Original articles
Alexander J. Trevarton, Yan Zhou, Dehua Yang, Gordon W. Rewcastle, Jack U. Flanagan, Antony Braithwaite, Peter R. Shepherd, Cristin G. Print, Ming-Wei Wang, Annette Lasham. Orthogonal assays for the identification of inhibitors of the single-stranded nucleic acid binding protein YB-1[J]. Acta Pharmaceutica Sinica B, 2019, 9(5): 997-1007

Orthogonal assays for the identification of inhibitors of the single-stranded nucleic acid binding protein YB-1
Alexander J. Trevartona, Yan Zhoub, Dehua Yangb, Gordon W. Rewcastlec,d, Jack U. Flanaganc,d, Antony Braithwaitee,f, Peter R. Shepherda,d, Cristin G. Printa,d, Ming-Wei Wangb,g, Annette Lashama,d
a Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand;
b The National Center for Drug Screening and the CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences(CAS), Shanghai 201203, China;
c Auckland Cancer Society Research Centre, Auckland, New Zealand;
d Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland, New Zealand;
e Department of Pathology, Dunedin School of Medicine, University of Otago, Dunedin, New Zealand;
f Maurice Wilkins Centre for Molecular Biodiscovery, University of Otago, Dunedin, New Zealand;
g School of Pharmacy, Fudan University, Shanghai 201203, China
We have previously shown that high expression of the nucleic acid binding factor YB-1 is strongly associated with poor prognosis in a variety of cancer types. The 3-dimensional protein structure of YB-1 has yet to be determined and its role in transcriptional regulation remains elusive. Drug targeting of transcription factors is often thought to be difficult and there are very few published high-throughput screening approaches. YB-1 predominantly binds to single-stranded nucleic acids, adding further difficulty to drug discovery. Therefore, we have developed two novel screening assays to detect compounds that interfere with the transcriptional activation properties of YB-1, both of which may be generalizable to screen for inhibitors of other nucleic acid binding molecules. The first approach is a cellbased luciferase reporter gene assay that measures the level of activation of a fragment of the E2F1 promoter by YB-1. The second approach is a novel application of the AlphaScreen system, to detect interference of YB-1 interaction with a single-stranded DNA binding site. These complementary assays examine YB-1 binding to two discrete nucleic acid sequences using two different luminescent signal outputs and were employed sequentially to screen 7360 small molecule compounds leading to the identification of three putative YB-1 inhibitors.
Key words:    Cancer    YB-1    Luciferase    AlphaScreen    Transcription factor    Single-stranded DNA   
Received: 2018-10-03     Revised: 2018-12-26
DOI: 10.1016/j.apsb.2018.12.011
Funds: We thank Yang Feng, Xiaoqing Cai, Margareta Sutija, Sandra Fitzgerald, Puja Bhatia and Ji Wu for technical assistance. This work was partially supported by grants from Health Research Council of New Zealand (IRF213-China, 13-1019 to PS, CP, AL and AB), the Ministry of Science and Technology of China (2014DFG32200 to M-WW), Shanghai Science and Technology Development Fund (15DZ2291600 to M-WW, China) and the Thousand Talents Program in China ([2011]166 to M-WW).
Corresponding author: Cristin G. Print, Ming-Wei Wang, Annette Lasham     Email:c.print@auckland.ac.nz;mwwang@simm.ac.cn;a.lasham@auckland.ac.nz
Author description:
PDF(KB) Free
Alexander J. Trevarton
Yan Zhou
Dehua Yang
Gordon W. Rewcastle
Jack U. Flanagan
Antony Braithwaite
Peter R. Shepherd
Cristin G. Print
Ming-Wei Wang
Annette Lasham

1. MacDonald GH, Itoh-Lindstrom Y, Ting JP. The transcriptional regulatory protein, YB-1, promotes single-stranded regions in the DRA promoter. J Biol Chem 1995;270:3527-33.
2. Evdokimova V, Ruzanov P, Imataka H, Raught B, Svitkin Y, Ovchinnikov LP, et al. The major mRNA-associated protein YB-1 is a potent 50 cap-dependent mRNA stabilizer. EMBO J 2001;20:5491-502.
3. Svitkin YV, Evdokimova VM, Brasey A, Pestova TV, Fantus D, Yanagiya A, et al. General RNA-binding proteins have a function in poly(A)-binding protein-dependent translation. EMBO J 2009;28:58-68.
4. Yadav BS, Singh S, Shaw AK, Mani A. Structure prediction and docking-based molecular insights of human YB-1 and nucleic acid interaction. J Biomol Struct Dyn 2016;34:2561-80.
5. Lasham A, Print CG, Woolley AG, Dunn SE, Braithwaite AW. YB-1:oncoprotein, prognostic marker and therapeutic target?. Biochem J 2013;449:11-23.
6. Eliseeva IA, Kim ER, Guryanov SG, Ovchinnikov LP, Lyabin DN. Ybox-binding protein 1(YB-1) and its functions. Biochemistry 2011;76:1402-33.
7. Lasham A, Samuel W, Cao H, Patel R, Mehta R, Stern JL, et al. YB-1, the E2F pathway, and regulation of tumor cell growth. J Natl Cancer Inst 2012;104:133-46.
8. Janz M, Harbeck N, Dettmar P, Berger U, Schmidt A, Jurchott K, et al. Y-box factor YB-1 predicts drug resistance and patient outcome in breast cancer independent of clinically relevant tumor biologic factors HER2, uPA and PAI-1. Int J Cancer 2002;97:278-82.
9. Yoshimatsu T, Uramoto H, Oyama T, Yashima Y, Gu C, Morita M, et al. Y-box-binding protein-1 expression is not correlated with p53 expression but with proliferating cell nuclear antigen expression in non-small cell lung cancer. Anticancer Res 2005;25:3437-43.
10. Chatterjee M, Rancso C, Stuhmer T, Eckstein N, Andrulis M, Gerecke C, et al. The Y-box binding protein YB-1 is associated with progressive disease and mediates survival and drug resistance in multiple myeloma. Blood 2008;111:3714-22.
11. Lasham A, Mehta SY, Fitzgerald SJ, Woolley AG, Hearn JI, Hurley DG, et al. A novel EGR-1 dependent mechanism for YB-1 modulation of paclitaxel response in a triple negative breast cancer cell line. Int J Cancer 2016;139:1157-70.
12. Lee C, Dhillon J, Wang MY, Gao Y, Hu K, Park E, et al. Targeting YB-1 in HER-2 overexpressing breast cancer cells induces apoptosis via the mTOR/STAT3 pathway and suppresses tumor growth in mice. Cancer Res 2008;68:8661-6.
13. Schittek B, Psenner K, Sauer B, Meier F, Iftner T, Garbe C. The increased expression of Y box-binding protein 1 in melanoma stimulates proliferation and tumor invasion, antagonizes apoptosis and enhances chemoresistance. Int J Cancer 2007;120:2110-8.
14. Sinnberg T, Sauer B, Holm P, Spangler B, Kuphal S, Bosserhoff A, et al. MAPK and PI3K/AKT mediated YB-1 activation promotes melanoma cell proliferation which is counteracted by an autoregulatory loop. Exp Dermatol 2012;21:265-70.
15. Kosnopfel C, Sinnberg T, Sauer B, Busch C, Niessner H, Schmitt A, et al. YB-1 expression and phosphorylation regulate tumorigenicity and invasiveness in melanoma by influencing EMT. Mol Cancer Res 2018;16:1149-60.
16. Ladomery M, Sommerville J. A role for Y-box proteins in cell proliferation. Bioessays 1995;17:9-11.
17. Jurchott K, Bergmann S, Stein U, Walther W, Janz M, Manni I, et al. YB-1 as a cell cycle-regulated transcription factor facilitating cyclin A and cyclin B1 gene expression. J Biol Chem 2003;278:27988-96.
18. Alemasova EE, Naumenko KN, Kurgina TA, Anarbaev RO, Lavrik OI. The multifunctional protein YB-1 potentiates PARP1 activity and decreases the efficiency of PARP1 inhibitors. Oncotarget 2018;9:23349-65.
19. Guryanov SG, Filimonov VV, Timchenko AA, Melnik BS, Kihara H, Kutyshenko VP, et al. The major mRNP protein YB-1:structural and association properties in solution. Biochim Biophys Acta 2013;1834:559-67.
20. Kloks CP, Spronk CA, Lasonder E, Hoffmann A, Vuister GW, Grzesiek S, et al. The solution structure and DNA-binding properties of the cold-shock domain of the human Y-box protein YB-1. J Mol Biol 2002;316:317-26.
21. Wu XN, Huang YD, Li JX, Yu YF, Qian Z, Zhang C, et al. Structure-based design, synthesis, and biological evaluation of novel pyrimidinone derivatives as PDE9 inhibitors. Acta Pharm Sin B 2018;8:615-28.
22. Johnson DG, Ohtani K, Nevins JR. Autoregulatory control of E2F1 expression in response to positive and negative regulators of cell cycle progression. Genes Dev 1994;8:1514-25.
23. Cohen SB, Ma W, Valova VA, Algie M, Harfoot R, Woolley AG, et al. Genotoxic stress-induced nuclear localization of oncoprotein YB-1 in the absence of proteolytic processing. Oncogene 2010;29:403-10.
24. Horwitz EM, Maloney KA, Ley TJ. A human protein containing a "cold shock" domain binds specifically to H-DNA upstream from the human γ-globin genes. J Biol Chem 1994;269:14130-9.
25. Baell JB, Holloway GA. New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays. J Med Chem 2010;53:2719-40.
26. Baell JB. Broad coverage of commercially available lead-like screening space with fewer than 350,000 compounds. J Chem Inf Model 2013;53:39-55.
27. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 2001;25:402-8.
28. Zhang JH, Chung TD, Oldenburg KR. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 1999;4:67-73.
29. Xiao T, Liu R, Proud CG, Wang MW. A high-throughput screening assay for eukaryotic elongation factor 2 kinase inhibitors. Acta Pharm Sin B 2016;6:557-63.
30. Singh H, Clerc RG, LeBowitz JH. Molecular cloning of sequencespecific DNA binding proteins using recognition site probes. BioTechniques 1989;7:252-61.
31. Gong Z, Hu G, Li Q, Liu Z, Wang F, Zhang X, et al. Compound libraries:recent advances and their applications in drug discovery. Curr Drug Discov Technol 2017;14:216-28.
32. Eglen RM, Reisine T, Roby P, Rouleau N, Illy C, Bosse R, et al. The use of AlphaScreen technology in HTS:current status. Curr Chem Genom 2008;1:2-10.
33. Mukherjee S, Hanson AM, Shadrick WR, Ndjomou J, Sweeney NL, Hernandez JJ, et al. Identification and analysis of hepatitis C virus NS3 helicase inhibitors using nucleic acid binding assays. Nucleic Acids Res 2012;40:8607-21.
34. Chen CZ, Sobczak K, Hoskins J, Southall N, Marugan JJ, Zheng W, et al. Two high-throughput screening assays for aberrant RNA-protein interactions in myotonic dystrophy type 1. Anal Bioanal Chem 2012;402:1889-98.
35. Cassel JA, Blass BE, Reitz AB, Pawlyk AC. Development of a novel nonradiometric assay for nucleic acid binding to TDP-43 suitable for high-throughput screening using AlphaScreen technology. J Biomol Screen 2010;15:1099-106.
36. Mills NL, Shelat AA, Guy RK. Assay Optimization and screening of RNA-Protein interactions by AlphaScreen. J Biomol Screen 2007;12:946-55.
37. Khageh Hosseini S, Kolterer S, Steiner M, von Manstein V, Gerlach K, Trojan J, et al. Camptothecin and its analog SN-38, the active metabolite of irinotecan, inhibit binding of the transcriptional regulator and oncoprotein FUBP1 to its DNA target sequence FUSE. Biochem Pharmacol 2017;146:53-62.
38. Kretov DA, Curmi PA, Hamon L, Abrakhi S, Desforges B, Ovchinnikov LP, et al. mRNA and DNA selection via protein multimerization:YB-1 as a case study. Nucleic Acids Res 2015;43:9457-73.