Genetically Encoded Optical Biosensors for Dissecting Brain Distribution and Metabolism of Cannabinoids

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ABSTRACT Cannabinoids are enjoying a resurgence of interest in potential therapeutic uses, such as pain treatment, but their clinical applications have been hindered due to pronounced psychoactive or other side effects. They can be locally metabolized to active metabolites in the brain and other tissues and it is unknown whether and to what extent they can impact therapeutic and side effects, such as analgesia and memory impairment. The long-term goal is to develop and use new genetically encoded drug and metabolite biosensors to elucidate cannabinoid pharmacology and guide the design of new analgesic therapeutics. The overall objective of this application is to apply new technologies (COMBINES-CID and SMI-seq) to create in vivo biosensors for the major psychoactive cannabis component, Δ9-tetrahydrocannabinol (THC), and its highly potent metabolite, 11-hydroxy-THC, and demonstrate their use in measuring single-cell and subcellular drug distribution and metabolism. The PI’s laboratory recently applied COMBINES-CID to create highly specific chemically induced dimerization systems (CIDs) for cannabidiol (CBD), a structural analog of THC. It is hypothesized that other cannabinoid-selective CIDs can be engineered similarly and then converted to in vivo biosensors, like GCaMP, by coupling to a fluorescence reporter. This hypothesis will be tested by pursuing two specific aims: 1) Generate THC- and 11- hydroxy-THC-induced CIDs with high sensitivity and fast kinetics; and 2) Engineer selected CIDs into fluorescent biosensors and validate their performance by measuring the perfused, photo-uncaged, and metabolized drugs in human iPSC-differentiated neurons. Under the first aim, phage-displayed combinatorial binder libraries constructed with nanobody, monobody, or computationally designed scaffolds will be screened to obtain CIDs with high sensitivity and selectivity (Aim 1A); their detection dynamic ranges will be further optimized by mutagenesis and SMI-seq (Aim 1B). For the second aim, CIDs will be coupled to an optical reporter, such as fluorescence resonance energy transfer or a circularly permuted fluorescent protein, to create the biosensors for fluorescence lifetime imaging (Aim 2A). The biosensors will be genetically encoded and localized to plasma membrane, ER, or Golgi apparatus to measure extracellular and intracellular THC and 11-hydroxy-THC concentrations (Aim 2B) and then study the impact of locally metabolized 11-hydroxy-THC on neuronal mitochondrial ATP synthesis (Aim 2C). The proposed project is innovative in that it will for the first time demonstrate a general solution for creating cannabinoid biosensors. It is significant because obtained biosensors will enable high spatiotemporal analysis of in vivo drug distribution and action. The new method will also have wide use in many other fields by largely expanding the biosensor toolkit for drug, metabolite, and signaling molecule detection.