Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

A combinatorial screen of the CLOUD uncovers a synergy targeting the androgen receptor


Approved drugs are invaluable tools to study biochemical pathways, and further characterization of these compounds may lead to repurposing of single drugs or combinations. Here we describe a collection of 308 small molecules representing the diversity of structures and molecular targets of all FDA-approved chemical entities. The CeMM Library of Unique Drugs (CLOUD) covers prodrugs and active forms at pharmacologically relevant concentrations and is ideally suited for combinatorial studies. We screened pairwise combinations of CLOUD drugs for impairment of cancer cell viability and discovered a synergistic interaction between flutamide and phenprocoumon (PPC). The combination of these drugs modulates the stability of the androgen receptor (AR) and resensitizes AR-mutant prostate cancer cells to flutamide. Mechanistically, we show that the AR is a substrate for γ-carboxylation, a post-translational modification inhibited by PPC. Collectively, our data suggest that PPC could be repurposed to tackle resistance to antiandrogens in prostate cancer patients.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Figure 1: The CLOUD.
Figure 2: A combinatorial HTS of the CLOUD uncovers the synergy between flutamide and PPC.
Figure 3: The combination of antiandrogens and vitamin K antagonists induces apoptosis of LNCaP prostate cancer cells.
Figure 4: The combination of flutamide and PPC induces AR degradation.
Figure 5: The AR is post-translationally modified by γ-glutamyl carboxylation.

Similar content being viewed by others

Accession codes

Primary accessions

Gene Expression Omnibus


  1. Paul, S.M. et al. How to improve R&D productivity: the pharmaceutical industry's grand challenge. Nat. Rev. Drug Discov. 9, 203–214 (2010).

    Article  CAS  PubMed  Google Scholar 

  2. Scannell, J.W., Blanckley, A., Boldon, H. & Warrington, B. Diagnosing the decline in pharmaceutical R&D efficiency. Nat. Rev. Drug Discov. 11, 191–200 (2012).

    Article  CAS  PubMed  Google Scholar 

  3. Ashburn, T.T. & Thor, K.B. Drug repositioning: identifying and developing new uses for existing drugs. Nat. Rev. Drug Discov. 3, 673–683 (2004).

    Article  CAS  PubMed  Google Scholar 

  4. Clavel, F. & Hance, A.J. HIV drug resistance. N. Engl. J. Med. 350, 1023–1035 (2004).

    Article  CAS  PubMed  Google Scholar 

  5. Holohan, C., Van Schaeybroeck, S., Longley, D.B. & Johnston, P.G. Cancer drug resistance: an evolving paradigm. Nat. Rev. Cancer 13, 714–726 (2013).

    Article  CAS  PubMed  Google Scholar 

  6. Bock, C. & Lengauer, T. Managing drug resistance in cancer: lessons from HIV therapy. Nat. Rev. Cancer 12, 494–501 (2012).

    Article  CAS  PubMed  Google Scholar 

  7. Chong, C.R., Chen, X., Shi, L., Liu, J.O. & Sullivan, D.J. Jr. A clinical drug library screen identifies astemizole as an antimalarial agent. Nat. Chem. Biol. 2, 415–416 (2006).

    Article  CAS  PubMed  Google Scholar 

  8. Huang, R. et al. The NCGC pharmaceutical collection: a comprehensive resource of clinically approved drugs enabling repurposing and chemical genomics. Sci. Transl. Med. 3, 80ps16 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Lamb, J. et al. The Connectivity Map: using gene-expression signatures to connect small molecules, genes, and disease. Science 313, 1929–1935 (2006).

    Article  CAS  PubMed  Google Scholar 

  10. Iskar, M. et al. Drug-induced regulation of target expression. PLoS Comput. Biol. 6, e1000925 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Overington, J.P., Al-Lazikani, B. & Hopkins, A.L. How many drug targets are there? Nat. Rev. Drug Discov. 5, 993–996 (2006).

    Article  CAS  PubMed  Google Scholar 

  12. Lipinski, C.A., Lombardo, F., Dominy, B.W. & Feeney, P.J. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Deliv. Rev. 46, 3–26 (2001).

    Article  CAS  PubMed  Google Scholar 

  13. Reiling, J.H. et al. A haploid genetic screen identifies the major facilitator domain containing 2A (MFSD2A) transporter as a key mediator in the response to tunicamycin. Proc. Natl. Acad. Sci. USA 108, 11756–11765 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Winter, G.E. et al. The solute carrier SLC35F2 enables YM155-mediated DNA damage toxicity. Nat. Chem. Biol. 10, 768–773 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Bliss, C.I. The toxicity of poisons applied jointly. Ann. Appl. Biol. 26, 585–615 (1939).

    Article  CAS  Google Scholar 

  16. Berenbaum, M.C. What is synergy? Pharmacol. Rev. 41, 93–141 (1989).

    CAS  PubMed  Google Scholar 

  17. Loewe, S. Die quantitativen Probleme der Pharmakologie. Ergeb. Physiol. 27, 48–187 (1928).

    Article  Google Scholar 

  18. Chou, T.C. & Talalay, P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv. Enzyme Regul. 22, 27–55 (1984).

    Article  CAS  PubMed  Google Scholar 

  19. Tang, J., Wennerberg, K. & Aittokallio, T. What is synergy? The Saariselkä agreement revisited. Front. Pharmacol. 6, 181 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Bürckstümmer, T. et al. A reversible gene trap collection empowers haploid genetics in human cells. Nat. Methods 10, 965–971 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  21. Carette, J.E. et al. Haploid genetic screens in human cells identify host factors used by pathogens. Science 326, 1231–1235 (2009).

    Article  CAS  PubMed  Google Scholar 

  22. Dehm, S.M. & Tindall, D.J. Alternatively spliced androgen receptor variants. Endocr. Relat. Cancer 18, R183–R196 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Veldscholte, J. et al. The androgen receptor in LNCaP cells contains a mutation in the ligand binding domain which affects steroid binding characteristics and response to anti-androgens. J. Steroid Biochem. Mol. Biol. 41, 665–669 (1992).

    Article  CAS  PubMed  Google Scholar 

  24. Alimirah, F., Chen, J., Basrawala, Z., Xin, H. & Choubey, D. DU-145 and PC-3 human prostate cancer cell lines express androgen receptor: implications for the androgen receptor functions and regulation. FEBS Lett. 580, 2294–2300 (2006).

    Article  CAS  PubMed  Google Scholar 

  25. Suttie, J.W. Vitamin K-dependent carboxylase. Annu. Rev. Biochem. 54, 459–477 (1985).

    Article  CAS  PubMed  Google Scholar 

  26. Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. USA 102, 15545–15550 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Feldman, B.J. & Feldman, D. The development of androgen-independent prostate cancer. Nat. Rev. Cancer 1, 34–45 (2001).

    Article  CAS  PubMed  Google Scholar 

  28. Gaddipati, J.P. et al. Frequent detection of codon 877 mutation in the androgen receptor gene in advanced prostate cancers. Cancer Res. 54, 2861–2864 (1994).

    CAS  PubMed  Google Scholar 

  29. Bhattacharyya, R.S., Krishnan, A.V., Swami, S. & Feldman, D. Fulvestrant (ICI 182,780) down-regulates androgen receptor expression and diminishes androgenic responses in LNCaP human prostate cancer cells. Mol. Cancer Ther. 5, 1539–1549 (2006).

    Article  CAS  PubMed  Google Scholar 

  30. Grad, J.M., Lyons, L.S., Robins, D.M. & Burnstein, K.L. The androgen receptor (AR) amino-terminus imposes androgen-specific regulation of AR gene expression via an exonic enhancer. Endocrinology 142, 1107–1116 (2001).

    Article  CAS  PubMed  Google Scholar 

  31. Eder, I.E. et al. Inhibition of LncaP prostate cancer cells by means of androgen receptor antisense oligonucleotides. Cancer Gene Ther. 7, 997–1007 (2000).

    Article  CAS  PubMed  Google Scholar 

  32. Liao, X., Tang, S., Thrasher, J.B., Griebling, T.L. & Li, B. Small-interfering RNA-induced androgen receptor silencing leads to apoptotic cell death in prostate cancer. Mol. Cancer Ther. 4, 505–515 (2005).

    Article  CAS  PubMed  Google Scholar 

  33. Martinez Molina, D. et al. Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Science 341, 84–87 (2013).

    Article  PubMed  Google Scholar 

  34. Hallgren, K.W., Zhang, D., Kinter, M., Willard, B. & Berkner, K.L. Methylation of γ-carboxylated Glu (Gla) allows detection by liquid chromatography-mass spectrometry and the identification of Gla residues in the γ-glutamyl carboxylase. J. Proteome Res. 12, 2365–2374 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Santos, R. et al. A comprehensive map of molecular drug targets. Nat. Rev. Drug Discov. 16, 19–34 (2017).

    Article  CAS  PubMed  Google Scholar 

  36. Taplin, M.-E. Drug insight: role of the androgen receptor in the development and progression of prostate cancer. Nat. Clin. Pract. Oncol. 4, 236–244 (2007).

    Article  CAS  PubMed  Google Scholar 

  37. de Bono, J.S. et al. Abiraterone and increased survival in metastatic prostate cancer. N. Engl. J. Med. 364, 1995–2005 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Tran, C. et al. Development of a second-generation antiandrogen for treatment of advanced prostate cancer. Science 324, 787–790 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Chen, C.D. et al. Molecular determinants of resistance to antiandrogen therapy. Nat. Med. 10, 33–39 (2004).

    Article  PubMed  Google Scholar 

  40. Balbas, M.D. et al. Overcoming mutation-based resistance to anti-androgens with rational drug design. eLife 2, e00499 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  41. Van Hemelrijck, M. et al. Cohort Profile: the National Prostate Cancer Register of Sweden and Prostate Cancer data Base Sweden 2.0. Int. J. Epidemiol. 42, 956–967 (2013).

    Article  PubMed  Google Scholar 

  42. Tagalakis, V., Tamim, H., Blostein, M., Hanley, J.A. & Kahn, S.R. Risk of prostate cancer death in long-term users of warfarin: a population-based case-control study. Cancer Causes Control 24, 1079–1085 (2013).

    Article  CAS  PubMed  Google Scholar 

  43. Tew, B.Y. et al. Vitamin K epoxide reductase regulation of androgen receptor activity. Oncotarget 8, 13818–13831 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  44. Itsumi, M. et al. PMA induces androgen receptor downregulation and cellular apoptosis in prostate cancer cells. J. Mol. Endocrinol. 53, 31–41 (2014).

    Article  CAS  PubMed  Google Scholar 

  45. Yu, Z. et al. Galeterone prevents androgen receptor binding to chromatin and enhances degradation of mutant androgen receptor. Clin. Cancer Res. 20, 4075–4085 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Licciardello, M.P. et al. NOTCH1 activation in breast cancer confers sensitivity to inhibition of SUMOylation. Oncogene 29, 319 (2014).

    Google Scholar 

  47. Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013).

    PubMed  PubMed Central  Google Scholar 

  48. Wang, L., Wang, S. & Li, W. RSeQC: quality control of RNA-seq experiments. Bioinformatics 28, 2184–2185 (2012).

    Article  CAS  PubMed  Google Scholar 

  49. Rudashevskaya, E.L. et al. A method to resolve the composition of heterogeneous affinity-purified protein complexes assembled around a common protein by chemical cross-linking, gel electrophoresis and mass spectrometry. Nat. Protoc. 8, 75–97 (2013).

    Article  CAS  PubMed  Google Scholar 

  50. Huber, M.L. et al. abFASP-MS: affinity-based filter-aided sample preparation mass spectrometry for quantitative analysis of chemically labeled protein complexes. J. Proteome Res. 13, 1147–1155 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references


We thank M. Iskar (EMBL) and P. Bork (EMBL) for providing us with DIPS scores, R. Schüle (Albert-Ludwigs-University Freiburg, Germany) for LAPC4 cells, and G. Winter (CeMM) and G. Superti-Furga (CeMM) for thoughtful discussions and initializing synergy screenings at CeMM. S.K. acknowledges support by a Marie Curie Career Integration Grant, the Austrian Federal Ministry of Science, Research and Economy and the National Foundation for Research, Technology, and Development and the Austrian Science Fund (FWF): F4701-B20.

Author information

Authors and Affiliations



P.M., F.K. and S.K. designed and assembled the CLOUD. M.P.L., F.K., C.-H.L. and S.K. designed and performed the screen of the CLOUD. M.P.L., F.K., M.C. and J.M. analyzed the data from the screen. M.P.L. and A.R. designed and performed viability, RT-qPCR, western blotting, immunoprecipitation and immunofluorescence experiments. S.S. performed immunofluorescence experiments. A.R. and E.S. designed and performed gene expression and immunoprecipitation experiments. A.R. and B.B. designed and performed knockdown experiments. A.C.M. and K.L.B. designed and performed proteomics experiments. A.W., R.H. and K.K. performed and analyzed 2D gel electrophoresis experiments. T.P., M.S. and C.B. performed and analyzed RNA-seq experiments. G.D. and J.C. performed DIPS score analyses. Y.F. and P.S. analyzed patient data in PCBaSe. V.I. synthesized, provided and quality controlled chemicals. M.P.L. and S.K. wrote the manuscript.

Corresponding author

Correspondence to Stefan Kubicek.

Ethics declarations

Competing interests

M.P.L. and S.K. have filed a patent based on findings described in this manuscript (WO2016170102 A1). V.I. is an employee of Enamine, Ltd and may also own shares in the company.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–4 and Supplementary Figures 1–21 (PDF 29615 kb)

Supplementary Data Set 1

STEAM and CLOUD drugs. (XLS 338 kb)

Supplementary Data Set 2

Data from CLOUD combinatorial screen. (XLS 4999 kb)

Supplementary Data Set 3

Synergies and antagonisms defined by both Bliss and Loewe scores. (XLS 207 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Licciardello, M., Ringler, A., Markt, P. et al. A combinatorial screen of the CLOUD uncovers a synergy targeting the androgen receptor. Nat Chem Biol 13, 771–778 (2017).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer