How much do we know about the way blood clots?
We already understand the many steps involved in blood clotting in great mechanistic detail. The process of blood vessels closing off in response to injury is necessary for preserving life, but blood platelets that are over-active, or activated inappropriately because of unstable plaque, can lead to heart attacks and strokes.
Drugs for treating thrombosis - or, blood clotting - are based on a variety of events leading to a clot. One of these events is blocked simply by taking an aspirin, but there are others that act in concert.
Interfering at various points in the thrombosis process with new medicines can reduce the risk of a thrombus forming. Thus, there are multiple approaches to designing antithrombotic drugs.
Could you please give a brief introduction to the receptor P2Y12 and explain why you were looking to discover its 3D structure?
Our work focuses at the atomic level on a protein called P2Y12 that acts as a receptor on the cell surface of the platelet. P2Y12 receives the chemical signal in the form of ADP (adenosine diphosphate, a nucleotide) that is released from surrounding cells and that normally promotes the formation of a thrombus.
The key concept is that it takes a signal from outside the cells and transmits it to the interior of the platelet, in order to form a thrombus. There are hundreds of such receptors, and each reacts to only its matched chemical signal.
Four antithrombotic drugs that act by blocking the P2Y12 receptor in various ways are already FDA-approved for use in patients. These antithrombotic drugs are effective because they prevent the amplification of the thrombus-forming process in arteries.
However, each has its side effects, liabilities and peculiarities that require caution in clinical use. Therefore, we need to be able to custom design new drugs for the P2Y12 target.
By knowing the 3D structure of the P2Y12 receptor, we expect to design improved antithrombotic drugs.
Prior to knowing this structure, P2Y receptor drug designers tried to discover effective chemicals to target this receptor by trial and error. In general, at any receptor for which the 3D structure is unknown, discovering new chemical structures to block that protein requires a very labor-intensive screening of large chemical libraries (into the millions), and sometimes logical guesses.
Having a high-resolution structure of the P2Y12 receptor enables drug discovery using computer methods either for broad screening or tailored fitting of individual ideas for new drugs to improve existing chemical modulators.
Another dimension of this work is that we now have a ‘window’ on the structures of the seven other P2Y receptors that belong to the same closely related family. The biology and disease relevance of these eight family members is vast and extends far beyond controlling platelets.
For example, we have reason to expect that some blockers of the P2Y12 receptor could also be used in treating pain. Moreover, there are concepts for many other disease treatments based on the other P2Y receptors that are just waiting for the right molecules to put them to the test.
A few of the diseases that could be potentially treated with P2Y receptor drugs include diabetes, cancer, memory deficit, bowel diseases, infectious diseases and osteoporosis. The uncovering of the new P2Y12 receptor structure may prove to be catalytic toward progress on all of these fronts.
What exactly did your research involve?
This work was truly an example of team science, involving two labs (Scripps Institute in La Jolla, California, and Shanghai Institute of Materia Medica in China) that are expert in determining the structures of the larger family of cell surface receptors (called G protein-coupled receptors, or GPCRs) and another lab in Bonn, Germany that already specializes, as we do at NIH, in the study of P2Y receptors.
We are medicinal chemists, meaning we design and synthesize new molecules for either blocking or activating the P2Y receptors, and we apply these molecules to pharmacology to understand the role of the receptors in living cells and tissues.
In this field the chemical contributions have proven critical to understanding of biology. We provide many of the probe molecules used by other laboratories studying P2Y receptors.
For this project, we supplied new molecules that target the P2Y12 receptor and the pharmacological component to show that the function indicated by the X-ray results corresponds to true biological phenomena.
We also interpreted the results of the new structures using computerized molecular modeling to try to understand better how this receptor functions as a tiny ‘machine.’ This machine can be turned on by the natural activator ADP and turned off by other molecules, including the antithrombotic drugs.
The new structures give ‘snapshots’ of the receptor in differently shaped forms depending on which small molecule or drug is stuck inside. So we begin to see a dynamic relationship, which greatly aids the drug discovery process.
What did you learn about the way the P2Y12 receptor structure changes when it interacts with antithrombotic drugs?
We learned that the forms (‘conformations’) of this receptor when exposed to activators or blockers are very different. Compared to the few other pairs of GPCRs in which we know the 3D structures of both forms, there is an enormous difference between the two in the case of the P2Y12 receptor.
We also see features of this receptor that were totally unanticipated from the other known receptors’ structures (certain parts are elongated or positioned very differently or a normal bridge bond is disconnected).
The form that is turned off by a blocker (we used an experimental drug from the pharmaceutical industry) is wide open to the outside of the cell. However, when the activator - the nucleotide - is presented to the receptor, it is as if the outer ‘arms’ of the receptor grab the nucleotide and hold it tight in its preferred location down in the receptor pocket. These ‘arms’ almost completely close off the opening to the pocket.
The explanation for this phenomenon is that the outer arms have many positive charges and the activator nucleotides are necessarily negatively charged, and opposite charges attract.
Were you surprised by this?
All these strikingly different features were quite unexpected. It seems that this receptor is especially dynamic, and our earlier attempts to model the structure by computerized guesses were not very accurate. This is a clear example where the determination of the X-ray structure altered our thinking fundamentally.
What impact will this understanding have on antithrombotic drug development going forward?
Because we want to ensure that the final product is safe and effective as possible, the drug development process can take many years. But our findings can be used immediately by academic and commercial labs to screen computationally, through large chemical databases, for novel drugs to act at the P2Y12 receptor.
Then the process of validating the predictions by testing the computer hits (actual chemicals that can be ordered from commercial suppliers) at the lab bench is straightforward.
When a good hit is identified, it will be up to chemists to remold the structure to fit better in the receptor. The X-ray structures will be invaluable in this process.
This shows the relevance of NIH basic research to future disease treatment. By exploring a receptor system in great depth to advance basic scientific knowledge, we create opportunities for commercial development, usually by the private sector.
Pharmaceutical companies look toward NIH as the source of new concepts that they can turn into products. Thus, our discoveries are the beginning of a process that has the potential to make a significant difference in patients' lives.
Is this research likely to affect the development of potential treatments for nervous system disorders?
Yes, there are potential applications of the P2Y receptor modulating drugs - either activators or blockers depending on the circumstances - for treatment of central nervous system disorders, including Alzheimer’s disease, Parkinson’s disease, pain, brain inflammation, traumatic brain injury and other conditions.
At least one of the P2Y receptors is involved in promoting neuronal regeneration from stem cells in the brain.
What are your future research plans for blood clotting receptors?
We are currently modeling how many other experimental drugs and molecular probes are recognized by the P2Y12 receptor. The expansion of this work to include structural predictions for other P2Y receptors is also a high priority for us.
These X-ray structures create many new research opportunities that we hope will eventually lead to new drug molecules for disease treatment. As medicinal chemists, we have the ability to synthesize new molecules as needed for a particular biological application.
Where can readers find more information?
The original publications in Nature are at: doi:10.1038/nature13083 and doi:10.1038/nature13288
Otherwise there is a wealth of information available about these receptors on the internet, for example: http://www.medscape.com/viewarticle/764760_3
About Dr. Jacobson
Kenneth A. Jacobson, Ph.D. is Chief of the Laboratory of Bioorganic Chemistry and the Molecular Recognition Section at the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health in Bethesda, Maryland, USA.
He is a medicinal chemist with interests in the structure and pharmacology of G protein-coupled receptors (GPCRs), in particular receptors for adenosine and for purine and pyrimidine nucleotides. GPCR structure is probed through computer modeling, mutatgenesis, and the structure activity relationships of novel small molecular ligands.
Dr. Jacobson originated various conceptual approaches to studying GPCRs, including neoceptors and functionalized congeners.
He graduated in 1975 from Reed College, Portland, Oregon with a B.A. in Liberal Arts, and received his Ph.D. in Chemistry with Prof. Murray Goodman at the University of California, San Diego in 1981.
He was a Bantrell Fellow at Weizmann Institute of Science in Rehovot, Israel before joining the NIH in 1983. Recent awards include the 2014 Goodman and Gilman Award in Receptor Pharmacology, the 2009 Pharmacia-ASPET Award in Experimental Therapeutics, the 2008 Sato Memorial International Award of the Pharmaceutical Society of Japan, and the 2003 Hillebrand Prize of the Chemical Society of Washington for original contributions to the science of chemistry.
In 2004, he ranked 4th among the most highly cited authors in pharmacology and toxicology during a ten year period (ISI).
In 2009, Dr. Jacobson was inducted into the Medicinal Chemistry Hall of Fame of the American Chemical Society, of which he is a Fellow, and served as Chair of the Medicinal Chemistry Division.
He has mentored >60 postdoctoral fellows and published approximately 600 scientific papers. Patents from his lab have been licensed to industry for commercialization of research tools and the development of therapeutic agents.
Thirty-six compounds invented in the Jacobson laboratory are now available commercially as research tools. Several nucleoside agonists of the A3 adenosine receptor invented by Dr. Jacobson are in advanced clinical trials for inflammatory diseases and cancer.
Interests: medicinal chemistry; organic chemistry; drug discovery; structure, function and pharmacology of G protein-coupled receptors (GPCRs), in particular receptors for adenosine and for purine and pyrimidine nucleotides; computer modeling; imaging; nanotechnology