Caffeine-operated synthetic modules for chemogenetic control of protein activities by life style

Introduction: A genetically-encoded caffeine-operated synthetic module (COSMO) is introduced herein as a robust chemically-induced dimerization (CID) system. COSMO enables chemogenetic manipulation of biological processes by caffeine, its major metabolites, as well as caffeinated beverages such as coffee, Coca-Cola, and energy drinks. This CID tool, evolved from an anti-caffeine nanobody via cell-based high throughput screening, permits caffeine-inducible gating of calcium channels, killing of cancer cells, activation of tyrosine receptor kinase signaling in the absence of growth factors, and boosting nanobody-mediated antigen recognition for the SARS-CoV-2 spike protein. Further rationalized engineering of this CID system led to 34~217-fold enhancement in the half maximal effective concentrations for caffeine (EC50: 16.9 nM) or its metabolites, which makes it among the most potent CID systems like the FKBP/FRB/rapamycin complex. Furthermore, the COSMO concatemer connected with a long linker (biCOSMO-L) favors intramolecular dimerization and acts as a versatile precision switch when inserted in host proteins to achieve tailored function. biCOSMO-L is further applied to enable rapid actin cytoskeleton remodeling with caffeine. Since COSMO operates as a homodimerization system, its potential applications are restricted across various fields, particularly in CAR-T therapy. To tackle this limitation, we pursued a solution by integrating biCOSMO-L with nanobodies. This integration allows us to exert indirect control over nanobodies, inducing caffeine-triggered heterodimerization. Our overarching objective with this COSMO innovation is to empower humanity in the battle against diseases and cancer.

Materials and Methods:
Molecular cloning and plasmids construction: Plasmids construction was performed using the standard restriction enzyme digestion and ligation method.
Cell culture and transfection: The HeLa cell line was purchased from ATCC. Cells were cultured at 37 °C with 5% CO2 in Dulbecco’s Modified Eagle medium (DMEM; Sigma-Aldrich; St. Louis, MO, USA) supplemented with 10% FBS and 1% penicillin/streptomycin cocktail.
Live-cell imaging and image analysis: Fluorescence imaging was performed on a Nikon Eclipse Ti-E microscope equipped with an A1R-A1 confocal module with LU-N4 laser sources (argon-ion: 405 and 488 nm; diode: 561 nm) and a live-cell culture cage (maintaining the temperate at 37 °C with 5% CO2). 60 × oil or 40 × oil lens was used for confocal imaging.

Results, Conclusions, and Discussions:
In summary, the COSMO and biCOSMO modules described in the study provide a novel platform for chemogenetic manipulation of biological processes with caffeine, its metabolites, or even caffeine-containing beverages. As caffeine is cheap and readily available in daily drinks, these chemical biology tools will greatly reduce the cost in engineering proteins or cells of therapeutic potentials. In theory, the amount of caffeine from a single cup of Starbucks coffee (100-200 mg in 350 ml; reaching an approximate peak plasma concentration of 2.5-5 mg/l or 13-26 μM in a 70 kg adult) or a can of soda (46 mg in a 355 ml Coke) is sufficient to activate COSMO or BiCOSMO, but far below the amount of caffeine addition or intoxication (75–100 cups of coffee for an adult; or over 1 gram) to cause severe insomnia or dizziness due to awakening and cardiotonic actions. Although caffeine is known to enhance calcium-induced calcium release through the ryanodine receptor, it requires high micromolar to low mM range of caffeine, which is at least 1000-fold higher than the EC50 of biCOSMO. Hence, the side effects associated with low-dose caffeine or its metabolites usage in the COSMO system are likely to be minimal. These desirable features pave the way for future in vivo application of the COSMO-based CID system.

Acknowledgments (Optional):
We thank the financial support from the National Institutes of Health (R01GM112003 to YZ, R21GM126532, R01CA232017 to YZ, R01HL134780 to YH, and R01HL146852 to YH), the Welch Foundation (BE-1913-20190330 to YZ), the John S. Dunn Foundation (to YZ), the Cancer Prevention and Research Institutes of Texas (RP190612 to FW), and the American Cancer Society (RSG-16-215-01-TBE to YZ and RSG-18-043-01-LIB to YH).