A collaboration of University of Pennsylvania and University of Wisconsin chemists and anesthesiologists have identified a fluorescent anesthetic compound that will assist researchers in obtaining more precise information about how anesthetics work in the body and will provide a means to more rapidly test new anesthetic compounds in the search for safer and more effective drugs.
The study is published online in the Proceedings of the National Academy of Sciences .
Using the fluorescing compound 1-aminoanthracene, or 1-AMA, the team developed a high-throughput assay to test for new anesthetic compounds. The assay will allow researchers to search for new anesthetic drugs and new molecular targets for anesthetics while at the same time creating high-resolution images of the compounds in action, a missing component that has hindered anesthetic research.
Researchers confirmed the compound as anesthetic 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 immobilized, 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 anesthetic administration and a key to improving efficacy and to reducing side effects. Because anesthetics 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 anesthetics work at a molecular level," said Roderic G. Eckenhoff, vice chair for research and the Austin Lamont Professor of Anesthesiology and Critical Care at Penn's School of Medicine. "Thus, the development of new anesthetics 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 anesthetics 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 anesthetics.
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. Anesthetics are known to positively modulate GABA signaling.
Because 1-AMA competes with other anesthetics to bind to apoferritin, researchers surmised that the protein likely binds to the same region of apoferritin as traditional anesthetics and thus shares their mechanism of action. Fluorescence of 1-AMA is enhanced when bound to apoferritin. Thus, displacement of 1-AMA by other anesthetics attenuates the fluorescence signal and allows determination of anesthetic affinity, that is, the drugs that bind tightly to the ferritin anesthetic site. In this way, 1-AMA fluorescence could be used to discover new anesthetics. This provides a unique fluorescence assay for compound screening and anesthetic discovery.
Using confocal microscopy to image the distribution of the protein, the team found that 1-AMA localizes largely in the brain and olfactory regions, unlike some general anesthetics which spread widely throughout the body. Ideally, clinical anesthetics would have a very focused target area in order to minimize systemic toxicity.