Laura E. Edgington

Noninvasive optical imaging of apoptosis by caspase-targeted activity-based probes

Imaging agents that enable direct visualization and quantification of apoptosis in vivo have great potential value for monitoring chemotherapeutic response as well as for early diagnosis and disease monitoring. We describe here the development of fluorescently labeled activity-based probes (ABPs) that covalently label active caspases in vivo. We used these probes to monitor apoptosis in the thymi of mice treated with dexamethasone as well as in tumor-bearing mice treated with the apoptosis-inducing monoclonal antibody Apomab (Genentech). Caspase ABPs provided direct readouts of the kinetics of apoptosis in live mice, whole organs and tissue extracts. The probes produced a maximum fluorescent signal that could be monitored noninvasively and that coincided with the peak in caspase activity, as measured by gel analysis. Overall, these studies demonstrate that caspase-specific ABPs have the potential to be used for noninvasive imaging of apoptosis in both preclinical and clinical settings.

Cathepsin B Inhibition Limits Bone Metastasis in Breast Cancer

Metastasis to bone is a major cause of morbidity in breast cancer patients, emphasizing the importance of identifying molecular drivers of bone metastasis for new therapeutic targets. The endogenous cysteine cathepsin inhibitor stefin A is a suppressor of breast cancer metastasis to bone that is coexpressed with cathepsin B in bone metastases. In this study, we used the immunocompetent 4T1.2 model of breast cancer which exhibits spontaneous bone metastasis to evaluate the function and therapeutic targeting potential of cathepsin B in this setting of advanced disease. Cathepsin B abundancy in the model mimicked human disease, both at the level of primary tumors and matched spinal metastases. RNA interference-mediated knockdown of cathepsin B in tumor cells reduced collagen I degradation in vitro and bone metastasis in vivo. Similarly, intraperitoneal administration of the highly selective cathepsin B inhibitor CA-074 reduced metastasis in tumor-bearing animals, a reduction that was not reproduced by the broad spectrum cysteine cathepsin inhibitor JPM-OEt. Notably, metastasis suppression by CA-074 was maintained in a late treatment setting, pointing to a role in metastatic outgrowth. Together, our findings established a prometastatic role for cathepsin B in distant metastasis and illustrated the therapeutic benefits of its selective inhibition in vivo.

Functional imaging of legumain in cancer using a new quenched activity-based probe

Legumain is a lysosomal cysteine protease whose biological function remains poorly defined. Legumain activity is up-regulated in most human cancers and inflammatory diseases most likely as the result of high expression in populations of activated macrophages. Within the tumor microenvironment, legumain activity is thought to promote tumorigenesis. To obtain a greater understanding of the role of legumain activity during cancer progression and inflammation, we developed an activity-based probe that becomes fluorescent only upon binding active legumain. This probe is highly selective for legumain, even in the context of whole cells and tissues, and is also a more effective label of legumain than previously reported probes. Here we present the synthesis and application of our probe to the analysis of legumain activity in primary macrophages and in two mouse models of cancer. We find that legumain activity is highly correlated with macrophage activation and furthermore that it is an ideal marker for primary tumor inflammation and early stage metastatic lesions.

A Nonpeptidic Cathepsin S Activity-Based Probe for Noninvasive Optical Imaging of Tumor-Associated Macrophages

Macrophage infiltration into tumors has been correlated with poor clinical outcome in multiple cancer types. Therefore, tools to image tumor-associated macrophages could be valuable for diagnosis and prognosis of cancer. Herein, we describe the synthesis and characterization of a cathepsin S-directed, quenched activity-based probe (qABP), BMV083. This probe makes use of an optimized nonpeptidic scaffold leading to enhanced in vivo properties relative to previously reported peptide-based probes. In a syngeneic breast cancer model, BMV083 provides high tumor-specific fluorescence that can be visualized using noninvasive optical imaging methods. Furthermore, analysis of probe-labeled cells demonstrates that the probe primarily targets macrophages with an M2 phenotype. Thus, BMV083 is a potential valuable in vivo reporter for tumor-associated macrophages that could greatly facilitate the future studies of macrophage function in the process of tumorigenesis.

Comparative Assessment of Substrates and Activity Based Probes as Tools for Non-Invasive Optical Imaging of Cysteine Protease Activity

Recent advances in the field of non-invasive optical imaging have included the development of contrast agents that report on the activity of enzymatic targets associated with disease pathology. In particular, proteases have proven to be ideal targets for development of optical sensors for cancer. Recently developed contrast agents for protease activity include both small peptides and large polymer-based quenched fluorescent substrates as well as fluorescently labeled activity based probes (ABPs). While substrates produce a fluorescent signal as a result of processing by a protease, ABPs are retained at the site of proteolysis due to formation of a permanent covalent bond with the active site catalytic residue. Both methods have potential advantages and disadvantages yet a careful comparison of substrates and ABPs has not been performed. Here we present the results of a direct comparison of commercially available protease substrates with several recently described fluorescent ABPs in a mouse model of cancer. The results demonstrate that fluorescent ABPs show more rapid and selective uptake into tumors as well as overall brighter signals compared to substrate probes. These data suggest that the lack of signal amplification for an ABP is offset by the increased kinetics of tissue uptake and prolonged retention of the probes once bound to a protease target. Furthermore, fluorescent ABPs can be used as imaging reagents with similar or better results as the commercially available protease substrates.

Treatment of arthritis by macrophage depletion and immunomodulation: testing an apoptosis-mediated therapy in a humanized death receptor mouse model.

OBJECTIVE To determine the therapeutic efficacy and immunomodulatory effect of an anti-human death receptor 5 (DR5) antibody, TRA-8, in eliminating macrophage subsets in a mouse model of type II collagen-induced arthritis (CIA). METHODS A human/mouse-chimeric DR5-transgenic mouse, under the regulation of a mouse 3-kb promoter and a loxP-flanked STOP cassette, was generated and crossed with an ubiquitous Cre (Ubc.Cre) mouse and a lysozyme M-Cre (LysM.Cre)-transgenic mouse to achieve inducible or macrophage-specific expression. Chicken type II collagen was used to induce CIA in mice, which were then treated with an anti-human DR5 antibody, TRA-8. Clinical scores, histopathologic severity, macrophage apoptosis and depletion, and T cell subset development were evaluated. RESULTS In human/mouse DR5-transgenic Ubc.Cre mice with CIA, transgenic DR5 was most highly expressed on CD11b+ macrophages, with lower expression on CD4+ T cells. In human/mouse DR5-transgenic LysM.Cre mice, transgenic DR5 was restrictively expressed on macrophages. Both in vivo near-infrared imaging of caspase activity and TUNEL staining demonstrated that TRA-8 rapidly induced apoptosis of macrophages in inflamed synovium. Depletion of pathogenic macrophages by TRA-8 led to significantly reduced clinical scores for arthritis; decreased macrophage infiltration, synovial hyperplasia, osteoclast formation, joint destruction, cathepsin activity, and inflammatory cytokine expression in joints; reduced numbers of Th17 cells; and an increased number of Treg cells in draining lymph nodes. CONCLUSION The anti-human DR5 antibody TRA-8 was efficacious in reducing the severity of arthritis via targeted depletion of macrophages and immunomodulation. Our data provide preclinical evidence that TRA-8 is a potential novel biologic agent for rheumatoid arthritis therapy.

Validation of the proteasome as a therapeutic target in Plasmodium using an epoxyketone inhibitor with parasite-specific toxicity

The Plasmodium proteasome has been suggested to be a potential antimalarial drug target; however, toxicity of inhibitors has prevented validation of this enzyme in vivo. We report a screen of a library of 670 analogs of the recent US Food and Drug Administration-approved inhibitor, carfilzomib, to identify compounds that selectively kill parasites. We identified one compound, PR3, that has significant parasite killing activity in vitro but dramatically reduced toxicity in host cells. We found that this parasite-specific toxicity is not due to selective targeting of the Plasmodium proteasome over the host proteasome, but instead is due to a lack of activity against one of the human proteasome subunits. Subsequently, we used PR3 to significantly reduce parasite load in Plasmodium berghei infected mice without host toxicity, thus validating the proteasome as a viable antimalarial drug target.

Non-Invasive Imaging of Cysteine Cathepsin Activity in Solid Tumors Using a 64Cu-Labeled Activity-Based Probe

The papain family of cysteine cathepsins are actively involved in multiple stages of tumorigenesis. Because elevated cathepsin activity can be found in many types of human cancers, they are promising biomarkers that can be used to target radiological contrast agents for tumor detection. However, currently there are no radiological imaging agents available for these important molecular targets. We report here the development of positron emission tomography (PET) radionuclide-labeled probes that target the cysteine cathepsins by formation of an enzyme activity-dependent bond with the active site cysteine. These probes contain an acyloxymethyl ketone (AOMK) functional group that irreversibly labels the active site cysteine of papain family proteases attached to a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) tag for labeling with 64Cu for PET imaging studies. We performed biodistribution and microPET imaging studies in nude mice bearing subcutaneous tumors expressing various levels of cysteine cathepsin activity and found that the extent of probe uptake by tumors correlated with overall protease activity as measured by biochemical methods. Furthermore, probe signals could be reduced by pre-treatment with a general cathepsin inhibitor. We also found that inclusion of a Cy5 tag on the probe increased tumor uptake relative to probes lacking this fluorogenic dye. Overall, these results demonstrate that small molecule activity-based probes carrying radio-tracers can be used to image protease activity in living subjects.

The Apoptosis Repressor with a CARD Domain (ARC) Gene Is a Direct Hypoxia-Inducible Factor 1 Target Gene and Promotes Survival and Proliferation of VHL-Deficient Renal Cancer Cells

ABSTRACT The induction of hypoxia-inducible factors (HIFs) is essential for the adaptation of tumor cells to a low-oxygen environment. We found that the expression of the apoptosis inhibitor ARC (apoptosis repressor with a CARD domain) was induced by hypoxia in a variety of cancer cell types, and its induction is primarily HIF1 dependent. Chromatin immunoprecipitation (ChIP) and reporter assays also indicate that the ARC gene is regulated by direct binding of HIF1 to a hypoxia response element (HRE) located at bp −190 upstream of the transcription start site. HIFs play an essential role in the pathogenesis of renal cell carcinoma (RCC) under normoxic conditions, through the loss of the Von Hippel-Lindau gene (VHL). Accordingly, our results show that ARC is not expressed in normal renal tissue but is highly expressed in 65% of RCC tumors, which also express high levels of carbonic anhydrase IX (CAIX), a HIF1-dependent protein. Compared to controls, ARC-deficient RCCs exhibited decreased colony formation and increased apoptosis in vitro. In addition, loss of ARC resulted in a dramatic reduction of RCC tumor growth in SCID mice in vivo. Thus, HIF-mediated increased expression of ARC in RCC can explain how loss of VHL can promote survival early in tumor formation.

Ferrous iron-dependent drug delivery enables controlled and selective release of therapeutic agents in vivo

Significance Selective drug delivery to diseased tissue is a promising approach to mitigate drug-related side effects while improving efficacy. This concept has been demonstrated in the case of photodynamic therapy, where activation of cytotoxic drugs at the tumor site provides a clear advantage compared with traditional chemotherapy. Here, we exploit the aberrantly high levels of mobile ferrous iron produced during the blood stage of malaria to selectively deliver a drug species to the parasite. The result is improved on-target vs. off-target action of the drug species and greater drug tolerability and efficacy. This approach should be applicable to other disease states associated with aberrant levels of ferrous iron. The precise targeting of cytotoxic agents to specific cell types or cellular compartments is of significant interest in medicine, with particular relevance for infectious diseases and cancer. Here, we describe a method to exploit aberrant levels of mobile ferrous iron (FeII) for selective drug delivery in vivo. This approach makes use of a 1,2,4-trioxolane moiety, which serves as an FeII-sensitive “trigger,” making drug release contingent on FeII-promoted trioxolane fragmentation. We demonstrate in vivo validation of this approach with the Plasmodium berghei model of murine malaria. Malaria parasites produce high concentrations of mobile ferrous iron as a consequence of their catabolism of host hemoglobin in the infected erythrocyte. Using activity-based probes, we successfully demonstrate the FeII-dependent and parasite-selective delivery of a potent dipeptidyl aminopeptidase inhibitor. We find that delivery of the compound in its FeII-targeted form leads to more sustained target inhibition with greatly reduced off-target inhibition of mammalian cathepsins. This selective drug delivery translates into improved efficacy and tolerability. These findings demonstrate the utility of a purely chemical means to achieve selective drug targeting in vivo. This approach may find useful application in parasitic infections and more broadly in any disease state characterized by aberrant production of reactive ferrous iron.