The study is published online in the Proceedings of the National Academy of Science.
Using the fluorescing compound 1-aminoanthracene, or 1-AMA, the team developed a high-throughput assay to test for new anaesthetic compounds. The assay will allow researchers to search for new anaesthetic drugs and new molecular targets for anaesthetics while at the same time creating high-resolution images of the compounds in action, a missing component that has hindered anaesthetic research.
Researchers confirmed the compound as anaesthetic after testing it successfully in tadpoles. By using transparent, albino tadpoles in the study, researchers were able to follow the fluorophore tag and image it in the brain of the immobilised, living animal.
Because the compound is fluorescent, researchers are able to image the compound in vivo in order to study its physiological effects. Where and how an anesthetic compound travels in an organism when administered and to what cells and concentrations are unknown in anaesthetic administration and a key to improving efficacy and to reducing side effects. Because anaesthetics bind weakly to their chemical targets, which may play a role in some of the unintended side effects, searching for new targets in the central nervous system is difficult.
"We don't know much about how anaesthetics work at a molecular level," said Roderic G. Eckenhoff, vice chair for research and the Austin Lamont Professor of Anaesthesiology and Critical Care at Penn's School of Medicine. "Thus, the development of new anaesthetics has become a stagnant field. This new tool will allow for the high-throughput screening of novel drugs."
Researchers from the School of Medicine and School of Arts and Sciences at Penn initiated the study in response to the health-care industry's need for new and more powerful tools to discover and test new anaesthetics and to learn more about how they work. The authors identified 1-AMA in a screen for compounds that bind to a cavity in horse spleen apoferritin, HSAF, that Eckenhoff and co-workers have shown to bind clinical anaesthetics.
Researchers noticed a resemblance in the crystal structure of the apoferritin protein to that of the transmembrane region of the superfamily of ligand-gated channels that includes the GABA receptor. Anaesthetics are known to positively modulate GABA signaling.
Because 1-AMA competes with other anaesthetics to bind to apoferritin, researchers surmised that the protein likely binds to the same region of apoferritin as traditional anaesthetics and thus shares their mechanism of action. Fluorescence of 1-AMA is enhanced when bound to apoferritin. Thus, displacement of 1-AMA by other anaesthetics attenuates the fluorescence signal and allows determination of anaesthetic affinity, that is, the drugs that bind tightly to the ferritin anaesthetic site. In this way, 1-AMA fluorescence could be used to discover new anaesthetics. This provides a unique fluorescence assay for compound screening and anaesthetic discovery.
Using confocal microscopy to image the distribution of the protein, the team found that 1-AMA localises largely in the brain and olfactory regions, unlike some general anaesthetics which spread widely throughout the body. Ideally, clinical anaesthetics would have a very focused target area in order to minimise systemic toxicity.
The Penn team will now collaborate with the National Chemical Genomics Center in Rockville, Md., to screen rapidly for novel anaesthetic compounds, allowing for the screening of hundreds of thousands of new compounds per week.
"The 1-AMA compound opens up new avenues for identifying the relevant biomolecular targets of general anaesthetics," Ivan J. Dmochowski, assistant professor in the Department of Chemistry at Penn, said. "1-AMA appears to be specific in its binding to proteins and also in its in vivo localisation, which should give us the opportunity to determine its mechanism of action," he said. "We hope to be able to extend our findings to learn how current general anaesthetics, such as propofol, work in human patients. There are many different and challenging aspects of trying to learn how anaesthetics work that involve medicinal chemistry, biochemistry, molecular modelling, imaging, cell electrophysiology, pharmacology, neurobiology and animal physiology."
According to the study, 1-AMA increases the transmission potential of the body's main neurotransmitter inhibitor, GABA. The compound also gives an appropriate dissociation constant, Kd 0.1 mM, for binding to the general anaesthetic site in horse spleen apoferritin, meaning the compound is behaving as traditional general anaesthetics would in humans.
In use for more than 150 years, general anaesthetics are one of medicine's greatest advances and yet there is still much to be learned about them. For many of the most commonly used anaesthetic compounds, the molecular mechanisms behind their numbing effects and the way these compounds travel the pathways of the body remain poorly understood or altogether unknown.
According to the study team, anaesthetics can bring on potentially harmful, even deadly, side effects for patients including rapid drops in blood pressure and heart rate, nausea and potentially irreversible cognitive problems, especially in older patients.
The study was funded by the National Science Foundation, the National Center for Research Resources, a Henry and Camille Dreyfus Teacher-Scholar Award, the National Institutes of Health and a University of Pennsylvania Institute for Medicine and Engineering Seed Grant.
The study was performed by Dmochowski and Christopher A. Butts of the Department of Chemistry at Penn, Eckenhoff and Jin Xib of the Department of Anaesthesiology and Critical Care at Penn, Grace Brannigan and Michael L. Klein of Penn's Center for Molecular Modeling and Abdalla A. Saad, Srinivasan P. Venkatachalan and Robert A. Pearce of the departments of Anaesthesiology, Anatomy and Physiology at the University of Wisconsin.
