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A novel pyruvate kinase M2 activator compound that suppresses lung cancer cell viability under hypoxia.

Kim DJ, Park YS, Kim ND, Min SH, You YM, Jung Y, Koo H, Noh H, Kim JA, Park KC, Yeom YI - Mol. Cells (2015)

Bottom Line: We demonstrate that PA-12 stimulates the pyruvate kinase activity of recombinant PKM2 in vitro, with a half-maximal activity concentration of 4.92 μM, and effectively suppresses both anchorage-dependent and -independent growth of lung cancer cells in non-essential amino acid-depleted medium.In addition, PA-12 blocked the nuclear translocalization of PKM2 in lung cancer cells, resulting in the inhibition of hypoxia response element (HRE)-mediated reporter activity as well as hypoxia-inducible factor 1 (HIF-1) target gene expression, eventually leading to the suppression of cell viability under hypoxia.Taken together, our data suggest that PA-12 is a novel and potent PKM2 activator that has therapeutic implications for lung cancer.

View Article: PubMed Central - PubMed

Affiliation: Medical Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 305-806, Korea.

ABSTRACT
Pyruvate kinase M2 isoform (PKM2), a rate-limiting enzyme in the final step of glycolysis, is known to be associated with the metabolic rewiring of cancer cells, and considered an important cancer therapeutic target. Herein, we report a novel PKM2 activator, PA-12, which was identified via the molecular docking-based virtual screening. We demonstrate that PA-12 stimulates the pyruvate kinase activity of recombinant PKM2 in vitro, with a half-maximal activity concentration of 4.92 μM, and effectively suppresses both anchorage-dependent and -independent growth of lung cancer cells in non-essential amino acid-depleted medium. In addition, PA-12 blocked the nuclear translocalization of PKM2 in lung cancer cells, resulting in the inhibition of hypoxia response element (HRE)-mediated reporter activity as well as hypoxia-inducible factor 1 (HIF-1) target gene expression, eventually leading to the suppression of cell viability under hypoxia. We also verified that the effects of PA-12 were dependent on PKM2 expression in cancer cells, demonstrating the specificity of PA-12 for PKM2 protein. Taken together, our data suggest that PA-12 is a novel and potent PKM2 activator that has therapeutic implications for lung cancer.

No MeSH data available.


Related in: MedlinePlus

Identification of PA-12 as a potent PKM2 activator. (A) Structure of potential PKM2 activator candidates. (B) Effects of the candidate compounds on the pyruvate kinase activity of PKM2. The activity was assessed by an in-vitro ATP assay using PKIII as the reference. (C) Effects of the candidate compounds on A549 lung cancer cell viability in an NEAA-depleted medium. (D) Cytotoxic effects of PA-12 on IMR-90 human lung fibroblast and NIH/3T3 mouse embryonic fibroblast cells. For B-D, all data are represented as the ± S.D. of values from three independent experiments with duplicate samples. The asterisk (*) indicates a significant difference (p < 0.05) of PA-12-treated samples or cells compared to untreated controls.
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f1-molce-38-4-373: Identification of PA-12 as a potent PKM2 activator. (A) Structure of potential PKM2 activator candidates. (B) Effects of the candidate compounds on the pyruvate kinase activity of PKM2. The activity was assessed by an in-vitro ATP assay using PKIII as the reference. (C) Effects of the candidate compounds on A549 lung cancer cell viability in an NEAA-depleted medium. (D) Cytotoxic effects of PA-12 on IMR-90 human lung fibroblast and NIH/3T3 mouse embryonic fibroblast cells. For B-D, all data are represented as the ± S.D. of values from three independent experiments with duplicate samples. The asterisk (*) indicates a significant difference (p < 0.05) of PA-12-treated samples or cells compared to untreated controls.

Mentions: To identify the initial group of candidate compounds targeting PKM2, we performed molecular docking-based virtual screening using published PKM2 structure (Kung, Hixon et al., 2012) and 8.4 × 105 compounds in the ChemBridge database, and identified 85 compounds that exhibited significant binding potentials to the allosteric binding site of tetrameric PKM2 protein (Supplementary Table and Supplementary Fig. 1A). We then experimentally evaluated these compounds for their effects on the pyruvate kinase activity in an in-vitro enzyme assay using recombinant PKM2 protein, and identified eight compounds showing > 2-fold activation compared to PKM2 enzyme alone as the candidate PKM2 activators (Fig. 1A and Supplementary Fig. 1B). The effects of these eight compounds in activating the pyruvate kinase activity of PKM2 were confirmed by repeating the in-vitro pyruvate kinase assay with PKIII included as the reference compound (Fig. 1B).


A novel pyruvate kinase M2 activator compound that suppresses lung cancer cell viability under hypoxia.

Kim DJ, Park YS, Kim ND, Min SH, You YM, Jung Y, Koo H, Noh H, Kim JA, Park KC, Yeom YI - Mol. Cells (2015)

Identification of PA-12 as a potent PKM2 activator. (A) Structure of potential PKM2 activator candidates. (B) Effects of the candidate compounds on the pyruvate kinase activity of PKM2. The activity was assessed by an in-vitro ATP assay using PKIII as the reference. (C) Effects of the candidate compounds on A549 lung cancer cell viability in an NEAA-depleted medium. (D) Cytotoxic effects of PA-12 on IMR-90 human lung fibroblast and NIH/3T3 mouse embryonic fibroblast cells. For B-D, all data are represented as the ± S.D. of values from three independent experiments with duplicate samples. The asterisk (*) indicates a significant difference (p < 0.05) of PA-12-treated samples or cells compared to untreated controls.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400313&req=5

f1-molce-38-4-373: Identification of PA-12 as a potent PKM2 activator. (A) Structure of potential PKM2 activator candidates. (B) Effects of the candidate compounds on the pyruvate kinase activity of PKM2. The activity was assessed by an in-vitro ATP assay using PKIII as the reference. (C) Effects of the candidate compounds on A549 lung cancer cell viability in an NEAA-depleted medium. (D) Cytotoxic effects of PA-12 on IMR-90 human lung fibroblast and NIH/3T3 mouse embryonic fibroblast cells. For B-D, all data are represented as the ± S.D. of values from three independent experiments with duplicate samples. The asterisk (*) indicates a significant difference (p < 0.05) of PA-12-treated samples or cells compared to untreated controls.
Mentions: To identify the initial group of candidate compounds targeting PKM2, we performed molecular docking-based virtual screening using published PKM2 structure (Kung, Hixon et al., 2012) and 8.4 × 105 compounds in the ChemBridge database, and identified 85 compounds that exhibited significant binding potentials to the allosteric binding site of tetrameric PKM2 protein (Supplementary Table and Supplementary Fig. 1A). We then experimentally evaluated these compounds for their effects on the pyruvate kinase activity in an in-vitro enzyme assay using recombinant PKM2 protein, and identified eight compounds showing > 2-fold activation compared to PKM2 enzyme alone as the candidate PKM2 activators (Fig. 1A and Supplementary Fig. 1B). The effects of these eight compounds in activating the pyruvate kinase activity of PKM2 were confirmed by repeating the in-vitro pyruvate kinase assay with PKIII included as the reference compound (Fig. 1B).

Bottom Line: We demonstrate that PA-12 stimulates the pyruvate kinase activity of recombinant PKM2 in vitro, with a half-maximal activity concentration of 4.92 μM, and effectively suppresses both anchorage-dependent and -independent growth of lung cancer cells in non-essential amino acid-depleted medium.In addition, PA-12 blocked the nuclear translocalization of PKM2 in lung cancer cells, resulting in the inhibition of hypoxia response element (HRE)-mediated reporter activity as well as hypoxia-inducible factor 1 (HIF-1) target gene expression, eventually leading to the suppression of cell viability under hypoxia.Taken together, our data suggest that PA-12 is a novel and potent PKM2 activator that has therapeutic implications for lung cancer.

View Article: PubMed Central - PubMed

Affiliation: Medical Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 305-806, Korea.

ABSTRACT
Pyruvate kinase M2 isoform (PKM2), a rate-limiting enzyme in the final step of glycolysis, is known to be associated with the metabolic rewiring of cancer cells, and considered an important cancer therapeutic target. Herein, we report a novel PKM2 activator, PA-12, which was identified via the molecular docking-based virtual screening. We demonstrate that PA-12 stimulates the pyruvate kinase activity of recombinant PKM2 in vitro, with a half-maximal activity concentration of 4.92 μM, and effectively suppresses both anchorage-dependent and -independent growth of lung cancer cells in non-essential amino acid-depleted medium. In addition, PA-12 blocked the nuclear translocalization of PKM2 in lung cancer cells, resulting in the inhibition of hypoxia response element (HRE)-mediated reporter activity as well as hypoxia-inducible factor 1 (HIF-1) target gene expression, eventually leading to the suppression of cell viability under hypoxia. We also verified that the effects of PA-12 were dependent on PKM2 expression in cancer cells, demonstrating the specificity of PA-12 for PKM2 protein. Taken together, our data suggest that PA-12 is a novel and potent PKM2 activator that has therapeutic implications for lung cancer.

No MeSH data available.


Related in: MedlinePlus