Development of Cysteine Protease Inhibitors and Their Application Towards Huntington's Disease and Malaria Therapeutic Models

Proteases are enzymes that catalyze the hydrolysis of amide bonds in peptides and proteins. Due to the vital role of proteases in various diseases, protease inhibitors have been aggressively pursued as therapeutic targets but most are peptidic structures. Although peptidic protease inhibitors are identified by traditional methods, drug candidacy is compromised with peptides because of poor metabolic stability and poor cell penetration. Therefore, a more viable approach to obtain drug-like structures is to identify and develop nonpeptidic protease inhibitors with improved pharmacokinetic properties. In this dissertation, approaches to nonpeptidic inhibitor development and studies of protease involvement in relevant diseases are described.Chapter 1. A brief introduction on proteases and traditional methods used to obtain protease inhibitors is discussed. Chapter 2. The identification of nonpeptidic pan-caspase inhibitors using the Substrate Activity Screening (SAS) method is described. Application of the SAS method against caspase-3 and caspase-6 resulted in the identification of three novel, pan-caspase inhibitors that block proteolysis of Htt at caspase-3 and -6 cleavage sites. In a Huntington's disease (HD) model, all three inhibitors rescued cell death in striatal and cortical neurons at nanomolar concentrations. Overall, these inhibitors have validated the correlation between blocking caspase Htt cleavage and rescue of HD-mediated neurodegeneration. Chapter 3. The development of dipeptidyl aminopeptidase (DPAP) inhibitors and application of a novel fragmenting hybrid approach is described. Homology modeling and computational docking were utilized to design and synthesize nonpeptidic DPAP1 inhibitors that kill Plasmodium falciparum at low nanomolar concentrations. A fragmenting hybrid was developed as an alternative to artemisinin combination therapy, which incorporated a trioxolane agent conjugated to our most potent nonpeptidic inhibitor of DPAP1. This strategy showed the slow release of our lead inhibitor and sustained DPAP1 inhibition in Plasmodium falciparum parasites. Overall, we validated DPAP1 as a valuable anti-malarial target and demonstrated that our fragmenting hybrid can be successfully used to deliver secondary anti-malarial agents into parasite-infected erythrocytes. Chapter 4. A summary of the projects described in Chapters 1-2 and a brief discussion of future directions is included.In summary, the projects described in this dissertation contribute to the traditional approaches to protease inhibitor development and enhance the tools available to study proteases in pertinent diseases