The proteasome is a new, interesting target in cancer drug therapy, and the proteasome inhibitor bortezomib has shown an effect in myeloma patients. It is of interest to efficiently discover and evaluate new proteasome inhibitors. The authors describe the development of an image-based screening assay for the identification of compounds with proteasome-inhibiting activity. The stably transfected human embryo kidney cell line HEK 293 ZsGreen Proteasome Sensor Cell Line expressing the ZsProSensor-1 fusion protein was used for screening and evaluation of proteasome inhibitors. Inhibition of the proteasome leads to accumulation of the green fluorescent protein ZsGreen, which is measured in the ArrayScan® High Content Screening system, in which cell morphology is studied simultaneously. When screening the LOPAC1280 substance library, several compounds with effect on the proteasome were found; among the hits were disulfiram and ammonium pyrrolidinedithiocarbamate (PDTC). Cytotoxic analysis of disulfiram and PDTC showed that the compounds induced cytotoxicity in the myeloma cell line RPMI 8226. The average Z' value for the assay was 0.66. The results indicate that the assay rapidly identifies new proteasome-inhibiting substances, and it will be further used as a tool for image-based screening of other chemically diverse compound libraries.
Peters JM, Cejka Z, Harris JR, Kleinschmidt JA, Baumeister W: Structural features of the 26 S proteasome complex. J Mol Biol1993; 234:932-937.
2.
Adams J: The proteasome: a suitable antineoplastic target. Nat Rev Cancer2004;4:349-360.
3.
Richardson PG, Barlogie B, Berenson J, Singhal S, Jagannath S, Irwin D, et al: A phase 2 study of bortezomib in relapsed, refractory myeloma. N Engl J Med2003;348:2609-2617.
4.
Lang P, Yeow K, Nichols A, Scheer A: Cellular imaging in drug discovery. Nat Rev Drug Discov2006;5:343-356.
5.
Ramm P: Image-based screening: a technology in transition. Curr Opin Biotechnol2005;16:41-48.
6.
Gasparri F, Mariani M, Sola F, Galvani A: Quantification of the proliferation index of human dermal fibroblast cultures with the ArrayScan highcontent screening reader. J Biomol Screen2004;9:232-243.
7.
Lovborg H, Nygren P, Larsson R: Multiparametric evaluation of apoptosis: effects of standard cytotoxic agents and the cyanoguanidine CHS 828. Mol Cancer Ther2004;3:521-526.
8.
Murakami Y, Matsufuji S, Kameji T, Hayashi S, Igarashi K, Tamura T, et al: Ornithine decarboxylase is degraded by the 26S proteasome without ubiquitination. Nature1992;360:597-599.
9.
Turpin PH, Yu F, inventors; Clontech Lab Inc., assignee. Sensitive proteasome sensor constructs and methods for their design and use. European patent CA20042514066 20040126. August 12, 2004. Retrieved from http://v3.espacenet.com/textdoc?DB=EPODOC&IDX=CA2514066.
10.
Rickardson L, Fryknas M, Haglund C, Lovborg H, Nygren P, Gustafsson MG, et al: Screening of an annotated compound library for drug activity in a resistant myeloma cell line. Cancer Chemother Pharmacol2006;58: 749-758.
11.
Larsson R, Nygren P, Ekberg M, Slater L: Chemotherapeutic drug sensitivity testing of human leukemia cells in vitro using a semiautomated fluorometric assay. Leukemia1990;4:567-571.
12.
Zhang JH, Chung TD, Oldenburg KR: A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen1999;4:67-73.
13.
Bridges JP, Xu Y, Na CL, Wong HR, Weaver TE: Adaptation and increased susceptibility to infection associated with constitutive expression of misfolded SP-C. J Cell Biol2006;172:395-407.
14.
Giuliano KA, DeBiasio RL, Dunlay RT, Gough A, Volosky JM, Zock J, et al: High-content screening: a new approach to easing key bottlenecks in the drug discovery process. J Biomol Screen1997;2:249-259.
15.
Wilson CJ, Si Y, Thompsons CM, Smellie A, Ashwell MA, Liu JF, et al: Identification of a small molecule that induces mitotic arrest using a simplified high-content screening assay and data analysis method. J Biomol Screen2006;11:21-28.
16.
Ramm P, Alexandrov Y, Cholewinski A, Cybuch Y, Nadon R, Soltys BJ: Automated screening of neurite outgrowth. J Biomol Screen2003;8:7-18.
17.
Bertelsen M, Sanfridson A: Inflammatory pathway analysis using a high content screening platform. Assay Drug Dev Technol2005;3:261-271.
18.
Lazo JS, Nemoto K, Pestell KE, Cooley K, Southwick EC, Mitchell DA, et al: Identification of a potent and selective pharmacophore for Cdc25 dual specificity phosphatase inhibitors. Mol Pharmacol2002;61:720-728.
19.
Lovborg H, Oberg F, Rickardson L, Gullbo J, Nygren P, Larsson R: Inhibition of proteasome activity, nuclear factor-κ B translocation and cell survival by the antialcoholism drug disulfiram. Int J Cancer2006;118:1577-1580.
20.
Chen D, Peng F, Cui QC, Daniel KG, Orlu S, Liu J, et al: Inhibition of prostate cancer cellular proteasome activity by a pyrrolidine dithiocarbamatecopper complex is associated with suppression of proliferation and induction of apoptosis. Front Biosci2005;10:2932-2939.
21.
Daniel KG, Chen D, Orlu S, Cui QC, Miller FR, Dou QP: Clioquinol and pyrrolidine dithiocarbamate complex with copper to form proteasome inhibitors and apoptosis inducers in human breast cancer cells. Breast Cancer Res2005;7:R897-R908.
22.
Chung KC, Park JH, Kim CH, Lee HW, Sato N, Uchiyama Y, et al: Novel biphasic effect of pyrrolidine dithiocarbamate on neuronal cell viability is mediated by the differential regulation of intracellular zinc and copper ion levels, NF-κB, and MAP kinases. J Neurosci Res2000;59:117-125.
23.
Stein RL, Melandri F, Dick L: Kinetic characterization of the chymotryptic activity of the 20S proteasome. Biochemistry1996;35:3899-3908.
24.
Achanta G, Modzelewska A, Feng L, Khan SR, Huang P: A boronicchalcone derivative exhibits potent anticancer activity through inhibition of the proteasome. Mol Pharmacol2006;70:426-433.
25.
Vaziri SA, Hill J, Chikamori K, Grabowski DR, Takigawa N, Chawla-Sarkar M, et al: Sensitization of DNA damage-induced apoptosis by the proteasome inhibitor PS-341 is p53 dependent and involves target proteins 14-3-3sigma and survivin. Mol Cancer Ther2005;4:1880-1890.
26.
Kristjansdottir K, Rudolph J: Cdc25 phosphatases and cancer. Chem Biol2004;11:1043-1051.
27.
Glickman MH, Rubin DM, Coux O, Wefes I, Pfeifer G, Cjeka Z, et al: A subcomplex of the proteasome regulatory particle required for ubiquitinconjugate degradation and related to the COP9-signalosome and eIF3. Cell1998;94:615-623.
28.
Groettrup M, Soza A, Eggers M, Kuehn L, Dick TP, Schild H, et al: A role for the proteasome regulator PA28α in antigen presentation. Nature1996;381:166-168.
29.
Kebabian JW, Britton DR, DeNinno MP, Perner R, Smith L, Jenner P, et al: A-77636: a potent and selective dopamine D1 receptor agonist with antiparkinsonian activity in marmosets. Eur J Pharmacol1992;229:203-209.
30.
Zafar KS, Siegel D, Ross D: A potential role for cyclized quinones derived from dopamine, DOPA, and 3,4-dihydroxyphenylacetic acid in proteasomal inhibition. Mol Pharmacol2006;70:1079-1086.
31.
McNaught KS, Olanow CW: Protein aggregation in the pathogenesis of familial and sporadic Parkinson's disease. Neurobiol Aging2006;27:530-545.
32.
Cen D, Brayton D, Shahandeh B, Meyskens FL Jr., Farmer PJ: Disulfiram facilitates intracellular Cu uptake and induces apoptosis in human melanoma cells. J Med Chem2004;47:6914-6920.
33.
Wang W, McLeod HL, Cassidy J: Disulfiram-mediated inhibition of NF-κB activity enhances cytotoxicity of 5-fluorouracil in human colorectal cancer cell lines. Int J Cancer2003;104:504-511.
34.
Wickström M, Danielsson K, Rickardson L, Gullbo J, Nygren P, Isaksson A, et al: Pharmacological profiling of disulfiram using human tumor cell lines and human tumor cells from patients. Biochem Pharmacol; in press.
35.
Bogyo M: Screening for selective small molecule inhibitors of the proteasome using activity-based probes. Methods Enzymol2005;399:609-622.
36.
Vivier M, Jarrousse AS, Bouchon B, Galmier MJ, Auzeloux P, Sauzieres J, et al: Preliminary studies of new proteasome inhibitors in the tumor targeting approach: synthesis and in vitro cytotoxicity. J Med Chem2005;48: 6731-6740.
