A GPCR-based yeast biosensor for biomedical, biotechnological, and point-of-use cannabinoid determination

Abstract

The decriminalization of cannabis and the growing interest in cannabinoids as therapeutics require efficient methods to discover novel compounds and monitor cannabinoid levels in human samples and products. However, current methods are limited by the structural diversity of the active compounds. Here, we construct a G-protein coupled receptor-based yeast whole-cell biosensor, optimize it to achieve high sensitivity and dynamic range, and prove its effectiveness in three real-life applications. First, we screen a library of compounds to discover two novel agonists and four antagonists and demonstrate that our biosensor can democratize GPCR drug discovery by enabling low-cost high-throughput analysis using open-source automation. Subsequently, we bioprospect 54 plants to discover a novel phytocannabinoid, dugesialactone. Finally, we develop a robust portable device, analyze body-fluid samples, and confidently detect illicit synthetic drugs like “Spice”/“K2”. Taking advantage of the extensive sensing repertoire of GPCRs, this technology can be extended to detect numerous other compounds.

Humanized CB1R and CB2R yeast biosensors enable facile screening of cannabinoid compounds

“…3). Interestingly, this latter phenomenon has been observed previously with other CB2R agonists in yeast 20 , and is consistent with known functional interactions between cholesterol and cannabinoids. We suspect that compound interactions at high concentrations with cholesterol and cholesterol-like membrane components may alter membrane fluidity or other features in a way that is deleterious to receptor function 32 .…”

“…Unlike CB1R, CB2R is functional in yeast in its native form 20,21 . To further optimize function, we compared three leader peptides to the native N-terminal sequence ( Fig.…”

“…The sheer speed at which many psychoactive mixtures can be created makes it difficult for regulatory bodies and health authorities to keep pace, highlighting the need for studies that can quickly provide insights into receptor binding and specificity. The availability of facile comparative assays via the dual yeast biosensors builds on earlier work expressing CB2R in yeast 20,21 and may potentially provide a straightforward basis for comparison suitable for both research and regulatory organizations.…”

Automation of yeast spot assays using an affordable liquid handling robot

“…The Opentrons OT-2 (OT-2; Opentrons Labworks Inc., New York, USA) is an automated pipetting machine that is a low-cost and open-source hardware, easy to cutomized by end-users, and is controlled by a populatr programming language Python [27]. The OT-2 is used for automating experimental procedures in yeast research, such as high-throughput screening [28], micro-cultivation experiments [29], DNA cloning [30], bioreactor assays [31], and proteomics [32]. If it is possible to automate yeast spot assays using OT-2, it is expected to be a solution with a low cost and few technical barriers that can be used in various laboratories, especially in academia.…”