Computer Modeling of G Protein-Coupled Receptors

G 蛋白偶联受体的计算机建模

• 批准号: 8939540
• 负责人: Kenneth Alan Jacobson
• 金额: $ 32.49万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8939540
• 关键词: ADORA3 gene; Adenosine; Adenosine A1 Receptor; Adenosine A2A Receptor; Adenosine A2B Receptor; Affinity; Agonist; Binding; Binding Proteins; Binding Sites; Biochemical; Biological Assay; CXCR4 gene; Characteristics; Charge; Clinical; Complex; Computational Technique; Computer Simulation; Data; Databases; Development; Docking; Drug Design; Elements; Family; Fibrinolytic Agents; G-Protein-Coupled Receptors; Homologous Gene; Homology Modeling; Human; Inflammation; Lead; Ligand Binding; Ligands; Light; Maps; Measures; Mediating; Membrane; Modeling; Molecular; Molecular Conformation; Mutagenesis; Nucleosides; Nucleotides; Performance; Pharmaceutical Preparations; Pharmacology; Platelet aggregation; Property; Protein Databases; Purinergic P1 Receptors; Purines; Pyrimidines; Receptor Activation; Reporting; Research; Resolution; Retrieval; Roentgen Rays; Sequence Alignment; Series; Shapes; Signal Transduction; Structure; Structure-Activity Relationship; Techniques; Testing; Triazoles; X-Ray Crystallography; analog; base; chemokine receptor; design; extracellular; farnesyl pyrophosphate; improved; inorganic phosphate; insight; molecular dynamics; molecular recognition; novel; nucleotide receptor; purine; purinoceptor P2Y1; radioligand; receptor; receptor binding; scaffold; screening; small molecule; small molecule libraries; three dimensional structure; three-dimensional modeling; tool; usability; virtual

Structure-based drug design strategies have been used to elucidate specific ligand recognition determinants and ultimately lead to the design of new small molecules for a specific target. The structure of the target receptor protein is the first requirement in structure-based drug design approaches. In the absence of a high resolution structure of a given G protein-coupled receptor (GPCR), computational techniques like homology modeling can be used to build a 3D model. Briefly, the best structural template is chosen from the Protein Database (PDB) mainly considering sequence identity and similarity with the target receptor and the quality of the crystallographic structure (e.g. resolution). The sequence of the target receptor is aligned to the template structure using highly conserved residues and the known shared structural features to guide the automated or semi-automated alignment. The sequence alignment and the template structures are the input for the homology modeling. Energy minimization or molecular dynamics can be used to further refine and optimize the resulting 3D models. The ligand-receptor interactions can be identified by means of structure-based approaches, e.g. molecular docking. The information contained in the ligand-receptor complexes from the docking can clarify structural elements for molecular recognition and lead to a further optimization of the compounds and the design of new derivatives. The binding site of a given GPCR can be mapped for each class of small molecule ligands. Also, virtual searching of chemically diverse databases for novel chemotypes to bind to a given GPCR structure has been productive using both structure-based and ligand-based strategies. We have applied mutagenesis and homology modeling to the study of GPCR families for extracellular purines and pyrimidines and used the structural insights gained to assist in the design of novel ligands. These families consist of the adenosine receptors (ARs) and the P2Y (nucleotide) receptors. The structures of the human A2AAR were recently reported in the antagonist-bound state and in the agonist-bound state. These structures can reliably serve as modeling templates, with some adjustment, for other ARs due to the relatively high sequence identity between ARs (average 47% between human subtypes). We achieved the structure-function analysis of P2YRs by indirect means, using mutagenesis and homology modeling based on a template of the high-resolution structure of similar GPCRs, such as the CXCR4 chemokine receptor. We have collaborated with one of the premier centers for X-ray crystallography of membrane-bound proteins, i.e. the lab of Prof. Ray Stevens (Scripps Research Inst.), to report the first X-ray structure of an agonist-bound A2AAR. Automatic docking of known potent nucleosides to the agonist-bound A2AAR crystallographic structure, and to homology models of other subtypes, was performed, resulting in new predictions of stabilizing interactions and a structural basis for previous empirical structure activity relationships. We predicted binding of novel C2 terminal and 5' derivatives of adenosine and used the models to interpret effects on measured binding affinity and efficacy of newly-synthesized agonists. Structures of G protein-coupled receptors (GPCRs) have a proven utility in the discovery of new antagonists and inverse agonists, which may modulate signaling of this important family of clinical targets. However, applicability of active-state GPCR structures to virtual screening and rational optimization of agonists, remains to be assessed. We have studied adenosine 5′ derivatives and evaluated the performance of an agonist-bound A2A adenosine receptor (AR) structure in retrieval of known agonists, and then employed the structure to screenfor new fragments optimally fitting the corresponding subpocket. Biochemical and functional assays demonstrate high affinity of new derivatives that include polar heterocycles. The binding models also explain a modest selectivity gain for some substituents toward the closely related A1AR subtype and the modified agonist efficacy of some of these ligands. The study suggested further applicability of in silico fragment screening to rational lead optimization for GPCRs in general. In order to investigate the usability of homology models and the inherent selectivity of a particular model in relation to close homologs, we constructed multiple homology models for the A1 adenosine receptor (A1AR) and docked, 2.2 M lead-like compounds. High-ranking molecules were tested on the A1AR as well as the close homologs A2AAR and A3AR. While the screen yielded numerous potent and novel ligands (hit rate 21% and highest affinity of 400 nM), it delivered few selective compounds. Moreover, most compounds appeared in the top ranks of only one model. The structure− activity relationship (SAR) for a novel class of 1,2,4- triazole antagonists of the human A2A adenosine receptor (hA2A AR) was explored. Thirty-three analogs of a ligand that was discovered in a structure-based virtual screen against the hA2A AR were tested in AR radioligand binding assays and in functional assays for the A2B AR subtype. As a series of closely related analogs of the initial lead did not display improved binding affinity or selectivity, molecular docking was used to guide the selection of more distantly related molecules. This resulted in the discovery of novel AR antagonists (Ki ≥200 nM) with high ligand efficiency. In light of the SAR for the 1,2,4-triazole scaffold, we also investigated the binding mode of these compounds based on docking to several A2AAR crystal structures The P2Y1 receptor (P2Y1R) is a G protein-coupled receptor naturally activated by extracellular ADP. Its stimulation is an essential requirement of ADP-induced platelet aggregation, thus making antagonists highly sought compounds for the development of antithrombotic agents. Here, through a virtual screening campaign based on a pharmacophoric representation of the common characteristics of known P2Y1R ligands and the putative shape and size of the receptor binding pocket, we have identified novel antagonist hits of microM affinity derived from a N,N-bis-arylurea chemotype. Unlike the vast majority of known P2Y1R antagonists, these drug-like compounds do not have a nucleotidic scaffold or highly negatively charged phosphate groups. Hence, our compounds may provide a direction for the development of receptor probes with altered physicochemical properties. The P2Y12R is an ADP-activated GPCR that is well validated clinically as an important target for antithrombotic drugs. Our new P2Y12R structure is the most predictive of the P2Y14R, a target for treating inflammation. Ligand docking to the P2Y12R-based model of the P2Y14R predicted poses of both reversibly-binding small molecules, consistent with observed pharmacology. We are using this approach to discover novel ligands of this receptor. Farnesyl pyrophosphate was recently identified as an insurmountable antagonist of ADP-induced platelet aggregation mediated by the P2Y12R. Docking of farnesyl pyrophosphate in a P2Y12R model revealed molecular similarities with ADP and a good fit into the binding pocket for ADP.

基于结构的药物设计策略已被用来阐明特定的配体识别决定因素，并最终导致针对特定靶点设计新的小分子。靶受体蛋白的结构是基于结构的药物设计方法的首要要求。在缺乏给定 G 蛋白偶联受体 (GPCR) 高分辨率结构的情况下，可以使用同源建模等计算技术来构建 3D 模型。简而言之，从蛋白质数据库（PDB）中选择最佳结构模板，主要考虑与目标受体的序列同一性和相似性以及晶体结构的质量（例如分辨率）。使用高度保守的残基和已知的共享结构特征将目标受体的序列与模板结构进行比对，以指导自动或半自动比对。序列比对和模板结构是同源建模的输入。能量最小化或分子动力学可用于进一步细化和优化生成的 3D 模型。配体-受体相互作用可以通过基于结构的方法来识别，例如分子对接。对接中配体-受体复合物中包含的信息可以澄清分子识别的结构元件，并导致化合物的进一步优化和新衍生物的设计。可以为每一类小分子配体绘制给定 GPCR 的结合位点。此外，使用基于结构和基于配体的策略，对化学多样性数据库进行虚拟搜索以寻找与给定 GPCR 结构结合的新化学型，已经取得了成效。 我们将诱变和同源建模应用于细胞外嘌呤和嘧啶 GPCR 家族的研究，并利用获得的结构见解来协助设计新型配体。这些家族由腺苷受体 (AR) 和 P2Y（核苷酸）受体组成。最近报道了拮抗剂结合状态和激动剂结合状态下的人 A2AAR 结构。由于 AR 之间的序列同一性相对较高（人类亚型之间的平均序列同一性为 47%），经过一些调整，这些结构可以可靠地用作其他 AR 的建模模板。我们通过间接手段实现了 P2YR 的结构功能分析，使用基于类似 GPCR（例如 CXCR4 趋化因子受体）高分辨率结构模板的诱变和同源建模。 我们与膜结合蛋白 X 射线晶体学的主要中心之一，即 Ray Stevens 教授（斯克里普斯研究所）的实验室合作，报告了激动剂结合 A2AAR 的第一个 X 射线结构。已知有效核苷与激动剂结合的 A2AAR 晶体结构以及其他亚型的同源模型的自动对接，产生了稳定相互作用的新预测以及先前经验结构活性关系的结构基础。我们预测了新型腺苷 C2 末端和 5' 衍生物的结合，并使用模型来解释对测量的结合亲和力和新合成激动剂功效的影响。 G 蛋白偶联受体 (GPCR) 的结构在发现新的拮抗剂和反向激动剂方面已被证明具有实用性，这些拮抗剂和反向激动剂可能会调节这一重要临床靶标家族的信号传导。然而，活性状态 GPCR 结构在激动剂虚拟筛选和合理优化中的适用性仍有待评估。我们研究了腺苷 5' 衍生物，并评估了激动剂结合的 A2A 腺苷受体 (AR) 结构在修复已知激动剂方面的性能，然后利用该结构筛选最适合相应子口袋的新片段。生化和功能测定证明包含极性杂环的新衍生物具有高亲和力。结合模型还解释了一些取代基对密切相关的 A1AR 亚型的适度选择性增益以及其中一些配体的改进的激动剂功效。该研究表明，计算机片段筛选可进一步应用于 GPCR 的合理先导优化。 为了研究同源模型的可用性以及特定模型相对于密切同源物的固有选择性，我们构建了 A1 腺苷受体 (A1AR) 和对接的 2.2 M 类先导化合物的多个同源模型。在 A1AR 以及密切同系物 A2AAR 和 A3AR 上测试了高级分子。虽然筛选产生了许多有效的新型配体（命中率为 21%，最高亲和力为 400 nM），但它提供的选择性化合物很少。此外，大多数化合物仅出现在一种模型的顶级行列中。 新型 1,2,4- 类的结构-活性关系 (SAR) 探索了人 A2A 腺苷受体 (hA2A AR) 的三唑拮抗剂。在针对 hA2A AR 的基于结构的虚拟筛选中发现的配体的 33 种类似物在 AR 放射性配体结合测定和 A2B AR 亚型的功能测定中进行了测试。由于初始先导化合物的一系列密切相关的类似物没有表现出改善的结合亲和力或选择性，因此使用分子对接来指导选择更远相关的分子。这导致了具有高配体效率的新型 AR 拮抗剂 (Ki ≥200 nM) 的发现。根据 1,2,4-三唑支架的 SAR，我们还基于与几种 A2AAR 晶体结构的对接研究了这些化合物的结合模式 P2Y1 受体 (P2Y1R) 是一种由细胞外 ADP 自然激活的 G 蛋白偶联受体。它的刺激是 ADP 诱导的血小板聚集的基本要求，因此拮抗剂成为抗血栓药物开发中备受追捧的化合物。在这里，通过基于已知 P2Y1R 配体的共同特征的药效团表示以及受体结合袋的假定形状和大小的虚拟筛选活动，我们已经确定了源自 N,N-bis- 的 microM 亲和力的新型拮抗剂命中。芳基脲化学型。与绝大多数已知的 P2Y1R 拮抗剂不同，这些药物样化合物不具有核苷酸支架或带高负电荷的磷酸基团。因此，我们的化合物可能为开发具有改变的物理化学性质的受体探针提供方向。 P2Y12R 是一种 ADP 激活的 GPCR，经过临床验证，可作为抗血栓药物的重要靶点。我们的新 P2Y12R 结构最能预测 P2Y14R（治疗炎症的靶点）。与基于 P2Y12R 的 P2Y14R 模型的配体对接预测了两种可逆结合小分子的姿势，与观察到的药理学一致。我们正在使用这种方法来发现该受体的新配体。 法呢基焦磷酸最近被确定为 P2Y12R 介导的 ADP 诱导的血小板聚集的不可克服的拮抗剂。 P2Y12R 模型中法呢基焦磷酸的对接揭示了其与 ADP 的分子相似性，并且与 ADP 的结合口袋非常吻合。