021/jm00065a012.
Inhibition and inactivation of two presynaptic cholinergic "markers", choline acetyltransferase and high affinity choline transporter, has been investigated using inhibitors designed with a redox-reactive catechol tethered to a quaternary ammonium group. Two quaternary ammonium alkyl-substituted catechols, 3[(trimethylammonio)methyl]catechol (TMC, 1) and N,N-dimethylepinephrine (catecholine, 2) were shown to bind weakly and noncompetitively to bovine choline acetyltransferase yet inactivated the enzyme in a time course consistent with the involvement of early intermediates in the spontaneous oxidation of these catechols. Both agents also inhibited high-affinity choline uptake. The time course of TMC and catecholine spontaneous oxidation-dependent inactivation of high affinity choline uptake sites was slower than, if it occurred at all, the spontaneous degradation of measurable choline transport in synaptosomes. When compared with inhibition of uptake of other neurotransmitters, it was shown that catecholine demonstrated more selectivity than TMC toward inhibition of choline transport. Km (microM) and Vmax (pmol/min per mg of protein) were measured for high affinity transport of choline, dopamine, and serotonin and were observed to be Km = 2.04 +/- 0.31, Vmax = 22 +/- 1; Km = 1.4, Vmax = 53; and Km = 0.15, Vmax = 23, respectively, in good agreement with published literature values. Ki's (mM) for catecholine and TMC, calculated from experimentally determined IC50's, were for catecholine 0.13 +/- 0.06, 0.53 +/- 0.09, and 0.39 +/- 0.10, and for TMC 0.06 +/- 0.03, 0.09 +/- 0.03, and 0.09 +/- 0.08, for choline, dopamine, and serotonin transport, respectively. In vivo studies using catecholine suggest that this compound impairs learning ability associated with long-term memory. Thus, catecholine represents a lead compound in a potential series of redox-reactive choline analogs, which may become useful irreversible antagonists of the critical cholinergic macromolecular targets underlying cholinergic hypofunction in disorders such as Alzheimer's disease.
利用设计的带有连接季铵基团的氧化还原活性儿茶酚的抑制剂，对两种突触前胆碱能“标记物”，即胆碱乙酰转移酶和高亲和力胆碱转运体的抑制和失活情况进行了研究。两种季铵烷基取代儿茶酚，3-[(三甲基铵基)甲基]儿茶酚（TMC，1）和N，N-二甲基肾上腺素（儿茶胆碱，2）被证明与牛胆碱乙酰转移酶弱结合且无竞争性，但能使该酶失活，其时间进程与这些儿茶酚自发氧化早期中间体的参与一致。这两种试剂还抑制高亲和力胆碱摄取。TMC和儿茶胆碱依赖自发氧化使高亲和力胆碱摄取位点失活的时间进程比突触体中可测量的胆碱转运的自发降解（若发生的话）要慢。与对其他神经递质摄取的抑制作用相比，结果表明儿茶胆碱对胆碱转运抑制的选择性比TMC更高。测定了胆碱、多巴胺和5-羟色胺高亲和力转运的Km（微摩尔）和Vmax（每毫克蛋白质每分钟皮摩尔），结果分别为Km = 2.04±0.31，Vmax = 22±1；Km = 1.4，Vmax = 53；以及Km = 0.15，Vmax = 23，与已发表的文献值高度一致。根据实验测定的IC50计算出的儿茶胆碱和TMC对胆碱、多巴胺和5-羟色胺转运的Ki（毫摩尔）分别为儿茶胆碱0.13±0.06、0.53±0.09和0.39±0.10，TMC分别为0.06±0.03、0.09±0.03和0.09±0.08。使用儿茶胆碱的体内研究表明，该化合物会损害与长期记忆相关的学习能力。因此，儿茶胆碱代表了潜在的一系列氧化还原活性胆碱类似物中的先导化合物，这可能成为阿尔茨海默病等疾病中胆碱能功能减退潜在关键胆碱能大分子靶点的有用不可逆拮抗剂。
