Zender Michael, Witzgall Florian, Drees Steffen L, Weidel Elisabeth, Maurer Christine K, Fetzner Susanne, Blankenfeldt Wulf, Empting Martin, Hartmann Rolf W
Helmholtz Institute for Pharmaceutical Research Saarland (HIPS) , Department Drug Design and Optimization, Campus E8.1, 66123 Saarbrücken, Germany.
Helmholtz Centre for Infection Research , Department Structure and Function of Proteins, Inhoffenstrasse 7, 38124 Braunschweig, Germany.
ACS Chem Biol. 2016 Jun 17;11(6):1755-63. doi: 10.1021/acschembio.6b00156. Epub 2016 Apr 28.
Pseudomonas aeruginosa uses quorum sensing (QS) as a cell-to-cell communication system to orchestrate the expression of virulence determinants. The biosynthesis of the important Pseudomonas quinolone signal (PQS) requires the pqsABCDE operon. Here, PqsE acts as a pathway-specific thioesterase, but it also contributes to the regulation of bacterial virulence via an unknown mechanism. In this manuscript, we report the discovery of PqsE inhibitors as tool compounds to gain further insights into its different functions. Differential scanning fluorimetry (DSF) was used to screen a fragment library, and isothermal titration calorimetry (ITC) was employed as a secondary filter. As proven by X-ray crystallography, hit molecules bound to the active center inhibiting PqsE's thioesterase activity in cell-based and in vitro assays. Notably, the ligands did not affect the levels of the PqsE-regulated virulence factor pyocyanin. These findings indicate that the regulatory function of PqsE is not linked to its thioesterase activity and must be encoded outside of the active center. This study highlights the potential of fragment-based screening for the discovery of tool compounds. This approach provided novel insight into complex biological systems, which could not be obtained by knockout studies.
铜绿假单胞菌利用群体感应（QS）作为细胞间通讯系统来协调毒力决定因素的表达。重要的铜绿假单胞菌喹诺酮信号（PQS）的生物合成需要pqsABCDE操纵子。在这里，PqsE作为一种途径特异性硫酯酶，但它也通过一种未知机制参与细菌毒力的调节。在本论文中，我们报告了发现PqsE抑制剂作为工具化合物，以进一步深入了解其不同功能。采用差示扫描荧光法（DSF）筛选片段库，并使用等温滴定量热法（ITC）作为二级筛选方法。经X射线晶体学证实，命中分子在基于细胞和体外试验中与活性中心结合，抑制PqsE的硫酯酶活性。值得注意的是，这些配体不影响PqsE调节的毒力因子绿脓菌素的水平。这些发现表明，PqsE的调节功能与其硫酯酶活性无关，并且必须在活性中心之外编码。本研究突出了基于片段筛选发现工具化合物的潜力。这种方法为复杂生物系统提供了新的见解，这是基因敲除研究无法获得的。
