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Abstract：
The D-aspartate ligase of Enterococcus faecium (Aslfm) is an attractive target for the development of narrow-spectrum antibacterial agents that are active against multidrug-resistant E. faecium. Although there is currently little available information regarding the structural characteristics of Aslfm, we exploited the knowledge that this enzyme belongs to the ATP-grasp superfamily to target its ATP binding site. In the first design stage, we synthesized and screened a small library of known ATP-competitive inhibitors of ATP-grasp enzymes. A series of amino-oxazoles derived from bacterial biotin carboxylase inhibitors showed low micromolar activity. The most potent inhibitor compound 12, inhibits Aslfm with a Ki value of 2.9 μM. In the second design stage, a validated ligand-based pharmacophore modeling approach was used, taking the newly available inhibition data of an initial series of compounds into account. Experimental evaluation of the virtual screening hits identified two novel structural types of Aslfm inhibitors with 7-amino-9H-purine (18) and 7-amino-1H-pyrazolo[3,4-d]pyrimidine (30 and 34) scaffolds, and also with Ki values in the low micromolar range. Investigation the inhibitors modes of action confirmed that these compounds are competitive with respect to the ATP molecule. The binding of inhibitors to the target enzyme was also studied using isothermal titration calorimetry (ITC). Compounds 6, 12, 18, 30 and 34 represent the first inhibitors of Aslfm reported to date, and are an important step forward in combating infections due to E. faecium.
摘要：
屎肠球菌的D-天冬氨酸连接酶（Aslfm）是开发对耐多药屎肠球菌具有活性的窄谱抗菌剂的有吸引力的靶标。尽管目前关于Aslfm的结构特征的可用信息很少，我们利用了该酶属于ATP-抓取超家族的知识来靶向其ATP结合位点。在第一个设计阶段，我们合成并筛选了已知ATP-抓取酶的ATP竞争性抑制剂的小型文库。一系列源自细菌生物素羧化酶抑制剂的氨基恶唑显示出低的微摩尔活性。最有效的抑制剂化合物12抑制Aslfm,Ki值为2.9μM。在第二设计阶段,使用了一种经过验证的基于配体的药效团建模方法，考虑到最初一系列化合物的新获得的抑制数据。虚拟筛选命中的实验评估确定了具有7-氨基-9H-嘌呤（18）和7-氨基-1H-吡唑并[3,4-d]嘧啶（30和34）支架的Aslfm抑制剂的两种新型结构类型，并且Ki值在低微摩尔范围内。研究抑制剂的作用模式证实这些化合物相对于ATP分子是竞争性的。还使用等温滴定量热法(ITC)研究了抑制剂与靶酶的结合。化合物6、12、18、30和34代表迄今为止报道的第一个Aslfm抑制剂，是对抗屎肠球菌感染的重要一步。
