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Bidirectional modulation of HIF-2 activity through chemical ligands

Abstract

Hypoxia-inducible factor-2 (HIF-2) is a heterodimeric transcription factor formed through dimerization between an oxygen-sensitive HIF-2α subunit and its obligate partner subunit ARNT. Enhanced HIF-2 activity drives some cancers, whereas reduced activity causes anemia in chronic kidney disease. Therefore, modulation of HIF-2 activity via direct-binding ligands could provide many new therapeutic benefits. Here, we explored HIF-2α chemical ligands using combined crystallographic, biophysical, and cell-based functional studies. We found chemically unrelated antagonists to employ the same mechanism of action. Their binding displaced residue M252 from inside the HIF-2α PAS-B pocket toward the ARNT subunit to weaken heterodimerization. We also identified first-in-class HIF-2α agonists and found that they significantly displaced pocket residue Y281. Its dramatic side chain movement increases heterodimerization stability and transcriptional activity. Our findings show that despite binding to the same HIF-2α PAS-B pocket, ligands can manifest as inhibitors versus activators by mobilizing different pocket residues to allosterically alter HIF-2α–ARNT heterodimerization.

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Fig. 1: Inhibition of HIF-2 by PT2385.
Fig. 2: Allosteric mechanism of PT2385 revealed by the crystal structure and H/D-ex MS.
Fig. 3: PT2385 disrupts subunit–subunit interactions in HIF-2 heterodimers.
Fig. 4: Newly identified antagonist T1001 points to a common inhibitory mechanism.
Fig. 5: The allosteric mechanism of agonist M1001.
Fig. 6: The agonistic effects of M1002.

Data availability

The three sets of structural data from HIF-2α–ARNT crystals in complex with PT2385, T1001 and M1001 are available from the PDB under accession codes 6E3S, 6E3T and 6E3U, respectively. Other data shown in the article are available from the correspond authors upon reasonable request.

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Acknowledgements

We thank members of the Structural Biology Center at Argonne National Laboratory for their help with data collection at the 19-ID beamline, D. Liu and I. Pass at Sanford Burnham Prebys for kindly providing materials, and G.N. Murshudov at University of Cambridge for the help with ligand building at the 2016 CCP4/APS summer school. This work was supported by the Wellcome Trust and by grants from the National Institutes of Health (R01GM117013, R01DK118297) and US ARMY Medical Research (W81XWH-16-1-0322) to F.R., as well as grants from Shandong University (Qilu Young Scholar 86963072), National Natural Science Foundation of China (31700114), Natural Science Foundation of Jiangsu Province (BK20170399), and the 111 Project (B16030) to D.W.

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D.W. purified proteins, carried out crystallization and solved the structures. X.S. conducted cell-based experiments. J.L. purified proteins and performed biochemical assays. S.L. executed the H/D-ex MS analysis. B.L.H. and S.V. performed the TR-FRET binding assays and thermal-shift-based screening experiments. N.P. produced the expression and mutation constructs. X.D. contributed to biochemical assays and structure refinement. Y.K. collected and processed synchrotron diffraction data. S.K. provided critical instruments and training for biochemical studies. All authors analyzed results. D.W. and F.R. conceived this study, designed experiments and wrote the manuscript.

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Correspondence to Dalei Wu or Fraydoon Rastinejad.

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Wu, D., Su, X., Lu, J. et al. Bidirectional modulation of HIF-2 activity through chemical ligands. Nat Chem Biol 15, 367–376 (2019). https://doi.org/10.1038/s41589-019-0234-5

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