November 15, 2009 (Vol. 29, No. 20)

Emerging Tools and Methodologies for Screening and Discovery Are Improving Odds for Success

In the organism, communication occurs at both the macro and the micro level. At the micro level—inside the cell—communication occurs between proteins relaying messages along signal-transduction pathways from both the internal and external to various compartments within the cell through the process of signal transduction. GPCRs are at the center of many signal-transduction pathways, and as a result, they are the subject of many basic biological research studies, as well as prime drug targets.

Historically, much of the pharmaceutical industry was only interested in discovering agonists or antagonists when it came to GPCR targets. Currently, however, the industry realizes that there are many more opportunities for targeting GPCRs if it screens for modulators as well as G-protein-dependent and G-protein-independent pathways.

Investigators are exploring a number of approaches, tools, and technologies related to GPCR drug discovery, and several scientists discussed their research in this area at the Cambridge Healthtech Discovery on Target conference “GPCR-Based Drug Discovery”, which was held in Boston earlier this month.

Structural Studies

Fiona H. Marshall, Ph.D., CSO of Heptares Therapeutics, spoke at the meeting about StaR—a thermostable GPCR locked into a chosen conformation by targeted mutagenesis. “GPCR drug discovery is hindered by the need to assay these receptors in cell membranes. This fact restricts the use of modern techniques in drug discovery such as biophysical and structural approaches.”

By introducing point mutations into GPCRs, Heptares has produced receptors that can be purified like soluble proteins, thus preparing them for biophysical analysis using methods such as SPR, NMR techniques, and x-ray crystallography.

“This approach enables fragment screening and structure-based design methods to discover drugs for previously intractable receptors.” In addition, other biophysical methods can be used to directly evaluate the biology of ligand-receptor interactions in native cell preparations (e.g., optical biosensors to measure intracellular mass redistributions or cellular dielectric spectroscopy to measure cell surface interactions).

GPCR-based drug discovery efforts at Heptares include a wide range of targets. In a discovery program targeted at the adenosine A2a receptor for Parkinson’s disease, Heptares has identified a lead compound series (and an unrelated backup series) with, among other pharmacologic properties, potent oral activity in rodent models of Parkinson’s disease.

Great as structure-based drug design programs may be, there is still a need for drug-screening platforms. DiscoveRx is battling the challenges of GPCR drug research using its screening platforms, which are based on its ß-galactosidase enzyme fragment complementation (EFC) technology.

G-protein dependent and G-protein independent pathway activation of activated GPCR (DiscoveRx)

Screening Platforms

One of those screening platforms is the HitHunter cAMP, which, according to the company’s senior director of cell biology Tom Wehrman, Ph.D., “is a sensitive and reliable platform that detects intracellular cyclic adenosine monophosphate (cAMP) using chemiluminescence.”

More recently, DiscoveRx used the EFC platform to develop an assay to detect ß-arrestin recruitment to activated GPCRs, a primary rather than secondary signaling event that enables rapid deorphanization of a GPCR target.

“Over the past five years, the rate of de-orphanization has declined, suggesting that the standard second messenger deorphanization platforms have not resulted in a large number of receptor-ligand pairings,” said Dr. Wehrman. “With the introduction of the PathHunter Arrestin technology, we are now seeing a renewed interest in deorphanization programs given that arrestin is known to interact with the vast majority of GPCRs.”

Dr. Wehrman’s presentation focused on the advantages of using multiple GPCR screening approaches such as G-protein-dependent cAMP/calcium screening along with G-protein-independent PathHunter ß-arrestin and generation of novel hits and pharmacology resulting from such comprehensive studies. His presentation also included a discussion of the next generation of tools developed based on PathHunter technology.

These tools provide either a quantitative measurement of GPCR trafficking (total endocytosis GPCR assay system) or GPCR heterodimerization (PathHunter GPCR dimerization assay). All of the PathHunter technologies are whole cell-based and use a single-step, no-wash, high-throughput screening-friendly, chemiluminescence-based assay format.

PathHunter GPCR dimerization assays are designed to study the interactions between different pairs of GPCR molecules or between GPCR heterodimers. “These assays are unique in that they measure the functional coupling of two GPCRs through the arrestin pathway and enable screens for modulators of GPCR activity,” commented Dr. Wehrman. The Endocytosis GPCR assay system measures GPCR internalization by quantifying the amount of receptor in the endosomal compartment, thus providing a nonimaging method to screen compounds for their ability to cause endocytosis of the receptor.

“A more complete view of compound activity is obtained using arrestin binding in conjunction with second messenger assays. The arrestin and second messenger pathways can lead to disparate biological effects, making it increasingly important to monitor arrestin binding, as well as G-protein activation.”

Charles Lunn, Ph.D., research fellow, department of new lead discovery, Schering-Plough, concured that “there is an interest in looking at GPCRs in ß-arrestin-dependent systems, partly to interrogate G-protein-independent as well as G-protein-dependent signal transduction mechanisms. But, we appreciate that every GPCR assay format can bias the types of modulators you identify. Label-free profiling of cell-ligand interactions may moderate this bias.” 

“People are also going into more label-free technologies, especially for GPCRs,” added Suresh Poda, Ph.D., senior scientist, Lundbeck Research. “The good thing about these label-free technologies is that you don’t need to overexpress your target. You don’t need to co-express any promiscuous G-proteins in your cell lines.”

PerkinElmer is developing tools and technologies for the screening and discovery of compounds targeting GPCRs. “Our flagship GPCR-related technology is the luminescent photo-protein, Aequorin,” said Martina Bielefeld Sévigny, Ph.D., vp and GM of drug discovery and research reagent solutions.

Aequorin-producing cell lines generate luminescence in response to an increase in intracellular calcium levels, thus enabling researchers to measure GPCR-induced signal-transduction pathways. Very high signal-to-noise ratios are typically obtained, enabling the discovery and development of allosteric modulators. PerkinElmer also offers over 300 GPCR cell lines, which are validated for different signaling pathways.

Photo protein assay principle: To reduce the M4 receptor expression level, CHO-aequorin-Ga16-M4 cells were serum-starved for two days. PerkinElmer used a double-injection protocol.

Drug Delivery

Stephen W. Hunt III, Ph.D., svp, discovery research at Ascent Therapeutics, presented data on the firm’s Pepducin® technology.

“Pepducin is a biotherapeutics platform that may overcome many of the obstacles to current drug discovery approaches. It provides an opportunity to pharmacologically address the entire family of GPCR targets including those that have been intractable to date,” Dr. Hunt said.

Its lead program targets CXCR4, a GPCR and chemokine receptor that has become an attractive drug target due to its involvement in cancer and HIV. Among the observations that make CXCR4 an important cancer target include the fact that it is the receptor for stromal cell-derived factor 1 (SDF1α), which is found in high concentrations in the bone marrow, it is expressed in hematopoietic stem cells, along with many mature leukocytes, and SDF1α-CXCR4 interaction leads to the retention of these cells in the marrow. “Interrupting this interaction can facilitate release of the cells into the systemic circulation,” explained Dr. Hunt.

“Pepducins work by a completely unique mechanism of cell entry and allosteric modulation of GPCR activity. Pepducins enter the cell in seconds via a mechanism that we call insertion and inversion. Pepducin’s lipid tail serves to insert and anchor the Pepducin in the external leaflet of the cell membrane. Charges in the peptidic portion of the molecule are neutralized, thus allowing the pepducin to traverse the membrane and anchor in the intracellular leaflet of the cell membrane. Once inside the cell, the Pepducin is in a position to encounter its target GPCR.”

GPCRs have become extremely popular drug targets for the pharmaceutical industry. Expansion beyond classical agonist and antagonist drug classes into the realm of GPCR modulators has created new opportunities for GPCR drug discovery. Structural studies utilizing state-of-the-art biophysical methods to analyze GPCRs have enabled enhanced drug design.

Additionally, current screening platforms allow for the measurement of GPCR-induced second messenger systems with higher throughput, higher sensitivity, and in a label-free format. Finally, new delivery technologies are enabling more accurate targeting of internal GPCR moieties.

Sidebar: Examining Compound Selectivity and Specificity

At Cambridge Healthtech’s “Discovery on Target” meeting, Blaine Armbruster, Ph.D., manager, GPCR drug discovery, Millipore, gave a talk on “Testing the Hypothesis of Allosteric Compound Selectivity through Large Scale Selectivity Profiling.”

Compound specificity (whether a compound interacts with unintended protein targets) is always a concern for drug safety and, therefore, is a prominent issue for drug development. GPCR compounds are no exception and certain off-target interactions contribute to the adverse reactions of marketed compounds.

Making an Assumption

The lack of selectivity has been attributed to the high degree of conservation of orthosteric binding sites, noted Dr. Armbruster.

“An assumption has been made that allosteric compounds, i.e., compounds that interact with less conserved, nonorthosteric sites, may have promise as more selective compounds,” he said. “However, we conducted a study that profiled kinase inhibitors through a large panel of GPCRs via Millipore’s GPCRProfiler® service. We demonstrated that these compounds can functionally interact with some GPCRs.”

The data highlights the inherent challenge that GPCRs present for compound selectivity, regardless of whether or not a compound was primarily designed for a GPCR target, added Dr. Armbruster.

He explained that Millipore has developed a service, AllostericProfiler, that tests the selectivity of both orthosteric and allosteric compounds at over 155 different GPCRs.

“The design of AllostericProfiler, which can detect three different activities (agonist, antagonist/NAM, and PAM activity) was applied to two different compounds from Jeffrey Conn’s lab at Vanderbilt University. Of the 30 receptors interrogated, the compounds were found to have PAM activity at a specific acetylcholine muscarinic receptor.

However, the screen reportedly also revealed a potential off-target agonist activity for one of the compounds at a bombesin receptor. “This suggests that, while family-specific selectivity may be enhanced for allosteric compounds, general selectivity across a diverse collection of GPCRs may still be a concern,” pointed out Dr. Armbruster. 

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