A New Drug Discovery Program Targeting Obesity Receives NIH Funding
By: Carol A. Rouzer, VICB Communications
Published: July 23, 2013
The first grant awarded to participants of the Vanderbilt-Leipzig Partnership will fund a drug discovery effort targeting the neuropeptide Y4 receptor, which suppresses appetite and regulates energy metabolism.
Recently recognized by the American Medical Association as a disease, obesity is one of the most serious public health threats currently challenging the developed world (Figure 1). Over 35% of adults and 17% of children in the United States are classified as obese, exposing them to an increased risk for diabetes, heart disease, high blood pressure, cancer, stroke, and osteoarthritis. The high cost of obesity-related medical care ($147 billion in 2008) will continue to place an enormous burden on our health care system in the future if nothing is done to stop and reverse the current trends. Although diet and exercise remain the most desirable treatment for obesity, it is becoming clear that this approach alone will not suffice for many patients. Thus, an attempt to identify specific causes of obesity that will lead to new therapeutic solutions is a high priority. To respond to this challenge, Vanderbilt Institute of Chemical Biology (VICB) members Jens Meiler and Dave Weaver have teamed up with Annette Beck-Sickinger of Leipzig University to launch a search for drugs that target the neuropeptide Y4 receptor, a protein known to regulate food intake and energy metabolism. Their collaboration, one of the many resulting from the Vanderbilt-Leipzig partnership, has now been awarded a grant from the National Institute of Diabetes and Digestive and Kidney Diseases. This is the first NIH funding to directly support research that has been fostered by this vibrant, trans-national alliance.
Figure 1. Rapid increase in obesity in the United States from 1990 through 2010. Reproduced from the Centers for Disease Control. Figure is in the public domain.
Pancreatic polypeptide (PP) is one of a family of 36 amino acid peptide hormones that also includes neuropeptide Y (NPY) and polypeptide YY (PYY). These peptides act by binding to a group of proteins, the neuropeptide Y receptors, which transduce the hormone signal through interaction with a G protein. G proteins are trimers comprising α, β, and γ subunits that bind GTP upon association with a hormone-bound receptor. The GTP-bound α subunit and the combined βγ subunits then act separately to trigger a range of cellular responses (Figure 2).
Figure 2. Example of how a G protein-coupled receptor transduces the signal from a receptor to an effector, in this case, an ion channel. Binding of a signaling molecule to the receptor leads to binding of GTP to the α subunit of the G protein. In pathway A, the alpha subunit dissociates from the β and γ subunits and activates a separate effector molecule, which then activates the ion channel. In pathway B, the α subunit activates the ion channel directly. In pathway C, the combined βγ subunits activate the ion channel. GIRK channels are activated by pathway C. Figure reproduced from Wikipedia and is in the public domain.
There are four neuropeptide Y receptor subtypes, Y1, Y2, Y4, and Y5. The different peptide hormones, PP, NPY, and PYY, have varying affinities for each of the subtypes, but PP only binds strongly to Y4 (Figure 3). PP, which is secreted after a meal by a subpopulation of cells in the pancreas, causes decreased appetite and food intake in both animals and humans. Mice genetically altered to overexpress PP exhibit decreased feeding accompanied by weight loss and reduced body fat. These results suggest that drugs that act like PP on the Y4 receptor could serve as appetite suppressants and help obese people lose weight. Indeed, a peptide similar to PP, Obinepitide, is currently being tested as an anti-obesity drug in clinical trials. However, even if Obinepitide is successful, peptides are generally not practical for use as drugs, as they can not be formulated into a pill and taken orally.
Figure 3. Side view of pancreatic polypeptide (purple) docked in the hY4R comparative model (cyan). Residues found to be important in the activation of hY4R by PP are highlighted. Predicted interactions are indicated with dotted red lines. Figure kindly provided by Dave Weaver and Jens Meiler.
Attempts to find selective small molecule agonists that mimic the action of PP have not, so far, been successful. One reason is that the binding site for PP on Y4, which is called the orthosteric site, is very similar to the orthosteric sites on the other Y receptors. Consequently, a small molecule that activates Y4 by binding to the orthosteric site will likely also activate one or more of the other receptors, leading to unwanted side effects. The Weaver and Meiler groups plan to overcome this obstacle by finding molecules that act as allosteric potentiators at Y4. An allosteric potentiator works by binding to the receptor at a site different from the orthosteric site. Allosteric potentiators have no effect on the receptor in the absence of the hormone, but when the hormone (in this case, PP) binds to the receptor, the potentiator increases the receptor’s response. Recent drug discovery efforts have shown that molecules that act at allosteric sites are often more selective for one receptor subtype than those that bind at the orthosteric site because allosteric binding sites vary more widely between receptor subtypes.
Funded by Pilot Project Awards from the VICB and the Diabetes Research and Training Center and spurred by visits of Dr. Jan Stichel from Leipzig and Greg Sliwoski from Vanderbilt to the respective partner institutions, the Weaver, Meiler, and Beck-Sickinger labs have already made considerable progress toward finding allosteric potentiators of the Y4 receptor. The Weaver lab exploited the Beck-Sickinger lab’s expertise on Y receptors to design a high-throughput screen (HTS) that tests for direct agonists, allosteric potentiators, and inhibitors of the Y4 receptor, all at one time. The screen uses COS 7 cells that express the human Y4 receptor and a G protein that activates an intracellular calcium flux upon binding of PP to the receptor. A calcium-sensitive fluorescent dye senses the flux and provides a signal that can be readily detected by the Hamamatsu Functional Drug Screening System (FDSS) in the VICB HTS Core laboratory. In the screen, the test compound is first added alone to see if it directly activates the receptor. Then, the hormone is added at a concentration that should produce a response that is 20% of the maximal response. If the test molecule is an allosteric potentiator, a greater than 20% response will result. Finally, more hormone is added to provide a concentration that should produce a response that is 80% of the maximal response. If the test molecule is an inhibitor, a less than 80% response will result. A preliminary screen of 2000 compounds has revealed two molecules that are allosteric potentiators of Y4 (Figure 4), and early medicinal chemistry efforts have shown that small structural changes can be used to fine-tune the selectivity of one of the molecules for the various Y receptors. These highly promising results indicate a strong likelihood that selective allosteric potentiators of Y4 can be found.
Figure 4. Fluorescence signal obtained from one of the hit molecules identified in the primary high-throughput screen. At the time indicated by the arrow labeled Cmpd/Vhl, either test compound (red line) or vehicle (blue line) was added to the cells. At the time indicated by the arrow labeled PP (EC20), PP was added at a concentration that normally provides 20% of the maximum response. Note that the response in the sample containing the test compound plus PP (red line) is much higher than the sample containing PP alone. At the time indicated by the arrow labeled PP (EC80), additional PP was added to give a concentration that normally provides 80% of the maximum response. At this concentration, an inhibitor would result in a lower response, which is not seen here. The green line represents results from a control well in which the test compound and PP were added to cells that do not express the Y4 receptor. Figure kindly provided by Dave Weaver and Jens Meiler.
With funding for the project now available, the Weaver and Meiler groups will immediately expand their screen to include all 160,000 compounds in the VICB’s chemical library. Hit molecules from the primary screen will be tested in multiple additional screens to eliminate false positives and evaluate the potency and selectivity of true positives. Then, the Meiler lab will use the results to develop a computational model that will identify the molecular characteristics (descriptors) of the active molecules. They will use the model to virtually screen a library of two million compounds that are readily available for acquisition and testing. The goal is to identify ~1,000 compounds that are structurally similar to the initial hits and are therefore likely to have allosteric potentiator activity. These compounds will be tested in the primary high-throughput screen, and those confirmed positive will be further evaluated for potency and selectivity.
The funded project will exploit the wide range of resources available through the VICB. These include the HTS Core facility, where most of the primary and secondary screens will be carried out, and the Chemical Synthesis Core, which will provide synthetic support as needed. Synthesis Core Director, Gary Sulikowski, and Craig Lindsley, Director of the Vanderbilt Specialized Chemistry Center for Accelerated Probe Development, will both be available for consultation, as needed. The Meiler/Weaver team will also benefit from collaborations with VICB members Vsevolod Gurevich and Roger Cone, who will provide expertise in G protein-coupled receptor signaling and the pathophysiology of obesity, respectively. Finally, the continuing collaboration with the Beck-Sickinger lab will guarantee that resources needed to fully explore the Y receptor pharmacology of promising hit molecules will be available. We look forward to hearing about the success of this highly promising, multidisciplinary, and trans-national project.