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

Coppola, Julia M.; Hamilton, Christin A.; Bhojani, Mahaveer S.; Larsen, Martha J.; Ross, Brian D.; Rehemtulla, Alnawaz

doi:10.1016/j.ab.2007.01.013

Cited by 36 publications

(32 citation statements)

References 55 publications

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“…B3 was dissolved from powder just prior to use. Plasmid pEF-GRAPfurin was as described (27). All HTS materials, including compound libraries (Chembridge DIVERSet, 10,000 compounds; ChemDiv, 20,000 compounds), were supplied by the Center for Chemical Genomics (Life Sciences Institute, University of Michigan).…”

Section: Methodsmentioning

confidence: 99%

“…Compounds B1, B2, B6, and B7 were from Chembridge (San Diego, CA). Compounds B3 (CCG8294) (27) and B10 were synthesized, purified, and characterized as described (32). All compounds were dissolved in dimethyl sulfoxide.…”

Section: Methodsmentioning

confidence: 99%

“…A preliminary report of the cell-based assay has been published elsewhere (27). Combining the results of the enzymatic screen with the cellular screen allowed identification of small molecule lead compounds with the desired properties of high affinity, high cell permeability, and low toxicity.…”

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confidence: 99%

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Inhibition of Furin/Proprotein Convertase-catalyzed Surface and Intracellular Processing by Small Molecules

Komiyama

Coppola

Larsen

et al. 2009

Journal of Biological Chemistry

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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 K i 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 K i 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.Furin, is a subtilisin-related serine protease and member of the proprotein convertase (PCs) 4 family that functions within the secretory and endocytic pathways and at the cell surface, cleaving proproteins at clusters of basic residues, typically of the form RX(K/R)R2 (for reviews see Refs. 1-3). The specificity of furin and its yeast homologue Kex2 correlate well with the three-dimensional structures of their catalytic domains (4, 5). Ubiquitously expressed, furin has numerous known or suspected physiological substrates that include growth factors, receptors, coagulation proteins, plasma proteins (e.g. pro-von Willebrand factor), extracellular matrix components, and protease precursors (e.g. matrix metalloproteases) (2). Although the homozygous furin knock-out mouse exhibits embryonic lethality (6), analysis of liver-specific ablation suggests functional overlap with other PCs, such as PACE4, PC5/6, and PC7, that are also widely expressed and act in the constitutive secretory pathway (7). Furin activity contributes to numerous chronic pathological conditions, including Alzheimer disease (8), other non-Alzheimer cerebral amyloidoses (9), osteoarthritis (10), atherosclerosis (11), and tumor progression and malignancy (12). Moreover, activation by host cells of bacterial toxins such as anthrax toxin, Pseudomonas exotoxin A, diphtheria toxin (13), Shiga toxin (14), and Bordetella dermonecrotic toxin (15), requires cleavage by furin or other PCs. Fu...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Inhibition of Furin/Proprotein Convertase-catalyzed Surface and Intracellular Processing by Small Molecules

Komiyama

Coppola

Larsen

et al. 2009

Journal of Biological Chemistry

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“…Andrographolide paniculata diterpines of the labdane family were the first example of such nonpeptide convertase inhibitors (Basak et al, 1999); however, inhibition was quite weak and nonspecific. Much more potent furin inhibitors have been generated recently by derivatization of 2,5-dideoxytryptamine with the addition of guanidinylated aryl groups (Jiao et al, 2006) and by using cell-based assays for screening (Coppola et al, 2007). For example, guanidinylated aryl compounds exhibit nanomolar K i values and possess excellent specificity (Jiao et al, 2006).…”

Section: Discussionmentioning

confidence: 99%

Synthetic Small-Molecule Prohormone Convertase 2 Inhibitors

et al. 2008

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The proprotein convertases are believed to be responsible for the proteolytic maturation of a large number of peptide hormone precursors. Although potent furin inhibitors have been identified, thus far, no small-molecule prohormone convertase 1/3 or prohormone convertase 2 (PC2) inhibitors have been described. After screening 38 small-molecule positional scanning libraries against recombinant mouse PC2, two promising chemical scaffolds were identified: bicyclic guanidines, and pyrrolidine bis-piperazines. A set of individual compounds was designed from each library and tested against PC2. Pyrrolidine bis-piperazines were irreversible, time-dependent inhibitors of PC2, exhibiting noncompetitive inhibition kinetics; the most potent inhibitor exhibited a K i value for PC2 of 0.54 M. In contrast, the most potent bicyclic guanidine inhibitor exhibited a K i value of 3.3 M. Cross-reactivity with other convertases was limited: pyrrolidine bis-piperazines exhibited K i values greater than 25 M for PC1/3 or furin, whereas the K i values of bicyclic guanidines for these other convertases were more than 15 M. We conclude that pyrrolidine bis-piperazines and bicyclic guanidines represent promising initial leads for the optimization of therapeutically active PC2 inhibitors. PC2-specific inhibitors may be useful in the pharmacological blockade of PC2-dependent cleavage events, such as glucagon production in the pancreas and ectopic peptide production in small-cell carcinoma, and to study PC2-dependent proteolytic events, such as opioid peptide production.

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“…Western blotting was done as described (25). Briefly, cells were washed in PBS and lysed with a buffer containing 50 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, and 1% NP40 supplemented with Complete protease inhibitor cocktail (Roche Diagnostics).…”

Section: Methodsmentioning

confidence: 99%

Noninvasive Imaging of Apoptosis and Its Application in Cancer Therapeutics

Coppola

Ross

Rehemtulla³

2008

Clinical Cancer Research

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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.

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

Cited by 36 publications

References 55 publications

Inhibition of Furin/Proprotein Convertase-catalyzed Surface and Intracellular Processing by Small Molecules

Inhibition of Furin/Proprotein Convertase-catalyzed Surface and Intracellular Processing by Small Molecules

Synthetic Small-Molecule Prohormone Convertase 2 Inhibitors

Noninvasive Imaging of Apoptosis and Its Application in Cancer Therapeutics

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