Date of Award
Summer 2018
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Chemistry
First Advisor
Dockendorff, Christopher
Second Advisor
St. Maurice, Martin
Third Advisor
Steinmetz, Mark
Abstract
Protease-activated receptors (PARs) are class A G protein-coupled receptors (GPCRs) with 4 subtypes (PAR 1 – 4) and with a unique mode of action. PARs are cleaved by extracellular proteases at the N-terminus, creating a new tethered ligand that activates the receptor and transduces biological signals into the cell. PARs have been implicated in various productive and pathological signals, including those related to thrombosis, inflammation, reperfusion injury, and cancer cell metastasis. Despite the fact that PARs are attractive as drug targets, their intramolecular mode of activation makes it challenging to modulate them with drugs in a selective manner, and only one PAR-targeting therapy, the PAR1 antagonist and antiplatelet drug vorapaxar, is currently approved by the FDA. Previously in the Dockendorff lab, compounds with a 1,3-diaminobenzene scaffold, called parmodulins, were identified as biased ligands of PAR1 that selectively inhibit Gαq-mediated signaling of PAR1 without significantly inhibiting Gα12/13-mediated signaling leading to platelet shape change. More recently, cytoprotective and anti-inflammatory effects of parmodulins in endothelial cells were identified that could be fruitful for the treatment of sepsis and myocardial infarction. We developed a Gαq-mediated intracellular Ca2+ mobilization (iCa2+) assay in endothelial cells to perform medium-throughput screening and characterization of parmodulins, and we demonstrated that they are reversible, negative allosteric modulators of PAR1, unlike ligands such as vorapaxar. We also extended structure-activity relationship (SAR) studies of parmodulins to identify analogues with potentially improved properties using medicinal chemistry, and these parmodulins are promising leads for safer antithrombotic and sepsis drugs. Additionally, we commenced investigations into the role of heteromeric PAR complexes in proliferative disorders such as restenosis and cancer cell metastasis. We synthesized a novel class of bivalent ligands targeting putative PAR1/2 heteromers and characterized them using our iCa2+ assay with endothelial cells (EA.hy926) and the breast cancer cell line MDA-MB-231. Such bivalent PAR ligands hold promise for the selective treatment of cancer and other proliferative disorders.