Li Hu, Huang Jianzhong, Jiang Xinhe, Seefeld Mark, McQueney Michael, Macarron Ricardo
Department of Molecular Screening, GlaxoSmithKline, King of Prussia, PA 19406, USA.
J Biomol Screen. 2003 Dec;8(6):712-5. doi: 10.1177/1087057103258185.
Undecaprenyl pyrophosphate synthase (UPPS) catalyzes the consecutive condensation of 8 molecules of isopentenyl pyrophosphate with farnesyl pyrophosphate to yield C55-undecaprenyl pyrophosphate, which is required for bacterial cell wall synthesis. UPPS is found in both gram-positive and gram-negative bacteria, and based on the differences between bacterial variants of UPPS and their human counterpart, dolicopyrophosphate synthase, it was identified as an attractive antibacterial target. An assay, which monitors the release of Pi by coupling the UPPS catalyzed reaction with inorganic pyrophosphatase, was employed to conduct an HTS campaign using an inhouse collection of compounds. A direct assay measuring the incorporation of 14C-IPP (isopentenyl pyrophosphate) was used as a secondary assay to evaluate the high-throughput screening (HTS) hits. From the HTS campaign, a few classes of UPPS inhibitors were identified. During the process of hit evaluation by the direct assay, the authors observed that Triton, an essential factor for the enzyme activity and accurate formation of the natural product, dramatically altered the inhibitory activity of a particular class of compounds. Above its critical micellar concentration (CMC), Triton abolished the inhibitory activity of these compounds. Further research will be required to establish the biophysical phenomenon that causes this effect. Meanwhile, it can be speculated that Triton (and other detergents) above CMC may hinder the identification in screening compounds of certain classes of hits.
十一异戊烯基焦磷酸合酶（UPPS）催化8分子异戊烯基焦磷酸与法尼基焦磷酸连续缩合，生成细菌细胞壁合成所需的C55 - 十一异戊烯基焦磷酸。UPPS存在于革兰氏阳性菌和革兰氏阴性菌中，基于UPPS的细菌变体与其人类对应物——多萜焦磷酸合酶之间的差异，它被确定为一个有吸引力的抗菌靶点。一种通过将UPPS催化反应与无机焦磷酸酶偶联来监测无机磷酸（Pi）释放的检测方法，被用于使用内部化合物库进行高通量筛选（HTS）。一种直接测量14C - 异戊烯基焦磷酸（IPP）掺入的检测方法被用作二级检测，以评估高通量筛选的命中化合物。通过高通量筛选，鉴定出了几类UPPS抑制剂。在通过直接检测进行命中评估的过程中，作者观察到Triton（一种对酶活性和天然产物准确形成至关重要的因素）显著改变了某一类化合物的抑制活性。高于其临界胶束浓度（CMC）时，Triton消除了这些化合物的抑制活性。需要进一步研究来确定导致这种效应的生物物理现象。同时，可以推测高于CMC的Triton（和其他洗涤剂）可能会阻碍在筛选化合物中识别某些类别的命中化合物。
