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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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.
The authors declare competing financial interests as employees of Amgen Inc.
Extended data figures and tables
Extended Data Figure 1 Optimization of the initial high-throughput screen hit (left) to produce AMG-1694 (right).
IC50 values were determined using the AlphaScreen GK–GKRP binding assay.
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. Kd = 0.005 ± 0.0003 μM; association rate constant (ka) = (2.7 × 105) ± (0.1 × 105) M−1 s−1; dissociation rate constant (kd) = (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 Kd = 0.029 ± 0.004 μM (ka = (8 × 104) ± (2 × 104) M−1s−1; kd = (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 (CCP4) refinement with both ligands removed from the calculations. Maps are contoured at ± 3σ. 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). Kd = 14 ± 0.4 μM; ka = (1.07 × 105) ± (0.02 × 105) M−1s−1; kd = 1.5 ± 0.1 s−1 (off-rate is outside of the range that can be measured accurately).
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).
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).
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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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