Skip to main content

Thank you for visiting nature.com. 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.

Allosteric inhibition of hypoxia inducible factor-2 with small molecules

Abstract

Hypoxia inducible factors (HIFs) are heterodimeric transcription factors induced in many cancers where they frequently promote the expression of protumorigenic pathways. Though transcription factors are typically considered 'undruggable', the PAS-B domain of the HIF-2α subunit contains a large cavity within its hydrophobic core that offers a unique foothold for small-molecule regulation. Here we identify artificial ligands that bind within this pocket and characterize the resulting structural and functional changes caused by binding. Notably, these ligands antagonize HIF-2 heterodimerization and DNA-binding activity in vitro and in cultured cells, reducing HIF-2 target gene expression. Despite the high sequence identity between HIF-2α and HIF-1α, these ligands are highly selective and do not affect HIF-1 function. These chemical tools establish the molecular basis for selective regulation of HIF-2, providing potential therapeutic opportunities to intervene in HIF-2–driven tumors, such as renal cell carcinomas.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Biophysical characterization of the HIF-2α PAS-B–2 complex.
Figure 2: Binding of 2 into HIF-2α PAS-B affects the heterodimeric β-sheet interface between HIF PAS-B domains.
Figure 3: 2 disrupts HIF-2 heterodimerization in vitro.
Figure 4: 2 binds selectively to HIF-2α over HIF-1α PAS-B.
Figure 5: 2 selectively antagonizes HIF-2 activity in cultured cells.

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. 1

    Semenza, G.L. Hypoxia-inducible factors: mediators of cancer progression and targets for cancer therapy. Trends Pharmacol. Sci. 33, 207–214 (2012).

    CAS  Article  Google Scholar 

  2. 2

    Xia, X. et al. Integrative analysis of HIF binding and transactivation reveals its role in maintaining histone methylation homeostasis. Proc. Natl. Acad. Sci. USA 106, 4260–4265 (2009).

    CAS  Article  Google Scholar 

  3. 3

    Schödel, J. et al. High-resolution genome-wide mapping of HIF-binding sites by ChIP-seq. Blood 117, e207–e217 (2011).

    Article  Google Scholar 

  4. 4

    Majmundar, A.J., Wong, W.J. & Simon, M.C. Hypoxia-inducible factors and the response to hypoxic stress. Mol. Cell 40, 294–309 (2010).

    CAS  Article  Google Scholar 

  5. 5

    Greer, S.N., Metcalf, J.L., Wang, Y. & Ohh, M. The updated biology of hypoxia-inducible factor. EMBO J. 31, 2448–2460 (2012).

    CAS  Article  Google Scholar 

  6. 6

    Kondo, K., Kim, W.Y., Lechpammer, M. & Kaelin, W.G. Jr. Inhibition of HIF2α is sufficient to suppress pVHL-defective tumor growth. PLoS Biol. 1, E83 (2003).

    Article  Google Scholar 

  7. 7

    Kondo, K., Klco, J., Nakamura, E., Lechpammer, M. & Kaelin, W.G. Jr. Inhibition of HIF is necessary for tumor suppression by the von Hippel-Lindau protein. Cancer Cell 1, 237–246 (2002).

    CAS  Article  Google Scholar 

  8. 8

    Maranchie, J.K. et al. The contribution of VHL substrate binding and HIF1-α to the phenotype of VHL loss in renal cell carcinoma. Cancer Cell 1, 247–255 (2002).

    CAS  Article  Google Scholar 

  9. 9

    Erbel, P.J., Card, P.B., Karakuzu, O., Bruick, R.K. & Gardner, K.H. Structural basis for PAS domain heterodimerization in the basic helix-loop-helix–PAS transcription factor hypoxia-inducible factor. Proc. Natl. Acad. Sci. USA 100, 15504–15509 (2003).

    CAS  Article  Google Scholar 

  10. 10

    Yang, J. et al. Functions of the Per/ARNT/Sim (PAS) domains of the hypoxia inducible factor (HIF). J. Biol. Chem. 280, 36047–36054 (2005).

    CAS  Article  Google Scholar 

  11. 11

    Partch, C.L., Card, P.B., Amezcua, C.A. & Gardner, K.H. Molecular basis of coiled coil coactivator recruitment by the aryl hydrocarbon receptor nuclear translocator (ARNT). J. Biol. Chem. 284, 15184–15192 (2009).

    CAS  Article  Google Scholar 

  12. 12

    Partch, C.L. & Gardner, K.H. Coactivators necessary for transcriptional output of the hypoxia inducible factor, HIF, are directly recruited by ARNT PAS-B. Proc. Natl. Acad. Sci. USA 108, 7739–7744 (2011).

    CAS  Article  Google Scholar 

  13. 13

    Henry, J.T. & Crosson, S. Ligand-binding PAS domains in a genomic, cellular, and structural context. Annu. Rev. Microbiol. 65, 261–286 (2011).

    CAS  Article  Google Scholar 

  14. 14

    Harper, S.M., Neil, L.C. & Gardner, K.H. Structural basis of a phototropin light switch. Science 301, 1541–1544 (2003).

    CAS  Article  Google Scholar 

  15. 15

    Wells, J.A. & McClendon, C.L. Reaching for high-hanging fruit in drug discovery at protein-protein interfaces. Nature 450, 1001–1009 (2007).

    CAS  Article  Google Scholar 

  16. 16

    Koehler, A.N. A complex task? Direct modulation of transcription factors with small molecules. Curr. Opin. Chem. Biol. 14, 331–340 (2010).

    CAS  Article  Google Scholar 

  17. 17

    Key, J., Scheuermann, T.H., Anderson, P.C., Daggett, V. & Gardner, K.H. Principles of ligand binding within a completely buried cavity in HIF2α PAS-B. J. Am. Chem. Soc. 131, 17647–17654 (2009).

    CAS  Article  Google Scholar 

  18. 18

    Scheuermann, T.H. et al. Artificial ligand binding within the HIF2α PAS-B domain of the HIF2 transcription factor. Proc. Natl. Acad. Sci. USA 106, 450–455 (2009).

    CAS  Article  Google Scholar 

  19. 19

    Qing, G. & Simon, M.C. Hypoxia inducible factor-2α: a critical mediator of aggressive tumor phenotypes. Curr. Opin. Genet. Dev. 19, 60–66 (2009).

    CAS  Article  Google Scholar 

  20. 20

    Morris, M.R. et al. Mutation analysis of hypoxia-inducible factors HIF1α and HIF2α in renal cell carcinoma. Anticancer Res. 29, 4337–4343 (2009).

    CAS  PubMed  Google Scholar 

  21. 21

    Kaelin, W.G. Jr. Treatment of kidney cancer: insights provided by the VHL tumor-suppressor protein. Cancer 115, 2262–2272 (2009).

    CAS  Article  Google Scholar 

  22. 22

    Shen, C. et al. Genetic and functional studies implicate HIF1α as a 14q kidney cancer suppressor gene. Cancer Discov. 1, 222–235 (2011).

    CAS  Article  Google Scholar 

  23. 23

    Amezcua, C.A., Harper, S.M., Rutter, J. & Gardner, K.H. Structure and interactions of PAS kinase N-terminal PAS domain: model for intramolecular kinase regulation. Structure 10, 1349–1361 (2002).

    CAS  Article  Google Scholar 

  24. 24

    Rogers, J.L. et al. Development of inhibitors of the PAS-B domain of the HIF-2α transcription factor. J. Med. Chem. 10.1021/jm301847z (30 January 2013).

  25. 25

    Krieg, M. et al. Up-regulation of hypoxia-inducible factors HIF-1α and HIF-2α under normoxic conditions in renal carcinoma cells by von Hippel-Lindau tumor suppressor gene loss of function. Oncogene 19, 5435–5443 (2000).

    CAS  Article  Google Scholar 

  26. 26

    Dioum, E.M. et al. Regulation of hypoxia-inducible factor 2α signaling by the stress-responsive deacetylase sirtuin 1. Science 324, 1289–1293 (2009).

    CAS  Article  Google Scholar 

  27. 27

    Scortegagna, M. et al. HIF-2α regulates murine hematopoietic development in an erythropoietin-dependent manner. Blood 105, 3133–3140 (2005).

    CAS  Article  Google Scholar 

  28. 28

    Semenza, G.L. Targeting HIF-1 for cancer therapy. Nat. Rev. Cancer 3, 721–732 (2003).

    CAS  Google Scholar 

  29. 29

    Keith, B., Johnson, R.S. & Simon, M.C. HIF1α and HIF2α: sibling rivalry in hypoxic tumour growth and progression. Nat. Rev. Cancer 12, 9–22 (2012).

    CAS  Article  Google Scholar 

  30. 30

    Franovic, A., Holterman, C.E., Payette, J. & Lee, S. Human cancers converge at the HIF-2α oncogenic axis. Proc. Natl. Acad. Sci. USA 106, 21306–21311 (2009).

    CAS  Article  Google Scholar 

  31. 31

    Holmquist-Mengelbier, L. et al. Recruitment of HIF-1α and HIF-2α to common target genes is differentially regulated in neuroblastoma: HIF-2α promotes an aggressive phenotype. Cancer Cell 10, 413–423 (2006).

    CAS  Article  Google Scholar 

  32. 32

    Pietras, A. et al. HIF-2α maintains an undifferentiated state in neural crest-like human neuroblastoma tumor-initiating cells. Proc. Natl. Acad. Sci. USA 106, 16805–16810 (2009).

    CAS  Article  Google Scholar 

  33. 33

    Mazumdar, J. et al. HIF-2α deletion promotes Kras-driven lung tumor development. Proc. Natl. Acad. Sci. USA 107, 14182–14187 (2010).

    CAS  Article  Google Scholar 

  34. 34

    Ivan, M. et al. HIFα targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. Science 292, 464–468 (2001).

    CAS  Article  Google Scholar 

  35. 35

    Jaakkola, P. et al. Targeting of HIF-α to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 292, 468–472 (2001).

    CAS  Article  Google Scholar 

  36. 36

    Lando, D. et al. FIH-1 is an asparaginyl hydroxylase enzyme that regulates the transcriptional activity of hypoxia-inducible factor. Genes Dev. 16, 1466–1471 (2002).

    CAS  Article  Google Scholar 

  37. 37

    Lando, D., Peet, D.J., Whelan, D.A., Gorman, J.J. & Whitelaw, M.L. Asparagine hydroxylation of the HIF transactivation domain: a hypoxic switch. Science 295, 858–861 (2002).

    CAS  Article  Google Scholar 

  38. 38

    Kaelin, W.G. Von Hippel-Lindau disease. Annu. Rev. Pathol. 2, 145–173 (2007).

    CAS  Article  Google Scholar 

  39. 39

    Halavaty, A.S. & Moffat, K. N- and C-terminal flanking regions modulate light-induced signal transduction in the LOV2 domain of the blue light sensor phototropin 1 from Avena sativa. Biochemistry 46, 14001–14009 (2007).

    CAS  Article  Google Scholar 

  40. 40

    Park, E.J. et al. Targeting the PAS-A domain of HIF-1α for development of small molecule inhibitors of HIF-1. Cell Cycle 5, 1847–1853 (2006).

    CAS  Article  Google Scholar 

  41. 41

    Lee, K. et al. Acriflavine inhibits HIF-1 dimerization, tumor growth, and vascularization. Proc. Natl. Acad. Sci. USA 106, 17910–17915 (2009).

    CAS  Article  Google Scholar 

  42. 42

    Semenza, G.L. HIF-1: upstream and downstream of cancer metabolism. Curr. Opin. Genet. Dev. 20, 51–56 (2010).

    CAS  Article  Google Scholar 

  43. 43

    Johnson, B.A. & Blevins, R.A. NMRView: a computer program for the visualization and analysis of NMR data. J. Biomol. NMR 4, 603–614 (1994).

    CAS  Article  Google Scholar 

  44. 44

    Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    CAS  Article  Google Scholar 

  45. 45

    Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    CAS  Article  Google Scholar 

  46. 46

    Schüttelkopf, A.W. & van Aalten, D.M. PRODRG: a tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr. D Biol. Crystallogr. 60, 1355–1363 (2004).

    Article  Google Scholar 

  47. 47

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    PubMed  PubMed Central  Google Scholar 

  48. 48

    Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    CAS  Article  Google Scholar 

  49. 49

    Winn, M.D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).

    CAS  Article  Google Scholar 

  50. 50

    Martí-Renom, M.A. et al. Comparative protein structure modeling of genes and genomes. Annu. Rev. Biophys. Biomol. Struct. 29, 291–325 (2000).

    Article  Google Scholar 

  51. 51

    Bookout, A.L. & Mangelsdorf, D.J. Quantitative real-time PCR protocol for analysis of nuclear receptor signaling pathways. Nucl. Recept. Signal. 1, e012 (2003).

    Article  Google Scholar 

  52. 52

    Scheuermann, T.H., Yang, J., Zhang, L., Gardner, K.H. & Bruick, R.K. Hypoxia-inducible factors Per/ARNT/Sim domains: structure and function. Methods Enzymol. 435, 3–24 (2007).

    CAS  PubMed  Google Scholar 

  53. 53

    Chen, R., Dioum, E.M., Hogg, R.T., Gerard, R.D. & Garcia, J.A. Hypoxia increases sirtuin 1 expression in a hypoxia-inducible factor-dependent manner. J. Biol. Chem. 286, 13869–13878 (2011).

    CAS  Article  Google Scholar 

  54. 54

    McNaney, C.A. et al. An automated liquid chromatography-mass spectrometry process to determine metabolic stability half-life and intrinsic clearance of drug candidates by substrate depletion. Assay Drug Dev. Technol. 6, 121–129 (2008).

    CAS  Article  Google Scholar 

  55. 55

    Drexler, D.M. et al. An automated high throughput liquid chromatography-mass spectrometry process to assess the metabolic stability of drug candidates. Assay Drug Dev. Technol. 5, 247–264 (2007).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

The authors thank S. Wang and members of the University of Texas Southwestern High-Throughput Screening Core Facility, D. Tomchick, C. Brautigam, J. MacMillan, N. Williams and members of our laboratories for their help. This work was funded by grants from the US National Institutes of Health (P01 CA095471, P30 CA142543) and the Cancer Prevention Research Institute of Texas (RP-100846). R.K.B. is the Michael L. Rosenberg Scholar in Medical Research and was supported by a Career Award in the Biomedical Sciences from the Burroughs Wellcome Fund; K.H.G. is the Virginia Lazenby O'Hara Chair in Biochemistry and W.W. Caruth Scholar in Biomedical Research; and U.K.T. is a W.W. Caruth Jr. Scholar in Biomedical Research. J.A.G. was supported by funds provided by the Department of Veterans Affairs. Results shown in this report are derived from work performed at Argonne National Laboratory, Structural Biology Center at the Advanced Photon Source. Argonne National Laboratory is operated by UChicago Argonne, LLC, for the US Department of Energy, Office of Biological and Environmental Research under contract DE-AC02-06CH11357. This investigation was conducted in a facility constructed with support from the Research Facilities Improvement Program (grant no. C06 RR 15437-01) from the National Center for Research Resources, US National Institutes of Health.

Author information

Affiliations

Authors

Contributions

R.K.B. and K.H.G. conceived and designed the experiments. T.H.S., Q.L., H.-W.M., L.Z., R.C., J.N. and J.L. performed the experiments. R.K.B., K.H.G., T.H.S., J.K., J.A.G., D.E.F. and U.K.T. analyzed the data. R.K.B., K.H.G. and T.H.S. wrote the paper.

Corresponding authors

Correspondence to Kevin H Gardner or Richard K Bruick.

Ethics declarations

Competing interests

Declaration: R.K.B., D.E.F., K.H.G., T.H.S. and U.K.T. have filed US Patent Application no. 61/583,662 covering the method described in the paper. R.K.B., K.H.G., T.H.S., J.K. and U.K.T. have received stock options and other financial compensation from Peloton Therapeutics, Inc.

Supplementary information

Supplementary Text and Figures

Supplementary Note, Supplementary Results (PDF 3816 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Scheuermann, T., Li, Q., Ma, HW. et al. Allosteric inhibition of hypoxia inducible factor-2 with small molecules. Nat Chem Biol 9, 271–276 (2013). https://doi.org/10.1038/nchembio.1185

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing