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A small molecule G6PD inhibitor reveals immune dependence on pentose phosphate pathway

Nature Chemical Biology volume 16, pages 731–739 (2020)Cite this article

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Abstract

Glucose is catabolized by two fundamental pathways, glycolysis to make ATP and the oxidative pentose phosphate pathway to make reduced nicotinamide adenine dinucleotide phosphate (NADPH). The first step of the oxidative pentose phosphate pathway is catalyzed by the enzyme glucose-6-phosphate dehydrogenase (G6PD). Here we develop metabolite reporter and deuterium tracer assays to monitor cellular G6PD activity. Using these, we show that the most widely cited G6PD antagonist, dehydroepiandosterone, does not robustly inhibit G6PD in cells. We then identify a small molecule (G6PDi-1) that more effectively inhibits G6PD. Across a range of cultured cells, G6PDi-1 depletes NADPH most strongly in lymphocytes. In T cells but not macrophages, G6PDi-1 markedly decreases inflammatory cytokine production. In neutrophils, it suppresses respiratory burst. Thus, we provide a cell-active small molecule tool for oxidative pentose phosphate pathway inhibition, and use it to identify G6PD as a pharmacological target for modulating immune response.

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Fig. 1: Cellular target engagement assays reveal lack of effective G6PD inhibition by DHEA.
Fig. 2: A nonsteroidal, cell-active inhibitor of G6PD.
Fig. 3: G6PDi-1 reveals T cells depend on oxPPP for maintaining cellular NADPH.
Fig. 4: G6PDi-1 suppresses T cell cytokine production while having a minimal effect on initial activation or proliferation.
Fig. 5: G6PDi-1 suppresses neutrophil oxidative burst.

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Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

References

  1. Voet, D., Voet, J. G. & Pratt, C. W. Fundamentals of Biochemistry: Life at the Molecular Level 5th edn (Wiley, 2016).

  2. Stanton, R. C. Glucose-6-phosphate dehydrogenase, NADPH, and cell survival. IUBMB Life 64, 362–369 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Uhlen, M. et al. Proteomics. Tissue-based map of the human proteome. Science 347, 1260419 (2015).

    Article  PubMed  CAS  Google Scholar 

  4. Kowalik, M. A., Columbano, A. & Perra, A. Emerging role of the pentose phosphate pathway in hepatocellular carcinoma. Front. Oncol. 7, https://doi.org/10.3389/fonc.2017.00087 (2017).

  5. Zhang, Q. et al. Overexpression of G6PD represents a potential prognostic factor in clear cell renal cell carcinoma. J. Cancer 8, 665–673 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Nagashio, R. et al. Prognostic significance of G6PD expression and localization in lung adenocarcinoma. Biochim. Biophys. Acta 1867, 38–46 (2019).

    Article  CAS  Google Scholar 

  7. Pu, H. et al. Overexpression of G6PD is associated with high risks of recurrent metastasis and poor progression-free survival in primary breast carcinoma. World J. Surgical Oncol. 13, 323–323 (2015).

    Article  Google Scholar 

  8. Longo, L. et al. Maternally transmitted severe glucose 6‐phosphate dehydrogenase deficiency is an embryonic lethal. EMBO J. 21, 4229–4239 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Cappellini, M. D. & Fiorelli, G. Glucose-6-phosphate dehydrogenase deficiency. Lancet 371, 64–74 (2008).

    Article  CAS  PubMed  Google Scholar 

  10. Chen, L. et al. NADPH production by the oxidative pentose-phosphate pathway supports folate metabolism. Nat. Metabolism 1, 404–415 (2019).

    Article  CAS  Google Scholar 

  11. Hamilton, N. M. et al. Novel steroid inhibitors of glucose 6-phosphate dehydrogenase. J. Med. Chem. 55, 4431–4445 (2012).

    Article  CAS  PubMed  Google Scholar 

  12. Preuss, J. et al. Identification and characterization of novel human glucose-6-phosphate dehydrogenase inhibitors. J. Biomol. Screening 18, 286–297 (2012).

    Article  CAS  Google Scholar 

  13. Mele, L. et al. A new inhibitor of glucose-6-phosphate dehydrogenase blocks pentose phosphate pathway and suppresses malignant proliferation and metastasis in vivo. Cell Death Dis. 9, 572 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  14. Marks, P. A. & Banks, J. Inhibition of mammalian glucose-6-phosphate dehydrogenase by steroids. Proc. Natl Acad. Sci. USA 46, 447–452 (1960).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Di Monaco, M. et al. Role of glucose-6-phosphate dehydrogenase inhibition in the antiproliferative effects of dehydroepiandrosterone on human breast cancer cells. Brit. J. Cancer 75, 589–592 (1997).

    Article  PubMed  PubMed Central  Google Scholar 

  16. Pashko, L. L., Lewbart, M. L. & Schwartz, A. G. Inhibition of 12-O-tetradecanoylphorbol-13-acetate-promoted skin tumor formation in mice by 16α-fluoro-5-androsten-17-one and its reversal by deoxyribonucleosides. Carcinogenesis 12, 2189–2192 (1991).

    Article  CAS  PubMed  Google Scholar 

  17. Girón, R. A., Montaño, L. F., Escobar, M. L. & López-Marure, R. Dehydroepiandrosterone inhibits the proliferation and induces the death of HPV-positive and HPV-negative cervical cancer cells through an androgen- and estrogen-receptor independent mechanism. FEBS J. 276, 5598–5609 (2009).

    Article  PubMed  CAS  Google Scholar 

  18. Ho, H. Y., Cheng, M. L., Chiu, H. Y., Weng, S. F. & Chiu, D. T. Dehydroepiandrosterone induces growth arrest of hepatoma cells via alteration of mitochondrial gene expression and function. Int. J. Oncol. 33, 969–977 (2008).

    CAS  PubMed  Google Scholar 

  19. Pacold, M. E. et al. A PHGDH inhibitor reveals coordination of serine synthesis and one-carbon unit fate. Nat. Chem. Biol. 12, 452 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Mullarky, E. et al. Identification of a small molecule inhibitor of 3-phosphoglycerate dehydrogenase to target serine biosynthesis in cancers. Proc. Natl Acad. Sci. USA 113, 1778 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Ducker, G. S. et al. Human SHMT inhibitors reveal defective glycine import as a targetable metabolic vulnerability of diffuse large B-cell lymphoma. Proc. Natl Acad. Sci. USA 114, 11404–11409 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Lu, W., Wang, L., Chen, L., Hui, S. & Rabinowitz, J. D. Extraction and quantitation of nicotinamide adenine dinucleotide redox cofactors. Antioxidant. Redox Signal. 28, 167–179 (2018).

    Article  CAS  Google Scholar 

  23. Gordon, G., Mackow, M. C. & Levy, H. R. On the mechanism of interaction of steroids with human glucose 6-phosphate dehydrogenase. Arch. Biochem. Biophys. 318, 25–29 (1995).

    Article  CAS  PubMed  Google Scholar 

  24. Zhang, Z., Chen, L., Liu, L., Su, X. & Rabinowitz, J. D. Chemical basis for deuterium labeling of fat and NADPH. J. Am. Chem. Soc. 139, 14368–14371 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Mercaldi, G. F., Ranzani, A. T. & Cordeiro, A. T. Discovery of new uncompetitive inhibitors of glucose-6-phosphate dehydrogenase. J. Biomol. Screening 19, 1362–1371 (2014).

    Article  CAS  Google Scholar 

  26. Schafer, Z. T. et al. Antioxidant and oncogene rescue of metabolic defects caused by loss of matrix attachment. Nature 461, 109–113 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Buck, M. D., O’Sullivan, D. & Pearce, E. L. T cell metabolism drives immunity. J. Experimental Med. 212, 1345–1360 (2015).

    Article  CAS  Google Scholar 

  28. Nobrega-Pereira, S. et al. G6PD protects from oxidative damage and improves healthspan in mice. Nat. Commun. 7, 10894 (2016).

  29. Ron-Harel, N. et al. Mitochondrial biogenesis and proteome remodeling promote one-carbon metabolism for T cell activation. Cell Metab. 24, 104–117 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Geiger, R. et al. l-Arginine modulates T cell metabolism and enhances survival and anti-tumor activity. Cell 167, 829–842.e813 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Sena, L. A. et al. Mitochondria are required for antigen-specific T cell activation through reactive oxygen species signaling. Immunity 38, 225–236 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Padgett, L. E. & Tse, H. M. NADPH oxidase-derived superoxide provides a third signal for CD4 T cell effector responses. J. Immunol. 197, 1733–1742 (2016).

    Article  CAS  PubMed  Google Scholar 

  33. Mak, T. W. et al. Glutathione primes T cell metabolism for inflammation. Immunity 46, 675–689 (2017).

    Article  CAS  PubMed  Google Scholar 

  34. Nguyen, G. T., Green, E. R. & Mecsas, J. Neutrophils to the ROScue: mechanisms of NADPH oxidase activation and bacterial resistance. Front. Cell Infect. Microbiol. 7, 373 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Goldfarb, A. H., McIntosh, M. K. & Boyer, B. T. Vitamin E attenuates myocardial oxidative stress induced by DHEA in rested and exercised rats. J. Appl. Physiol. 80, 486–490 (1996).

    Article  CAS  PubMed  Google Scholar 

  36. Fan, J. et al. Quantitative flux analysis reveals folate-dependent NADPH production. Nature 510, 298–302 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Morelli, A., Benatti, U., Gaetani, G. F. & De Flora, A. Biochemical mechanisms of glucose-6-phosphate dehydrogenase deficiency. Proc. Natl Acad. Sci. USA 75, 1979–1983 (1978).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Marks, P. A., Johnson, A. B. & Hirschberg, E. Effect of age on the enzyme activity in erythrocytes. Proc. Natl Acad. Sci. USA 44, 529–536 (1958).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Cunningham, A. D., Colavin, A., Huang, K. C. & Mochly-Rosen, D. Coupling between protein stability and catalytic activity determines pathogenicity of G6PD variants. Cell Rep. 18, 2592–2599 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Morellini, M., Colonna-Romano, S., Meloni, T., Battistuzzi, G. & Gandini, E. Glucose-6-phosphate dehydrogenase of leukocyte subpopulations in normal and enzyme deficient individuals. Haematologica 70, 390–395 (1985).

    CAS  PubMed  Google Scholar 

  41. Ardati, K. O., Bajakian, K. M. & Tabbara, K. S. Effect of glucose-6-phosphate dehydrogenase deficiency on neutrophil function. Acta Haematologica 97, 211–215 (1997).

    Article  CAS  PubMed  Google Scholar 

  42. Vives Corrons, J. L. et al. Severe-glucose-6-phosphate dehydrogenase (G6PD) deficiency associated with chronic hemolytic anemia, granulocyte dysfunction, and increased susceptibility to infections: description of a new molecular variant (G6PD Barcelona). Blood 59, 428–434 (1982).

    Article  CAS  PubMed  Google Scholar 

  43. Macintyre, A. N. et al. The glucose transporter Glut1 is selectively essential for CD4 T cell activation and effector function. Cell Metab. 20, 61–72 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Ho, P. C. et al. phosphoenolpyruvate is a metabolic checkpoint of anti-tumor T cell responses. Cell 162, 1217–1228 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Cham, C. M. & Gajewski, T. F. Glucose availability regulates IFN-gamma production and p70S6 kinase activation in CD8+ effector T cells. J. Immunol. 174, 4670–4677 (2005).

    Article  CAS  PubMed  Google Scholar 

  46. Shi, L. Z. et al. HIF1α–dependent glycolytic pathway orchestrates a metabolic checkpoint for the differentiation of TH17 and Treg cells. J. Experimental Med. 208, 1367–1376 (2011).

    Article  CAS  Google Scholar 

  47. Chang, C. H. et al. Posttranscriptional control of T cell effector function by aerobic glycolysis. Cell 153, 1239–1251 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. 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  CAS  Google Scholar 

  49. Ron-Harel, N. et al. T cell activation depends on extracellular alanine. Cell Rep. 28, 3011–3021.e3014 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Xiao, H. et al. HDAC5 controls the functions of Foxp3(+) T-regulatory and CD8(+) T cells. Internat. J. Cancer 138, 2477–2486 (2016).

    Article  CAS  Google Scholar 

  51. Wang, R. et al. The transcription factor Myc controls metabolic reprogramming upon T lymphocyte activation. Immunity 35, 871–882 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Katz, J. & Wood, H. G. The use of C14O2 yields from glucose-1- and -6-C14 for the evaluation of the pathways of glucose metabolism. J. Biol. Chem. 238, 517–523 (1963).

    CAS  PubMed  Google Scholar 

  53. Lu, W. et al. Metabolomic analysis via reversed-phase ion-pairing liquid chromatography coupled to a stand alone orbitrap mass spectrometer. Anal. Chem. 82, 3212–3221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Seim, G. L. et al. Two-stage metabolic remodelling in macrophages in response to lipopolysaccharide and interferon-γ stimulation. Nat. Metabolism 1, 731–742 (2019).

    Article  CAS  Google Scholar 

  55. Su, X., Lu, W. & Rabinowitz, J. D. Metabolite spectral accuracy on orbitraps. Anal. Chem. 89, 5940–5948 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank C. DeCoste of the Princeton University flow cytometry resource facility and the Cytomics Unit of the IIS-La Fe for experimental set-up and design; R.S. O’Connor of University of Pennsylvania for assistance in setting up T cell experiments and for comments and suggestions on the figures; J. Jiao of the Children’s Hospital of Philadelphia for technical assistance with the Treg experiments; I. Babic of Nerdbio for assistance with the CETSA experiments; Y. Huang of Peking University for helpful suggestions pertaining to structure–activity relationship analysis and C. Bartman and the rest of members of the Rabinowitz laboratory for comments and suggestions. This work was supported by National Institutes of Health grant nos. 1DP1DK113643 and R01 CA163591. J.C.G.C. is supported by funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement no. 751423.

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  1. These authors contributed equally: Jonathan M. Ghergurovich, Juan C. García-Cañaveras.

Authors and Affiliations

  1. Lewis Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA

    Jonathan M. Ghergurovich, Juan C. García-Cañaveras, Emily Schmidt, Zhaoyue Zhang, Tara TeSlaa, Harshel Patel, Li Chen & Joshua D. Rabinowitz

  2. Department of Molecular Biology, Princeton University, Princeton, NJ, USA

    Jonathan M. Ghergurovich

  3. Department of Chemistry, Princeton University, Princeton, NJ, USA

    Juan C. García-Cañaveras, Joshua Wang, Emily Schmidt, Zhaoyue Zhang, Tara TeSlaa, Harshel Patel, Li Chen, Hahn Kim & Joshua D. Rabinowitz

  4. Morgridge Institute for Research, Department of Nutritional Sciences, University of Wisconsin-Madison, Madison, WI, USA

    Emily C. Britt & Jing Fan

  5. Biomarkers and Precision Medicine Unit, Instituto de Investigación Sanitaria Fundación Hospital La Fe, Valencia, Spain

    Marta Piqueras-Nebot & Agustín Lahoz

  6. Freshage Research Group, Department of Physiology, Faculty of Medicine, University of Valencia, Valencia, Spain

    Mari Carmen Gomez-Cabrera

  7. Centro de Investigación Biomédica en Red Fragilidad y Envejecimiento Saludable, Fundación Investigación Hospital Clínico Universitario/INCLIVA, Valencia, Spain

    Mari Carmen Gomez-Cabrera

  8. Division of Nephrology, Department of Pediatrics, Children’s Hospital of Philadelphia, University of Pennsylvania, Philadelphia, PA, USA

    Ulf H. Beier

  9. Princeton University Small Molecule Screening Center, Princeton University, Princeton, NJ, USA

    Hahn Kim

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Contributions

J.D.R., J.M.G., J.C.G.-C. and H.K. conceived the study and designed the experiments. J.M.G. developed the in vitro and cell-based assays, conducted the biochemical characterization of G6PDi-1 and characterized the metabolic effects of G6PDi-1 HCT116, HepG2 and other adherent cell lines. J.C.G.C. characterized the metabolic effects of G6PDi-1 in suspension cell lines and the functional effects of G6PDi-1 in T cells and macrophages. J.W., E.S. and H.P. conducted protein expression and purification, in vitro activity assays and western blotting. L.C. isolated mPGD HCT116 cells. Z.Z. and T.T. isolated and cultured primary murine hepatocytes. U.H.B. designed and conducted the Treg experiments. E.C.B. and J.F. designed and conducted the neutrophil experiments. M.C.G.-C. provided the G6PD transgenic mice. J.C.G.C., M.P.-N., M.C.G.-C. and A.L. designed and conducted the experiments with G6PD transgenic mice. H.K. and J.M.G. conducted the structure–activity relationship analysis. H.K. designed and oversaw the chemical synthetic strategy. J.D.R., J.M.G. and J.C.G.-C. wrote the paper. All authors edited and approved the manuscript.

Corresponding author

Correspondence to Joshua D. Rabinowitz.

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Competing interests

Princeton University has filed a patent relating to the new G6PD inhibitors and their uses. J.D.R. is a cofounder of Raze Therapeutics, advisor and stock owner in Kadmon, Agios, CRP, LEAF and Bantam Pharmaceuticals and consultant to Pfizer. No competing interests were disclosed by the other authors.

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Ghergurovich, J.M., García-Cañaveras, J.C., Wang, J. et al. A small molecule G6PD inhibitor reveals immune dependence on pentose phosphate pathway. Nat Chem Biol 16, 731–739 (2020). https://doi.org/10.1038/s41589-020-0533-x

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