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

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.

Data availability

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

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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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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.

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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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