变构调节
化学
药物发现
G蛋白偶联受体
可药性
生物正交化学
马拉维洛克
连接器
计算生物学
生物化学
点击化学
受体
组合化学
生物
人类免疫缺陷病毒(HIV)
免疫学
操作系统
基因
计算机科学
作者
Jordan M. Mattheisen,Chris Limberakis,Roger B. Ruggeri,Matthew S. Dowling,Christopher W Am Ende,Emilie Ceraudo,Thomas Huber,Christopher L. McClendon,Thomas P. Sakmar
摘要
G protein-coupled receptors (GPCRs) modulate diverse cellular signaling pathways and are important drug targets. Despite the availability of high-resolution structures, the discovery of allosteric modulators remains challenging due to the dynamic nature of GPCRs in native membranes. We developed a strategy to covalently tether drug fragments adjacent to allosteric sites in GPCRs to enhance their potency and enable fragment-based drug screening in cell-based systems. We employed genetic code expansion to site-specifically introduce noncanonical amino acids with reactive groups in C-C chemokine receptor 5 (CCR5) near an allosteric binding site for the drug maraviroc. We then used molecular dynamics simulations to design heterobifunctional maraviroc analogues consisting of a drug fragment connected by a flexible linker to a reactive moiety capable of undergoing a bioorthogonal coupling reaction. We synthesized a library of these analogues and employed the bioorthogonal inverse electron demand Diels-Alder reaction to couple the analogues to the engineered CCR5 in live cells, which were then assayed using cell-based signaling assays. Tetherable low-affinity maraviroc fragments displayed an increase in potency for CCR5 engineered with reactive unnatural amino acids that were adjacent to the maraviroc binding site. The strategy we describe to tether novel drug fragments to GPCRs should prove useful to probe allosteric or cryptic binding site functionality in fragment-based GPCR-targeted drug discovery.
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