Metformin, the first drug chosen to be tested in a clinical trial aimed to target the biology of aging per se, has been clinically exploited for decades in the absence of a complete understanding of its therapeutic targets or chemical determinants. We here outline a systematic chemoinformatics approach to computationally predict biomolecular targets of metformin. Using several structure- and ligand-based software tools and reference databases containing 1,300,000 chemical compounds and more than 9,000 binding sites protein cavities, we identified 41 putative metformin targets including several epigenetic modifiers such as the member of the H3K27me3-specific demethylase subfamily, KDM6A/UTX. AlphaScreen and AlphaLISA assays confirmed the ability of metformin to inhibit the demethylation activity of purified KDM6A/UTX enzyme. Structural studies revealed that metformin might occupy the same set of residues involved in H3K27me3 binding and demethylation within the catalytic pocket of KDM6A/UTX. Millimolar metformin augmented global levels of H3K27me3 in cultured cells, including reversion of global loss of H3K27me3 occurring in premature aging syndromes, irrespective of mitochondrial complex I or AMPK. Pharmacological doses of metformin in drinking water or intraperitoneal injection significantly elevated the global levels of H3K27me3 in the hepatic tissue of low-density lipoprotein receptor-deficient mice and in the tumor tissues of highly aggressive breast cancer xenograft-bearing mice. Moreover, nondiabetic breast cancer patients receiving oral metformin in addition to standard therapy presented an elevated level of circulating H3K27me3. Our biocomputational approach coupled to experimental validation reveals that metformin might directly regulate the biological machinery of aging by targeting core chromatin modifiers of the epigenome.

CNRS UMR 8104 Paris France

Girona Biomedical Research Institute Girona Spain

INSERM U1016 Institut Cochin Paris France

Institute of Biotechnology Czech Academy of Sciences Prague West Czech Republic

Institute of Chemical Technology Prague Czech Republic

Mind the Byte Barcelona Spain

ProCURE Metabolism and Cancer Group Catalan Institute of Oncology Girona Catalonia Spain

School of Medical Science Menzies Health Institute Queensland Griffith University Southport Queensland Australia

Unit of Clinical Research Catalan Institute of Oncology Girona Spain

Unitat de Recerca Biomèdica Hospital Universitari de Sant Joan IISPV Rovira i Virgili University Reus Spain

Université Paris Descartes Sorbonne Paris Cité Paris France

Zobrazit více v PubMed

Barzilai, N. , Crandall, J. P. , Kritchevsky, S. B. , & Espeland, M. A. (2016). Metformin as a tool to target aging. Cell Metabolism, 23, 1060–1065. 10.1016/j.cmet.2016.05.011 PubMed DOI PMC

Benayoun, B. A. , Pollina, E. A. , & Brunet, A. (2015). Epigenetic regulation of ageing: Linking environmental inputs to genomic stability. Nature Reviews Molecular Cell Biology, 16, 593–610. 10.1038/nrm4048 PubMed DOI PMC

Booth, L. N. , & Brunet, A. (2016). The aging epigenome. Molecular Cell, 62, 728–744. 10.1016/j.molcel.2016.05.013 PubMed DOI PMC

Boukalova, S. , Stursa, J. , Werner, L. , Ezrova, Z. , Cerny, J. , Bezawork‐Geleta, A. , … Neuzil, J. (2016). Mitochondrial targeting of metformin enhances its activity against pancreatic cancer. Molecular Cancer Therapeutics, 15, 2875–2886. 10.1158/1535-7163.MCT-15-1021 PubMed DOI

Bridges, H. R. , Jones, A. J. , Pollak, M. N. , & Hirst, J. (2014). Effects of metformin and other biguanides on oxidative phosphorylation in mitochondria. The Biochemical Journal, 462, 475–487. 10.1042/BJ20140620 PubMed DOI PMC

Bridges, H. R. , Sirviö, V. A. , Agip, A. N. , & Hirst, J. (2016). Molecular features of biguanides required for targeting of mitochondrial respiratory complex I and activation of AMP‐kinase. BMC Biology, 14, 65 10.1186/s12915-016-0287-9 PubMed DOI PMC

Chance, B. , & Hollunger, G. (1963). Inhibition of electron and energy transfer in mitochondria. II. The site and the mechanism of guanidine action. Journal of Biological Chemistry, 238, 432–438. PubMed

Chandel, N. S. , Avizonis, D. , Reczek, C. R. , Weinberg, S. E. , Menz, S. , Neuhaus, R. , … Pollak, M. (2016). Are metformin doses used in murine cancer models clinically relevant? Cell Metabolism, 23, 569–570. 10.1016/j.cmet.2016.03.010 PubMed DOI

Cufi, S. , Corominas‐Faja, B. , Vazquez‐Martin, A. , Oliveras‐Ferraros, C. , Dorca, J. , Bosch‐Barrera, J. , … Menendez, J. A. (2012). Metformin‐induced preferential killing of breast cancer initiating CD44 + CD24‐/low cells is sufficient to overcome primary resistance to trastuzumab in HER2 +  human breast cancer xenografts. Oncotarget, 3, 395–398. PubMed PMC

Cuyàs, E. , Fernández‐Arroyo, S. , Verdura, S. , García, R. Á. , Stursa, J. , Werner, L. , … Menendez, J. A. (2017). Metformin regulates global DNA methylation via mitochondrial one‐carbon metabolism. Oncogene, 37(7), 963 10.1038/onc.2017.367. PubMed DOI

Deligezer, U. , Yaman, F. , Darendeliler, E. , Dizdar, Y. , Holdenrieder, S. , Kovancilar, M. , & Dalay, N. (2010). Post‐treatment circulating plasma BMP6 mRNA and H3K27 methylation levels discriminate metastatic prostate cancer from localized disease. Clinica Chimica Acta, 411, 1452–1456. 10.1016/j.cca.2010.05.040 PubMed DOI

Dowling, R. J. , Lam, S. , Bassi, C. , Mouaaz, S. , Aman, A. , Kiyota, T. , … Stambolic, V. (2016). Metformin pharmacokinetics in mouse tumors: Implications for human therapy. Cell Metabolism, 23, 567–568. 10.1016/j.cmet.2016.03.006 PubMed DOI

Egesipe, A. L. , Blondel, S. , Cicero, A. L. , Jaskowiak, A. L. , Navarro, C. , Sandre‐Giovannoli, A. , … Nissan, X. (2016). Metformin decreases progerin expression and alleviates pathological defects of Hutchinson‐Gilford progeria syndrome cells. NPJ Aging and Mechanisms of Disease, 2, 16026 10.1038/npjamd.2016.26 PubMed DOI PMC

Genheden, S. , & Ryde, U. (2015). The MM/PBSA and MM/GBSA methods to estimate ligand‐binding affinities. Expert Opinion on Drug Discovery, 10, 449–461. 10.1517/17460441.2015.1032936 PubMed DOI PMC

Gezer, U. , Yörüker, E. E. , Keskin, M. , Kulle, C. B. , Dharuman, Y. , & Holdenrieder, S. (2015). Histone methylation marks on circulating nucleosomes as novel blood‐based biomarker in colorectal cancer. International Journal of Molecular Sciences, 16, 29654–29662. 10.3390/ijms161226180 PubMed DOI PMC

Glossmann, H. , & Reider, N. (2013). A marriage of two “Methusalem” drugs for the treatment of psoriasis?: Arguments for a pilot trial with metformin as add‐on for methotrexate. Dermato‐endocrinology, 5, 252–263. 10.4161/derm.23874 PubMed DOI PMC

Griss, T. , Vincent, E. E. , Egnatchik, R. , Chen, J. , Ma, E. H. , Faubert, B. , … Jones, R. G. (2015). Metformin antagonizes cancer cell proliferation by suppressing mitochondrial‐dependent biosynthesis. PLoS Biology, 13, e1002309 10.1371/journal.pbio.1002309 PubMed DOI PMC

Han, S. , & Brunet, A. (2012). Histone methylation makes its mark on longevity. Trends in Cell Biology, 22, 42–49. 10.1016/j.tcb.2011.11.001 PubMed DOI PMC

Heublein, S. , Kazi, S. , Ogmundsdóttir, M. H. , Attwood, E. V. , Kala, S. , Boyd, C. A. , … Goberdhan, D. C. (2010). Proton‐assisted amino‐acid transporters are conserved regulators of proliferation and amino‐acid‐dependent mTORC1 activation. Oncogene, 29, 4068–4079. 10.1038/onc.2010.177 PubMed DOI PMC

Holm, K. , Grabau, D. , Lövgren, K. , Aradottir, S. , Gruvberger‐Saal, S. , Howlin, J. , … Ringnér, M. (2012). Global H3K27 methylation and EZH2 abundance in breast tumor subtypes. Molecular Oncology, 6, 494–506. 10.1016/j.molonc.2012.06.002 PubMed DOI PMC

Jin, C. , Li, J. , Green, C. D. , Yu, X. , Tang, X. , Han, D. , … Han, J. D. (2011). Histone demethylase UTX‐1 regulates C. elegans life span by targeting the insulin/IGF‐1 signaling pathway. Cell Metabolism, 14, 161–172. 10.1016/j.cmet.2011.07.001 PubMed DOI

König, J. , Müller, F. , & Fromm, M. F. (2013). Transporters and drug‐drug interactions: Important determinants of drug disposition and effects. Pharmacological Reviews, 65, 944–966. 10.1124/pr.113.007518 PubMed DOI

Kruidenier, L. , Chung, C. W. , Cheng, Z. , Liddle, J. , Che, K. , Joberty, G. , … Wilson, D. M. (2012). A selective jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response. Nature, 488, 404–408. 10.1038/nature11262 PubMed DOI PMC

Kumar, S. , Pamulapati, H. , & Tikoo, K. (2016). Fatty acid induced metabolic memory involves alterations in renal histone H3K36me2 and H3K27me3. Molecular and Cellular Endocrinology, 422, 233–242. 10.1016/j.mce.2015.12.019 PubMed DOI

Lockwood, T. D. (2010). The lysosome among targets of metformin: New anti‐inflammatory uses for an old drug? Expert Opinion on Therapeutic Targets, 14, 467–478. 10.1517/14728221003774135 PubMed DOI

Logie, L. , Harthill, J. , Patel, K. , Bacon, S. , Hamilton, D. L. , Macrae, K. , … Rena, G. (2012). Cellular responses to the metal‐binding properties of metformin. Diabetes, 61, 1423–1433. 10.2337/db11-0961 PubMed DOI PMC

López‐Otín, C. , Blasco, M. A. , Partridge, L. , Serrano, M. , & Kroemer, G. (2013). The hallmarks of aging. Cell, 153, 1194–1217. 10.1016/j.cell.2013.05.039 PubMed DOI PMC

López‐Otín, C. , Galluzzi, L. , Freije, J. M. , Madeo, F. , & Kroemer, G. (2016). Metabolic control of longevity. Cell, 166, 802–821. 10.1016/j.cell.2016.07.031 PubMed DOI

Martin‐Castillo, B. , Dorca, J. , Vazquez‐Martin, A. , Oliveras‐Ferraros, C. , Lopez‐Bonet, E. , Garcia, M. , … Menendez, J. A. (2010). Incorporating the antidiabetic drug metformin in HER2‐positive breast cancer treated with neo‐adjuvant chemotherapy and trastuzumab: An ongoing clinical‐translational research experience at the Catalan Institute of Oncology. Annals of Oncology, 21, 187–189. 10.1093/annonc/mdp494 PubMed DOI

Martin‐Castillo, B. , Oliveras‐Ferraros, C. , Vazquez‐Martin, A. , Cufí, S. , Moreno, J. M. , Corominas‐Faja, B. , … Menendez, J. A. (2013). Basal/HER2 breast carcinomas: Integrating molecular taxonomy with cancer stem cell dynamics to predict primary resistance to trastuzumab (Herceptin). Cell Cycle, 12, 225–245. 10.4161/cc.23274 PubMed DOI PMC

Maures, T. J. , Greer, E. L. , Hauswirth, A. G. , & Brunet, A. (2011). The H3K27 demethylase UTX‐1 regulates C. elegans lifespan in a germline‐independent, insulin‐dependent manner. Aging Cell, 10, 980–990. 10.1111/j.1474-9726.2011.00738.x PubMed DOI PMC

McAnena, P. , Brown, J. A. , & Kerin, M. J. (2017). Circulating nucleosomes and nucleosome modifications as biomarkers in cancer. Cancers, 9, pii: E5 10.3390/cancers9010005 PubMed DOI PMC

McCauley, B. S. , & Dang, W. (2014). Histone methylation and aging: Lesson learned from model systems. Biochimica et Biophysica Acta, 1839, 1454–1462. 10.1016/j.bbagrm.2014.05.008 PubMed DOI PMC

McCord, R. P. , Nazario‐Toole, A. , Zhang, H. , Chines, P. S. , Zhan, Y. , Erdos, M. R. , … Cao, K. (2013). Correlated alterations in genome organization, histone methylation, and DNA‐lamin A/C interactions in Hutchinson‐Gilford progeria syndrome. Genome Research, 23, 260–269. 10.1101/gr.138032.112 PubMed DOI PMC

Memmott, R. M. , Mercado, J. R. , Maier, C. R. , Kawabata, S. , Fox, S. D. , & Dennis, P. A. (2010). Metformin prevents tobacco carcinogen–induced lung tumorigenesis. Cancer Prevention Research (Philadelphia, Pa.), 3, 1066–1076. 10.1158/1940-6207.CAPR-10-0055 PubMed DOI PMC

Menendez, J. A. , Martin‐Castillo, B. , & Joven, J. (2016). Metformin and cancer: Quo vadis et cui bono? Oncotarget, 7, 54096–54101. PubMed PMC

Mishur, R. J. , Khan, M. , Munkácsy, E. , Sharma, L. , Bokov, A. , Beam, H. , … Rea, S. L. (2016). Mitochondrial metabolites extend lifespan. Aging Cell, 15, 336–348. 10.1111/acel.12439 PubMed DOI PMC

Newman, J. C. , Milman, S. , Hashmi, S. K. , Austad, S. N. , Kirkland, J. L. , Halter, J. B. , & Barzilai, N. (2016). Strategies and challenges in clinical trials targeting human aging. Journals of Gerontology. Series A, Biological Sciences and Medical Sciences, 71, 1424–1434. 10.1093/gerona/glw149 PubMed DOI PMC

Nigsch, F. , & Mitchell, J. B. (2008). How to winnow actives from inactives: Introducing molecular orthogonal sparse bigrams (MOSBs) and multiclass Winnow. Journal of Chemical Information and Modeling, 48, 306–318. 10.1021/ci700350n PubMed DOI

Oliveras‐Ferraros, C. , Corominas‐Faja, B. , Cufí, S. , Vazquez‐Martin, A. , Martin‐Castillo, B. , Iglesias, J. M. , … Menendez, J. A. (2012). Epithelial‐to‐mesenchymal transition (EMT) confers primary resistance to trastuzumab (Herceptin). Cell Cycle, 11, 4020–4032. 10.4161/cc.22225 PubMed DOI PMC

Pal, S. , & Tyler, J. K. (2016). Epigenetics and aging. Science Advances, 2, e1600584 10.1126/sciadv.1600584 PubMed DOI PMC

Pernas, S. , Dorca, J. , Álvarez‐López, I. , Martínez, S. , Saura, C. , Batista López, N. , … Martin‐Castillo, B. 2017. Safety and efficacy of neoadjuvant metformin with trastuzumab and chemotherapy in women with HER2‐positive early breast cancer: A randomized, open‐label, multicenter, phase 2 trial. Poster presented at: 2017 San Antonio Breast Cancer Symposium (SABCS); December 5–9, 2017; San Antonio, TX.

Pernicova, I. , & Korbonits, M. (2014). Metformin–mode of action and clinical implications for diabetes and cancer. Nature Reviews. Endocrinology, 10, 143–156. 10.1038/nrendo.2013.256 PubMed DOI

Piñero, J. , Bravo, À. , Queralt‐Rosinach, N. , Gutiérrez‐Sacristán, A. , Deu‐Pons, J. , Centeno, E. , … Furlong, L. I. (2017). DisGeNET: A comprehensive platform integrating information on human disease‐associated genes and variants. Nucleic Acids Research, 45, D833–D839. PubMed PMC

Reagan‐Shaw, S. , Nihal, M. , & Ahmad, N. (2008). Dose translation from animal to human studies revisited. FASEB Journal, 22, 659–661. 10.1096/fj.07-9574LSF PubMed DOI

Riera‐Borrull, M. , García‐Heredia, A. , Fernández‐Arroyo, S. , Hernández‐Aguilera, A. , Cabré, N. , Cuyàs, E. , … Joven, J. (2017). Metformin potentiates the benefits of dietary restraint: A metabolomic study. International Journal of Molecular Sciences, 18, pii: E2263 10.3390/ijms18112263 PubMed DOI PMC

Sakaki, H. , Okada, M. , Kuramoto, K. , Takeda, H. , Watarai, H. , Suzuki, S. , … Kitanaka, C. (2015). GSKJ4, a selective jumonji H3K27 demethylase inhibitor, effectively targets ovarian cancer stem cells. Anticancer Research, 35, 6607–6614. PubMed

Scaffidi, P. , & Misteli, T. (2005). Reversal of the cellular phenotype in the premature aging disease Hutchinson‐Gilford progeria syndrome. Nature Medicine, 11, 440–445. 10.1038/nm1204 PubMed DOI PMC

Sen, P. , Dang, W. , Donahue, G. , Dai, J. , Dorsey, J. , Cao, X. , … Berger, S. L. (2015). H3K36 methylation promotes longevity by enhancing transcriptional fidelity. Genes & Development, 29, 1362–1376. 10.1101/gad.263707.115 PubMed DOI PMC

Sengoku, T. , & Yokoyama, S. (2011). Structural basis for histone H3 Lys 27 demethylation by UTX/KDM6A. Genes & Development, 25, 2266–2277. 10.1101/gad.172296.111 PubMed DOI PMC

Shah, P. P. , Donahue, G. , Otte, G. L. , Capell, B. C. , Nelson, D. M. , Cao, K. , … Berger, S. L. (2013). Lamin B1 depletion in senescent cells triggers large‐scale changes in gene expression and the chromatin landscape. Genes & Development, 27, 1787–1799. 10.1101/gad.223834.113 PubMed DOI PMC

Shpargel, K. B. , Sengoku, T. , Yokoyama, S. , & Magnuson, T. (2012). UTX and UTY demonstrate histone demethylase‐independent function in mouse embryonic development. PLoS Genetics, 8, e1002964 10.1371/journal.pgen.1002964 PubMed DOI PMC

Shumaker, D. K. , Dechat, T. , Kohlmaier, A. , Adam, S. A. , Bozovsky, M. R. , Erdos, M. R. , … Goldman, R. D. (2006). Mutant nuclear lamin A leads to progressive alterations of epigenetic control in premature aging. Proceedings of the National Academy of Sciences of the United States of America, 103, 8703–8708. 10.1073/pnas.0602569103 PubMed DOI PMC

Sweeney, D. , Raymer, M. L. , & Lockwood, T. D. (2003). Antidiabetic and antimalarial biguanide drugs are metal‐interactive antiproteolytic agents. Biochemical Pharmacology, 66, 663–677. 10.1016/S0006-2952(03)00338-1 PubMed DOI

Tan, A. S. , Baty, J. W. , Dong, L. F. , Bezawork‐Geleta, A. , Endaya, B. , Goodwin, J. , … Berridge, M. V. (2015). Mitochondrial genome acquisition restores respiratory function and tumorigenic potential of cancer cells without mitochondrial DNA. Cell Metabolism, 21, 81–94. 10.1016/j.cmet.2014.12.003 PubMed DOI

Tan, L. , Ke, Z. , Tombline, G. , Macoretta, N. , Hayes, K. , Tian, X. , … Gorbunova, V. (2017). Naked mole rat cells have a stable epigenome that resists iPSC reprogramming. Stem Cell Reports, 9, 1721–1734. 10.1016/j.stemcr.2017.10.001 PubMed DOI PMC

Tarhonskaya, H. , Nowak, R. P. , Johansson, C. , Szykowska, A. , Tumber, A. , Hancock, R. L. , … Kawamura, A. (2017). Studies on the interaction of the histone demethylase KDM5B with tricarboxylic acid cycle intermediates. Journal of Molecular Biology, 429, 2895–2906. 10.1016/j.jmb.2017.08.007 PubMed DOI PMC

Thorne, D. P. , & Lockwood, T. D. (1991). Effect of Zn2 +  on the proteolytic inhibitory action of insulin and biguanide antihyperglycemic drugs. Diabetes, 40, 612–620. 10.2337/diab.40.5.612 PubMed DOI

Tikoo, K. , Sharma, E. , Amara, V. R. , Pamulapati, H. , & Dhawale, V. S. (2016). Metformin improves metabolic memory in high fat diet (HFD)‐induced renal dysfunction. Journal of Biological Chemistry, 291, 21848–21856. 10.1074/jbc.C116.732990 PubMed DOI PMC

Vazquez‐Martin, A. , Cufi, S. , Lopez‐Bonet, E. , Corominas‐Faja, B. , Oliveras‐Ferraros, C. , Martin‐Castillo, B. , & Menendez, J. A. (2012). Metformin limits the tumourigenicity of iPS cells without affecting their pluripotency. Scientific Reports, 2, 964 10.1038/srep00964 PubMed DOI PMC

Vella, S. , Gnani, D. , Crudele, A. , Ceccarelli, S. , De Stefanis, C. , Gaspari, S. , … Alisi, A. (2013). EZH2 down‐regulation exacerbates lipid accumulation and Inflammation in in vitro and in vivo NAFLD. International Journal of Molecular Sciences, 14, 24154–24168. 10.3390/ijms141224154 PubMed DOI PMC

Wan, L. , Xu, K. , Wei, Y. , Zhang, J. , Han, T. , Fry, C. , … Wei, W. (2018). Phosphorylation of EZH2 by AMPK suppresses PRC2 methyltransferase activity and oncogenic function. Molecular Cell, 69, 279–291.e5. 10.1016/j.molcel.2017.12.024 PubMed DOI PMC

Watarai, H. , Okada, M. , Kuramoto, K. , Takeda, H. , Sakaki, H. , Suzuki, S. , … Kitanaka, C. (2016). Impact of H3K27 demethylase inhibitor GSKJ4 on NSCLC cells alone and in combination with metformin. Anticancer Research, 36, 6083–6092. 10.21873/anticanres PubMed DOI

Wei, Y. , Xia, W. , Zhang, Z. , Liu, J. , Wang, H. , Adsay, N. V. , … Hung, M. C. (2008). Loss of trimethylation at lysine 27 of histone H3 is a predictor of poor outcome in breast, ovarian, and pancreatic cancers. Molecular Carcinogenesis, 47, 701–706. 10.1002/mc.20413 PubMed DOI PMC

Yan, N. , Xu, L. , Wu, X. , Zhang, L. , Fei, X. , Cao, Y. , & Zhang, F. (2017). GSKJ4, an H3K27me3 demethylase inhibitor, effectively suppresses the breast cancer stem cells. Experimental Cell Research, 359, 405–414. 10.1016/j.yexcr.2017.08.024 PubMed DOI

Zhang, W. , Li, J. , Suzuki, K. , Qu, J. , Wang, P. , Zhou, J. , … Belmonte, J. C. (2015). Aging stem cells. A Werner syndrome stem cell model unveils heterochromatin alterations as a driver of human aging. Science, 348, 1160–1163. 10.1126/science.aaa1356 PubMed DOI PMC