Antidiabetic effects of glucokinase regulatory protein small-molecule disruptors (original) (raw)

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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.

References

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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

Authors and Affiliations

  1. Department of Metabolic Disorders, Amgen Inc., One Amgen Center Drive, Thousand Oaks, 91320, California, 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, 91320, California, 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, 91320, California, USA
    Gwyneth Van, Elizabeth J. Galbreath & Steven L. Vonderfecht

Authors

  1. David J. Lloyd
  2. David J. St Jean Jr
  3. Robert J. M. Kurzeja
  4. Robert C. Wahl
  5. Klaus Michelsen
  6. Rod Cupples
  7. Michelle Chen
  8. John Wu
  9. Glenn Sivits
  10. Joan Helmering
  11. Renée Komorowski
  12. Kate S. Ashton
  13. Lewis D. Pennington
  14. Christopher Fotsch
  15. Mukta Vazir
  16. Kui Chen
  17. Samer Chmait
  18. Jiandong Zhang
  19. Longbin Liu
  20. Mark H. Norman
  21. Kristin L. Andrews
  22. Michael D. Bartberger
  23. Gwyneth Van
  24. Elizabeth J. Galbreath
  25. Steven L. Vonderfecht
  26. Minghan Wang
  27. Steven R. Jordan
  28. Murielle M. Véniant
  29. Clarence Hale

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.

Corresponding authors

Correspondence toDavid J. Lloyd or Clarence Hale.

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Competing interests

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

Extended data figures and tables

Extended Data Figure 2 SPR and crystallographic studies of GK, GKRP, S6P and AMG1694.

a, The binding of GKRP to GK was determined by SPR spectroscopy, using GKRP-biotin immobilized on a streptavidin biosensor surface. Binding of untagged GK was monitored at a highest concentration of 0.125 μM. _K_d = 0.005 ± 0.0003 μM; association rate constant (_k_a) = (2.7 × 105) ± (0.1 × 105) M−1 s−1; dissociation rate constant (_k_d) = (1.35 × 10−3) ± (0.01 × 10−3) s−1. b, Determination of the binding of S6P on GKRP. GKRP-biotin was immobilized at high density onto the SPR biosensor surface to allow small-molecule detection, and increasing concentrations of S6P (0.062, 0.185, 0.556, 1.667 and 5 μM indicated with arrows) were passed over the GKRP surface in the absence and presence of 50 µM S6P in the running buffer. S6P bound GKRP with a _K_d = 0.029 ± 0.004 μM (_k_a = (8 × 104) ± (2 × 104) M−1s−1; _k_d = (2.2 × 10−3) ± (0.4 × 10−3) s−1), whereas no binding was observed when S6P was pre-bound, in agreement with the sugar binding pocket being blocked by S6P. c, d, Difference electron density maps for AMG-1694 (c) and S6P (d). Maps were calculated by 10 cycles of REFMAC (CCP_4) refinement with both ligands removed from the calculations. Maps are contoured at ± 3_σ. e, Ribbon drawing showing AMG-1694 in its binding site making hydrogen bonds to the backbone nitrogen of Ile 11 and the side chain of Arg 525. Illustrated using PyMOL (Schrödinger). f, Interaction map for AMG-1694 as generated by the program MOE (Chemical Computing Group). g, AMG-1694 requires residues 1–19 of GKRP for efficient binding. Immobilized biotinylated GKRP (20–625) on a biosensor surface was exposed to increasing concentrations of AMG-1694 (arrows; 0.247, 0.741, 2.2, 6.6 and 20 μM). _K_d = 14 ± 0.4 μM; _k_a = (1.07 × 105) ± (0.02 × 105) M−1s−1; _k_d = 1.5 ± 0.1 s−1 (off-rate is outside of the range that can be measured accurately).

Extended Data Figure 3 GK translocation in a primary rat hepatocyte assay by glucose and AMG-1694.

a, Hepatocytes incubated with increasing concentrations of glucose resulted in clear nuclear disappearance and cytoplasmic appearance of GK as detected by immunocytochemistry and visualized in pseudo colour using the ArrayScan platform. Scale bars, 50 μm. b, Image analysis of a permitted the GK nuclear/cytoplasmic difference to be calculated, illustrating a dose response with glucose exposure. c, GK translocation in a hepatocyte assay visualized in psuedo colour using an Operetta platform. Hepatocytes were incubated with increasing concentrations of AMG-1694, resulting in clear nuclear disappearance and cytoplasmic appearance of GK. Scale bars, 15 μm. d, Image analysis of c assessing the nuclear/cytoplasmic difference demonstrated GK translocation dose response with AMG-1694.

Extended Data Figure 4 Effects of AMG-1694 on GK translocation, insulin and triglyceride levels in Wistar and ZDF rats.

a, Wistar rats were dosed with AMG-1694, and 1 h later livers were analysed. Both 30 and 100 mg kg−1 doses elicited GK translocation that was greater than that observed with glucose/fructose (gluc/fruc; 2 g per 0.25 g per /kg). b, e, Insulin and triglyceride levels after 1 h in Wistar rats dosed with multiple doses of AMG-1694 as presented in a. c, f, Insulin and triglyceride levels in Wistar rats dosed with AMG-1694 as presented in Fig. 3b. d, g, Insulin and triglyceride levels in ZDF rats dosed with AMG-1694 as presented in Fig. 3c. Error bars denote s.e.m.; n = 3. *P < 0.05, ***P < 0.001 versus vehicle (ANOVA).

Extended Data Figure 5 A GK–GKRP disruptor piperazine derivative (AMG-3969).

a, Optimization of AMG-1694 (left) to produce AMG-3969 (right). b, Potency of AMG-3969 using GK translocation in a primary rat hepatocyte assay visualized using an Operetta platform.

Extended Data Figure 6 GK immunoblotting in AMG1694 dosed ZDF rats and GKRP–S6P cocrystal structure overlay with and without AMG-1694.

a, Expression of GK and GKRP levels were unaltered in livers of ZDF rats dosed with 100 mg kg−1 AMG-1694 for 4 days. The blots were stripped and reprobed with anti-β-actin antibody to ensure equal loading. b, The GKRP–S6P cocrystal structure overlay with and without AMG-1694. Crystals were originally grown as a complex of GKRP and S6P (shown in red). The crystals were then soaked in a solution containing AMG-1694 (shown in green).

Extended Data Table 1 Small-molecule library and screening data

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Extended Data Table 2 Crystallographic data collection and refinement statistics of GKRP, S6P and AMG-1694

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Lloyd, D., St Jean, D., Kurzeja, R. et al. Antidiabetic effects of glucokinase regulatory protein small-molecule disruptors.Nature 504, 437–440 (2013). https://doi.org/10.1038/nature12724

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