A team of Chinese and US scientists has determined the high-resolution atomic structure of a cell-surface receptor that plays a critical role in thrombosis, or blood clot, formation. This research unexpectedly discloses many new structural features, which challenge the conventional concepts of drug action at G protein-coupled receptors (GPCRs) and open a new door for future drug discovery. 

In a paper published in Nature on March 30, the researchers at the Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, collaborating with research groups from the National Institutes of Health (NIH, U.S.), the Bridge Institute at the University of Southern California (U.S.) and the iHuman Institute of ShanghaiTech University (China), provide a detailed molecular map of the human P2Y₁ receptor (P2Y₁R), a GPCR, in complex with a nucleotide antagonist MRS2500 and a non-nucleotide antagonist BPTU.  

Human purinergic receptors P2Y₁R and P2Y₁₂R play a major physiological role in adenosine 5′-diphosphate (ADP)-mediated platelet aggregation, an important component of thrombosis, which can subsequently result in life-threatening diseases, such as heart attack and stroke. While most of the available antithrombotic drugs act on P2Y₁₂R, P2Y₁R has been suggested as a new promising antithrombotic drug target and may offer a safety advantage over P2Y₁₂R inhibitors in terms of reduced bleeding liabilities. However, efforts to develop new drugs have been impeded by poor understanding of receptor-ligand interaction. 

“The P2Y₁R structures help us understand how this receptor and different types of experimental drugs interact at the molecular level, and could enable further exploration to design new and safer antithrombotic drugs with reduced adverse effects,” said team leader Dr. WU Beili, professor at SIMM.  

The new study provides many valuable insights into the structure of P2Y₁R. One very exciting finding is that the P2Y₁R structures reveal two completely distinct ligand-binding sites. The nucleotide ligand MRS2500 recognizes a binding site within the transmembrane bundle of P2Y₁R; however, it is different in shape and location from the nucleotide-binding site in the P2Y₁₂R structure previously determined by the same collaboration. Although recognized by the same endogenous ligand ADP, P2Y₁R and P2Y₁₂R structures reveal very different features in binding their nucleotide ligands. “It is amazing to observe that two GPCRs recognize the same ligand in such different ways,” said Dr. WU. “The finding highlights the diversity of signal recognition mechanisms in GPCRs, and this is of great value to drug design for each receptor with high selectivity.” 

The most surprising finding of this research is that, instead of interacting within the transmembrane bundle, the non-nucleotide ligand BPTU binds to a pocket on the outer interface of the receptor that is embedded in the cell membrane. This is the first structurally characterized selective and high-affinity GPCR ligand located entirely outside of the helical bundle, and it represents a new paradigm in ligand binding to alter signaling in GPCRs. The P2Y₁R structure reveals new opportunities for broadening the scope of future GPCR drug discovery to target novel sites outside of the conventional GPCR ligand-binding pocket, which may greatly improve drug selectivity and reduce side effects, and will thus facilitate the development of new pharmaceuticals for the treatment of many severe diseases.  

“The new structures will allow drug designers to work more efficiently and with greater precision to build new molecules to modulate the function of this receptor and other closely related receptors, many of which have potential for treating cancer and inflammation,” said Dr. Kenneth Jacobson, chief of the Laboratory of Bioorganic Chemistry at the National Institute of Diabetes and Digestive and Kidney Diseases at NIH. Jacobson coauthored the study and invented the antagonist MRS2500 that was crystallized with P2Y₁R. 

In addition to WU and Jacobson, the contributors to the study, “Two disparate ligand-binding sites in the human P2Y₁ receptor,” include lead author Dandan Zhang, Kaihua Zhang, Jiang Wang, Cuiying Yi, Limin Ma, Wenru Zhang, Hong Liu, Hualiang Jiang and Qiang Zhao, all of SIMM; Zhan-Guo Gao, Evgeny Kiselev, Steven Crane, and Silvia Paoletta of NIH; Gye Won Han, Vadim Cherezov, Vsevolod Katritch and Raymond Stevens of the Bridge Institute at the University of Southern California; and Raymond Stevens from the iHuman Institute, ShanghaiTech University. 

The study was funded in part by the National Basic Research Program of China, the CAS Strategic Priority Research Program, the National Science Foundation of China, the National Science and Technology Major Projects Program, ShanghaiTech University, Shanghai Municipal Government, the National Institutes of Health, and the National Institutes of Health Protein Structure Initiative. To view the study, visit http://www.nature.com. 

During the implementation of the “One-Three-Five” strategic plan, SIMM has identified “significant progress in GPCRs as drug targets” as one of the “3-in-1” focuses of the institute. Since 2013, SIMM has achieved several breakthroughs in research on the structure and function of drug targets. There is no doubt that determination of the three-dimensional structure of P2Y₁R is another breakthrough for SIMM’s “One-Three-Five” development plan. Currently, SIMM is vigorously working to establish the Institutes for Drug Research Innovation with relevant drug discovery R&D capability within the Chinese Academy of Sciences. As one of the development directions of the Institutes for Drug Research Innovation, basic research aimed at new targets, new biomarkers, new mechanisms and novel molecular entities is critical to meeting the needs of drug innovation. Therefore, determination of the three-dimensional structure of P2Y₁R and the discovery of related drug candidates will definitely lay the foundation for the establishment of the Institutes for Drug Research Innovation. 

Figure: The new Nature study reveals the structure of the human P2Y1 receptor, which mediates thrombosis formation. The image shows that two different ligands that inhibit platelet aggregation interact with P2Y1R by binding to two completely distinct sites on the receptor. The receptor structure is shown as a grey ribbon and surface. The ligands MRS2500 and BPTU are shown as magenta and yellow spheres, respectively. (Image courtesy of Yekaterina Kadyshevskaya of the Bridge Institute at the University of Southern California.)