April 15, 2015 (Vol. 35, No. 8)

Lisa Heiden Ph.D. Director of Business Development MyBioSource

The Cell, Like an Engine, May Need a Tune-Up for Optimal Performance, or Even a Smooth Idle

G protein coupled receptors (GPCRs) are essential drug targets for therapeutic intervention due to their integral roles in a plethora of fundamental signal transduction pathways.

Indeed, GPCRs have a fruitful history in the pharmaceutical industry and are the targets of over half of all prescribed drugs today. Yet discovering, designing, and synthesizing GPCR-targeted compounds for modulating signaling has historically been fraught with challenges. GPCRs and their signaling pathways are extremely complex and nuanced.

“About 800 proteins have been classified as GPCRs,” says Alexander Heifetz, Ph.D., principal scientist, computational chemistry, Evotec. “But drugs have been developed against only 50 of these.” Moreover, existing, effective drugs are associated with problematic side effects.

Dr. Heifetz and his colleagues in drug discovery and development are exploring innovative strategies in biased signaling and other advances in hopes of refining GPCR-targeted approaches. As part of a broader effort to embrace challenges, develop solutions, and facilitate new precision medicine paradigms, scientists are participating in a number of GPCR-related drug discovery conferences.

Notable events include CHI’s Drug Discovery Chemistry (San Diego, April 21–23), particularly the Protein-Protein Interactions section; Global Technology Community’s European Pharma Summit (Berlin, May 5–8), GPCR Targeted Screening; and CHI’s World Preclinical Congress (Boston, June 10–12), Targeting GPCRs.

Presenters at these events will run the gamut from academic research to clinical application. For example, at the Targeting GPCRs conference, one of the academically oriented presenters is Jeffrey L. Benovic, Ph.D., professor and chair, department of biochemistry and molecular biology, Thomas Jefferson University. (He will discuss novel strategies for biasing GPCR signaling.) Another presenter at the same event, Conrad Cowan, Ph.D., head of biology at Trevena, is expected to describe clinical applications. (He will discuss his company’s most advanced biased ligand programs.)


The Hierarchical GPCR Modeling Protocol (HGMP) represents Evotec’s GRPC drug discovery strategy. The interative drug discovery life cycle ranges from computational modeling through experimental verification and involves both computational and medicinal chemists.

Biased Ligand Signaling

Biased ligand signaling as an approach to GPCR drug discovery “is something very new,” says Dr. Heifetz, who is scheduled to present at the GPCR Targeted Screening conference. “The concept is simply promoting selective interaction of the receptor through a smaller number of proteins than it normally interacts with, that is, biasing the signaling that normally occurs in a cell,” adds Dr. Benovic.

“Historically we knew agonist ligands activate the G protein signaling pathway, and now we know there’s an additional signaling pathway being activated via β-arrestin,” explains Dr. Heifetz. He is adamant GPCR nomenclature should be updated. For example, he says that the term “G protein-β-arrestin coupled receptors” should be used because it reflects the receptor’s ability to signal through both G protein and β-arrestin pathways.

“These signaling pathways are involved in practically every aspect of cellular function, and some agonists activate both pathways through one receptor,” Dr. Heifetz emphasizes. “One pathway may be beneficial whereas side effects, previously thought to result from nonspecific binding, can originate from the other pathway.”

Dr. Benovic’s example is key: “In asthma, the β-agonist is the major treatment and is absolutely critical for someone having an asthma attack. However, β-agonists actually have a black box warning because they can induce a severe fatal asthmatic attack. It is thought that the β-arrestin signaling pathway is mediating that side effect.”

Dr. Heifetz summarizes, “We need ligands that will be biased and activate one pathway but not the other; not inhibit, just not activate.”


Trevena’s TRV027 is a biased ligand that binds the angiotensin II type 1 (AT1) receptor to stimulate the ß-arrestin pathway while inhibiting the G protein pathway. Preclinical studies have shown that TRV027 reduces blood pressure and cardiac workload while increasing cardiac contractility and preserving renal function. TRV027 is currently being evaluated for potential benefits in acute heart failure patients in the Phase IIb BLAST-AHF study.

Computational Modeling Tools

Dr. Heifetz continues: “Over the last 20 years, the pharmaceutical industry has moved from trial-and-error approaches to rational drug discovery. This is especially true for GPCRs with major breakthroughs in crystallography, modeling, and functional selectivity (biased signaling). GPCRs are very dynamic, and our modeling protocol technology is a set of computational tools for exploring dynamically how ligands interact with proteins, elucidating GPCR structures, and defining structure-function relationships.”

Membrane protein simulation modeling shows that the moment an agonist ligand binds, distinguishable populations of active forms of G protein and β-arrestin are induced. “Modeled ligands can also be antagonists and inverse agonists,” notes Dr. Heifetz. “Our tools modify their properties to make them more potent or agonistic, stronger binders, more or less biased, or even switch them between agonist and antagonist. The computational chemist is the ‘eye’ of the drug discovery project, and modeling tools help assign desired properties to small molecules.”

The medicinal chemist “moves” the computational design to synthesis, experimentally validating or challenging the design. The feedback regarding outcomes is only a matter of weeks, and then an iterative process may ensue between the chemists to precisely tune the compound.


Structure-Function Interactions

“The goal of everyone’s research is to see what you are working on be useful in treating a disease,” remarks Dr. Benovic. “A critical stage is the very basic research and understanding of mechanisms from an academic perspective that ultimately helps develop better drugs.”

“GPCRs interact with three different protein families in an activation- or agonist-dependent matter: heterotrimeric G proteins, GPCR kinases (GRKs), and arrestins,” he continues. Dr. Benovic uses structural approaches to define functional consequences of these interactions between receptors and signaling proteins in cell, animal, and disease processes. He explains that the knowledge gained can ultimately be applied to come up with more selective drugs that have fewer or less severe side effects.

“We developed a strategy to target the surface of the receptor with pepducins, compounds made by synthesizing pieces of the receptor from intracellular loops and putting a lipid on one end and amidating the other end,” informs Dr. Benovic. “These peptides appear to bind the lipid bilayer, get inside the cell, stay associated with the membrane, and via an unknown mechanism interact with the receptor they were made from and can bias signaling.”

The concept is that the pepducin interacts selectively with, for example, the β2-adrenergic receptor and biases the signaling. Dr. Benovic focuses on the β2-adrenergic receptor because “it plays a central role in the treatment of asthma, and something that biases the signaling through Gs (Gα2βγ heterotrimer) and not β-arrestin would most likely be an improved asthma therapeutic.”


This image depicts a model of ß-adrenergic receptor signaling that was developed by Jeffrey Benovic, Ph.D., at Thomas Jefferson University. The left panel shows how an unbiased agonist induces balanced signaling by promoting receptor coupling to G proteins, GPCR kinases (GRKs), and ß-arrestins. The other panels show how a biased agonist can stabilize a receptor conformation that selectively couples to a particular G protein, GRK, or ß-arrestin, resulting in different functional consequences and potential therapeutics.

Receptor Binding Assay Technology

“Back-scattering interferometry (BSI) is a label-free solution-based interaction platform typically used to measure the affinity of a ligand for a protein target,” conveys Richard “Jake” Isaacs, Ph.D., applied research supervisor, Molecular Sensing. Dr. Isaacs, who is scheduled to present at the Targeting GPCR conference, says, “the reason we see so much interest for GPCRs is they are notoriously difficult to analyze by conventional means biophysically.

“BSI is binding site agnostic and able to do a host of allosteric binding experiments that you just can’t do with other means,” continues Dr. Isaacs. “Allosteric compounds are a hot area for GPCRs now.”

Allosteric compounds can achieve functional selectivity since they bind to different sites than endogenous ligands, modulating signals through various mechanisms. “You may have a family of GPCRs with multiple members all binding the same ligand in different tissues, associated with different diseases,” he elaborates. “Allosteric sites are much less conserved, so you can achieve specificity by targeting those sites, which are attractive both pharmacologically and from an intellectual property perspective.”

The technology works by drawing the sample into a microfluidic chip that is interrogated with a laser beam. The laser light that is reflected backwards forms an interferometric or fringe pattern that changes in response to ligand binding, thereby measuring changes between the refractive indices of unbound and ligand bound protein.

“BSI can determine whether a putative allosteric compound is effective in modulating the binding of an orthosteric agonist or antagonist or whether it is competing with them for binding,” adds Dr. Isaac. “BSI can also differentiate different modes of action for different types of compounds, and whether the compounds are competing for the same binding site or they are binding elsewhere.”


Compound Screening Libraries

“Finding high-quality starting points for drug discovery programs is a constant challenge facing our industry,” remarks Jackie Macritchie, Ph.D., senior director, lead optimization, Charles River. Charles River is expected to present at the Drug Discovery conference, in the Protein-Protein Interactions section.

“The better the quality of a hit compound, the more efficient the onward drug discovery, and the higher the chance of a successful outcome,” asserts Dr. Macritchie. “GPCR SoftFocus® libraries, which include family A GPCRs and family C mGluR receptors, are collections of compounds pre­designed to interact with biological target proteins within the broad GPCR family space. A typical SoftFocus library is based around a novel chemical scaffold designed to interact with multiple GPCRs possessing commonality in primary sequence and secondary structural features.”

“The scaffolds,” Dr. Macritchie continues, “are then decorated with functionality that is likely to be recognized by the target proteins of specific interest to the industry. This decoration is key to deliver the selectivity required across the different members of the GPCR family to ensure the desired profile can be achieved in drug candidates.”

The libraries come into play at the very early point of the drug discovery process, facilitating efficiency and success in identifying clinical candidates. The compounds are designed to have the requisite physicochemical properties to be highly attractive starting points for drug discovery programs.

“When a SoftFocus library is screened, structure-activity relationships are generated directly from the primary screen, which speeds up subsequent drug discovery phases,” Dr. Macritchie insists. “We are proud of our track record of success, with compounds from our SoftFocus libraries being the source of multiple clinical candidates, compounds currently in clinical trials, and numerous patents filed by pharma and biotech companies.”


Compounds in the Clinic

“Although many publications and scientific talks discuss the role of bias in GPCR signaling and what bias is, we are not aware of any other companies that have actually taken compounds forward in the clinic,” relates Dr. Cowan. “Trevena has three compounds in the clinic with a fourth compound in preclinical development.” They are TRV027 (Phase II) for heart failure; TRV130 (Phase II) and TRV734 (Phase I) for acute moderate-to-severe pain (these drugs are administered intravenously and orally, respectively); and TRV250 (preclinical) for refractory migraine and other indications.

Dr. Cowan explains that Trevena turns the concept of bias signaling into products, and that all four compounds are based on a biased ligand platform. A series of in-house and commercial assays are employed to identify and understand bias in the compounds and how they interact with receptors and signaling pathways.

“Pathways are teased apart through the use of β-arrestin knockout mice or other techniques that target the desirable signaling pathway,” Dr. Cowan details. “Then we identify compounds that activate the desirable pathway while avoiding the undesirable pathway. In many cases, from literature mining and internal discovery efforts, we can elucidate that one set of signaling events leads to a beneficial effect, and a different set of signaling events leads to the undesirable or the side effects.

“Once we have the compound that has the signaling profiles we want in our in vitro cell models, we move it very quickly into animal models. Each compound is tested in various in vivo models to ensure it has the desired differentiation from unbiased ligands as well as, importantly, required drug characteristics such as pharmacokinetics and safety margins prior to clinical trials.”

Dr. Cowan reflects that past GPCR drugs have treated the receptor like a light switch, turning it all on or all off. “However, we now realize there are multiple switches for each GPCR,” he concludes. “We can selectively turn them off or on to get a more precise and desirable effect.”

























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