High-throughput screening using the differential radial capillary action of ligand assay identifies ebselen as an inhibitor of diguanylate cyclases.
High-throughput screening using the differential radial capillary action of ligand assay identifies ebselen as an inhibitor of diguanylate cyclases.
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DOI：
10.1021/cb400485k
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发表时间：
2014-01-17
影响因子：
4
通讯作者：
Lee VT
中科院分区：
文献类型：
--
作者：
Lieberman OJ;Orr MW;Wang Y;Lee VT
细菌对传统抗生素耐药性的上升促使最近的努力，以确定新的候选药物的目标毒力因子或其调控途径。一种这样的抗毒性靶标是环状二GMP（cdiGMP）信号传导途径，其调节生物膜形成、运动性和发病机制。铜绿假单胞菌是一种重要的条件致病菌，其利用cdiGMP调节的多糖（包括藻酸盐和表膜多糖（PEL））来介导毒力和抗生素抗性。CdiGMP通过与包括PelD和Alg 44的特异性受体结合来激活PEL和藻酸盐生物合成。消除cdiGMP与这些受体结合的突变阻止多糖产生。鉴定能够抑制cdiGMP与这些蛋白质上的变构位点结合的小分子可以模拟结合缺陷突变体并潜在地减少生物膜形成或藻酸盐分泌。在这里，我们报告了一个快速和定量的高通量筛选抑制剂的蛋白质-cdiGMP相互作用的基础上的差分径向毛细管作用的配体测定（DRaCALA）的发展。使用这种方法，我们将依布硒啉鉴定为与含有RxxD结构域的受体结合的cdiGMP抑制剂，所述RxxD结构域包括PelD和二鸟苷酸环化酶（DGC）。依布硒啉通过共价修饰半胱氨酸残基降低二鸟苷酸环化酶活性。依布硒啉氧化物（依布硒啉的硒酮类似物）也通过相同的共价机制抑制cdiGMP结合。依布硒啉和依布硒啉氧化物通过抑制二鸟苷酸环化酶来抑制铜绿假单胞菌中生物膜形成和鞭毛介导的运动性的cdiGMP调节。依布硒啉的鉴定提供了一种原理证明，即DRaCALA高通量筛选方法可用于鉴定通过靶向cdiGMP结合结构域逆转cdiGMP信号传导调节的生物活性剂。
The rise of bacterial resistance to traditional antibiotics has motivated recent efforts to identify new drug candidates that target virulence factors or their regulatory pathways. One such antivirulence target is the cyclic-di-GMP (cdiGMP) signaling pathway, which regulates biofilm formation, motility, and pathogenesis. Pseudomonas aeruginosa is an important opportunistic pathogen that utilizes cdiGMP-regulated polysaccharides, including alginate and pellicle polysaccharide (PEL), to mediate virulence and antibiotic resistance. CdiGMP activates PEL and alginate biosynthesis by binding to specific receptors including PelD and Alg44. Mutations that abrogate cdiGMP binding to these receptors prevent polysaccharide production. Identification of small molecules that can inhibit cdiGMP binding to the allosteric sites on these proteins could mimic binding defective mutants and potentially reduce biofilm formation or alginate secretion. Here, we report the development of a rapid and quantitative high-throughput screen for inhibitors of protein-cdiGMP interactions based on the differential radial capillary action of ligand assay (DRaCALA). Using this approach, we identified ebselen as an inhibitor of cdiGMP binding to receptors containing an RxxD domain including PelD and diguanylate cyclases (DGC). Ebselen reduces diguanylate cyclase activity by covalently modifying cysteine residues. Ebselen oxide, the selenone analogue of ebselen, also inhibits cdiGMP binding through the same covalent mechanism. Ebselen and ebselen oxide inhibit cdiGMP regulation of biofilm formation and flagella-mediated motility in P. aeruginosa through inhibition of diguanylate cyclases. The identification of ebselen provides a proof-of-principle that a DRaCALA high-throughput screening approach can be used to identify bioactive agents that reverse regulation of cdiGMP signaling by targeting cdiGMP-binding domains.