April 1, 2017 (Vol. 37, No. 7)

Kathy Liszewski

Novel Drugs May Exploit Previously Overlooked GPCR Openings

Drugs that target G-coupled protein receptors (GPCRs) constitute the therapeutic establishment. They account for more than one-third of all drugs on the market, and they know how to court the GPCR block, a large, diverse, and influential molecular constituency.

More than 800 GPCR genes have been identified in the human genome, and they encode members of the largest family of transmembrane proteins.

GPCRs are heterogeneously distributed master communicators. Woven into the cell membrane with seven transmembrane domains, GPCRs are well positioned to control key physiologic functions such as neurotransmission, hormone release, and immune responses.

While much is known about GPCRs, much remains unclear. GPCRs are hard to “read” because they have structures that are complicated, changeable, and transportable. Different GPCR conformations, which may be triggered by different ligands, and different localizations may lead to the activation of different signaling pathways.

Such GPCR complexities will be addressed at an upcoming event, the GPCR-Targeted Drug Design Conference. Organized by Cambridge Healthtech Institute and scheduled to take place April 24–25 in San Diego, the conference will highlight several innovations in the GPCR field. For example, it will cover intriguing findings that GPCR signaling runs deeper than we might have guessed. That is, GPCRs within the cell, embedded within endosomal and nuclear membranes, may contribute as much to the cell’s signaling complexity as the GPCRs in the plasma membrane.

The conference’s coverage of alternative GPCR localization and other topics of current interest can be previewed in this article. These additional topics include the social nature of GPCRs. (New findings have revealed unsuspected associations in functional megaplexes.) Finally, this article anticipates the conference’s coverage of GPCR-ready analytical techniques. (New and inventive platforms have been developed to help researchers handle the volume and integrate the variety of GPCR data.)


Novel and inventive investigative platforms have been developed to help researchers handle the volume and integrate the variety of GPCR data. POBA/Getty Images

Second Binding Pocket

Opioid receptors are among the most studied of GPCRs, primarily owing to the roles of their agonists, such as morphine, in pain management. But the success of morphine and other such drugs comes at a cost. They have significant side effects such as constipation and respiratory suppression, and they pose serious risks for dependency and abuse.

These problems could become less serious, however, if drug discovery were to exploit GPCR structural knowledge that has emerged over the past decade. A particularly important development is the realization that members of every mammalian receptor superfamily have a “second” binding pocket. The evolutionarily conserved endogenous first ligand binding region in the receptor is called the “orthosteric site,” whereas the second site is called the “allosteric site.”

“Positive allosteric modulators, or PAMs, are compounds that not only enhance the effect of the agonist, they also confer better specificity,” says Andrew Alt, Ph.D., associate director, biology, Arvinas. “They act only in the presence of the orthosteric ligand.”

“The discovery of PAMs,” he continues, “represents a significant opportunity to develop new and better medicines that reduce side effects and engage a more natural response in the body.”

Most drugs affect the whole body and not only the precise region where needed. However, PAMs bind to a site on the receptor that is topographically distinct from the orthosteric site. According to Dr. Alt, PAMs offer a unique advantage.

“We now know that development of distinct PAMs can amplify the effect of endogenous signaling molecules while not disrupting normal physiological regulation of receptor activation,” he explains. “This could provide superior efficacy and reduce side-effect profiles, as compared to use of the traditional orthosteric agonists.”

These studies are at an early preclinical phase, and will have to be succeeded by fuller assessments. Nonetheless, they have already generated encouraging findings. For example, as Dr. Alt points out, opioid PAMs have been shown to work in vivo.

“This is still a new field, but we are finding that it offers the possibility of a relatively exciting way to treat not only pain, but also depression and addiction,” says Dr. Alt. “As safety profiles of compounds continue to be improved, PAMs could conceivably someday replace many drugs in the market with ones that are more targeted.”

Positive allosteric modulators (PAMs) enhance natural agonists. (A) During pain, endogenous opioid agonists are released only in relevant regions of the brain and spinal cord. (B) Exogenous agonists (morphine, oxycodone, etc.) indiscriminately activate opioid receptors throughout the body. (C) PAMs enhance the effects of native agonists, thus conferring more natural specificity. (Adapted with permission from Burford et al. 2015. Br. J. Pharmacol. 172: 277–286. [© 2014 The British Pharmacological Society]

Journey to the Center of the Cell

Although GPCRs are well-known denizens of the plasma membrane, at least 30 of them have been found to be situated in the cell nucleus. “This suggests the plasma membrane can no longer be considered the exclusive signaling site of GPCRs,” states Sylvain Chemtob, M.D., Ph.D., professor of pediatrics, ophthalmology, and pharmacology, University of Montreal.

The new picture emerging is that some GPCRs can journey to the center of the cell, to the nucleus, where they may participate in therapeutically important intracrine signaling. However, this picture relies on early findings that are in need of reinforcement and elaboration. At present, the specific physiological functions of nuclear GPCRs are largely unknown.

Dr. Chemtob and colleagues recently found distinct but complementary functions depending on the location of the same GPCR. “We studied the GPCR receptor called protease-activated receptor 2 (PAR2),” he details. “On the cell surface, PAR2 participates in maturation of vascular network.” That is, plasma membrane–located PAR2 promotes vessel maturation. Yet, nuclear PAR2, the investigators found, promotes an early proliferative vascular phenotype.

“We found two nuclear localization signal motifs important for its translocation to the nucleus,” notes Dr. Chemtob. “Once inside, its proximity to the genome allowed PAR2 to form a transcriptional complex to directly bind DNA.”

The studies utilized chromatin immunoprecipitation sequencing of DNA fragments (ChiP-Seq) to confirm a unique transcriptional nuclear program. They also confirmed that the same in vivo phenotypes were conferred, using PAR2-deficient mice transfected with PAR2.

“Overall, these studies demonstrated that the same GPCR in different subcellular locations orchestrates distinct yet complementary responses important for retinal vascular development, as we recently confirmed for a different GPCR, namely the platelet-activating factor receptor,” asserts Dr. Chemtob.

Specifically targeting intracellular GPCRs raises a unique set of challenges. According to Dr. Chemtob, promising intracellular drug-delivery systems include lipid-coated nanoparticles and cell-penetrating peptides.

“This is an entirely new dimension,” concludes Dr. Chemtob. “Our hope is to eventually expand the therapeutic repertoire of current drugs to include delivery strategies and new therapeutics that will harness the power of subcellular GPCR signaling.”

GPCR Megaplexes

Classically, GPCR stimulation promotes G-protein signaling at the plasma membrane. This process is rapidly followed by the desensitization of G-protein signaling mediated by β-arrestin that binds to and blocks the receptor’s G-protein binding site and subsequently mediates endosomal receptor internalization. However, for some GPCRs this is not the end of the story as they continue G-protein signaling from the endosome, a phenomenon termed sustained G-protein signaling. A new study determined that “megaplexes,” assemblies consisting of a single GPCR, G protein, and β-arrestin, may be responsible.

“For about the last five to eight years, studies showed that some GPCRs signal from internalized compartments,” observes Alex R.B. Thomsen, Ph.D., a postdoctoral fellow in the Duke University laboratory of Robert J. Lefkowitz, M.D. “Yet, association with β-arrestin, which drives the internalization, should prevent activation of G protein.

“We tested the hypothesis that both G proteins and β-arrestin simultaneously interact with the receptor to provide sustained G-protein signaling from the endosome. To do this, we utilized a variety of cellular, biochemical, and biophysical approaches.”

Employing both confocal microscopy and bioluminescence resonance energy transfer biosensors, Dr. Thomsen and colleagues found that some GPCRs interact with both G proteins and β-arrestins in endosomes. The investigators were able to form such megaplexes in vitro and to then illustrate their architecture by performing single-particle electron microscopy.

“We demonstrated that a single GPCR simultaneously binds through its core region with G protein and through its phosphorylated C-terminal tail with β-arrestin,” reports Dr. Thomsen. “Why β-arrestin association leads to sustained G-protein activation in some GPCRs, while desensitizing others, is a good question.

“It’s possible that in order to form megaplexes, GPCRs may need a C-terminal tail with phosphorylation clusters that can promote a strong interaction with β-arrestin following phosphorylation. For GPCRs with such phosphorylation clusters, β-arrestin may be able to form a stable complex with the receptor without interacting with the G protein-binding region at the core of the receptor.”

There is still much to explore, as Dr. Thomsen explains: “Right now, it’s too early to see the impact of these findings therapeutically. We need to establish specific functions of G-protein signaling from the endosome versus that from the cell surface. It’s possible that in the future, compounds may be discovered that can modulate these processes differentially. Such compounds could be used to treat certain diseases with fewer side effects.”

Protein Superfamily Analysis

Making sense of and utilizing the overwhelming amount of data on GPCRs and other protein superfamilies remains a daunting task. To assist with managing and integrating this growing body of information, Bio-Prodict has developed a software platform, the 3DM system, that can combine many types of (publically available) information.

Although collecting and curating current information may seem straightforward and simple at first glance, that’s not really the case. “Just take the literature, for example. There are tens of thousands of articles on GPCRs,” notes Henk-Jan Joosten, Ph.D., Bio-Prodict’s CEO. “Thus, the amount of data that needs to be collected from heterogeneous sources, converted to syntactic and semantic homogeneity, validated, curated, stored, and indexed is enormous.

“We’ve custom-built a system that is unlike any other in the world. Our linkage of all of this information is unique.”

Examples of data types that are used by 3DM include sequences, structural information, protein-ligand and protein-protein contact information, mutational data, and information extracted from multiple sequence alignments. All these different data types are stored in specialized protein superfamily systems that are centered around structure-based multiple-sequence alignments. To stay up to date, the system automatically retrieves information from all data sources, including the primary literature.

According to Dr. Joosten, combining data and information extracted from sequences, structural information, and the literature into one searchable source can greatly enhance the understanding of not only GPCRs, but many other proteins and their functions. “Aside from drug design, 3DM can also be applied to protein engineering and DNA diagnostics. It can be used to make changes in enzyme specificity, to increase activity or thermostability, to evaluate ligand binding properties, or even to design better inhibitors.”

Dr. Joosten says Bio-Prodict can make 3DM systems that automatically process information for all the proteins in a complete genome. “However, we still keep the system simple and easy to use,” he remarks. “We designed this for biologists, not just bioinformaticians.”

Future GPCR Landscape

New and more comprehensive structural insights into novel binding sites on GPCRs are on the horizon, suggests Sujata Sharma, Ph.D., director, screening and protein science, Merck & Co. She adds, however, that several obstacles must be overcome before GPCR structural insights become routine elements in drug discovery.

“One major challenge is to obtain sufficient expression of the receptor in the desired oligomeric state. Progress toward this goal can be accelerated by leveraging high-throughput expression technologies and rapid identification and characterization of reconstituted materials,” comments Dr. Sharma. “Another challenge is to obtain conformationally stabilized receptors to obtain ligand-bound structures in a desired pharmacological state to enable drug discovery.”

Several tricks, such as scanning mutagenesis, ligands, and G-protein mimics, have been employed by experts to generate stabilized receptors for structural and biophysical studies. “Receptors exist in a spectrum of different conformations, and crystal structures are frozen snapshots,” notes Dr. Sharma. Consequently, structural findings would be more useful if they were validated “via orthogonal approaches, such mutagenesis coupled with biochemical and biophysical assays.”

Dr. Sharma is optimistic about the GPCR field. She believes it will benefit from technological advances in single-molecule methods, nuclear magnetic resonance spectroscopy, and chemical biology.

“These technologies are likely to provide a deeper understanding of protein conformational changes, receptor-ligand interactions, and GPCR dynamics,” she comments. “Getting structural insights into novel ligand-binding pockets and how molecules binding in those pockets affect downstream signaling can potentially allow us to design better GPCR-targeted drugs.” 

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