Protease-activated receptors (PARs) are G-protein coupled receptors (GPCRs) with a unique mode of activation involving the cleavage of the N-terminus of the receptor by various proteases. Among numerous activities, PARs play a vital role in the activation of platelet cells in hemostasis, as well as regulating immune responses in endothelial and immune cells. The pleiotropic effects mediated by PARs are governed by signaling pathways involving different G proteins and B-arrestins, and evidence has accumulated with PARs, and GPCRs in general, that different ligands can bias their balance of signals in beneficial ways that could lead to safer and more effective drugs. In previous years, we identified a new class of PAR1 ligands (called parmodulins) that have been found to be allosteric and biased ligands for PAR1. Unusually, we have found that they are effective inhibitors of platelet activation, but can also induce protective effects in endothelial and other cell types that may have broad applications to the treatment of inflammation-related disorders. This proposal aims to continue our efforts to discover new and improved parmodulins and to determine their precise intracellular signaling pathways.
In Aim 1, libraries of compounds with a relatively unexplored heterocyclic scaffold will be prepared, potentially using a multistep one pot synthesis from readily available building blocks.
In Aim 2, novel PAR1 G protein and B-arrestin assays will be developed in collaboration with Dr. John McCorvy (Medical College of Wisconsin), with a focus on the use of G protein-BRET sensors to elucidate which of the numerous G proteins may contribute to the protective signaling of parmodulins. A high throughput assay measuring phospho-Akt formation in endothelial cells will also be validated, which is relevant to the protective effects of parmodulins.
In Aim 3, these assays will be used to characterize the novel analogs of Aim 1 in an iterative fashion, and to define structure-activity and structure-functional selectivity relationships (SARs and SFSRs) of these parmodulins. In collaboration with Dr. Hartmut Weiler (Blood Research Institute), select compounds will undergo more detailed study to characterize their utility as anti-inflammatory and cytoprotective compounds. Certain parmodulins will also be subjected to standard profiling experiments to identify compounds with safety, stability, and selectivity profiles suitable for future in vivo studies.

Public Health Relevance

The goal of this research is to develop novel small molecules for the control of protease-activated receptors (PARs), and to determine how PARs can be modulated by certain molecules (called parmodulins) in a way that can protect endothelial cells within blood vessels from damage during stressful events such as severe infection or stroke. PARs are cell-surface receptors that regulate many disease-relevant processes, including hemostasis (blood clotting) and immune responses. The molecules and assays developed in this proposal will be used to study exactly how PAR signaling can be modulated and how productive signals propagate within cells. This information and the compounds synthesized in this project could support new strategies for the treatment of cardiovascular disease, infection, and proliferative disorders.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Academic Research Enhancement Awards (AREA) (R15)
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Biochemistry and Biophysics of Membranes Study Section (BBM)
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Sarkar, Rita
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Marquette University
Schools of Arts and Sciences
United States
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