Proteases are crucial components of life. They regulate numerous biological pathways, such blood coagulation, neurotransmission, cell proliferation and apoptosis. They are also found to be involved in the development cancer, Alzheimer’s and many infectious diseases. Proteases, as a post-translational modification enzyme, regulate biological processes through proteolysis. And like many other enzymes, one of the key features of proteases is their substrate specificity, which is tightly controlled by specific interactions between the amino acid sequences of the substrates and their binding pockets. It’s important to characterize proteases’ specificities as it is a key to understand their biological functions. This work focused on the development and applications of a mass spectrometry-based technology, named Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS), to characterize the specificities of proteases, and the design of fluorescent substrates and inhibitors based on protease specificity profiles.

Chapter II demonstrates the application of MSP-MS in uncovering the substrate specificity of a bacterial protease, Pd_dinase, which is a putative C1B-like cysteine protease commonly secreted by the human gut commensal Parabacteroidetes distasonis. We designed and synthesized a potent protease inhibitor glycine-arginine-AOMK based on its specificity profile. Furthermore, we revealed that Pd_dinase hydrolyzes several human antimicrobial peptides, such as β-defensin 2 and keratin-derived antimicrobial peptides, indicating that it may be secreted into the extracellular milieu to assist in gut colonization by inactivation of host antimicrobial peptides.

Chapter III presents the study to extend the application of the MSP-MS technology in studying complex biological samples. In this study, we isolated bovine chromaffin granules (CGs) and quantified the endogenous proteins and peptides through proteomics and peptidomics. In addition, we performed degradation assays to profile proteolytic processing of proneuropeptides and MSP-MS assays to characterize proteas activities. With comprehensive profile of proteases, substrates and proteolytic specificities, we were able to discover six catalytically active proteases and assign their activities to some of the cleavages on proneuropeptides.

Chapter IV presents the study of developing quantitative Multiplex Substrate Profiling by Mass Spectrometry (qMSP-MS) method by combining the quantitative power of tandem mass tags (TMTs) with our previously established peptide cleavage assay, MSP-MS. We validated the method with papain, a well-characterized cysteine peptidase and uncovered the substrate specificity of two minimally characterized intramembrane rhomboid proteases. We further showed that activity from multiple peptidases in complex biological samples can be deciphered, including secretions from lung cancer cell lines. Discovery of the protease specificity at the site of the disease highlights the potential for qMSP-MS to guide the development of protease-activating drugs for cancer and infectious disease.

MG-132 is a tripeptide-aldehyde (Cbz-leu-leu-leucinal) and is extensively used in cellular studies to understand the role of the proteasome in cells as it is a potent inhibitor of the human proteasome. Early articles of MG-132 revealed its ability to inhibit cysteine cathepsins and calpains (1) and advised the community that controls such as a cysteine protease inhibitor also be used distinguish between cellular function of the proteasome and that of cysteine proteases. Many papers in the last 25 years have made conclusions about the proteasome based on phenotypic changes that have occurred following treatment with MG-132 but without a control compound. We previously determined that MG-132 is not only a potent inhibitor of cathepsin B and cathepsin L, but also potently inhibits the aspartyl proteases, cathepsin D and E. We then assayed HeLa and A549 lysates for protease activity at pH 3.5 and 5.5 with MG-132 and observed complete inhibition with 10 µM of MG-132 while other proteasome inhibitors such as marizomib, bortezomib and carfilzomib did not significantly inhibit this enzyme activity. In dose response assays with A549 lysate, we determined that the IC50 value for MG-132 at pH 3.5 and pH 5.5 was 5.0 nM and 8.7 nM, respectively. In summary, MG-132 is a potent inhibitor of aspartyl protease and cysteine protease within the concentration range that is used in cellular studies. Therefore, the phenotypic and biochemical changes that occur upon treatment of cells with this inhibitor are unlikely to be solely due to inhibition of the proteasome.

We are developing a rapid, point-of-care (POC) test for detection of Candidemia using giant magnetoresistive (GMR) biosensors. Candidemia is a fungal infection caused by the overgrowth of a yeast called Candida albicans. It is the 4th leading cause of bloodstream infections in hospitalized patients in the U.S. and severely affects immunocompromised individuals. Candidemia can be fatal if not treated rapidly and is often only detected post-mortem. The current gold standard for its detection is a blood culture, which typically takes 3-5 days, wasting precious treatment time. Therefore rapid, unambiguous diagnosis is required to improve prognosis. This technology aims to detect a unique enzyme that is secreted by C. albicans during infection. This enzyme, Sap6, is a protease that can degrade a synthetic peptide. The POC test works by immobilizing magnetic nanoparticles onto the GMR biosensor via the peptide. Once peptide cleavage by Sap6 occurs, the magnetic nanoparticle is released away from the sensor surface. This change in the magnetic field of the GMR sensors is detected in real-time. We have demonstrated this proof-of-concept in a 96-well plate format using a fluorescent sensor. This assay immobilizes approximately 1.8 g of peptide per well which can then be released by 13.34 g of protease in 10 minutes at room temperature. We have translated the assay to the GMR biosensors and detected up to 60% reduction in signal within 60 minutes. We hope to optimize this technology by reducing the detection time and expand it further to test other diseases like cystic fibrosis.

Meeting the nutritional demand for high‐risk infants is challenging and problems such as necrotizing enterocolitis, growth faltering, and neurodevelopmental impairment persist inneonatal intensive care. The use of pasteurized donor human milk is increasing for high‐risk infants particularly when mother’s milk is insufficient, or breastfeeding is not possible. Proteins are a major source of nutrients in milk and are degraded into peptides and subsequently into nutritious amino acids by proteases. Proteomics studies have identified at least 38 different proteases in human milk, but little research has been performed to biochemically characterize these enzymes. We utilized our mass spectrometry-based peptide digestion assay to detect and characterize the active proteases in human milk. We first acidified the milk to

Proteases are fundamental to successful parasitism, including for the schistosome flatworm parasite, which causes the disease schistosomiasis in 200 million people worldwide. One protease complex, the proteasome, is receiving much attention as a potential drug target for treatment of a variety of infectious parasitic diseases but has been relatively understudied in the schistosome. Adult Schistosoma mansoni was incubated in the presence of 1 µM of the commercial proteasome inhibitors bortezomib, carfilzomib and MG132. After 24 h, bortezomib and carfilzomib decreased worm motility by more than 85% and inhibited endogenous proteasome activity by >75% relative to DMSO controls. The clear association between the engagement of the proteasome target, and the phenotypic and biochemical effects recorded, encouraged the two-step chromatographic enrichment of the S. mansoni proteasome (Sm20S). Proteomic analysis of Sm20S identified 14 subunits each with an ortholog in the constitutive human proteasome. Activity assays with standard fluorescent substrates for the β1, β2 and β5 proteasome subunits revealed that Sm20S contains caspase-type (β1), trypsin-type (β2) and chymotrypsin-type (β5) activities. Sm20S was screened with 11 peptide epoxyketone inhibitors derived from the marine natural product, carmaphycin B. Analog 17 exhibited preferential inhibition of the Sm20S β2 and β5 subunits over that of c20S and i20S. Furthermore, 1 µM of 17 decreased both worm motility and endogenous Sm20S activity by more than 90% after 24 h. We provide direct evidence of the proteasome’s importance to schistosome viability and identify a lead for which future studies will aim to improve the potency, selectivity and safety.

Cathepsin B (CatB) is a cysteine protease that is abundant in the lysosome of cells and is responsible for degradation of proteins to maintain cellular homeostasis. However, in certain pathogenic conditions such as cancer, neurodegenerative brain disorders, autoinflammatory, and pathogen-induced pyroptosis, CatB can be found outside the lysosome. Most lysosomal enzymes are optimally active at the acidic pH of lysosomes and become inactivated at neutral pH outside the lysosome. However, CatB retains good stability and catalytic activity at extra-lysosomal pH conditions such as the cytosol where it has been shown to participate in cellular apoptotic and inflammatory pathways. This has led to the hypothesis that translocation of CatB from the lysosome to either the cytosol, nucleus, or extracellular environment contributes to the pathogenesis of various diseases. To investigate such hypothesis, we sought to develop inhibitor probes of CatB that are selective for this protease at either neutral pH or acidic pH. In parallel, we aimed to develop inhibitor probes that are specific to CatB over related cathepsins. We found that established inhibitors such as E64c target CatB but also potently inhibit other cysteine cathepsins. CA-074 is an established selective inhibitor for CatB but we discovered that it is over 100-fold more potent at pH 4.6 than pH 7.2. We then proposed that it is possible to develop an inhibitor that is the converse of CA-074—being more potent at pH 7.2 than pH 4.6. This is based on the hypothesis that CatB may have distinct substrate and inhibitor interactions at different pH conditions. To evaluate this hypothesis, we profiled the cleavage specificity of CatB at pH 4.6 and pH 7.2, along with other related cathepsins, and developed substrates that are cleaved by CatB better at neutral pH than at acidic pH and not cleaved by other related cathepsins. We then modified the substrate with a cysteine reactive warhead to successfully develop a novel inhibitor that potently and selectively inhibits CatB at neutral pH to be used as a molecular probe for the investigation of extra-lysosomal CatB pathogenic role in various cell-death and inflammatory pathways of human diseases.

Worldwide, there are about 1,400 pathogens that infect humans. In particular, for the neglected diseases of poverty that are caused by eukaryotic pathogens, chemotherapy often relies on a small number of antiquated drugs that can be toxic, and for which resistance is either established or a concern. Chapter 1 introduces the 20S proteasome, its biology and the use of small molecule inhibitors to treat various cancers, such as multiple myeloma. The chapter then describes the current state-of-the-art regarding 20S proteasome inhibitor development for a range of parasitic diseases, not least, malaria and leishmaniasis, with the key point being that in spite of its conservation in life, inhibitor specificity to a particular 20S target can be achieved to deliver exciting new treatments. Chapter 2 focuses on the synthesis and application of two novel activity-based probes, LJL-1 and LJL-2, each with a different specificity for the 20 proteasome of two parasitic organisms, Leishmania donovani and Trichomonas vaginalis. The probes were designed to enable the attachment of a bifunctional fluorescent-biotin handle via click chemistry to visualize and formally identify the component catalytic β subunits for each proteasome. Also, the probes are more specific to the 20S target and offer greater functionality compared to a well-described and commercially-available product. Chapter 3 focuses on the whole-organism screening of T. brucei with and the development of a structure activity relationship (SAR) for a series of peptidyl, epoxyketone 20S inhibitors derived from the marine natural product, carmaphycin B (CPB). The compound collection stems from a campaign to develop Plasmodium falciparum 20S (Pf20S) inhibitors and the SAR arising is similar to that described for Pf20S. Not least is the strong toxicity differential between T. brucei and mammalian cells generated by the introduction of D stereochemistry at the P3 position of the CPB backbone. In a mouse model of T. brucei infection, one such inhibitor extended the survival of mice, thus, supporting the further development of this compound class as a potential treatment for African trypanosomiasis.