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1.  Applications of Molecular Imaging 
Today molecular imaging technologies play a central role in clinical oncology. The use of imaging techniques in early cancer detection, treatment response and new therapy development is steadily growing and has already significantly impacted clinical management of cancer. In this chapter we will overview three different molecular imaging technologies used for the understanding of disease biomarkers, drug development, or monitoring therapeutic outcome. They are (1) optical imaging (bioluminescence and fluorescence imaging) (2) magnetic resonance imaging (MRI), and (3) nuclear imaging (e.g, single photon emission computed tomography (SPECT) and positron emission tomography (PET)). We will review the use of molecular reporters of biological processes (e.g. apoptosis and protein kinase activity) for high throughput drug screening and new cancer therapies, diffusion MRI as a biomarker for early treatment response and PET and SPECT radioligands in oncology.
PMCID: PMC4327770  PMID: 21075334
2.  DCE and DW-MRI Monitoring of Vascular Disruption following VEGF-Trap Treatment of a Rat Glioma Model 
NMR in biomedicine  2011;25(7):935-942.
Vascular-targeted therapies have shown promise as adjuvant cancer treatment. As these agents undergo clinical evaluation, sensitive imaging biomarkers are need to assess drug target interaction and treatment response. In this study, dynamic contrast enhanced MRI (DCE-MRI) and diffusion-weighted MRI (DW-MRI) were evaluated for detecting response of intracerebral 9L gliosarcomas to the antivascular agent VEGF-Trap, a fusion protein designed to bind all forms of Vascular Endothelial Growth Factor-A (VEGF-A) and Placental Growth Factor (PGF). Rats with 9L tumors were treated twice weekly for two weeks with vehicle or VEGF-Trap. DCE- and DW-MRI were performed one day prior to treatment initiation and one day following each administered dose. Kinetic parameters (Ktrans: volume transfer constant, kep: efflux rate constant from extravascular/extracellular space to plasma, and vp: blood plasma volume fraction) and the apparent diffusion coefficient (ADC) over the tumor volumes were compared between groups. A significant decrease in kinetic parameters was observed 24 hours following the first dose of VEGF-Trapin treated versus control animals (p<0.05) and was accompanied by a decline in ADC values. In addition to the significant hemodynamic effect, VEGF-Trap treated animals exhibited significantly longer tumor doubling times (p<0.05) compared to the controls. Histological findings were found to support imaging response metrics. In conclusion, kinetic MRI parameters and change in ADC have been found to serve as sensitive and early biomarkers of VEGF-Trapanti-vascular targeted therapy.
PMCID: PMC4307830  PMID: 22190279
anti-angiogenic therapy; VEGF-Trap; glioma; DCE-MRI; DW-MRI; hemodynamics; diffusion; preclinical
3.  Glycogen Synthase Kinase 3β in Pancreatic Cancer and its Implications in Chemotherapy and Radiation Therapy 
Pancreatic cancer is a highly lethal disease with a poor prognosis characterized by local and systemic disease progression. Both radiation and chemotherapy play important roles in the management of this disease. However, in order to improve standard therapy many molecularly targeted agents are being developed. Glycogen synthase kinase 3β (GSK3β) participates in a multitude of cellular processes and is a newly proposed therapeutic target in pancreatic cancer. This review will discuss both the oncogenic and tumor suppressor functions of GSK3β in pancreatic cancer with an emphasis on the roles of GSK3β in tumor cell survival and sensitivity to radiation and chemotherapy.
PMCID: PMC4059685  PMID: 24944842
GSK3β; Pancreatic cancer; c-Met; Radiation
4.  Inhibition of N-Linked Glycosylation Disrupts Receptor Tyrosine Kinase Signaling in Tumor Cells 
Cancer research  2008;68(10):3803-3809.
Receptor tyrosine kinases (RTK) are therapeutic targets for the treatment of malignancy. However, tumor cells develop resistance to targeted therapies through the activation of parallel signaling cascades. Recent evidence has shown that redundant or compensatory survival signals responsible for resistance are initiated by nontargeted glycoprotein RTKs coexpressed by the cell. We hypothesized that disrupting specific functions of the posttranslational machinery of the secretory pathway would be an effective strategy to target both primary and redundant RTK signaling. Using the N-linked glycosylation inhibitor, tunicamycin, we show that expression levels of several RTKS (EGFR, ErbB2, ErbB3, and IGF-IR) are exquisitely sensitive to inhibition of N-linked glycosylation. Disrupting this synthetic process reduces both cellular protein levels and receptor activity in tumor cells through retention of the receptors in the endoplasmic reticulum/Golgi compartments. Using U251 glioma and BXPC3 pancreatic adenocarcinoma cell lines, two cell lines resistant to epidermal growth factor receptor–targeted therapies, we show that inhibiting N-linked glycosylation markedly reduces RTK signaling through Akt and radiosensitizes tumor cells. In comparison, experiments in nontransformed cells showed neither a reduction in RTK-dependent signaling nor an enhancement in radiosensitivity, suggesting the potential for a therapeutic ratio between tumors and normal tissues. This study provides evidence that enzymatic steps regulating N-linked glycosylation are novel targets for developing approaches to sensitize tumor cells to cytotoxic therapies.
PMCID: PMC3696581  PMID: 18483264
5.  Molecular Imaging of N-linked Glycosylation Suggests Glycan Biosynthesis is a Novel Target for Cancer Therapy 
Redundant receptor tyrosine kinase (RTK) signaling is a mechanism for therapeutic resistance to EGFR inhibition. A strategy to reduce parallel signaling by co-expressed RTKs is inhibition of N-linked glycosylation (NLG), an endoplasmic reticulum (ER) co-translational protein modification required for receptor maturation and cell surface expression. We therefore investigated the feasibility of blocking NLG in vivo to reduce over-expression of RTKs.
Experimental design
We developed a model system to dynamically monitor NLG in vitro and in vivo using bioluminescent imaging techniques. Functional imaging of NLG is accomplished with a luciferase reporter (ER-LucT) modified for ER-translation and glycosylation. After in vitro validation, this reporter was integrated with D54 glioma xenografts to perform non-invasive imaging of tumors, and inhibition of NLG was correlated with RTK protein levels and tumor growth.
The ER-LucT reporter demonstrates the ability to sensitively and specifically detect NLG inhibition. Using this molecular imaging approach we performed serial imaging studies to determine safe and efficacious in vivo dosing of the GlcNAc-1-phosphotransferase inhibitor, tunicamycin, which blocks N-glycan precursor biosynthesis. Molecular analyses of tunicamycin treated tumors showed reduced levels of EGFR and Met, two RTKs over-expressed in gliomas. Furthermore, D54 and U87MG glioma xenograft tumor experiments demonstrated significant reductions in tumor growth following NLG inhibition and radiation therapy, consistent with an enhancement in tumor radiosensitivity.
This study suggests NLG inhibition is a novel therapeutic strategy for targeting EGFR and RTK signaling in both gliomas and other malignant tumors.
PMCID: PMC3413408  PMID: 20413434
Glycosylation; Radiation; EGFR; Met
6.  Molecular Imaging of c-Met Kinase Activity 
Analytical Biochemistry  2011;412(1):1-8.
The receptor tyrosine kinase c-Met and its ligand, hepatocyte growth factor/scatter factor (HGF/SF) modulate signaling cascades implicated in cellular proliferation, survival, migration, invasion, and angiogenesis. Therefore, dysregulation of HGF/c-Met signaling can compromise the cellular capacity to moderate these activities, and lead to tumorigenesis, metastasis, and therapeutic resistance in various human malignancies. To facilitate studies investigating HGF/cMet receptor coupling or c-Met signaling events in real time and in living cells and animals, we here describe a genetically engineered reporter wherein bioluminescence can be used as a surrogate for c-Met tyrosine kinase activity. C-Met kinase activity in cultured cells and tumor xenografts was monitored quantitatively and dynamically in response to the activation or inhibition of the HGF/c-Met signaling pathway. Treatment of tumor bearing animals with a c-Met inhibitor and the HGF neutralizing antibody stimulated the reporter’s bioluminescence activity in a dose dependent manner and led to a regression of U-87 MG tumor xenografts. Results obtained from these studies provide unique insights into the pharmacokinetics and pharmacodynamics of agents that modulate c-Met activity and validate c-Met as a target for human glioblastoma therapy.
PMCID: PMC3265038  PMID: 21276769
c-Met; non-invasive molecular imaging; bioluminescence; kinase activity
7.  Targeted Imaging and Therapy of Brain Cancer using Theranostic Nanoparticles 
Molecular Pharmaceutics  2010;7(6):1921-1929.
The past decade has seen momentous development in brain cancer research in terms of novel imaging-assisted surgeries, molecularly targeted drug-based treatment regimens or adjuvant therapies and in our understanding of molecular footprints of initiation and progression of malignancy. However, mortality due to brain cancer has essentially remained unchanged in the last three decades. Thus, paradigm-changing diagnostic and therapeutic reagents are urgently needed. Nanotheranostic platforms are powerful tools for imaging and treatment of cancer. Multifunctionality of these nanovehicles offers a number of advantages over conventional agents. These include targeting to a diseased site thereby minimizing systemic toxicity, the ability to solubilize hydrophobic or labile drugs leading to improved pharmacokinetics and their potential to image, treat and predict therapeutic response. In this article, we will discuss the application of newer theranostic nanoparticles in targeted brain cancer imaging and treatment.
PMCID: PMC3291122  PMID: 20964352
Nanoparticle; MRI; Photodynamic therapy; F3 and RGD peptides; toxicity; cancer therapy
8.  Assessment of Multi-Exponential Diffusion Features as MRI Cancer Therapy Response Metrics 
The aim of this study was to empirically test the effect of chemotherapy-induced tissue changes in a glioma model as measured by several diffusion indices calculated from non-monoexponential formalisms over a wide range of b-values. We also compared these results to the conventional two-point apparent diffusion coefficient (ADC) calculation using nominal b-values.
Diffusion weighted imaging was performed over an extended range of b-values (120–4000s/mm2) on intracerebral rat 9L gliomas prior to and following a single dose of 1,3-bis(2-chloroethyl)-1-nitrosourea. Diffusion indices from three formalisms of DW signal decay ((a) two-point analytical calculation using either low or high b-values, (b) a stretched exponential formalism and (c) a biexponential fit) were tested for responsiveness to therapy-induced differences between control and treated groups.
Diffusion indices sensitive to “fast diffusion” produced the largest response to treatment, which resulted in significant differences between groups. These trends were not observed for “slow diffusion” indices. Although the highest rate of response was observed from the biexponential formalism, this was not found to be significantly different from the conventional monoexponential ADC method. In conclusion, parameters from the more complicated non-monoexponential formalisms did not provide additional sensitivity to treatment response in this glioma model beyond that observed from the two-point conventional monoexponential ADC method.
PMCID: PMC2965786  PMID: 20860004
9.  Molecular Imaging of GSK3β and CK1α kinases 
Analytical biochemistry  2010;405(2):246-254.
Glycogen synthase kinase-3 (GSK3 β) and casein kinase-1 alpha (CK-1α) are multifunctional kinases that play critical role in the regulation of a number of cellular processes. In spite of their importance, molecular imaging tools for non-invasive and real-time monitoring of their kinase activities have not been devised. Here, we report development of bioluminescent GSK3β and CK-1α reporter (BGCR) based on firefly luciferase complementation. Treatment of SW620 cells stably expressing the reporter with inhibitors of GSK3β (SB415286 and LiCl) or CK1α (CKI-7) resulted in dose and time dependent increase in BGCR activity which were validated using western blotting. No increase in bioluminescence was observed in case of S37A mutant (GSK3β inhibitors) or with S45A mutant (CKI-7) demonstrating the specificity of the reporter. Imaging of mice tumor xenograft generated with BGCR expressing SW620 cells following treatment with LiCl showed unique oscillations in GSK3β activity which were corroborated by phospho-GSK3β immunoblotting. Taken together, BGCR is novel molecular imaging tool that reveals unique insight into GSK3β and CK1α kinase activities and may provide powerful tool in experimental therapeutics for rapid optimization of dose and schedule of targeted therapies and for monitoring therapeutic response.
PMCID: PMC2922438  PMID: 20561505
Molecular imaging; GSK3 β; CK1α; split luciferase; reporter
10.  Molecular imaging of protein kinases 
Cell cycle (Georgetown, Tex.)  2007;7(3):314-317.
Protein kinases are important regulators of signal-transduction pathways. Dysregulated kinase activity is observed in a variety of human diseases such as cancer, making them targets for the development of molecular therapies. Identification of new drugs is greatly aided by molecular imaging tools which enable real time, non-invasive, dynamic and quantitative imaging of kinase activity in vivo. We have recently described a new reporter platform based on conformation dependent complementation of firefly luciferase to monitor serine/threonine kinase (Akt) activity. The reporter system provides unique insights into the pharmacokinetics and pharmacodynamics of drugs that modulate kinase activity in living subjects and also provide a platform for cell based high-throughput drug screening for modulators of kinase activity.
PMCID: PMC3160745  PMID: 18235250
protein kinase; non-invasive imaging; kinase activity
11.  Synthesis and Investigation of a Radioiodinated F3 Peptide Analog as a SPECT Tumor Imaging Radioligand 
PLoS ONE  2011;6(7):e22418.
A radioiodinated derivative of the tumor-homing F3 peptide, (N-(2-{3-[125I]Iodobenzoyl}aminoethyl)maleimide-F3Cys peptide, [125I]IBMF3 was developed for investigation as a SPECT tumor imaging radioligand. For this purpose, we custom synthesized a modified F3 peptide analog (F3Cys) incorporating a C-terminal cysteine residue for site-specific attachment of a radioiodinated maleimide conjugating group. Initial proof-of-concept Fluorescence studies conducted with AlexaFluor 532 C5 maleimide-labeled F3Cys showed distinct membrane and nuclear localization of F3Cys in MDA-MB-435 cells. Additionally, F3Cys conjugated with NIR fluorochrome AlexaFluor 647 C2 maleimide demonstrated high tumor specific uptake in melanoma cancer MDA-MB-435 and lung cancer A549 xenografts in nude mice whereas a similarly labeled control peptide did not show any tumor uptake. These results were also confirmed by ex vivo tissue analysis. No-carrier-added [125I]IBMF3 was synthesized by a radioiododestannylation approach in 73% overall radiochemical yield. In vitro cell uptake studies conducted with [125I]IBMF3 displayed a 5-fold increase in its cell uptake at 4 h when compared to controls. SPECT imaging studies with [125I]IBMF3 in tumor bearing nude mice showed clear visualization of MDA-MB-435 xenografts on systemic administration. These studies demonstrate a potential utility of F3 peptide-based radioligands for tumor imaging with PET or SPECT techniques.
PMCID: PMC3139646  PMID: 21811604
12.  A transgenic mouse for imaging caspase-dependent apoptosis within the skin 
Apoptosis is an essential process for the maintenance of normal physiology. The ability to non-invasively image apoptosis in living animals would provide unique insights into its role in normal and disease processes. Herein, a recombinant reporter consisting of beta galactosidase gene flanked by two ER regulatory domains and intervening DEVD sequences was constructed to serve as a tool for invivo assessment of apoptotic activity. The results demonstrate that when expressed in its intact form, the hybrid reporter had undetectable LACZ activity. Caspase 3 activation in response to an apoptotic stimulus resulted in cleavage of the reporter, and thereby reconstitution of LACZ activity. Enzymatic activation of the reporter during an apoptotic event enabled non-invasive measurement of LACZ activity in living cells, which correlated with traditional measures of apoptosis in a dose and time dependent manner. Furthermore, using a near-infrared fluorescent substrate of beta galactosidase (DDAOG), non-invasive invivo imaging of apoptosis was achieved using a xenograft tumor model in response to pro-apoptotic therapy. Lastly, a transgenic mouse model was developed expressing the ER-LACZ-ER reporter within the skin. This reporter and transgenic mouse could serve as a unique tool for the study of apoptosis in living cells and animals especially in context of skin biology.
PMCID: PMC3097416  PMID: 20357819
Molecular imaging; proteaseiImaging; caspase 3; beta galactosidase
13.  High Throughput Molecular Imaging for the Identification of FADD Kinase Inhibitors 
Journal of biomolecular screening  2010;15(9):1063-1070.
Fas-Associated protein with Death Domain (FADD) was originally reported as a pro-apoptotic adaptor molecule that mediates receptor induced apoptosis. Recent studies have revealed a potential role of FADD in NF-κB activation, embryogenesis, and cell cycle regulation and proliferation. Over-expression of FADD and its phosphorylation have been associated with the transformed phenotype in many cancers and is therefore a potential target for therapeutic intervention. In an effort to delineate signaling events that lead to FADD phosphorylation and to identify novel compounds that impinge on this pathway, we developed a cell based reporter for FADD kinase activity. The reporter assay, optimized for a high throughput screen (HTS), measures bioluminescence in response to modulation of FADD kinase activity in live cells. In addition, the potential use of the reporter cell line in the rapid evaluation of pharmacologic properties of HTS hits in mouse models has been demonstrated.
PMCID: PMC3108567  PMID: 20855560
FADD; phosphorylation; non-invasive molecular imaging; bioluminescence; kinase activity
14.  Molecular Imaging of Akt Enables Early Prediction of Response to Molecular Targeted Therapy12 
Translational Oncology  2011;4(3):122-125.
Development of noninvasive, real-time molecular imaging tools to assess responsiveness of a given therapy may be a critical component of the success of individualized therapy approach for patients. Toward this, we have previously developed and validated molecular sensors for Akt and caspase-3 activity, and in this report, we have explored the utility of these reporters in assessing the responsiveness of tumors to a combination of gemcitabine (Gem) and cetuximab (Cet) delivered in two opposite schedules. We found that human head and neck cancer (UMSCC1) xenografts responded significantly better in a schedule where cetuximab was administered after gemcitabine when compared with the schedule of cetuximab followed by gemcitabine. Wilcoxon two-sample tests suggested that the difference in tumor volumes in two schedules became significant on day 7 (P > .05 on day 4, and P < .05 on days 7 and 10), and the difference in activity of Akt in two schedules became significant on day 4 (P < .05 on days 4, 6, and 10). Using Akt reporter activity and cubic spline interpolation, the distinction between the two schedules could be detected 2 days before using the tumor volume, suggesting that molecular imaging of Akt may allow early prediction of therapy responsiveness. We did not observe a significant difference between the two schedules in the caspase-3 activity. In summary, this proof-of-concept study provides a basis for using molecular imaging of Akt as an early indicator of therapeutic efficacy.
PMCID: PMC3104693  PMID: 21633667
15.  Evaluation of Treatment Associated Inflammatory Response on Diffusion Weighted-MRI and FDG-PET Imaging Biomarkers 
Functional imaging biomarkers of cancer treatment response offer the potential for early determination of outcome through assessment of biochemical, physiological, and micro-environmental readouts. Cell death may result in an immunological response thus complicating interpretation of biomarker readouts. This study evaluated the temporal impact of treatment-associated inflammatory activity on diffusion-MRI and FDG-PET imaging biomarkers to delineate the effects of the inflammatory response on imaging readouts.
Experimental Design
Rats with intracerebral 9L gliosarcomas were separated into four groups consisting of control, an immunosuppressive agent dexamethasone (Dex), 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), and BCNU+Dex (BCNU+Dex). Animals were imaged using diffusion-weighted MRI and FDG-PET at 0, 3 and 7 days post-treatment.
In the BCNU and BCNU+Dex treated animal groups, diffusion values increased progressively over the 7 day study period to about 23% over baseline. FDG %SUV decreased at day 3 (−30.9%) but increased over baseline levels at day 7 (+20.1%). FDG-PET of BCNU+Dex treated animals were found to have %SUV reductions of −31.4% and −24.7% at days 3 and 7, respectively following treatment. Activated macrophages were observed on day 7 in the BCNU treatment group with much fewer found in the BCNU+Dex group.
Results revealed treatment-associated inflammatory response following tumor therapy resulted in accentuation of tumor diffusion response along with a corresponding increase in tumor FDG uptake due to the presence of glucose-consuming activated macrophages. The dynamics and magnitude of potential inflammatory response should be considered when interpreting imaging biomarker results.
PMCID: PMC2843556  PMID: 20160061
Diffusion; MRI; ADC; positron emission tomography; FDG; brain tumor
16.  Novel molecular imaging platform for monitoring oncological kinases 
Recent advances in oncology have lead to identification of a plethora of alterations in signaling pathways that are critical to oncogenesis and propagation of malignancy. Among the biomarkers identified, dysregulated kinases and associated changes in signaling cascade received the lion's share of scientific attention and have been under extensive investigations with goal of targeting them for anti-cancer therapy. Discovery of new drugs is immensely facilitated by molecular imaging technology which enables non-invasive, real time, dynamic imaging and quantification of kinase activity. Here, we review recent development of novel kinase reporters based on conformation dependent complementation of firefly luciferase to monitor kinase activity. Such reporter system provides unique insights into the pharmacokinetics and pharmacodynamics of drugs that modulate kinase signaling and have a huge potential in drug discovery, validation, and drug-target interactions.
PMCID: PMC2914645  PMID: 20615241
17.  Curcumin Promotes Apoptosis, Increases Chemosensitivity, and Inhibits Nuclear Factor κB in Esophageal Adenocarcinoma1 
Translational Oncology  2010;3(2):99-108.
The transcription factor, nuclear factor κB (NF-κB), plays a central role as a key mediator of cell survival and proliferation, and its activation may confer increased tumor chemoresistance. Curcumin, an orally available naturally occurring compound, has been shown to inhibit NF-κB and has a potential role in cancer chemoprevention. We investigated the effects of curcumin on NF-κB activity, on cell viability, and as a chemosensitizing agent with 5-fluorouracil (5-FU) or cisplatin (CDDP) in esophageal adenocarcinoma (EAC). Oligonucleotide microarray analysis of 46 cases, consisting of Barrett metaplasia, low-grade dysplasia, high-grade dysplasia and EAC, showed increased expression of NF-κB and IκB kinase subunits and decreased effector caspase expression in EAC compared with Barrett metaplasia. Stromal expression of both IκB and phospho-IκB was detected in several EAC samples by tissue microarray analysis. Curcumin alone inhibited NF-κB activity and induced apoptosis in both Flo-1 and OE33 EAC cell lines as determined by Western blot analysis, NF-κB reporter assays, and Caspase-Glo 3/7 assays. It also increased 5-FU- and CDDP-induced apoptosis in both cell lines. These data suggest that activation of NF-κB and inhibition of apoptosis may play a role in the progression from Barrett metaplasia to EAC. In addition, curcumin, a well-known inhibitor of NF-κB activity, was shown to increase apoptosis and enhance both 5-FU- and CDDP-mediated chemosensitivity, suggesting that it may have potential application in the therapy of patients with EAC.
PMCID: PMC2847317  PMID: 20360934
18.  Enhancing Akt imaging through targeted reporter expression 
Molecular imaging  2008;7(4):168-174.
The serine/threonine kinase PKB/Akt is a key mediator of survival and resistance to cancer therapy. Pharmacological inhibition of Akt and its biologic sequelae may significantly impact the treatment of cancer. The use of molecular imaging technologies has contributed significantly to drug discovery research with emphasis on drug efficacy, on the mechanism of action and on target validation studies. We have constructed a genetically engineered hybrid bioluminescent reporter molecule (BAR) which reports on Akt serine/throenine kinase activity. Based on the fact that Akt is recruited to the plasma membrane upon activation, we here describe a modified version of this reporter molecule (MyrPalm-BAR) that is membrane bound and whose bioluminescence activity can be used to monitor Akt activity at the cell membrane. Utilizing changes in Akt activation status with small molecule inhibitors of Akt we demonstrated that the membrane targeted Akt reporter was more sensitive and quantitative. In addition, inhibition of upstream signaling kinases such as EGFR and PI-3-Kinase activity resulted in changes in Akt activity which was quantitatively monitored by bioluminescence imaging. Based on these results we propose that the membrane associated Akt reporter may be better suited for identification of novel compounds that modulate the Akt pathway by high throughput screening.
PMCID: PMC2659640  PMID: 19123987
Akt; non-invasive molecular imaging; bioluminescence; kinase activity
19.  Identification of inhibitors using a cell based assay for monitoring golgi-resident protease activity 
Analytical biochemistry  2007;364(1):19-29.
Non-invasive 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, upon 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, and in activation of anthrax, pseudomonas, and diphtheria toxins. This technology was adapted for high throughput screening of 30,000 compound small molecule libraries, leading to identification of furin inhibitors. Further, this strategy was utilized to identify inhibitors of another Golgi protease, the β-site APP-cleaving enzyme (BACE). BACE cleavage of the amyloid precursor protein leads to formation of the Aβ peptide, a key event that leads to Alzheimer’s disease. In conclusion, we describe a customizable, non-invasive technology for real time assessment of Golgi protease activity used to identify inhibitors of furin and BACE.
PMCID: PMC1995463  PMID: 17316541
Furin; BACE; TGN; Prohormone Convertase; Alzheimers; SEAP; Alkaline Phosphatase; NSAIDs
20.  A Small-Molecule Furin Inhibitor Inhibits Cancer Cell Motility and Invasiveness1 
Neoplasia (New York, N.Y.)  2008;10(4):363-370.
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 Ki and IC50 of 12µM. 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).
PMCID: PMC2288536  PMID: 18392131

Results 1-20 (20)