The human herpesviruses (HHVs) make up one of the most prevalent families of human viral pathogens. Infection with HHV is associated with several serious illnesses, including oral/genital herpes, chickenpox/shingles, infectious mononucleosis, cancer and neurodegeneration. In patients who are immunocompromised, including HIV/AIDs patients, organ transplant recipients and newborn infants, HHV infection can be life threatening and lead to serious complications. The current antiviral treatments for HHV infection almost exclusively target the viral DNA polymerase, and these drugs suffer from toxicity and emergent resistance. To develop new routes to managing HHV infection, there is a critical need for the identification and validation of new viral therapeutic targets. We seek to pharmacologically validate the viral maturational protease as a new therapeutic target. The human herpesvirus protease (HHV Pr) is essential for viral replication and infectivity, and inhibition of proteolysis arrests the viral replication cycle and disrupts infection. All HHV Prs are obligate homodimers, and undergo significant allosteric rearrangement that causes a disorder-to-order conformational switch within the dimer interface and active site, leading to catalytic activation. Here we present new methods for exploiting the HHV Pr monomer/dimer equilibrium to isolate HHV Pr in various conformational states that are catalytically inactive. These methods present routes for HHV Pr inhibitor development and have produced new tools for managing HHV infection.

Serine proteases are an abundant class of enzymes that are vital to many biological processes. Proteolysis must be tightly controlled, and this regulation is frequently carried out through serine protease inhibitors. Dysregulation of either the protease or the inhibitor can have severe biological consequences, such as the development of cancer. Additionally, the role of proteases can be challenging to study, since the active form or proteases can be difficult to isolate. In this thesis, the role of the naturally occurring protease inhibitor PI-9 is examined in the development of prostate cancer. The findings presented here show that PI-9 protects prostate cancer cells from immunosurveillance early in cancer progression. Additionally, a chemical biology approach is taken to develop small molecule activity based probes for serine proteases, in order to better study active proteases in complex systems. Principles governing the design and potency of these probes were discovered, and new imaging applications for these probes were developed. These new techniques could be applied to the study of proteases in any biological system. The work described here provides new information on how to identify proteases involved in cancer and bacterial development, and such insights could be used to design new therapeutic strategies.

Caspases execute programmed cell death, where low levels of caspase activity are linked to cancer. Chemotherapies utilize induction of apoptosis as a key mechanism for cancer treatment, where caspase-3 is a major player involved in dismantling these aberrant cells. The ability to sensitively measure the initial caspase-3 cleavage events during apoptosis is important for understanding the initiation of this complex cellular process, however, current ensemble methods are not sensitive enough to measure single cleavage events in cells. By utilizing the optical properties of plasmon coupling, peptide-linked gold nanoparticles were developed to enable single molecule imaging of caspase-3 activity in two different cancer systems. Au crown nanoparticles were assembled in a multimeric fashion to overcome the high and heterogeneous background scattering of live cells. In a colon cancer (SW620) cell line challenged with tumor necrosis factor-alpha (TNF-alpha), single molecule trajectories show early stage caspase-3 activation within minutes, which was not detectable by ensemble assays until 23 hours. Variability in caspase-3 activation among the population of cells was identified and likely a result of each cell's specific resistance to death receptor-induced apoptosis. Following these studies, improvements by way of sensitivity and selectivity were tailored into an improved nanosensor construct. Au nanoshell dimers were prepared as a comparably bright construct with 1) reduced heterogeneity compared to the synthesis of the crown nanoparticles and 2) a peptide sequence highly selective for caspase-3. Chronic myeloid leukemia (CML) K562 cells were assessed for their early apoptotic response upon treatment with dasatinib, a clinically approved tyrosine kinase inhibitor that specifically targets BCR-ABL. It has been demonstrated that inhibition of BCR-ABL by dasatinib commits K562 cells to apoptosis. Single molecule experiments with Au nanoshell dimers show caspase-3 activation as early as 8 hours than previously reported. This suggests an early commitment to apoptosis that precedes the competing fate of growth factor mediated survival in CML patient-derived BCR-ABL cells. These nanosensors are sensitive and selective in observing caspase-3 activation compared to ensemble methods; and allow the possibility to detect caspase-3 activity for use as a drug screening or diagnostic tool for personalized care in the treatment of cancer.

Proteases play important roles in human biology and disease, and the development of selective tools is essential for studying their function and structure. Members of the same protease family have structurally similar active sites and closely-related substrate specificities. This makes targeting individual proteases particularly challenging. We are interested in using recombinant antibody technology to generate protease-specific tools that are inhibitory, selective, and compatible for translational applications. The focus of our work has been on the design and development of antibodies to study serine proteases involved in cancer pericellular proteolysis. The type II transmembrane serine protease (TTSP) family includes members, matriptase, hepsin, TMPRSS2, TMPRSS4, and DESC1, which are involved in this process. Using a naïve phage display antibody library, we identified a new inhibitory antibody A11 against matriptase. A11 uses a novel reverse binding motif found in a long antibody loop to inhibit matriptase in a mechanism different from another antibody E2 that uses a substrate-like binding motif. We designed synthetic antibody libraries around these two motifs to create biased libraries for identifying antibody-based inhibitors of serine protease targets more efficiently than other general approaches. The libraries were validated against matriptase and tested against a second protease target TMPRSS2, chosen for its increasing importance in aggressive and metastatic prostate cancer. Multiple expression systems were tested to produce reagent quantities of active TMPRSS2 for antibody selection as well as future structural efforts. Using recombinant protein expressed from Pichia pastoris with our libraries, we identified and characterized a number of inhibitory TMPRSS2 antibodies and observed a higher ratio of inhibitors selected from the biased libraries. These antibodies were used in flow cytometry and immunofluorescence microscopy applications to follow TMPRSS2 expression and localization in prostate cancer cells. Since TMPRSS2 is an androgen receptor regulated gene, we explored using our antibodies for monitoring androgen receptor signaling, critical for prostate cancer cell survival and treatment resistance mechanisms, for in vivo molecular imaging. The work presented here advances our knowledge of protease-targeting mechanisms of antibodies, demonstrates the importance of selective tools for studying proteases, and contributes to ongoing work in understanding the role of TTSPs in cancer.

The urokinase plasminogen activator receptor (uPAR) is involved in the proliferation and migration of cells from diverse lineages. As a heavily glycosylated, glycosyl phosphotidyl inositol (GPI) linked protein of approximately 300 amino acids, its ubiquitousness in many aggressive cancer types-triple negative breast cancer (TNBC) in particular-makes it an attractive target for uPAR-directed therapies. uPAR participates in many protein/ protein interactions, which leads to pericellular proteolysis and "outside-in" signaling that is mediated by specific integrins. Here, the Craik Lab's human antigen-binding fragment (Fab) phage display library was panned for anti-uPAR antibodies. In total, 22 unique anti-uPAR Fabs were found. Two of these, 2G10 and 3C6, were found to antagonize the the uPAR/uPA and uPAR/β1 integrin interactions. While antagonism of these interactions did not effect cytotoxic responses, the antagonism did prevent uPAR over-expressing H1299 cells from migrating through a layer of cross-linked extracellular matrix. Further investigation revealed coarse-level binding sites of 2G10 and 3C6, as well as a unique feature of 3C6's ability to antagonize the uPAR/β1 integrin interaction by binding uPAR in an extended conformation. While surface plasmon resonance (SPR) revealed high binding affinities of 2G10 and 3C6 for uPAR, apparent affinities of 2G10 and 3C6 for the uPAR over-expressing MDA-MB-231 cells were different, and suggested that 3C6 Fab has a very poor affinity for uPAR. Regardless, both antibodies preferentially accumulated in MDA-MB-231-derived tumors, and demonstrated favorable pharmacokinetics and high tumor uptake. As a promising result, 2G10 IgG labeled with 177Lu demonstrated potent anti-tumor activity. The results of my research provide both reagents and a framework for the development of a multi-modal clinical probe and therapeutic for cancers, and other disorders, where uPAR over-expression plays a role.

Human herpesviruses (HHVs) make up one of the most prevalent viral families and are the etiological agents of a variety of devastating human illnesses that lack safe and effective treatments. All HHVs express a dimeric serine protease that is essential to the viral life cycle. Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr), a gamma subfamily member, was chosen as the model system for developing broad-spectrum herpesviral inhibitors. Of particular interest are KSHV Pr inhibitors that act allosterically, either by dimer dissociation or other mechanisms. A small-molecule KSHV Pr inhibitor has been identified that traps a transient allosteric pocket at the protease dimer interface. The binding of this molecule (DD2) has been shown to prevent the dimerization of KSHV Pr, which is required for its activation. This novel binding pocket is bioinformatically predicted to be conserved across all HHV proteases and DD2 has been shown to have activity against the serine protease from cytolomegalovirus (CMV) a member of the beta herpesvirus subfamily. Additionally, methodologies have been developed to identify new inhibitors via a high throughput screen, elucidate the mechanism and binding modes of screening hits, and test for efficacy in a cell culture viral replication assay. This work presents methodologies and demonstrates the feasibility for identifying and using small-molecule allosteric inhibitors to target a conserved enzyme family that is conserved across a viral family.

Enzyme function is ubiquitous in living organisms. Assays and biomarkers utilizing enzyme activity have become a pillar of chemical biology with relevance to the discovery of new biology, clinical diagnostics, and streamlining research and industrial pipelines. This thesis project seeks to develop assays and probes harnessing enzyme activity to expand the current repertoire of biomarkers and inhibitory drugs.Chapter1 describes restricted interaction peptides (RIPs) as a platform technology for tracking protease activity in vivo in real time. We show the proof of concept for this, as we harness the catalytic activity of proteases for tracking various biological events and disease states. This chapter evinces the most characterized and furthest along adaptation of the RIP technology, tracking immune activation via imaging with positron emission tomography (PET) by harnessing granzyme B (GZMB) catalysis Chapter 2 continues the concept of harnessing enzyme activity to aid the field of nuclear medicine and radioimaging by detailing the development of a lipoic acid ligase that conjugates radiofluorine onto proteins of interest that contain a specific peptide substrate sequence. This approach would potentially expand the toolbox and broaden the pipelines of proteins amenable to radioimaging. Chapter 3 describes the inhibition, as opposed to the amplification, of enzyme activity by developing substrates and enzyme functional assays for screening inhibitory drugs against the SARS-CoV-2 major protease (Mpro).

Due to the conserved nature of protease active sites, it has been difficult to develop protease inhibitors that are both potent and specific. The vast majority of both small molecule and naturally-occuring protein protease inhibitors target the active site, and while potent, are often promiscuous. With the goal of developing novel classes of protease inhibitors that would be both potent and specific, a phage-displayed antibody library was panned against the cancer-associated protease membrane-type serine protease 1 (MT-SP1). Two inhibitors were discovered from the screen, and their mechanism of inhibition was further studied to understand the basis of their potency and specificity.

A number of kinetic and biochemical experiments revealed that while the two inhibitors, E2 and S4, bound to the enzyme with different binding mechanisms, they were both competitive inhibitors of both small-molecule and macromolecular MT-SP1 substrates. Mutational analysis by alanine scanning of the surface loops surrounding the protease active site revealed that the antibodies made numerous contacts with these loops. The surface loops are sites of natural diversity among closely related serine proteases, and created the epitope that allowed for antibody specificity. Taken together, these results suggest that these antibody inhibitors gain a measure of potency by binding in the protease active site, and additional potency and specificity through interactions with the protease surface loops. These strategies could be adapted in efforts to develop specific inhibitors against other proteases.

A structure of the Fab construct of E2 in complex with the protease domain of MT-SP1 was determined by x-ray crystallography. The structure revealed that the inhibitor has a novel mechanism of inhibition; it gains potency and specificity through interactions with the protease surface loops, and inhibits by binding a very large H3 loop in the active site in a catalytically non-competent manner.

The results here illustrate that antibodies can be effective inhibitors because they can bind to features that traditional protease inhibitors do not. These lessons can be used to guide the development of molecules that can specifically inhibit any protease.

Misregulated proteolysis has been linked to the initiation and progression of a large number of pathological processes. Transcriptional analysis of primary tumors and cell lines in breast cancer identified differential expression of proteases and protease inhibitors associated with different molecular subtypes. However, due to the tight regulation of protease activity post-translationally, it is not sufficient to know protease expression levels alone. Here, we used an unbiased global protease profiling approach to analyze changes in extracellular proteolysis in an isogenic cell line model, MCF10A HER2, representative of the HER2 subtype, with the goal of identifying proteases contributing to the oncogenic potential of HER2 in breast cancer. HER2 expression revealed changes consistent with activation of TGF-beta signaling. Specifically, we determined that a metalloprotease involved in TGF-beta activation, BMP1, was upregulated at both the protein and activity level. In summary, through global identification of extracellular proteolysis, we elucidate proteolysis associated with HER2-mediated signaling.