Fei Xiang, Holmes Thomas, Diddle Julianna, Hintz Lauren, Delaney Dan, Stock Alex, Renner Danielle, McDevitt Molly, Berkowitz David B, Soukup Juliane K
Department of Chemistry, University of Nebraska , Lincoln, Nebraska 68588, United States.
ACS Chem Biol. 2014 Dec 19;9(12):2875-82. doi: 10.1021/cb500458f. Epub 2014 Oct 27.
The glmS riboswitch is unique among gene-regulating riboswitches and catalytic RNAs. This is because its own metabolite, glucosamine-6-phosphate (GlcN6P), binds to the riboswitch and catalytically participates in the RNA self-cleavage reaction, thereby providing a novel negative feedback mechanism. Given that a number of pathogens harbor the glmS riboswitch, artificial actuators of this potential RNA target are of great interest. Structural/kinetic studies point to the 2-amino and 6-phosphate ester functionalities in GlcN6P as being crucial for this actuation. As a first step toward developing artificial actuators, we have synthesized a series of nine GlcN6P analogs bearing phosphatase-inert surrogates in place of the natural phosphate ester. Self-cleavage assays with the Bacillus cereus glmS riboswitch give a broad SAR. Two analogs display significant activity, namely, the 6-deoxy-6-phosphonomethyl analog (5) and the 6-O-malonyl ether (13). Kinetic profiles show a 22-fold and a 27-fold higher catalytic efficiency, respectively, for these analogs vs glucosamine (GlcN). Given their nonhydrolyzable phosphate surrogate functionalities, these analogs are arguably the most robust artificial glmS riboswitch actuators yet reported. Interestingly, the malonyl ether (13, extra O atom) is much more effective than the simple malonate (17), and the "sterically true" phosphonate (5) is far superior to the chain-truncated (7) or chain-extended (11) analogs, suggesting that positioning via Mg coordination is important for activity. Docking results are consistent with this view. Indeed, the viability of the phosphonate and 6-O-malonyl ether mimics of GlcN6P points to a potential new strategy for artificial actuation of the glmS riboswitch in a biological setting, wherein phosphatase-resistance is paramount.
glmS核糖开关在基因调控核糖开关和催化RNA中独树一帜。这是因为其自身的代谢物6-磷酸葡萄糖胺（GlcN6P）与核糖开关结合并催化参与RNA自我切割反应，从而提供了一种新型的负反馈机制。鉴于许多病原体都含有glmS核糖开关，这种潜在RNA靶点的人工激活剂备受关注。结构/动力学研究表明，GlcN6P中的2-氨基和6-磷酸酯官能团对于这种激活至关重要。作为开发人工激活剂的第一步，我们合成了一系列九个GlcN6P类似物，它们带有磷酸酶惰性替代物以取代天然磷酸酯。用蜡样芽孢杆菌glmS核糖开关进行的自我切割试验给出了广泛的构效关系。两个类似物显示出显著活性，即6-脱氧-6-膦酰甲基类似物（5）和6-O-丙二酰醚（13）。动力学曲线表明，与葡萄糖胺（GlcN）相比，这些类似物的催化效率分别高出22倍和27倍。鉴于它们不可水解的磷酸替代官能团，这些类似物可以说是迄今报道的最稳定的人工glmS核糖开关激活剂。有趣的是，丙二酰醚（13，额外的氧原子）比简单的丙二酸酯（17）有效得多，“空间真实”的膦酸酯（5）远优于链截短的（7）或链延长的（11）类似物，这表明通过镁配位进行定位对活性很重要。对接结果与这一观点一致。事实上，GlcN6P的膦酸酯和6-O-丙二酰醚模拟物的可行性指出了在生物环境中人工激活glmS核糖开关的一种潜在新策略，其中抗磷酸酶能力至关重要。
