Novel Molecules as a Window to Anesthetic Mechanisms

Project Summary The introduction of general anesthesia for “painless” surgery is one of the largest advancements ever made in medicine and has transformed surgery from being a barbaric last resort to an event that occurs over 40 million times a year in US operating rooms (ORs). There is an irony that one of the greatest medical advancements is still one of the least understood and anesthetics are still said to be among the most dangerous drugs in clinical use due to their remarkably narrow therapeutic indices. Perhaps related to their toxicity, anesthetics act on many targets and are likely to have multiple mechanisms of action in addition to agonism of the GABAA receptor. Identification of a broader cache of anesthetic targets would allow a novel approach to rational design where multiple targets are actually required. But a challenge is differentiating the many “non-specific” anesthetic interactions from the mechanistically important (or desirable) interactions. This work seeks to uncover anesthetic mechanistic targets by exploiting a propofol derivative that was incidentally discovered to antagonize anesthesia, and use it as a novel approach to the “reverse engineering” of anesthetic action. My research has blossomed out of observations made with a fluorinated derivative of propofol, called propofluor (aka fropofol). This molecule, initially hypothesized to be an anesthetic, but instead elicited propofol antagonism in tadpoles and larval zebrafish. Further experiments strongly suggest competitive antagonism, and we have now identified multiple additional compounds that have increased antagonistic potency. We have shown that sensitivity to this antagonist changes throughout early development in parallel with sensitivity to propofol, but the antagonism only persists until the age of sexual differentiation in zebrafish (21-15 days post fertilization). Preliminary findings have recapitulated this developmental effect in mice. In addition to further pharmacologic investigations, we propose to use bifunctional photoaffinity derivatives of propofol and its antagonists to identify the molecular interactomes during development. The relevance of these targets will be further refined via comparison to large developmental transcriptomic databases (for mice and zebrafish) that have just recently become available. This analysis will truncate the list of potential targets so that functional validation in animal models can be most efficiently pursued. This multidisciplinary program to utilize anesthetic antagonists as research tools to reveal relevant anesthetic mechanisms represents an entirely innovative approach to the study of anesthetic mechanisms.