Prostanoids are a class of oxygenated lipids that provoke diverse biological actions in many tissues and cell types through interaction with nine G-protein-coupled receptors (GPCRs). The prostanoid thromboxane A2 (TXA2) is a key activator of platelet. It is crucial in the amplification and maintenance of the platelet response and in the recruitment of new platelets to the growing thrombus.

Stimulated by TXA2, the human TXA2 receptor (TP) plays a pivotal role in cardiovascular homeostasis. Thus, TP is considered as an important drug target for cardiovascular disease. Over recent decades, a number of TP inhibitors have been developed as drug leads. However, limited success has been made in their clinical application due to efficacy and toxicity issues.

In a paper published in Nature Chemical Biology on December 03, 2018 (16:00PM, London time) titled “Structural basis for ligand recognition of the human thromboxane A2 receptor”, a team of scientists from the Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences (CAS) reported the high-resolution crystal structures of human TP bound to two non-prostanoid antagonists ramatroban and daltroban. These structures reveal, for the first time, the molecular details of TP binding to its ligands at atomic level, and offer new opportunities for drug optimization and discovery targeting this physiologically important receptor.

This study provides some valuable insights into the ligand binding of TP. One exciting finding is the unique arrangement of extracellular loops that differs from all the other known GPCR structures. The extracellular region of TP forms a two-layer “roof” structure constrained by two pairs of disulfide bonds. One of the disulfide bonds connects the N terminus and the extracellular tip of the third transmembrane helix of the receptor. This has not been observed in any other GPCR structures.

This double-layer cover stacks on top of the ligand-binding pocket in the TP structures. It provides a suitable binding environment for the ligand, and most likely blocks the access of the ligand from the extracellular milieu. The hydrophobic endogenous ligands, and their synthetic mimics, likely enter the ligand-binding site through the lipid bilayer and transmembrane helices.

"The TP structures deepen our knowledge about how the ligands gain their route to regulate the receptor function. This helps us to better understand the mechanisms of this receptor playing its roles in human cells." said team leader and SIMM professor Dr. WU Beili.

The TP structures provide a detailed molecular map of interactions between TP and the two non-prostanoid antagonists, ramatroban and daltroban. These molecular details not only help to integrate previous mutagenesis and structure-activity relationship data, but also provide an accurate template for drug design targeting TP.

In addition, to further investigate the ligand-binding behavior of TP, the researchers performed molecular docking of two prostanoid-like ligands into the TP structure. Combined with extensive studies of mutagenesis, ligand binding and cell signaling assays, the docking models suggest a prostanoid binding mode that may also be adopted by other prostanoid receptors. Furthermore, it was found that the first and second transmembrane helices of the prostanoid receptors are key determinants for prostanoid ligand specificity.

These insights into the prostanoid receptors deepen the understanding about ligand recognition and selectivity mechanisms of this important GPCR sub-family, and would enable development of new anti-cardiovascular disease drugs with better selectivity and efficacy.

In addition to Dr. WU, other notable investigators included Dr. ZHAO Qiang, Dr. XU Yechun, Dr. HAN Shuo and graduate students FAN Hengxin (the first author), CHEN Shuanghong (the co-first author), YUAN Xiaojing and ZHANG Hui from SIMM and Dr. XIA Weiliang from Shanghai Jiao Tong University.

The study was funded by National Key R&D Program of China, Chinese Academy of Sciences and the National Science Foundation of China.

Link to the article: https://www.nature.com/articles/s41589-018-0170-9

Figure: The binding mode of TP in complex with two inhibitors ramatroban and daltroban. The receptor structure is shown as green ribbon and grey surface. The TP ligands ramatroban, daltroban, SQ-29548 and U46619 are shown as spheres and colored magenta, cyan, green and yellow. (Image courtesy of Dr. WU Beili’s lab from the Shanghai Institute of Materia Medica, Chinese Academy of Sciences)

Contact:
WU Beili (beiliwu@simm.ac.cn)