GPCRs Remain “Hot” Drug Targets

GPCRs represent the largest family of genes expressed in the human genome, according to Jeremy Richman, Ph.D., associate director, cardiovascular biology, at Arena Pharmaceuticals. “Their cell membrane localization, the diversity of tissue expression, and the fact that they have the potential to elicit essentially every signaling pathway, makes these proteins ideal therapeutic targets.”

Informa Life Sciences’ “6th Annual Congress: G Protein-Coupled Receptors in Drug Discovery” conference in Berlin in mid-March showcased some spectacular discoveries in this scientific arena that, although years in the making, have only recently come to fruition.

“It is a really good time to be in this field,” said P. Jeffrey Conn, Ph.D., professor and director, Vanderbilt University program in drug discovery, and a keynote speaker at the conference. This well-established conference assembles leading experts from both academia and industry in the field of GPCR research, and as evidenced by the presentations, there was plenty of good news to share.

Allosteric Modulators

Dr. Conn’s presentation focused on his lab’s research. First, he spoke about selective activators of specific subtypes of metabotropic glutamate receptors (mGluRs) that have potential as therapeutic agents for psychiatric and neurological disorders. Specifically, he outlined studies suggesting the utility of mGluR5 activators as a treatment for schizophrenia. Unfortunately, the search for selective agonists for this receptor was unsuccessful. Successful antagonists were discovered though, and currently, three companies are pursuing clinical trials with these drugs.

Dr. Conn’s group developed allosteric potentiators of multiple mGluR subtypes that offer high selectivity for the targeted receptor. “We reasoned that allosteric antagonists inhibit coupling of receptors, and that was where we started. The result was that we developed allosteric compounds that showed an increased response, and the data we presented showed that this was a viable approach.”

More recently, Dr. Conn’s group discovered allosteric potentiators for M1 and M4 muscarinic receptors that have unprecedented subtype selectivity. Until recently, it was virtually impossible to activate the M1 and M4 muscarinic receptors without activating the M2 and M3 receptors, inducing devastating results.

“We used a combination of cheminformatics, functional screening, and medicinal chemistry to develop some of the first truly specific activators of M1 and also of M4 muscarinic receptors that have nice drug-like properties,” said Dr. Conn. “The results build on strong clinical data that provided proof of concept for utility of muscarinic agents in Alzheimer’s patients and in schizophrenia.”

Subtype-selective allosteric potentiators of multiple GPCR subtypes have also been discovered by the team. According to Dr. Conn, these have robust effects in animal models used to predict efficacy for novel antipsychotic agents (mGluR2, mGluR5, and M1), anxiolytics (mGluR2), and antiparkinsonian agents (mGluR4).

“This has generated a lot of excitement,” explained Dr. Conn, “because compounds, developed for at least five different GPCR targets, that are amenable to optimization show high selectivity and have robust effects in animal models. Ultimately, it will be important to fully optimize these compounds for use in clinical studies.”

Differentially Activated Signal-Transduction Pathways

Arena Pharmaceuticals is geared specifically to studying GPCRs, and Dr. Richman’s team is primed to look at therapeutic targets in cardiovascular health. One such therapeutic target is the high-affinity, nicotinic acid receptor GPR109A. This Gi-coupled receptor, expressed on adipocytes, macrophages, and Langerhans cells, is presumed to mediate the therapeutic actions of nicotinic acid. A water-soluble vitamin, nicotinic acid, at high doses favorably affects all lipid and lipoprotein parameters believed to be cardiovascular risk factors.

The problem with niacin formulations, Dr. Richman said, is their side effects, the worst of which is an uncomfortable flushing response. Dr. Richman’s group hypothesized that the signal transduction pathways mediating the antilipolytic and prostaglandin D2/flushing pathways are distinct, and that agonists may be identified that are capable of selectively eliciting the therapeutic, antilipolytic pathway while avoiding the activation of the parallel flush-inducing pathway.

The conclusion they arrived at, according to Dr. Richman, is that separation of downstream effector signals, in order to achieve a therapeutic effect without the unwanted side effects, does not necessarily require the development of partial agonists.

“The drug design and identification process should be focused on the identification of agonists that potently stimulate responses downstream of effectors known to mediate therapeutic responses and that lack the ability to stimulate effectors that mediate untoward side effects,” Dr. Richman added.

“On a fundamental level, we support the concept that you can’t take it as a given that you won’t get the signal based on what agonist you use. You have to look at the larger therapeutic picture and be careful and aware of the questions you are asking with regard to what you are expecting a drug to do.”

GPCR ligands with subtle structural differences can activate different signaling pathways via the same receptor, a phenomenon termed functional selectivity. Matt Peters, Ph.D., principle scientist at AstraZeneca, said that this phenomenon is not widely exploited in drug discovery, possibly owing to the difficulty of assessing structure activity relationships (SAR) versus multiple pathways.

Dr. Peters’ talk centered on cellular dielectric spectroscopy (CDS), an emerging technology that can distinguish Gs, Gi/o, and Gq signal transduction in a whole-cell, label-free format, enabling one to monitor activation pathways by different ligands within the same assay condition.

“Cellular dielectric spectroscopy and other plate forms based on cellular impedance technology offer sufficient sensitivity that you don’t need to use radio ligands, so radioactivity is not involved. There are no dyes and there are no recombinant receptors, so you don’t have to overexpress the genes just to get a signal.”

Dr. Peters reported that his group was able to show that GPCR agonists, antagonists, inverse agonists, and allosteric modulators can be quantified by CDS with precision suitable for drug discovery SAR studies. “Furthermore, agonist ligands for one GPCR reveal Gs-dominant coupling in HEK cells versus Gi-dominant coupling in CHO cells,” noted Dr. Peters. “Other ligands showed inverse agonism against Gi coupling only, consistent with either ligand-specific trafficking or pathway-specific constitutive activity.

“This technology has great potential, but the difficulty right now is its modest throughput—96 wells. The company is planning to take it to 384 wells later this year. In addition, broader usage will help us understand the technology’s intricacies.”

In Silico Screening

Approaches to in silico drug design for soluble proteins are becoming straightforward, albeit nontrivial, said Sid Topiol, Ph.D., associate director of computational chemistry at Lundbeck Research USA.

“The most common pharmaceutical targets are GPCRs, and what we’ve been doing is taking advantage of a lot of ligand-only based computational methods. We’ve complemented that with structure-based approaches taking advantage of the crystal structures that were available, which generally meant using Rhodopsin for transmembrane homology modeling.”

For GPCR targets, x-ray structure-based approaches are novel, reported Dr. Topiol. The recently reported x-ray structure of the b2-adrenergic receptor, the first available crystal structure of a ligand-mediated GPCR, was recently investigated for its utility in computer-aided drug design.

“We were fortunate in that the Kobilka group [Brian Kobilka, Ph.D., Stanford University] has done the scientifically noble thing of making the beta2 x-ray coordinates available to the public.” According to Dr. Topiol, such approaches provide valuable results for lead finding, optimization, and novel template identification, particularly when coupled to a broad platform of ligand-based approaches. “Really, the best way to do structure-based design is to use x-ray crystallography,” he said.

Dr. Topiol’s team conducted validations with known beta blockers, followed by high-throughput docking studies with proprietary and commercial databases, to further validate the x-ray structure’s usefulness as a design tool and to explore the potential for discovery of novel chemical classes acting as b2 inhibitors.

“Our results include the finding of ligands with traditional beta-blocker motifs as well as new motifs, which both validates the approach and projects its usefulness in the finding and design of novel compounds,” Dr. Topiol noted. “Thanks to the recently published x-ray structures of extracellular domains, class 3 GPCRs such as the mGluR receptors are now open to exploration.

“We’re excited about the hard work that has made the x-ray structures possible. We are able to take advantage of them because everything has been geared up and primed for x-ray structure-based design in areas such as soluble proteins. All of the machinery is now in place and the real work can begin.”

Crystal Structures

EPIX Pharmaceuticals is also advancing in silico modeling. “Our approach is unique in that we use PREDICT™, nonhomology algorithms, to model 3-D structures of GPCRs. We base lead identification and optimization on what the binding site looks like,” said Sharon Shacham, Ph.D., svp of drug development.

“Our discovery platform is based on GPCR models created in silico with PREDICT, structure-based in silico high-throughput screening of GPCRs, and structure-guided lead optimization using the technology. All of this requires collaboration between our computational and medical chemists.

“Each compound goes through several tiers of affinity and selectivity evaluation in silico before any of the compounds are synthesized by the medicinal chemists. To maintain this discipline and drive efficiency, requires a tight and focused interaction between computational and medicinal chemists as well as the biologists and pharmacologists working on the team.”

PREDICT generates multiple transmembrane bundle configurations, and the subsequent selection is based on energy and/or binding-site location, size, or composition. Molecular dynamics simulations are then used to optimize the binding-site conformation in the presence of known ligands.

Dr. Shacham’s group synthesizes a small number of compounds. “We evaluate about the same number of compounds as big pharma,” she said. “But, the compounds we analyze are mainly evaluated in silico, so optimization occurs more rapidly than if you’re doing a high-throughput screen and usual wet-lab-based iterative medicinal chemical syntheses. Combine this with collaboration between the computational and medicinal chemists, and the result is a solid lead-optimization program that is efficient and often successful.”

Marion Blomenrohr, Ph.D., senior research scientist at Organon (now a part of Schering-Plough), noted that many pharmaceutical industries have embarked on high-throughput screening of large collections of compound libraries to identify new low molecular weight modulators of GPCRs.

“Traditional methods for high-throughput screening of GPCRs rely on the coupling of the ligand-bound receptor to heterotrimeric G proteins” she said. “New assay methods, however, have become available that are not based on G-protein activation but that apply the molecular mechanism underlying the attenuation of G-protein signaling mediated by beta-arrestin.”

Beta-arrestin is a cytoplasmic protein that targets receptors to endocytotic vesicles for degradation or recycling. This process has been visualized and quantified in high-content imaging assays using receptor or beta-arrestin fusion proteins with green fluorescent protein. Dr. Blomenrohr compared these technologies in her talk.

“Recently, methods have become available that do not require expensive high-content imaging equipment but instead use enzyme- fragment complementation or reporter genes and can be read on conventional multimode readers available in most laboratories,” said Dr. Blomenrohr. “In the screening world, there is great interest in these new technologies. Not only do beta-arrestin recruitment assays provide a generic assay for GPCR drug screening, they also allow the identification of previously unknown pharmacologies of existing drugs.”