Abstract

The macrolactone rapamycin (RAP) presents a broad range of bioactivities, but its clinical applications are compromised due to the poor water solubility and low bioavailability, which could probably be overcome by glycosylation. In this study, we tested a set of promiscuous glycosyltransferases (GTs) to modify rapamycin with four different sugar donors. BsGT-1 displayed the best glycosylation activity with a preference for UDP-glucose, and the glycosylation happened at C-28 or C-40 of rapamycin, producing rapamycin-40-O-β-d-glucoside (RG1), and two new compounds rapamycin-28-O-β-d-glucoside (RG2) and rapamycin-28,40-O-β-d-diglucoside (RG3). The glycosylation remarkably improved water solubility and almost completely abolished cytotoxicity but simultaneously attenuated the antifungal, antitumor, and immunosuppression bioactivities of rapamycin. We found the glycosylation at C-40 had less effect on the bioactivities than that at C-28. The molecular docking analysis revealed that the glycosylation, especially the glycosylation at C-28, weakened the hydrophobic and hydrogen bonding contacts between the rapamycin glucosides and the binding proteins: the FK506-binding protein (FKBP12) and the FKBP12-rapamycin binding (FRB) domain. This study highlights a succinct approach to expand the chemical diversity of the therapeutically important molecule rapamycin by using promiscuous glycosyltransferases. Moreover, the fact that glycosyl moieties at different positions of rapamycin affect bioactivity to different extents inspires further glycosylation engineering to improve properties of rapamycin.

Key points

• Rapamycin was glycosylated efficiently by some promiscuous GTs.

• Glycosylation improved water solubility, attenuated cytotoxicity, and bioactivities.

• Glycosylation affected the interactions between ligand and binding proteins.

中文翻译：

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糖基化对雷帕霉素生物活性的影响。

摘要

大内酯雷帕霉素（RAP）具有广泛的生物活性，但由于水溶性差和生物利用度低，其临床应用受到了损害，这很可能可以通过糖基化来克服。在这项研究中，我们测试了一组混杂的糖基转移酶（GTs），用四种不同的糖供体修饰雷帕霉素。BsGT-1表现出最好的糖基化活性，偏爱UDP葡萄糖，且糖基化发生在雷帕霉素的C-28或C-40处，产生雷帕霉素40- O - β - d-葡萄糖苷（RG1），另外两个化合物雷帕霉素28- ö -β- d葡糖苷（RG2）和雷帕霉素- 28,40- ö -β- d-二葡糖苷（RG3）。糖基化显着改善了水溶性，几乎完全消除了细胞毒性，但同时减弱了雷帕霉素的抗真菌，抗肿瘤和免疫抑制生物活性。我们发现C-40处的糖基化对生物活性的影响比C-28处的小。分子对接分析显示，糖基化，尤其是C-28处的糖基化，减弱了雷帕霉素糖苷与结合蛋白之间的疏水键和氢键接触：FK506结合蛋白（FKBP12）和FKBP12-雷帕霉素结合（FRB）域。这项研究突出了一种简洁的方法，即通过使用混杂的糖基转移酶来扩大治疗上重要的分子雷帕霉素的化学多样性。此外，

关键点

•雷帕霉素被一些混杂的GT有效地糖基化。

•糖基化改善了水溶性，减弱了细胞毒性和生物活性。

•糖基化影响配体与结合蛋白之间的相互作用。