Inhibition of furin/proprotein convertase-catalyzed surface and intracellular processing by small molecules.

Furin is a ubiquitously expressed proprotein convertase (PC) that plays a vital role in numerous disease processes including cancer metastasis, bacterial toxin activation (e.g. anthrax and Pseudomonas), and viral propagation (e.g. avian influenza and human immunodeficiency virus). To identify small molecule inhibitors of furin and related processing enzymes, we performed high-throughput screens of chemical diversity libraries utilizing both enzyme-based and cell-based assays. The screens identified partially overlapping sets of compounds that were further characterized for affinity, mechanism, and efficacy in additional cellular processing assays. Dicoumarols were identified as a class of compounds that inhibited furin non-competitively and reversibly with Ki values in the micromolar range. These compounds inhibited furin/furin-like activity both at the cell surface (protecting against anthrax toxin) and in the secretory pathway (blocking processing of the metastasis factor membrane-type 1 matrix metalloproteinase/MT1-MMP) at concentrations close to Ki values. Compounds tested exhibited distinct patterns of inhibition of other furin-family PCs (rat PACE4, human PC5/6 and human PC7), showing that dicoumarol derivatives might be developed as either generic or selective inhibitors of the PCs. The extensive clinical use, high bioavailability and relatively low toxicity of dicoumarols suggests that the dicoumarol structure will be a good starting point for development of drug-like inhibitors of furin and other PCs that can act both intracellularly and at the cell surface.

Noninvasive imaging of apoptosis and its application in cancer therapeutics.

PURPOSE: Activation of the apoptotic cascade plays an important role in the response of tumors to therapy. Noninvasive imaging of apoptosis facilitates optimization of therapeutic protocols regarding dosing and schedule and enables identification of efficacious combination therapies. EXPERIMENTAL DESIGN: We describe a hybrid polypeptide that reports on caspase-3 activity in living cells and animals in a noninvasive manner. This reporter, ANLucBCLuc, constitutes a fusion of small interacting peptides, peptide A and peptide B, with the NLuc and CLuc fragments of luciferase with a caspase-3 cleavage site (DEVD) between pepANLuc (ANLuc) and pepBCLuc (BCLuc). During apoptosis, caspase-3 cleaves the reporter, enabling separation of ANLuc from BCLuc. A high-affinity interaction between peptide A and peptide B restores luciferase activity by NLuc and CLuc complementation. Using a D54 glioma model, we show the utility of the reporter in imaging of apoptosis in living subjects in response to various chemotherapy and radiotherapy regimens. RESULTS: Treatment of live cells and mice carrying D54 tumor xenografts with chemotherapeutic agents such as temozolomide and perifosine resulted in induction of bioluminescence activity, which correlated with activation of caspase-3. Treatment of mice with combination therapy of temozolomide and radiation resulted in increased bioluminescence activity over individual treatments and increased therapeutic response due to enhanced apoptosis. CONCLUSION: The data provided show the utility of the ANLucBCLuc reporter in dynamic, noninvasive imaging of apoptosis and provides a rationale for use of this technology to optimize dose and schedule of novel therapies or to develop novel combination therapies using existing drugs.

A small-molecule furin inhibitor inhibits cancer cell motility and invasiveness.

Furin, a member the proprotein convertase (PC) family, processes inactive precursor proteins to functional proteins within the Golgi/trans-Golgi network secretory pathway. Furin and other PC family members (furin/PCs) activate proteins vital to proper physiological functioning, including growth factors and hormones, receptors, plasma proteins, and matrix metalloproteases (MMPs). Additionally, the expression and activity of furin/PC are necessary for processing substrates important for cell transformation and tumor progression, metastasis, and angiogenesis. Furin processing of the remodeling protease membrane type-1 matrix metalloproteinase (MT1-MMP) enhances cellular motility and invasiveness, contributing to aggression and metastatic potential cancer cells. Whereas overexpression and activity of furin/PC exacerbate the cancer phenotype, inhibition of its activity decreases or nullifies furin/PC-mediated effects, and thus, inhibition of furin may be a viable route to cancer therapy. Recently, we identified a small-molecule inhibitor of furin, named B3, by high-throughput screening with a K(i) and IC(50) of 12 microM. Here, we show that this cell-permeable, small-molecule compound inhibits furin-mediated cleavage of proMT1-MMP, resulting in decreased MMP-2 activation and cell motility in CHO cells expressing proMT1-MMP. Additionally, this molecule inhibited proMT1-MMP processing, complete MMP-2 maturation, and invasiveness of human fibrosarcoma cells (HT1080).

Identification of inhibitors using a cell-based assay for monitoring Golgi-resident protease activity.

Noninvasive real-time quantification of cellular protease activity allows monitoring of enzymatic activity and identification of activity modulators within the protease's natural milieu. We developed a protease activity assay based on differential localization of a recombinant reporter consisting of a Golgi retention signal and a protease cleavage sequence fused to alkaline phosphatase (AP). When expressed in mammalian cells, this protein localizes to Golgi bodies and, on protease-mediated cleavage, AP translocates to the extracellular medium where its activity is measured. We used this system to monitor the Golgi-associated protease furin, a pluripotent enzyme with a key role in tumorigenesis, viral propagation of avian influenza, ebola, and HIV as well as in activation of anthrax, pseudomonas, and diphtheria toxins. This technology was adapted for high-throughput screening of 39,000-compound small molecule libraries, leading to identification of furin inhibitors. Furthermore, this strategy was used to identify inhibitors of another Golgi protease, the beta-site amyloid precursor protein (APP)-cleaving enzyme (BACE). BACE cleavage of the APP leads to formation of the Abeta peptide, a key event that leads to Alzheimer's disease. In conclusion, we describe a customizable noninvasive technology for real-time assessment of Golgi protease activity used to identify inhibitors of furin and BACE.

Imaging of proteolytic activity using a conditional cell surface receptor.

Programmed cell death (apoptosis) is a ubiquitous means utilized by multicellular organisms for elimination of unwanted cells during development and homeostasis. Dysregulated apoptosis is implicated in an array of clinical disorders including cancer, autoimmune diseases, neurodegenerative disorders, and ischemia. During programmed cell death, a series of proteases, known as caspases, with different specificities play crucial roles in the apoptotic process. Caspase-3, a group II cysteine aspartate protease, recognizes and cleaves substrates harboring the amino acid sequence aspartic acid-glutamic acid-valine-aspartic acid (DEVD), and it plays an important role in the terminal phase of apoptosis. Here we report the development of a novel imaging platform for sensing the activation of cellular proteases. A recombinant chimeric protein was constructed, composed of a cell-surface-targeted single-chain antibody (sFv) fused to a Golgi retention signal. The DEVD tetrapeptide sequence was included between the single-chain antibody and the Golgi retention signal as a caspase-3 protease cleavage site. When expressed in cultured cells this fusion protein was localized to Golgi bodies and was not detected on the cell surface. Induction of apoptosis resulted in cleavage of the fusion protein releasing the single-chain antibody from the Golgi retention signal in a caspase-dependent manner. As a result, in cells undergoing apoptosis the single-chain antibody was visualized at the cell surface by immunofluorescence microscopy. The expression of sFv on the surface of cells in a protease-dependent manner provides a unique opportunity for real-time imaging through the use of targeted nanoparticles. This methodology may provide for a multimodal noninvasive real-time imaging of apoptosis and a new opportunity for high-throughput screening of cell-death-modulating therapeutic agents.

Phosphorylated FADD induces NF-kappaB, perturbs cell cycle, and is associated with poor outcome in lung adenocarcinomas.

In an effort to identify a clinical biomarker for lung cancer, we used cDNA microarray and 2D protein analyses to demonstrate that increased Fas-associated death domain (FADD) mRNA and protein were significantly associated with poor survival. Analyses of copy number and sequence of the FADD gene in 24 independent tumors ruled out the existence of an amplified and/or mutated FADD gene in aggressive lung cancers. Immunohistochemistry-based tissue microarray analysis showed that nuclear localization of FADD and elevation of the phosphorylated form of FADD (p-FADD) correlated with poor outcome (P = 0.003). Tumors with increased p-FADD expression showed elevated NF-kappaB (P = 0.004) activation, a frequent molecular alteration associated with tumorigenesis and metastasis in a variety of cancers. To provide a link between p-FADD and NF-kappaB, cell culture studies demonstrated that overexpression of p-FADD leads to an increase in NF-kappaB activity and a decrease in the number of cells in the G2 phase of the cell cycle, compared with cells expressing the nonphosphorylatable form of FADD or the vector control. Furthermore, cDNA microarray analyses of lung tumor samples showed that increased levels of FADD transcripts were significantly correlated with overexpression of cyclins D1 (P < 0.01) and B1 (P < 0.01), genes that are involved in the regulation of cell cycle progression and are inducible by NF-kappaB. These studies demonstrate that induction of NF-kappaB activity and its effects on cell-cycle progression may represent a molecular basis underlying the aggressive tumor behavior associated with elevated p-FADD expression in lung adenocarcinoma.