Development and Applications of Bioorthogonal Chemistry ABSTRACT This MIRA application combines two research efforts in our lab in tackling the critical barriers in the study of class B GPCR biophysics and signaling in live cells, i.e., the lack of suitable tools for constructing functional GPCR biosensors as well as capturing the transient and highly dynamic GPCR-interacting proteins involved in biased agonism. We have a long-standing interest in developing the reactivity-based chemical tools to address significant biological problems that are difficult to solve using conventional molecular biology techniques. In the past five years we continued to make progress in both tool development and the applications of these tools to address important biological problems. For instance, we optimized several bioorthogonal reactions, including the photoinduced tetrazole?alkene cycloaddition reaction (?photoclick? chemistry), the spiroalkene?tetrazine ligation reaction, the palladium-mediated cross-coupling reactions, and the sequence-specific 2-cyanobenzo- thiazole?cysteine ligation reaction. Together with the genetic code expansion involving a spiroalkene amino acid, two of these reactions (photoclick chemistry and tetrazine ligation) were harnessed for site-specific introduction of organic fluorophore (fluorescein, Cy3 and Cy5) at the extracellular loop 3 of GLP-1R and GCGR, two members of the class B GPCRs implicated in diabetes and obesity, for an ongoing single-cell FRET study of the domain movement during ligand-induced receptor activation in live cells. In addition, we made a serendipitous discovery that 2-aryl-5-carboxytetrazole (ACT) offers a new proximity-dependent photo-cross- linker, which was then used in the design of the photo-affinity labels that enabled in situ capture and subsequent identification of the drug targets as well as a genetically encoded amino acid for site-specific incorporation and subsequent capture of the transient EGFR?Grb2 interaction complex in mammalian cells. Built upon these results, in this application we plan to continue our studies of orthogonal chemical reactivity at the chemistry-biology interface and pursue the following two related projects. In Project 1, we will construct the FRET-based biosensors of GLP-1R and GCGR via bioorthogonal labeling to probe the conformational dynamics involved in the receptor activation and signaling in live cells. A new set of fluorescence ?turn-on? reagents will be designed for bioorthogonal, fluorescent labeling of the intracellular loop 3 (ICL3) of GLP-1R and GCGR to allow single-cell intra- and intermolecular FRET analysis of receptor conformations in live cells. In Project 2, we will develop a genetically encoded ACT photo-cross-linker containing an alkyne group and apply this photo-cross-linker to map the time-dependent GLP-1R and ?-arrestins interactomes by mass spectrometry in response to ligand stimulation. We expect that these studies will not only validate new chemical tools for real-time monitoring of protein conformations and protein-protein interactions in live cells but also provide novel insights into the GLP-1R and GCGR activation and biased signaling that are crucial for the development of targeted therapies for the treatment of diabetes and obesity.

Public Health Relevance

Development and Applications of Bioorthogonal Chemistry Narrative This application aims to develop and apply new reactivity-based chemical tools that, together with the genetic encoding of unique chemical functionalities, enable the in situ construction of the functional GPCR biosensors as well as the capture of the transient and highly dynamic GPCR-interacting proteins involved in biased signaling in living cells. The biophysical and chemical proteomic studies will provide novel insights into the GLP-1R and GCGR activation and signaling, which are particularly valuable for the development of biased agonists of GLP- 1R and GCGR as potential therapies for the treatment of diabetes and obesity.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Unknown (R35)
Project #
1R35GM130307-01
Application #
9626052
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Fabian, Miles
Project Start
2019-01-01
Project End
2023-12-31
Budget Start
2019-01-01
Budget End
2019-12-31
Support Year
1
Fiscal Year
2019
Total Cost
Indirect Cost
Name
State University of New York at Buffalo
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
038633251
City
Amherst
State
NY
Country
United States
Zip Code
14228