Petrov K
Int J Risk Saf Med. 2015;27 Suppl 1:S72-3. doi: 10.3233/JRS-150695.
Acetylcholinesterase (AChE) inhibitors are widely used in medicine for pharmacological correction of cholinergic neurotransmission pathologies such as myasthenia gravis (MG) and Alzheimer's disease [1, 2]. The efficacy of anti-AChE drugs is based on their ability to potentiate the effects of acetylcholine (ACh) due to a decrease in the rate of AChE-catalyzed hydrolysis of ACh. Crystallographic studies showed that the active site of AChE is located at the bottom of a deep gorge [3]. It was shown that, in addition to its catalytic center, AChE has other sites that are crucial for the proper functioning of the enzyme. In particular, the so-called peripheral anionic site (PAS) located at the entrance of the active site gorge is responsible for: 1) allosteric modulation of the catalytic center; 2) enzyme inhibition at high substrate concentration; 3) and non-catalytic functions such as enhancement of cell adhesion and neurite outgrowth.
Especially interesting is the relationship between the PAS and pathological beta-amyloid deposition. This led to a new hypothesis for rational design of more effective anti-Alzheimer drugs [4].
Concentration of drug producing 50% of AChE activity inhibition (IC50) was measured using the method of Ellman et al. [5]. Toxicological experiments were performed using IP injection of the different compounds in mice. LD50, dose (in mg/kg) causing lethal effects in 50% of animals was taken as a criterion of toxicity [6]. Molecular docking was performed with Autodock 4.2.6 software.
We described previously a new class of selective mammalian AChE vs. butyrylcholinesterase (BChE) inhibitors based on alkylammonium derivatives of 6-methyluracil of acyclic topology [7]. In the present study, taking acyclic derivatives of 6-methyluracil as a model AChE inhibitor, we attempted to develop AChE inhibitors that specifically bind to the PAS with weak binding to the active site of AChE. We attempted to increase the size of AChE ligands to restrict specific binding to the PAS of AChE. To this aim we synthesized pyrimidinophanes bearing two o-nitrobenzylethyldialkylammonium heads. Almost all of synthesized pyrimidinophanes inhibited AChE in the nanomolar range. Based on molecular docking simulations, it was suggested that compounds bind AChE to the active center as well as to the PAS or only to the PAS. Thus, we found that introduction of the spacer, flexible or rigid, between [5-(o-nitrobenzylethylammonium)pentyl] units at N atoms of the 6-methyluracil moiety allows tuning the binding of 6-methyluracil derivatives with AChE.
In conclusion, it can be stated that pyrimidinophanes are promising lead scaffold structures for further design of specific ligands for the PAS of AChE. Also AChE inhibitors with a 6-methyluracil moiety may be considered as potential drugs for the treatment of pathological muscle weakness syndromes.
乙酰胆碱酯酶（AChE）抑制剂在医学上被广泛用于药理学纠正胆碱能神经传递病理学，如重症肌无力（MG）和阿尔茨海默病[1,2]。抗AChE药物的疗效基于其因降低AChE催化ACh水解速率而增强乙酰胆碱（ACh）作用的能力。晶体学研究表明，AChE的活性位点位于一个深峡谷的底部[3]。研究表明，除了其催化中心外，AChE还有其他对酶的正常功能至关重要的位点。特别是，位于活性位点峡谷入口处的所谓外周阴离子位点（PAS）负责：1）催化中心的变构调节；2）高底物浓度下的酶抑制；3）以及非催化功能，如增强细胞粘附和神经突生长。
特别有趣的是PAS与病理性β-淀粉样蛋白沉积之间的关系。这导致了一种关于合理设计更有效抗阿尔茨海默病药物的新假设[4]。
采用埃尔曼等人的方法[5]测量产生50% AChE活性抑制（IC50）的药物浓度。使用不同化合物腹腔注射小鼠进行毒理学实验。将导致50%动物产生致死效应的剂量（以mg/kg计）LD50作为毒性标准[6]。使用Autodock 4.2.6软件进行分子对接。
我们之前描述了一类基于无环拓扑结构的6-甲基尿嘧啶烷基铵衍生物的新型选择性哺乳动物AChE与丁酰胆碱酯酶（BChE）抑制剂[7]。在本研究中，以6-甲基尿嘧啶的无环衍生物作为模型AChE抑制剂，我们试图开发与PAS特异性结合且与AChE活性位点弱结合的AChE抑制剂。我们试图增加AChE配体的大小以限制与AChE的PAS的特异性结合。为此，我们合成了带有两个邻硝基苄基乙基二烷基铵头的嘧啶环番。几乎所有合成的嘧啶环番在纳摩尔范围内抑制AChE。基于分子对接模拟，表明化合物与AChE的活性中心以及PAS结合或仅与PAS结合。因此，我们发现，在6-甲基尿嘧啶部分的N原子处的[5-（邻硝基苄基乙基铵）戊基]单元之间引入柔性或刚性间隔基，可以调节6-甲基尿嘧啶衍生物与AChE的结合。
总之，可以说嘧啶环番是用于进一步设计AChE的PAS特异性配体的有前景的先导支架结构。此外，带有6-甲基尿嘧啶部分的AChE抑制剂可被视为治疗病理性肌肉无力综合征的潜在药物。
