Fragment-Based Discovery of Subtype-Selective Adenosine Receptor Ligands from Homology Models

Fragment-based lead discovery (FBLD) holds great promise for drug discovery, but applications to G protein-coupled receptors (GPCRs) have been limited by a lack of sensitive screening techniques and scarce structural information. If virtual screening against homology models of GPCRs could be used to identify fragment ligands, FBLD could be extended to numerous important drug targets and contribute to efficient lead generation. Access to models of multiple receptors may further enable the discovery of fragments that bind specifically to the desired target. To investigate these questions, we used molecular docking to screen >500 000 fragments against homology models of the A3 and A1 adenosine receptors (ARs) with the goal to discover A3AR-selective ligands. Twenty-one fragments with predicted A3AR-specific binding were evaluated in live-cell fluorescence-based assays; of eight verified ligands, six displayed A3/A1 selectivity, and three of these had high affinities ranging from 0.1 to 1.3 μM. Subsequently, structure-guided fragment-to-lead optimization led to the identification of a >100-fold-selective antagonist with nanomolar affinity from commercial libraries. These results highlight that molecular docking screening can guide fragment-based discovery of selective ligands even if the structures of both the target and antitarget receptors are unknown. The same approach can be readily extended to a large number of pharmaceutically important targets.基于片段的先导化合物发现（Fragment-based lead discovery, FBLD）在药物研发领域拥有巨大应用潜力，但针对G蛋白偶联受体（G protein-coupled receptors, GPCRs）的相关应用却因缺乏高灵敏度筛选技术与结构信息匮乏而受到诸多限制。若可通过针对GPCR同源模型的虚拟筛选识别片段配体，FBLD便能拓展至众多重要的药物靶点，为高效先导化合物的生成提供助力。获取多受体同源模型还有望进一步发现能够特异性结合预期靶点的片段分子。为探究上述问题，我们采用分子对接技术，针对A3与A1型腺苷受体（adenosine receptors, ARs）的同源模型筛选了逾50万个片段化合物，以期发现选择性靶向A3AR的配体。我们对21个预测具备A3AR特异性结合能力的片段开展了活细胞荧光检测实验；在8个得到验证的配体中，6个展现出A3/A1受体选择性，其中3个具有0.1至1.3 μM的高亲和力。后续通过结构导向的片段-先导化合物优化，从商业化合物库中筛选得到了一个选择性提升超100倍、纳摩尔级亲和力的拮抗剂。上述结果表明，即便靶标受体与非靶标受体的结构均未知，分子对接筛选仍可指导基于片段的选择性配体发现。该方法同样可便捷地拓展至大量具有药学研究价值的靶点。