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Identification of Epigallocatechin-3- Gallate as an Inhibitor of Phosphoglycerate Mutase 1.

ABSTRACT: Targeting metabolic enzymes is believed to provide new therapeutic opportunities for cancer therapy. Phosphoglycerate mutase 1 (PGAM1) is a glycolytic enzyme that importantly coordinates glycolysis, pentose phosphate pathway (PPP) flux and serine biosynthesis in cancer cells and hence gains increasing interest of inhibitor discovery. Only few PGAM1 inhibitors have been reported and the molecular potency remains very limited. In an effort to discover new PGAM1 inhibitors, we carried out a biochemical assay-based screen that was focused on natural products derived small molecule compounds. (-)-Epigallocatechin-3-gallate (EGCG), the major natural catechins of green tea extract, was identified as a PGAM1 inhibitor that was tremendously more potent than known PGAM1 inhibitors. Further studies combining molecular docking and site-specific mutagenesis revealed that EGCG inhibited PGAM1 enzymatic activity in a manner independent of substrate competition. EGCG modulated the intracellular level of 2-phosphoglycerate, impaired glycolysis and PPP and inhibited proliferation of cancer cells. This study suggested EGCG as a chemical scaffold for the discovery of potent PGAM1 inhibitors and gained mechanistic insights to understand the previously appreciated anticancer properties of EGCG.

SUBMITTER: Li X

PROVIDER: S-EPMC5447756 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Identification of Epigallocatechin-3- Gallate as an Inhibitor of Phosphoglycerate Mutase 1.

Li Xiaoguang X Tang Shuai S Wang Qian-Qian QQ Leung Elaine L-H EL Jin Hongyue H Huang Yongzhuo Y Liu Jia J Geng Meiyu M Huang Min M Yuan Shengtao S Yao Xiao-Jun XJ Ding Jian J

Frontiers in pharmacology 20170530

Targeting metabolic enzymes is believed to provide new therapeutic opportunities for cancer therapy. Phosphoglycerate mutase 1 (PGAM1) is a glycolytic enzyme that importantly coordinates glycolysis, pentose phosphate pathway (PPP) flux and serine biosynthesis in cancer cells and hence gains increasing interest of inhibitor discovery. Only few PGAM1 inhibitors have been reported and the molecular potency remains very limited. In an effort to discover new PGAM1 inhibitors, we carried out a biochem ...[more]

PMID: 28611670

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Project description:Phosphoglycerate mutase 2 (PGAM2) is a critical glycolytic enzyme that is highly expressed in skeletal muscle. In humans, naturally occurring mutations in Phosphoglycerate mutase 2 have been etiologically linked to glycogen storage disease X (GSDX). Phosphoglycerate mutase 2 activity is regulated by several posttranslational modifications such as ubiquitination and acetylation. Here, we report that Phosphoglycerate mutase 2 activity is regulated by sumoylation-a covalent conjugation involved in a wide spectrum of cellular events. We found that Phosphoglycerate mutase 2 contains two primary SUMO acceptor sites, lysine (K)49 and K176, and that the mutation of either K to arginine (R) abolished Phosphoglycerate mutase 2 sumoylation. Given that K176 is more highly evolutionarily conserved across paralogs and orthologs than K49 is, we used the CRISPR-mediated homologous recombination technique in myogenic C2C12 cells to generate homozygous K176R knock-in cells (PGAM2K176R/K176R). Compared with wild-type (WT) C2C12 cells, PGAM2K176R/K176R C2C12 cells exhibited impaired myogenic differentiation, as indicated by decreased differentiation and fusion indexes. Furthermore, the results of glycolytic and mitochondrial stress assays with the XF96 Extracellular Flux analyzer revealed a reduced proton efflux rate (PER), glycolytic PER (glycoPER), extracellular acidification rate (ECAR), and oxygen consumption rate (OCR) in PGAM2K176R/K176R C2C12 cells, both at baseline and in response to stress. Impaired mitochondrial function was also observed in PGAM2K176R/K176R P19 cells, a carcinoma cell line. These findings indicate that the PGAM2-K176R mutation impaired glycolysis and mitochondrial function. Gene ontology term analysis of RNA sequencing data further revealed that several downregulated genes in PGAM2K176R/K176R C2C12 cells were associated with muscle differentiation/development/contraction programs. Finally, PGAM2 with either of two naturally occurring missense mutations linked to GSDX, E89A (conversion of glutamic acid 89 to alanine) or R90W (conversion of arginine 90 to tryptophan), exhibited reduced Phosphoglycerate mutase 2 sumoylation. Thus, sumoylation is an important mechanism that mediates Phosphoglycerate mutase 2 activity and is potentially implicated in Phosphoglycerate mutase 2 mutation-linked disease in humans.

Project description:BackgroundHepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with poor prognosis due to resistance to conventional chemotherapy and limited efficacy of radiotherapy. There is an urgent need to develop novel biomarkers for early diagnosis, as well as to identify new drug targets for therapeutic interventions.Patients and methods54 paired HCC samples and 21 normal liver tissues were obtained from West China Hospital of Sichuan University. Informed consent was obtained from all the patients or their relatives prior to analysis, and the project was approved by the Institutional Ethics Committee of Sichuan University. Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC)-based proteomics was employed to profile the differentially expressed proteins between a HepG2 human hepatoma cell line and an immortal hepatic cell line L02. Validation of PGAM1 expression was performed by semi-quantitative RT-PCR, immunoblot and immunohistochemistry using clinical samples. shRNA expressing plasmids specifically targeting PGAM1 were designed and constructed by GenePharma Corporation (Shanghai, China), and were utilized to silence expression of PGAM1 in vitro and in vivo. Cell proliferation was measured by a combination of colony formation assay and Ki67 staining. Apoptosis was examined by flow cytometry and TUNEL assay.ResultsA total of 63 dysregulated proteins were identified, including 51 up-regulated proteins, and 12 down-regulated proteins (over 2-fold, p < 0.01). Phosphoglycerate mutase 1 (PGAM1) was found markedly upregulated. Clinico-pathological analysis indicated that overexpression of PGAM1 was associated with 66.7% HCC, and strongly correlated with poor differentiation and decreased survival rates (p < 0.01). shRNAs-mediated repression of PGAM1 expression resulted in significant inhibition in liver cancer cell growth both in vitro and in vivo.ConclusionOur studies suggested that PGAM1 plays an important role in hepatocarcinogenesis, and should be a potential diagnostic biomarker, as well as an attractive therapeutic target for hepatocellular carcinoma.

Dataset Information

Identification of Epigallocatechin-3- Gallate as an Inhibitor of Phosphoglycerate Mutase 1.

Publications

Identification of Epigallocatechin-3- Gallate as an Inhibitor of Phosphoglycerate Mutase 1.

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