HONG KONG – A Sino-U.S. research collaboration has determined the atomic structure of the G protein-coupled (GPC) P2Y1 receptor, which plays a key role in thrombus formation, challenging conventional concepts of drug action at GPC receptors and opening the door for future new drug discovery and development.

The P2Y1 and P2Y12 purinergic receptors play a central physiological role in adenosine 5'-diphosphate (ADP)-mediated platelet aggregation, which is an important component of thrombosis that can have potentially fatal consequences, including heart attack and stroke.

Currently, most of the available antithrombotic drugs such as Plavix (clopidogrel, Bristol-Myers Squibb Co. and Sanofi SA), Brilinta (ticagrelor, Astrazeneca plc) and Effient (prasugrel, Eli Lilly and Co.) act on the P2Y12 receptor, but those agents have been associated with safety concerns.

"The main safety concern [with clopidogrel] is lack of efficacy in patients unable to metabolize it effectively to the active compound," said Brian Tomlinson, professor of medicine and therapeutics and head of the Division of Clinical Pharmacology at The Chinese University of Hong Kong, adding there may also be interactions between clopidogrel and drugs such as proton pump inhibitors.

"Although these problems have been largely overcome with prasugrel and ticagrelor, all of these antithrombotic drugs have the side effect of increased risk of bleeding," warned Tomlinson.

The P2Y1 receptor has been proposed as a new antithrombotic target that may offer safety advantages, particularly in terms of reduced bleeding, although development of such new agents has been hampered by a poor understanding of the interactions between the P2Y1 receptor and its ligand.

In a study published in the March 30, 2015, issue of Nature, researchers at the Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, and at the U.S. NIH, the University of Southern California and ShanghaiTech University used X-ray crystallography to derive detailed molecular maps of the P2Y1 receptor in complex with a nucleotide and non-nucleotide antagonist.

"This helps us to understand better how the receptor interacts with different types of antagonists," explained lead researcher Beili Wu, a principal investigator and professor at SIMM. "In future, researchers could design new drugs with more efficacy by strengthening or blocking any particular interactions between the drugs and the receptor, based on this structural information."

Moreover, knowing the detailed P2Y1 receptor structures "helps 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," she added.

TWO BINDING SITES

Perhaps the most valuable insight into the structure of the P2Y1 receptor was that there are two fundamentally distinct ligand-binding sites.

The nucleotide ligand, known as MRS2500, recognizes a binding site within the transmembrane bundle of the P2Y1 receptor, which is different in shape and location from the nucleotide-binding site in the P2Y12 receptor structure that was previously determined by the same researcher collaboration.

Moreover, although recognized by the same endogenous ligand ADP, X-ray crystallography demonstrated that the P2Y1 and P2Y12 GPC receptor structures reveal very different features when they are bound with their nucleotide ligands.

"It is amazing to observe that two GPC receptors recognize the same ligand in such different ways," noted Wu. "The finding highlights the diversity of signal recognition mechanisms in GPC receptors, and this is of great value to drug design for each receptor with high selectivity."

However, perhaps an even more surprising finding is that the non-nucleotide ligand, known as BPTU, binds to a pocket on the outer interface of the receptor embedded in the cell membrane, rather than interacting within the transmembrane bundle.

This is the first structurally characterized selective and high-affinity GPC receptor ligand that has been shown to be located entirely outside of the helical bundle, and as such represents a new paradigm in ligand binding to alter signaling in GPC receptors.

"In all of the previous GPC receptor structural studies, the ligands at least partially occupy the conventional binding pocket within the transmembrane bundle," said Wu, pointing out that most of the GPC receptor drug discovery efforts to date have therefore focused on targeting that pocket.

"This is the first time that a ligand-binding site has been found outside of the bundle," she said. That is important in terms of drug discovery, since "researchers could start looking for novel drug-binding sites on the outer surface of the receptor. This may improve drug selectivity and make the drug development more efficient. "

The P2Y1 receptor structure therefore opens new opportunities to broaden the scope of future GPC receptor drug discovery to target novel sites outside of the conventional GPC receptor ligand-binding pocket, which may greatly improve drug selectivity and reduce side effects, thereby facilitating development of new pharmaceuticals for the treatment of diverse severe diseases.

"Our research may help to design new drugs to modulate the functions of the P2Y1 receptor and other closely related receptors, many of which have potential for treating cancer and inflammation," Wu told BioWorld Today.

On her group's future research plans, she said, "Besides thrombosis, we are trying to solve the structures of some GPC receptors involved in other human diseases, such as diabetes and viral infection.

"By understanding how these GPC receptors recognize their ligands and how they transduct the signal into the cells, we will have a much better idea about the mechanisms of disease development and perform structure-based drug design."