Development and Applications of Bioorthogonal Chemistry ABSTRACT A major hurdle in biophysical studies of class B GPCR conformational transitions, particularly the movements of the two large domains during ligand-induced activation, is that there are very few techniques available that allow site-specific introduction o biophysical probes into these two domains without altering the receptor function. To overcome this limitation, our long-term goal is to develop bioorthogonal chemistry tools that enable biophysical studies of the multi-domain signaling proteins such as class B GPCRs in living cells. In our previous studies, we have optimized a bioorthogonal, photoinduced tetrazole-alkene cycloaddition reaction ('photoclick chemistry'), and developed the palladium-mediated cross-coupling reactions for selective protein labeling in living systems as well as a phage-assisted interrogation of reactivity strategy for evolving the sequence-specific bioorthogonal reactions. Built upon these results, in this application we plan to integrate the bioorthogonal chemistry tools with the genetic encoding of unique chemical functionalities to generate in situ the chemically modified GLP-1R/GCGR?two members of the class B GPCRs that are implicated in diabetes and obesity, and study their conformational transitions and photo-regulation in living cells.
The specific aims are as follows: 1) Apply photoclick chemistry to generate in situ the environment-sensitive fluorescent probes on GCGR/GLP-1R and probe the ligand-induced conformational changes in living cells. A spiro[2,3]hex-1-ene or fumarate-derived lysine will be site-specifically incorporated at the extracellular loop 3 region of GCGR/GLP- 1R to direct the photoclick chemistry, and the resulting fluorescent labeled GCGR/GLP-1R will be used in the studies of the conformational transitions induced by the specific ligands;2) Develop binary bioorthogonal chemistry for dual-labeling of GCGR/GLP-1R to probe the ligand-induced conformational changes by FRET in living cells. The photoclick chemistry will be used in tandem with the sequence-specific palladium-mediated cross-coupling or the cysteine-nitrile condensation reaction to enable the simultaneous introduction of two fluorophores at the juxtamembrane domain and the N-terminal extracellular domain, respectively. The dynamic movements of these two interconnected domains upon perfusion of the specific peptide ligand in living cells will be monitored by FRET using confocal microscope;3) Develop the azobenzene-based optochemical genetic tools for optical regulation of GLP-1R activation in living cells. A biocompatible inverse azo-coupling reaction based on the condensation of phenylhydrazines with fluoroquinols will be developed, which together with the genetic encoding of fluoroquinolalanine, will allow us to introduce the azobenzenes site-specifically into the two regulatory regions of GLP-1R. The effect of reversible photoswitching on GLP-1R activity in the absence or presence of GLP-1 will be assessed using b-arrestin-mCherry as a reporter for receptor activation. These studies will provide the key insights into GLP-1R/GCGR activation mechanisms, which are crucial for the development of GLP-1R/GCGR dual agonists as therapeutic agents for the treatment of diabetes and obesity.

Public Health Relevance

Development and Applications of Bioorthogonal Chemistry Narrative To probe the conformational movements of the two domains of the class B GPCRs in living cells, this project aims to develop and apply new bioorthogonal chemistry tools that, together with the genetic encoding of unique chemical functionalities, enable the in situ synthesis of the chemically modified GLP-1R/GCGR and the studies of their conformational transitions and photo-regulation in living cells. This study will provide the key insights into the GLP-1R/GCGR activation mechanisms, which are crucial for the development of GLP-1R/GCGR dual agonists as pharmaceutical agents for the treatment of diabetes and obesity.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
2R01GM085092-06A1
Application #
8759491
Study Section
Synthetic and Biological Chemistry A Study Section (SBCA)
Program Officer
Fabian, Miles
Project Start
Project End
Budget Start
Budget End
Support Year
6
Fiscal Year
2014
Total Cost
Indirect Cost
Name
State University of New York at Buffalo
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Buffalo
State
NY
Country
United States
Zip Code
14260
Lim, Reyna K V; Li, Nan; Ramil, Carlo P et al. (2014) Fast and sequence-specific palladium-mediated cross-coupling reaction identified from phage display. ACS Chem Biol 9:2139-48
Yu, Zhipeng; Lin, Qing (2014) Design of spiro[2.3]hex-1-ene, a genetically encodable double-strained alkene for superfast photoclick chemistry. J Am Chem Soc 136:4153-6
Ramil, Carlo P; Lin, Qing (2014) Photoclick chemistry: a fluorogenic light-triggered in vivo ligation reaction. Curr Opin Chem Biol 21:89-95
Cheng, Gang; Lim, Reyna K V; Ramil, Carlo P et al. (2014) Storable N-phenylcarbamate palladacycles for rapid functionalization of an alkyne-encoded protein. Chem Commun (Camb) 50:11679-82
Ramil, Carlo P; Lin, Qing (2013) Bioorthogonal chemistry: strategies and recent developments. Chem Commun (Camb) 49:11007-22
Yu, Zhipeng; Ohulchanskyy, Tymish Y; An, Peng et al. (2013) Fluorogenic, two-photon-triggered photoclick chemistry in live mammalian cells. J Am Chem Soc 135:16766-9
Cheng, Gang; Lim, Reyna K V; Li, Nan et al. (2013) Storable palladacycles for selective functionalization of alkyne-containing proteins. Chem Commun (Camb) 49:6809-11
An, Peng; Yu, Zhipeng; Lin, Qing (2013) Design and synthesis of laser-activatable tetrazoles for a fast and fluorogenic red-emitting 1,3-dipolar cycloaddition reaction. Org Lett 15:5496-9
An, Peng; Yu, Zhipeng; Lin, Qing (2013) Design of oligothiophene-based tetrazoles for laser-triggered photoclick chemistry in living cells. Chem Commun (Camb) 49:9920-2
Yu, Zhipeng; Ho, Lok Yin; Lin, Qing (2011) Rapid, photoactivatable turn-on fluorescent probes based on an intramolecular photoclick reaction. J Am Chem Soc 133:11912-5

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