Project Grant F31DA060678

OPTIMIZING CHEMICAL- AND LIGHT-DEPENDENT PROTEIN SWITCHES FOR CONTROLLING OPIOID PEPTIDE ACTIVITY WITH TEMPORAL CONTROL - PROPOSAL SUMMARY THE STUDY OF OPIOID RECEPTORS IS ESSENTIAL TO GAIN INSIGHT INTO THE MECHANISMS UNDERLYING THE EFFECTS OF OPIOID MEDICATIONS SUCH AS PAIN RELIEF AND ADDICTION. THE MU OPIOID RECEPTOR (MOR) IS PARTICULARLY IMPORTANT IN PAIN MODULATION, WITH ACTIVATION REDUCING PAIN SENSATION AS WELL AS INDUCING EUPHORIA AND RELAXATION. UNDERSTANDING THE MOLECULAR AND CELLULAR INTERACTIONS OF OPIOID RECEPTORS HAS THE POTENTIAL TO INFORM THE DEVELOPMENT OF SAFER PAIN MANAGEMENT STRATEGIES, NOVEL OPIOID MEDICATIONS, AND INTERVENTIONS TO ADDRESS THE HARMFUL ADDICTIVE PROPERTIES. PROTEIN TOOLS HAVE BEEN INSTRUMENTAL IN PROVIDING INSIGHTS INTO OPIOID RECEPTOR FUNCTION AND HAVE AIDED IN THE DEVELOPMENT OF SELECTIVE OPIOID RECEPTOR MODULATORS AS WELL AS FURTHERING NEUROSCIENCE RESEARCH. CHEMOGENETICS AND OPTOGENETICS ARE TWO POWERFUL TOOLS USED TO MANIPULATE AND STUDY SPECIFIC NEURAL CIRCUITS WITH HIGH TEMPORAL AND SPATIAL PRECISION. BOTH TECHNIQUES HAVE UNIQUE ADVANTAGES. CHEMOGENETIC INVOLVES THE USE OF SMALL MOLECULES TO CONTROL CELLULAR ACTIVITY GENETICALLY. ONE SIGNIFICANT ADVANTAGE OF CHEMOGENETICS IS ITS VERSATILITY. IT CAN BE USED IN A WIDE RANGE OF EXPERIMENTAL SYSTEMS, INCLUDING CELL CULTURES, SLICE PREPARATIONS, AND INTACT ANIMALS, ALLOWING TO STUDY NEURAL CIRCUITS IN A VARIETY OF CONTEXTS. OPTOGENETICS, ON THE OTHER HAND, INVOLVES THE USE OF GENETICALLY ENCODED PROTEINS THAT CAN BE ACTIVATED BY LIGHT. THIS ALLOWS FOR PRECISE, MILLISECOND-SCALE CONTROL OF NEURAL ACTIVITY, ENABLING THE PRECISE MANIPULATION OF NEURAL CIRCUITS WITH HIGH TEMPORAL RESOLUTION. OPTOGENETICS CAN ALSO BE USED TO TARGET SPECIFIC CELL TYPES, SUCH AS EXCITATORY OR INHIBITORY NEURONS, ALLOWING FOR THE SELECTIVE MANIPULATION OF SPECIFIC NEURAL CIRCUITS. THE CHEMOGENETIC AND OPTOGENETIC APPROACH CAN BE USED TO STUDY A WIDE RANGE OF BIOLOGICAL PROCESSES, INCLUDING INTRACELLULAR SIGNALING, TRANSCRIPTIONAL REGULATION, AND PROTEIN-PROTEIN INTERACTIONS. IN MY LABORATORY, WE HAVE DEVELOPED A CHEMOGENETIC TOOL CALLED CHEMICAL ACTIVATED PROTEIN DOMAIN (CAPS), WHICH OFFERS SEVERAL ADVANTAGES OVER OTHER CHEMOGENETIC APPROACHES. THE ADVANTAGES OF CAPS INCLUDE PRECISE, NON-TOXIC CONTROL OF PROTEIN ACTIVATION, OFFERING A POTENT TOOL FOR PEPTIDE THERAPY DEVELOPMENT AND BIOLOGICAL STUDIES. HERE, WE DEVELOPED A PLATFORM TO IMPROVE PEPTIDE CAGING OF CHEMOGENETIC AND OPTOGENETIC TOOLS, SPECIFICALLY CHEMICAL ACTIVATED PROTEIN (CAP) TO CONTROL OPIOID PEPTIDES. WE SUCCESSFULLY EVOLVED CAPC AND CAPN LIBRARIES AND DEMONSTRATED THAT IT CAGES THE PEPTIDE MORE EFFICIENTLY, RESULTING IN LESS LEAKY BINDING IN THE ABSENCE OF SHIELD-1. THESE TOOLS ARE INTENDED TO BE USED IN ANIMAL MODELS TO ADVANCE OUR UNDERSTANDING OF PEPTIDE-RELATED MECHANISMS IN VIVO AND TO POTENTIALLY DEVELOP NEW INTERVENTIONS FOR VETERINARY USE.