Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada.
FEMS Microbiol Lett. 2009 Nov;300(1):97-106. doi: 10.1111/j.1574-6968.2009.01777.x. Epub 2009 Aug 31.
The emergence of bacterial antibiotic resistance poses a significant challenge in the pursuit of novel therapeutics, making new strategies for drug discovery imperative. We have developed a yeast growth-defect phenotypic screen to help solve this current dilemma. This approach facilitates the identification and characterization of a new diphtheria toxin (DT) group, ADP-ribosyltransferase toxins from pathogenic bacteria. In addition, this assay utilizes Saccharomyces cerevisiae, a reliable model for bacterial toxin expression, to streamline the identification and characterization of new inhibitors against this group of bacterial toxins that may be useful for antimicrobial therapies. We show that a mutant of the elongation factor 2 target protein in yeast, G701R, confers resistance to all DT group toxins and recovers the growth-defect phenotype in yeast. We also demonstrate the ability of a potent small-molecule toxin inhibitor, 1,8-naphthalimide (NAP), to alleviate the growth defect caused by toxin expression in yeast. Moreover, we determined the crystal structure of the NAP inhibitor-toxin complex at near-atomic resolution to provide insight into the inhibitory mechanism. Finally, the NAP inhibitor shows therapeutic protective effects against toxin invasion of mammalian cells, including human lung cells.
细菌对抗生素的耐药性的出现给新型疗法的研发带来了巨大挑战，因此迫切需要新的药物发现策略。我们开发了一种酵母生长缺陷表型筛选方法，以帮助解决这一当前困境。该方法有助于鉴定和表征来自致病菌的新型白喉毒素 (DT) 组，即 ADP-核糖基转移酶毒素。此外，该检测方法利用酿酒酵母（一种用于细菌毒素表达的可靠模型）来简化针对该组细菌毒素的新型抑制剂的鉴定和表征，这些抑制剂可能对抗菌治疗有用。我们证明，酵母中伸长因子 2 靶蛋白的突变体 G701R 赋予了对所有 DT 组毒素的抗性，并恢复了酵母中的生长缺陷表型。我们还证明了一种有效的小分子毒素抑制剂 1,8-萘二甲酰亚胺 (NAP) 能够缓解毒素在酵母中表达引起的生长缺陷。此外，我们还确定了 NAP 抑制剂-毒素复合物的晶体结构，达到了近乎原子分辨率，从而深入了解了抑制机制。最后，NAP 抑制剂在哺乳动物细胞（包括人肺细胞）中对抗毒素侵袭具有治疗保护作用。
