Scientists constantly push the frontiers of knowledge. For every question answered, there are at least a dozen more questions. At the recent IBC “Assay and Cellular Conference” in Las Vegas, scientists convened to assess the state of target identification and related areas of drug discovery.
High-throughput screening assays for G-protein coupled receptors (GPCRs) traditionally determine a compound’s ability to interact at the ligand binding site. However, researchers at Caden Biosciences (formerly Cue Biotech; www.cadenbiosciences.com) recognized that targeting alternative—or allosteric—sites can be advantageous. “Our presentation demonstrated our development of a rational screen for allosteric modulators of GPCRs and the data we generated using the technology to identify enhancers and inhibitors of the thrombin receptor,” CSO Annette Gilchrist noted.
The idea of screening for allosteric modulators has been around for a while. “People have been talking about this for about 10 years, but we’ve actually been doing it for four,” Gilchrist said. “The implications are far-reaching in pharmacology, as it means that a GPCR could have a different pharmacological profile depending on which G protein is activated. Simply put, the same GPCR could have different roles depending on the G proteins present in the local environment. With this added complexity it becomes important to know which signal transduction pathway is responsible for the physiological response, and be able to measure multiple pathway responses.”
Traditionally agonists and antagonists were identified by their ability to bind to the receptor’s orthosteric ligand site, that is, the site recognized by the cell’s own endogenous agonist. It is now evident that GPCRs possess additional allosteric binding sites. “Allosteric modulators offer many advantages over orthosteric ligands as therapeutic agents, including the potential for greater GPCR-subtype selectivity and safety,” Gilchrist said.
“Functional screens for GPCRs measure signal-transduction events further downstream from receptor activation, but the consequence is that they are more subject to false positives than those assays that measure more proximal events,” said Gilchrist. “Moreover, the window to identify allosteric modulators using cAMP assays is very small.”
Endogenous Characterization of GPCR Modulators
Berlex (www.berlex.com) presented a study that compared a series of modulators for a GPCR target using the CellKey system in an endogenous versus transfected and over-expressed GPCR setting.
“Target identification wasn’t the point of this presentation, rather, it was more about the drug discovery aspect,” said research scientist Sofia Ribeiro. “What we did was an evaluation of the small molecule in an endogenous setting. Target identification is usually done in reverse, in that you have up and down regulators, and you have to prove that the target you are using is valid.”
In this case, Berlex had already identified the target; they were looking at a drug that could allay some of the symptoms for multiple sclerosis. “Knowing the target already was the focus of the presentation,” said Ribeiro. “Our goal was to develop the assay that would demonstrate stimulation of the target using CellKey (MDS Sciex; www.mdssciex.com) system.”
The CellKey system is relatively new. It is based on Cellular Dielectric Spectroscopy technology and is a fully automated system designed for target validation and secondary screening laboratories. Receptor stimulation (agonist) produces changes in cellular impedance. The CellKey system has an integrated 96-well pipetting head, which allows simultaneous compound addition and measurement of cellular impedance. Kinetic measurements of impedance are taken in real-time with all wells of the microplate read in parallel.
“When your goal is to find an agonist, you need to express a receptor. Ideally this is studied in an endogenous GPCR setting, so that it most closely mimics actual human cellular activity,” Ribeiro said.
GPR30: Another Possible Drug Target
“For many years, steroids and estrogens were thought to function solely through nuclear hormone receptors,” said Eric Prossnitz, professor of cell biology and physiology at the University of New Mexico. “More recently, evidence has accumulated that GPCRs might also be involved in steroid signaling, especially for estrogen, and it has been suggested that GPR30 might mediate certain cellular responses.”
The novel G protein-coupled estrogen receptor GPR30 is a classic 7-transmembrane domain GPCR that, unlike most GPCRs, resides predominantly in the endoplasmic reticulum. From this location, it binds estrogen and activates numerous cellular-signaling pathways including calcium mobilization and PI3K activation.
“Although classical estrogen receptors can activate many of the same cellular effectors as GPR30,” Prossnitz said, “the signaling pathways differ for the two receptors. To quantitate and localize estrogen binding, we have developed a novel fluorescent estrogen analog that localizes with both classical estrogen receptors and GPR30.”
Consequently, GPR30 is gaining recognition as a potential target for the treatment of estrogen-dependent diseases. “The challenge, though, is that GPR30 binds to both agonists and antagonists of the classical nuclear estrogen receptors,” said Prossnitz. “You have to be able to investigate the individual functions of each receptor to understand their biology.”
Through virtual screening, a group of New Mexico scientists, led by Prossnitz, identified a high-affinity agonist for GPR30 that shows no significant binding to classical estrogen receptors. “The first paper we published showed that GPR30 could bind to estrogen-like compounds,” Prossnitz said. “Our more recent work has focused on the characterization of novel ligands that bind exclusively to either GPR30 or the classical estrogen receptors.”
Having identified a GPR30-specific agonist, Prossnitz and his group are beginning to evaluate the physiological functions of GPR30. “At this point, it is unclear what the pharmaceutical market for GPR30-targeted drugs might be, but we believe current and future work will show GPR30 to be involved in numerous physiological processes important to disease,” he concluded.
Odor Receptors as Drug Targets
Using odor receptors (OR) as drug targets is not as far-fetched as it sounds, says Stuart Firestein, professor of biological sciences at Columbia University. “The family of odor receptors is the largest family of GPCRs, numbering around 350 in humans and approximately 1,200 in the mouse and other mammals. Not all of these are expressed in the nose, or only in the nose. So it is very possible these ORs may be the new orphan GPCRs that serve critical functions in other tissues.”
The activity of all cells in the nervous system is regulated by the interaction of various chemicals, such as neurotransmitters, hormones, and peptides with membrane receptors, explained Firestein. “The way in which these substances exert their influence is known generally as signal transduction,” he said.
“We use the vertebrate olfactory receptor neuron as a model for investigating general principles and mechanisms of signal transduction/receptor-ligand interactions, modulation by second messengers, ion-channel gating, and the long-term mechanisms of adaptation and desensitization. The olfactory neuron is uniquely suited for these studies since it is designed specifically for the detection and discrimination of a wide variety of small organic molecules, or odors.”
Firestein’s most recent work makes use of adenovirus vectors to drive over-expression of cloned odor receptors in olfactory neurons. “They are excellent receptors to try and understand the relation between amino acid sequence and ligand-binding affinities,” said Firestein.
It is a novel approach, and one worth investigating, although Firestein notes that it might be awhile before industry investigates this avenue of research in a drug discovery context.
EpiCept(www.epicept.com) is applying cell-based assays to apoptosis and oncology for new molecular targets, said Ben Tseng, vp of research and development. “Cell-based assays that measure apoptosis provide the advantage that multiple pathways are interrogated and identified and new pathways that induce apoptosis are discovered,” Tseng noted. “We identify small molecule compounds that are active in the induction of apoptosis as anticancer agents.”
According to Tseng, EpiCept looks at the biology behind the compound. “Using cell-based high-throughput screening and a proprietary substrate, we identify hits and analogs for which we carry out some early biological assessments as to potential mechanisms, and if we find an interesting compound, we’ll carry out animal efficacy studies. We then use chemical genetics to find the molecular target of the compound.”
At the end of the process, the goal is to have identified a clinical candidate compound and its cellular target. “We’ve identified four different compounds, and one of these has been licensed and is expected to enter Phase II soon. Another compound, we have developed ourselves,” said Tseng, “and expect to enter Phase I soon.”
Two other candidates are being advanced internally, while both are available for partnership or licensing. “These have interesting molecular targets that have unique roles for activating apoptosis,” Tseng noted. One compound targets the transferrin receptor, which is highly overexpressed in many cancers. The other affects the IGF-I growth factor pathway through the IGF II receptor. These are targets that would not have been readily identified using more traditional genetic approaches, Tseng noted. “There are enabling technologies that have brought cell-based assays into an approachable form for target identification, which provides a biomarker to ascertain whether the drug is having the desired effect.”
RNAi and High-content Screening
“It is easy enough to generate targets— the bottleneck has been the target validation step,” said Stephen Walker, research scientist, applied genomics, at Bristol-Myers Squibb (BMS; www.bms.com). Walker works with the oncology group and external collaborators doing oncology-specific high-content screens that generate potential new targets. “The combination of RNAi and high-content screening allows us to rapidly test these targets in disease-relevant cell lines and come up with a list of validated targets to hand off to BMS oncology.”
Walker and his group screened focused sets of RNAi across a variety of high-content screening assays in multiple cell lines leading to the discovery of potential new drug targets for cancer. “I presented on two high-content screening assays,” Walker said. “They were used to study and validate genes from both the IGFR/AKT/PTEN and myc pathways. In both instances, we started with hundreds of genes and narrowed the list to a handful of targets that will now move on to further validation. Hopefully some of these targets will lead to new cancer drugs.”
Potential targets were first identified in pathway-specific model organism genetic screens. Working with multiparameter high-content screening endpoints combined with siRNA knockdown, enabled his group to quickly identify, validate, and classify novel oncology drug targets. The assays that Walker reported on were run across a number of different cancer cell lines. Assay endpoints included transcription factor translocation, apoptosis, and cell cycle arrest, all of which are desired oncology readouts. “If we can stop tumor cells from dividing or causing apoptosis, we may have found a legitimate target to treat cancer,” Walker said.