G proteins ?? subunits play a central role in G-protein coupled receptor (GPCR)-mediated signal transduction. They act as cofactors in the receptor-mediated activation process as well as playing direct roles in signal transfer to downstream targets. Considerable data has accumulated in number of systems that excess ?? signaling has pathological consequences and that manipulation of ?? subunit signaling could be an effective therapeutic strategy in heart failure as well as other diseases. We developed a novel targeting strategy for selective manipulation of G protein ?? subunit signaling pathways by selectively blocking ?? -subunit binding interactions with functional protein partners using small molecules. In the previous funding period we defined the binding modes for several compounds by surface plasmon resonance (SPR) coupled with site directed mutagenesis and solved the co-crystal structure of M201 bound to the hot spot of G??. These data confirmed a direct mechanism for binding to G?? that influences protein-protein interactions and support our overall hypothesis that small molecules selectively modulate downstream effectors signaling by binding to different subsites on the G?? hotspot. Additionally, we published results demonstrating efficacy and specificity of these compounds in cellular and animal models of heart failure, inflammation and morphine- dependent analgesia. In the experiments proposed in this application we will continue to explore the fundamental mechanisms underlying binding and selectivity of these ?? binding compounds.
Specific aim 1 will focus on mutagenesis and x-ray crystallography to identify multiple binding modes within the G?? hotspot that contribute to selectivity.
Specific aim 2 will explore the mechanism for compound-dependent G?? subunit activation.
Specific aim 3 will explore specificity and mechanism of action in intact cells. Successful completion of the proposed experiments will lead to a thorough understanding of a the mechanism of action of a new family of molecules that target G23 signaling that have potential uses in dissecting the mechanisms of action of GPCR stimulated signaling and providing the basis for novel therapeutic approaches.

Public Health Relevance

G protein coupled receptors (GPCRs) are a major class of transmembrane receptors responsible for recognition of a large class of diverse ligands. Here we propose investigation of selective small molecule inhibitors of G protein ? subunits identified in our laboratory which could be used to inhibit multiple GPCRs and modify actions of existing GPCR directed pharmaceuticals. Results of these experiments will help to validate this alternate approach to modification of signaling pathways downstream of GPCRs that could ultimately lead to development of novel therapeutics.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM081772-07
Application #
8640950
Study Section
Molecular and Integrative Signal Transduction Study Section (MIST)
Program Officer
Dunsmore, Sarah
Project Start
2008-04-01
Project End
2016-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
7
Fiscal Year
2014
Total Cost
$302,851
Indirect Cost
$106,831
Name
University of Rochester
Department
Pharmacology
Type
Schools of Dentistry
DUNS #
041294109
City
Rochester
State
NY
Country
United States
Zip Code
14627
Surve, Chinmay R; To, Jesi Y; Malik, Sundeep et al. (2016) Dynamic regulation of neutrophil polarity and migration by the heterotrimeric G protein subunits Gαi-GTP and Gβγ. Sci Signal 9:ra22
Rangel-Moreno, Javier; To, Jesi Y; Owen, Teresa et al. (2016) Inhibition of G Protein βγ Subunit Signaling Abrogates Nephritis in Lupus-Prone Mice. Arthritis Rheumatol 68:2244-56
Brand, Cameron S; Sadana, Rachna; Malik, Sundeep et al. (2015) Adenylyl Cyclase 5 Regulation by Gβγ Involves Isoform-Specific Use of Multiple Interaction Sites. Mol Pharmacol 88:758-67
Malik, S; deRubio, R G; Trembley, M et al. (2015) G protein βγ subunits regulate cardiomyocyte hypertrophy through a perinuclear Golgi phosphatidylinositol 4-phosphate hydrolysis pathway. Mol Biol Cell 26:1188-98
Smrcka, Alan V (2015) Regulation of phosphatidylinositol-specific phospholipase C at the nuclear envelope in cardiac myocytes. J Cardiovasc Pharmacol 65:203-10
Surve, Chinmay R; Lehmann, David; Smrcka, Alan V (2014) A chemical biology approach demonstrates G protein βγ subunits are sufficient to mediate directional neutrophil chemotaxis. J Biol Chem 289:17791-801
Kamal, Fadia A; Mickelsen, Deanne M; Wegman, Katherine M et al. (2014) Simultaneous adrenal and cardiac g-protein-coupled receptor-gβγ inhibition halts heart failure progression. J Am Coll Cardiol 63:2549-57
Le, Nhat-Tu; Takei, Yuichiro; Izawa-Ishizawa, Yuki et al. (2014) Identification of activators of ERK5 transcriptional activity by high-throughput screening and the role of endothelial ERK5 in vasoprotective effects induced by statins and antimalarial agents. J Immunol 193:3803-15
Sun, Zhizeng; Smrcka, Alan V; Chen, Songhai (2013) WDR26 functions as a scaffolding protein to promote Gβγ-mediated phospholipase C β2 (PLCβ2) activation in leukocytes. J Biol Chem 288:16715-25
Smrcka, Alan V (2013) Molecular targeting of Gα and Gβγ subunits: a potential approach for cancer therapeutics. Trends Pharmacol Sci 34:290-8

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