Dr. GPCR Ecosystem
Identification of GPCRs Modulating Flow-induced Signaling Pathways in Vascular Endothelial Cells
About this episode
The endothelium is an important regulator of vascular homeostasis through its ability to regulate vasoconstriction, vascular permeability, and inflammatory processes. In the healthy state, the endothelium behaves as an anti-inflammatory and anti-thrombotic barrier preventing the passage of inflammatory cells into the vascular media while also contributing to blood fluidity through the expression of thrombomodulin (TM) and endothelial nitric oxide synthase (eNOS). It is well known that the physiological state of endothelial cells is regulated by the pattern of blood flowing over the cell surface. Specifically, a high shear force, the laminar pattern of blood flow supports a healthy endothelial phenotype. One of the key transcriptional mediators of laminar flow effects on endothelial cells is kruppel- like factor 2 (KLF2). Laminar flow induces KLF2 expression resulting in increased expression of eNOS, TM, and tissue plasminogen activator while decreasing expression of VEGF, PAI-1, and tissue factor.
We sought to identify druggable targets capable of modulating KLF2 expression in endothelial cells. Utilizing siRNA and bioinformatics we identified known GPCRs expressed on the surface of human endothelial cells. Targeted knockdown of these receptors revealed several GPCRs capable of modulating KLF2 expression. We will present LGR4 (the R-spondin receptor) as an example of the utility of this approach to identifying novel drug discovery targets and how one can translate the identification of targets into drug discovery.
Expression of LGR4 was found to be positively correlated with increased KLF2 expression. Using a β-arrestin assay, we identified approximately 1000 small molecule compounds capable of stimulating KLF2 expression in an LGR-dependent fashion. These LGR4 ligands were found to increase KLF2 and KLF2 target gene expression in human endothelial cells. These compounds did not stimulate β-catenin expression thus suggesting they are biased agonists of LGR4. Functionally, LGR4 biased agonists were found to promote endothelial barrier function and to prevent monocyte adhesion to monolayers of human endothelial cells. Lastly, these compounds were also found to induce KLF2 expression in ex vivo culture of rat aorta.
In summary, we have identified LGR4 as an endothelial GPCR capable of modulating KLF2 expression. Furthermore, we have identified the first known synthetic agonists to LGR4 and shown that they can positively affect endothelial function both in vitro and ex vivo.
About Dr. Brian Arey
Brian is Senior Director of Leads Discovery and Optimization at Bristol-Myers, Squibb Co. in Lawrenceville, NJ. He obtained both his MS and Ph.D. in Neuroendocrine Physiology at Florida State University before completing his postdoctoral training at Northwestern University. He then moved to work in the pharmaceutical industry where he held various positions. These make him an experienced physiologist and a successful drug discovery scientist.
Dr. Arey's work led him to contribute to the development of some of the first described allosteric agonists of FSHR, the discovery of numerous clinical candidate compounds, 5 marketed medicines as well as editing books including Biased Signaling in Physiology, Pharmacology, and Therapeutics available on Amazon.
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