Proteolytic activity is vital to organism homeostasis. Perturbations in host protease activity can be debilitating and result in pathological conditions. Proteolytic activity is also required for pathogenesis of viral and bacterial diseases. Thus, developing protease inhibitors as therapeutic agents is an active area of investigation. Development of cell-based assays to monitor proteolytic activity permits assessment of function within the context of the cell and enables identification of activity modulators, pointing to compounds that may serve as potential pharmaceutical agents. In this work, cell based assays were constructed to monitor the activity of three proteases important for disease pathogenesis: beta-site amyloid precursor protein (APP) cleaving enzyme (BALE), furin, and caspase-3. To monitor activity and identify inhibitors of the Golgi resident proteases, BACE and furin, a cell-based reporter protein was created. Cleavage of APP by BACE is required for Alzheimer's disease (AD) progression. Chronic use of non-steroidal anti-inflammatory drugs (NSAIDs) inhibits AD development. The BACE reporter assay was utilized to determine that certain NSAIDs inhibit BACE activity. Furin activity is implicated in cancer, AD, infectivity of HIV and avian influenza, and toxicity of anthrax and diphtheria. The furin cell-based assay was utilized to identify small molecule inhibitors using high throughput screening (HTS) of chemical diversity libraries. Novel non-competitive inhibitors with structural similarity to dicoumarol were identified and shown to inhibit processing of MT1-MMP, important for cancer, and anthrax toxicity. A cell-based reporter system for caspase-3 enabled non-invasive monitoring of apoptosis in live cells and animals. This assay system was used to demonstrate that administration of the chemotherapeutic agent, temozolomide, in combination with radiation, provides enhanced apoptosis over individual treatments in a glioma model. This system validated the role of apoptosis in response to targeted therapies, provided a platform for discovery of novel cancer therapeutics, and allowed optimization of dosing and scheduling of established anti-cancer therapies. This work demonstrates that cell-based assays provide novel avenues to study medically important proteases and identify modulators in a cellular context. The assay systems developed here may be adapted to monitor other proteases and may pave the way for discovery of future generations of protease activity altering therapies.Ph.D.BiochemistryBiological SciencesHealth and Environmental SciencesMolecular biologyPharmacologyPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/126395/2/3253248.pdf