Date of Award
Doctor of Philosophy (PhD)
Protease-activated receptors (PARs) are a unique family of G Protein-Coupled Receptors (GPCRs) that are activated through the proteolytic cleavage of their N-terminus. This forms a tethered ligand that binds to the receptor, causing a conformational change and eliciting a plethora of biological signals. The most well-researched of the PARs, PAR1, is involved in platelet activation and clot formation and has been shown to have an important role in inflammation, as its activation by different enzymes can either elicit pro- or anti-inflammatory signals. Currently the only FDA approved ligand of PAR1 is vorapaxar, an orthosteric antagonist that binds nearly irreversibly. We have discovered and reported a new class of small molecule modulators, known as parmodulins, that bind to PAR1 allosterically and elicit biased signaling of the receptor. Parmodulins selectively inhibit signaling of the Gαq pathway while potentially increasing cytoprotective signaling through PAR1, similar to the cytoprotective actions of Activated Protein C (aPC). Chapter 1 of this dissertation provides a general introduction to GPCRs, PARs, and strategies used to modulate PAR1. Chapter 2 introduces the type of assays used in GPCR-directed medicinal chemistry, with emphasis placed on the calcium mobilization assay we use as a primary screen. Chapter 3 reports and updates our previously published calcium assay protocols. The update improves reproducibility, decreases costs, and drastically reduces the amount of time required to test a 96-well plate with a plate reader. Also in Chapter 3 are studies of apoptosis and necrosis assays that will be used to discern if parmodulins are cytoprotective, indicating their potential as useful therapies for disorders such as stroke and diabetic nephropathy. Chapter 4 interrogates the structure-activity relationships (SAR) of the 1,3-diaminobenzene parmodulin scaffold. We report a new highly potent analog of our current lead compound, NRD-21, and details of a study attempting to synthesize a prodrug version of NRD-21. Chapter 5 reports an SAR study of a new parmodulin scaffold based on a cyanopyridone structure. The parmodulin JTD-69 was identified, that has greatly increased aqueous solubility while retaining much of the activity of parmodulins of the 1,3-diaminobenzene scaffold.