Abstract
Aerobic glycolysis, also known as the Warburg effect, is a hallmark of cancer cell glucose metabolism and plays a crucial role in the activation of various types of immune cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of D-glyceraldehyde 3-phosphate to D-glycerate 1,3-bisphosphate in the 6th critical step in glycolysis. GAPDH exerts metabolic flux control during aerobic glycolysis and therefore is an attractive therapeutic target for cancer and autoimmune diseases. Recently, GAPDH inhibitors were reported to function through common suicide inactivation by covalent binding to the cysteine catalytic residue of GAPDH. Herein, by developing a high-throughput enzymatic screening assay, we discovered that the natural product 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose (PGG) is an inhibitor of GAPDH with Ki = 0.5 μM. PGG blocks GAPDH activity by a reversible and NAD+ and Pi competitive mechanism, suggesting that it represents a novel class of GAPDH inhibitors. In-depth hydrogen deuterium exchange mass spectrometry (HDX-MS) analysis revealed that PGG binds to a region that disrupts NAD+ and inorganic phosphate binding, resulting in a distal conformational change at the GAPDH tetramer interface. In addition, structural modeling analysis indicated that PGG probably reversibly binds to the center pocket of GAPDH. Moreover, PGG inhibits LPS-stimulated macrophage activation by specific downregulation of GAPDH-dependent glucose consumption and lactate production. In summary, PGG represents a novel class of GAPDH inhibitors that probably reversibly binds to the center pocket of GAPDH. Our study sheds new light on factors for designing a more potent and specific inhibitor of GAPDH for future therapeutic applications.
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Acknowledgements
We are grateful to the National Centre for Protein Science Shanghai (Protein Expression and Purification system) for their instrument support and technical assistance. We gratefully acknowledge the financial support from the National Natural Science Foundation of China (81728020 to KHZ, 91853205, 81625022, and 81821005 to CL, 21820102008 to HLJ), K. C. Wong Education to CL, the Science and Technology Commission of Shanghai Municipality (19XD1404700 to CL and 18431907100 to HLJ), the Chinese Academy of Sciences (CASIMM0120184015 to CL). KHZ is supported by Taishan Scholar. Part of the study is funded by the Top Talents Program for One Case Discussion of Shandong Province.
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WL, LPL, KHZ and CL conceived the project, provided intellectual contributions, designed experiments, performed and analyzed experiments, and wrote the paper. NS and JZ performed the HDX-MS experiment and described the method used to perform this experiment. YLD and NXZ performed the NMR experiment and described the method used to perform the NMR experiment. YJL provided advice for the animal experiment. CPL provided expert advice on enzymology. YYZ, ZYS, XRZ, JL, HBW, and SHX provided help with the protein expression and high-throughput assays. KXC and HLJ read the paper and provided valuable suggestions about the paper.
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Li, W., Liao, Lp., Song, N. et al. Natural product 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose is a reversible inhibitor of glyceraldehyde 3-phosphate dehydrogenase. Acta Pharmacol Sin 43, 470–482 (2022). https://doi.org/10.1038/s41401-021-00653-0
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DOI: https://doi.org/10.1038/s41401-021-00653-0
