Abstract

Glucose homeostasis is a vital and complex process, and its disruption can cause hyperglycaemia and type II diabetes mellitus1. Glucokinase (GK), a key enzyme that regulates glucose homeostasis, converts glucose to glucose-6-phosphate2,3 in pancreatic β-cells, liver hepatocytes, specific hypothalamic neurons, and gut enterocytes4. In hepatocytes, GK regulates glucose uptake and glycogen synthesis, suppresses glucose production3,5, and is subject to the endogenous inhibitor GK regulatory protein (GKRP)6,7,8. During fasting, GKRP binds, inactivates and sequesters GK in the nucleus, which removes GK from the gluconeogenic process and prevents a futile cycle of glucose phosphorylation. Compounds that directly hyperactivate GK (GK activators) lower blood glucose levels and are being evaluated clinically as potential therapeutics for the treatment of type II diabetes mellitus1,9,10. However, initial reports indicate that an increased risk of hypoglycaemia is associated with some GK activators11. To mitigate the risk of hypoglycaemia, we sought to increase GK activity by blocking GKRP. Here we describe the identification of two potent small-molecule GK–GKRP disruptors (AMG-1694 and AMG-3969) that normalized blood glucose levels in several rodent models of diabetes. These compounds potently reversed the inhibitory effect of GKRP on GK activity and promoted GK translocation both in vitro (isolated hepatocytes) and in vivo (liver). A co-crystal structure of full-length human GKRP in complex with AMG-1694 revealed a previously unknown binding pocket in GKRP distinct from that of the phosphofructose-binding site. Furthermore, with AMG-1694 and AMG-3969 (but not GK activators), blood glucose lowering was restricted to diabetic and not normoglycaemic animals. These findings exploit a new cellular mechanism for lowering blood glucose levels with reduced potential for hypoglycaemic risk in patients with type II diabetes mellitus.

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Accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the GKRP–AMG-1694 complex are deposited in the Protein Data Bank under accession code 4LY9.

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Acknowledgements

We would like to thank J. Calahan, J. Laubacher, M. Moore and D. Reid for pharmaceutics support, J. Civet for in vivo assistance, J. Han and R. Fachini for recombinant protein production, K. Kim for LC–MS/MS technical assistance, N. Nishimura and K. Yang for scale-up of AMG-3969, J. Chen for pharmacokinetic support, and A. Shaywitz and L. Rice for critical reading and editorial support of the manuscript.

Author information

Affiliations

  1. Department of Metabolic Disorders, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320, USA

    • David J. Lloyd
    • , Rod Cupples
    • , Michelle Chen
    • , John Wu
    • , Glenn Sivits
    • , Joan Helmering
    • , Renée Komorowski
    • , Minghan Wang
    • , Murielle M. Véniant
    •  & Clarence Hale
  2. Department of Therapeutic Discovery, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320, USA

    • David J. St Jean Jr
    • , Robert J. M. Kurzeja
    • , Robert C. Wahl
    • , Klaus Michelsen
    • , Kate S. Ashton
    • , Lewis D. Pennington
    • , Christopher Fotsch
    • , Mukta Vazir
    • , Kui Chen
    • , Samer Chmait
    • , Jiandong Zhang
    • , Longbin Liu
    • , Mark H. Norman
    • , Kristin L. Andrews
    • , Michael D. Bartberger
    •  & Steven R. Jordan
  3. Department of Comparative Biology & Safety Sciences, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320, USA

    • Gwyneth Van
    • , Elizabeth J. Galbreath
    •  & Steven L. Vonderfecht

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Contributions

D.J.L., S.R.J., M.M.V. and C.H. designed experiments, analysed data and wrote the manuscript. K.M. performed SPR spectroscopy. D.J.S., K.S.A., K.L.A., L.D.P., C.F., L.L., M.D.B. and M.H.N. were responsible for the design and synthesis of AMG-1694 and AMG-3969. R.C. collected data and performed biochemical assays. M.C. developed and collected data for the hepatocyte GK translocation assay. J.W. validated and collected data for the hepatocyte 2DG uptake assay. K.C. and R.C.W. designed, and K.C. performed, the high-throughput screen. R.C.W. developed the GK–GKRP binding assay. M.W. designed experiments. M.V. and R.J.M.K. generated protein reagents. S.C., J.Z. and S.R.J. conducted crystallographic studies. G.V., E.J.G. and S.L.V. performed IHC. G.S., J.H. and R.K. conducted in vivo experiments.

Competing interests

The authors declare competing financial interests as employees of Amgen Inc.

Corresponding authors

Correspondence to David J. Lloyd or Clarence Hale.

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https://doi.org/10.1038/nature12724

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