Thursday, April 25, 2024
4/25/2024
PROJECT SUMMARY Over the past decade, synthetic cannabinoids that intend to mimic the effects of cannabis use have emerged as an important group of New psychoactive substances (NPSs) sold as “legal high” products under brand names such as “Spice”, “Black Mamba”, and “Annihilation”. These compounds are more potent and dangerous agonists than those found in cannabis and they have caused a variety of adverse health effects, including psychoses, hospitalizations, and deaths. In response, the DEA has placed 43 synthetic cannabinoids under Schedule I classification; however, new synthetic cannabinoid ingredients are regularly created and released to both evade legal restrictions and provide a product that avoids detection by routine drug screening tests. Although GC/LC-MSn can be used to comprehensively detect these agents, the vast majority of forensic and clinical labs rely on immunoassays for their drug tests due to simplicity and cost. New methods that accelerate the development of diagnostic for the ever-changing substances of abuse would be useful, both for addressing the synthetic cannabinoid problem and as a general technology. We propose to address this challenge by developing a new platform for rapid assay development that functions orthogonally to immunoassays. To do this, we propose to use directed evolution to reprogram the ligands sensed by the plant PYR1-PP2C stress hormone sensing system. This sensor functions through a naturally occurring chemical-induced dimerization (CID) mechanism that couples ligand recognition by PYR1 to the formation of a stable PYR1-PP2C complex; this feature facilitates genetic selection experiments for receptors that recognize new ligands. We hypothesize that our platform will enable rapid development cycles for new diagnostics. To prove this hypothesis, this exploratory study will (1) Develop receptors for selective recognition of synthetic cannabinoids through directed evolution, (2) Integrate target recognition and signal output using protein fragment complementation assays, and (3) Establish and validate synthetic cannabinoid detection systems in biological matrices. Sensors will be designed to detect multiple synthetic cannabinoids, prioritizing the most prevalent synthetic cannabinoid of 2020 (5F-MDMB-PICA), which is not detected by current clinical immunoassays. The feasibility of this proposal is backed by extensive preliminary data from the PI’s laboratory, which demonstrates that the PYR1-PP2C system can be used to evolve high-affinity synthetic cannabinoid sensors. Our long-term goal is to establish this new system for the rapid development of diagnostic tools and deliver reagents that enable fast and selective detection of synthetic cannabinoids in low volumes of biological specimens. These enabling tools will be valuable for biomedical and clinical analyses of synthetic cannabinoids to control drug abuse, improve treatment and diagnosis of intoxication, to characterize their pharmacological and health effects, and as a general platform for rapidly evolving new diagnostic assays.
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