Design and synthesis of nucleoside-based small molecules to inhibit phosphoglycosyl transferases

项目总结/摘要 抗生素耐药性正在成为对全球健康的日益严重的威胁。该奖学金旨在产生 针对与细菌存活和最终抗生素抗性相关的酶的抑制剂文库。 核苷类天然产物对细菌细胞壁合成具有生物活性， 抑制剂发展的极好支架。然而，这些天然产物的复杂性使得 合成、控制选择性和使毒性最小化是一个挑战。酶家族的目标是 该建议是单位磷酸糖基转移酶（PGTs）。这些PGTs是催化 合成与细菌毒力相关的糖缀合物的初始步骤。一旦一组抑制剂 合成后，将用各种单位PGTs进行生物化学测定以确定其IC 50， Kd值。然而，抗生素开发的一个主要限制是一些抑制剂不能穿过 革兰氏阴性菌的膜和积累。出于这个原因，将测试抑制剂的细胞毒性。 在革兰氏阴性菌中的摄取，并使用液相色谱-质谱联用进行定量 （LC-MS）。最后，将进行UDP-糖的化学酶促合成，并与以下组合使用： 生物化学测定以帮助表征单聚体PGT。建议的工作的最终结果是， 这项研究将有助于开发针对单位PGTs的有效抑制剂， 提供研究糖缀合物生物合成的工具，并最终帮助对抗抗生素耐药性。

Project Summary/Abstract Antibiotic resistance is becoming an increasing threat to global health. This fellowship aims to produce inhibitor libraries that target enzymes associated with bacterial survival and ultimately antibiotic resistance. Nucleoside natural products have shown biological activity towards bacterial cell wall synthesis, making them excellent scaffolds for inhibitor development. However, the complexity of these natural products makes synthesis, controlling selectivity, and minimizing toxicity a challenge. The family of enzymes that are targeted in this proposal are monotopic phosphoglycosyl transferases (PGTs). These PGTs are enzymes that catalyze the initial step in the synthesis of glycoconjugates that are linked with bacterial virulence. Once a set of inhibitors are synthesized, they will be tested with biochemical assays with various monotopic PGTs to establish their IC50 and Kd values. However, a major limitation with antibiotic development is the inability of some inhibitors to traverse the membrane and accumulate in Gram-negative bacteria. For that reason, the inhibitors will be tested for cellular uptake in Gram-negative bacteria and quantified using liquid chromatography coupled with mass spectroscopy (LC-MS). Lastly, chemoenzymatic syntheses of a UDP-sugars, will be performed and used in combination with biochemical assays to aid in the characterization of a monotopic PGT. The culmination of the work proposed in this fellowship will contribute to the development of effective inhibitors targeting monotopic PGTs, which will provide tools to study glycoconjugate biosynthesis and ultimately aid in the fight against antibiotic resistance.