Ligand-Gated Ion Channels
ChanTest focuses on ion channels as a primary target for drug discovery and safety, according to Glenn E. Kirsch, Ph.D., senior director for pharmacology and program management. “There are currently, a wide range of possibilities, including well-worn paths such as hypertension, areas still in early development such as pain, and largely unexplored conditions, of which autoimmune disease is a tantalizing representative.”
Current investigations at ChanTest include 5-HT3a, the ionotropic serotonin receptor, a target for the treatment of nausea and vomiting. It figures largelyS in pain disorders and drug addiction. A second area of concern is the nAChR α3/β4 or nicotinic acetylcholine receptor, a long-known target for Alzheimer disease and alcohol abuse.
Two other appealing choices are the ASIC1a acid-sensitive ion channel and the Nav1.7 voltage-gated sodium channel, which both respond to pain in damaged tissues. Finally, the HERG cardiac potassium channel, which performs as a safety antitarget for drug-induced torsade arrhythmias, is also under investigation.
The ion-channel protocols take advantage of genetically modified, transfected HEK293 and CHO cells that were subjected to automated patch-clamp assays to measure their responses to a number of drug candidates. A planar patch-clamp electrode array can be used for recording whole-cell ion-channel activity simultaneously from multiple cells in a 384-well format.
When coupled with robotic liquid handling, the technique can screen thousands of compounds per day against both ligand- and voltage-gated ion channels.
Dr. Kirsch describes this technology as “a second-generation Ion Works® (Molecular Devices) instrument with the capability to improve high-throughput ion-channel assays. This is brought about by extending the range of targets to include fast desensitizing ligand-gated channels via continuous recording during rapid compound addition.”
“Allosteric modulators are regulatory molecules that do not bind to the same site as the receptor’s natural ligand, but rather to alternative sites on the protein, modulating the binding and signaling of the natural ligand,” explains Patricia McDonald, Ph.D., associate director at the Scripps Research Institute. This characteristic offers unique opportunities for the development of a new class of molecular pharmacological agents.
Dr. McDonald and her team focus on the GPCR family of transmembrane spanning receptors that transmit signals from outside the cell to inside the cell through interactions with their cognate G proteins. They are activated by a diverse array of ligands that include light, odorants, hormones, and neurotransmitters. They are noted for their participation in many disease processes, making them important candidates for drug development.
Dr. McDonald has studied arrestin, which binds to activated receptors in a phosphorylation-dependent manner. Arrestin binding to the receptor blocks further G protein mediated signaling, terminating the signal, and desensitizing the receptor, an important regulatory process that prevents the cell from overstimulation.
“The CellKey System is a cell-based label-free technology that measures changes in cellular impedance (Z) using cellular dielectric spectroscopy (CDS),” Dr. McDonald explains.
CellKey measures the integrated response of the cell to receptor activation, permitting interrogation of the mechanism of action of candidate compounds. Following receptor activation, changes in cell morphology, cell adherence, and cell-to-cell interactions contribute to changes in Z. Using this technology the team has developed a strategy to monitor the efficacies of drugs on both receptor activation and desensitization in real time.
“We can detect complex receptor behavior in response to multiple classes of pharmacological agents including allosteric modulators and evaluate their potential therapeutic effectiveness providing valuable insight into how these agents may behave in vivo,” Dr. McDonald says.
Thomas Briese, Ph.D., associate director of the Center for Infection and Immunity and a faculty member at Columbia University, says that the center builds on a bioinformatics approach for sequence analysis and data evaluation, and employs MassTag multiplex PCR, GreeneChip cDNA microarrays, and next-generation sequencing to build an overall picture of the universe of pathogens that threaten global health.
Investigators from the center have created sequence analysis algorithms that deliver automated retrieval, filtering, and alignment of sequences from databases. The goals of the program include characterization of microflora, investigation of outbreaks and pathogenesis of chronic diseases, design and construct field systems for on-site deployment in the developing world, and assessment of animals for potential human-risk agents.
The need for such efforts was amply demonstrated by the West Nile outbreak in New York in 1999, the 2003 SARS outbreak, and more recently, in a nosocomial hemorrhagic fever cluster caused by the Lujo virus.
Pathogen Detection and Discovery
“The discovery process for new viruses is still in its infancy,” says Dr. Briese, and indeed, the causative agent is never identified in a vast number of enteric and respiratory infections.
“There are approximately 50,000 vertebrate species. Even if each has only 20 endemic viruses, we can expect the existence of one million vertebrate viruses. Given the current state of knowledge, this means that more than 99 percent of vertebrate viruses remain to be discovered.”
The center’s pathogen discovery platform expands on classical culture-based methods by bringing the speed and sensitivity of modern molecular diagnostics to bear. As Dr. Briese explains, to balance costs, sensitivity, and breadth, clinical specimens are first screened by multiplex MassTag PCR, which uses small photocleavable reporter tags detected by mass spectroscopy to test for up to 30 known pathogens in a single assay.
If no agent is identified or if no candidate agents are obvious, samples are analyzed by GreeneChip microarrays, which can identify viruses, bacteria, fungi, and parasites for which sequence information is available. If an agent is not indicated by either method, unbiased high-throughput sequencing analysis, the only current method capable of detecting uncharacterized agents, is used.
In all cases, analysis ideally culminates in sequencing and identification. However, proving causation requires extensive subsequent analyses and isolation of the candidate pathogen through classical methods.
According to Yu-Tsueng Liu, M.D., Ph.D., assistant adjunct professor, medicine at UC San Diego, the multiplexing technologies, which have co-evolved with the human genome project, will have a great impact in the next revolution of pathogen diagnosis. Because nearly all current tests rely on prior knowledge of the pathogen and the experience of the clinician, microarrays and next-generation sequencers will bring a vast assortment of new options and the possibility of precise identification, without which effective treatment cannot proceed.