Discovery of Small-Molecule Glucokinase Regulatory Protein Modulators That Restore Glucokinase Activity.
In the nuclei of hepatocytes, glucokinase regulatory protein (GKRP) modulates the activity of glucokinase (GK), a key regulator of glucose homeostasis. Currently, direct activators of GK (GKAs) are in development for the treatment of type 2 diabetes. However, this approach is generally associated with a risk of hypoglycemia. To mitigate such risk, we target the GKRP regulation, which indirectly restores GK activity. Here we describe a screening strategy to look specifically for GKRP modulators, in addition to traditional GKAs. Two high-throughput screening campaigns were performed with our compound libraries using a luminescence assay format, one with GK alone and the other with a GK/GKRP complex in the presence of sorbitol-6-phosphate (S6P). By a subtraction method in the hit triage process of these campaigns, we discovered two close analogs that bind GKRP specifically with sub-µM potency to a site distinct from where fructose-1-phosphate binds. These small molecules are first-in-class allosteric modulators of the GK/GKRP interaction and are fully active even in the presence of S6P. Activation of GK by this particular mechanism, without altering the enzymatic profile, represents a novel pharmacologic modality of intervention in the GK/GKRP pathway.
Amgen, Inc, Molecular Structure and Characterization, Thousand Oaks, CA, USA chen@amgen.com.
Kui
K
Chen
Amgen, Inc, Molecular Structure and Characterization, Cambridge, MA, USA.
Klaus
K
Michelsen
Amgen, Inc, Protein Technologies, Thousand Oaks, CA, USA.
Robert J M
RJ
Kurzeja
Amgen, Inc, Biologic Discovery, Thousand Oaks, CA, USA.
Joon
J
Han
Amgen, Inc, Protein Technologies, Thousand Oaks, CA, USA.
Mukta
M
Vazir
Amgen, Inc, Medicinal Chemistry, Thousand Oaks, CA, USA.
David J
DJ
St Jean
Amgen, Inc, Metabolic Disorders, Thousand Oaks, CA, USA.
Clarence
C
Hale
Amgen, Inc, Molecular Structure and Characterization, Thousand Oaks, CA, USA.
Robert C
RC
Wahl
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000737:chemistry; D000494:Allosteric Regulation; D000818:Animals; D001786:Blood Glucose / Q000032:analysis; D002151:Calorimetry; D003924:Diabetes Mellitus, Type 2 / Q000188:drug therapy; D055808:Drug Discovery / Q000379:methods*; D005453:Fluorescence; D005470:Fluorometry; D005636:Fructosephosphates / Q000737:chemistry; D005941:Glucokinase / Q000737:chemistry*; D022781:Hepatocytes / Q000378:metabolism; D006600:Hexosephosphates / Q000737:chemistry; D006706:Homeostasis; D006801:Humans; D007003:Hypoglycemia / Q000517:prevention & control; D020128:Inhibitory Concentration 50; D049449:Luminescence; D011485:Protein Binding; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D051381:Rats; D020349:Surface Plasmon Resonance
D048868:Adaptor Proteins, Signal Transducing; D001786:Blood Glucose; D005636:Fructosephosphates; C485100:GCKR protein, human; D006600:Hexosephosphates; C032284:fructose-1-phosphate; C035752:sorbitol 6-phosphate; C027618:fructose-6-phosphate; D000255:Adenosine Triphosphate; D005941:Glucokinase
NADPH-coupled fluorescence assay; differential scanning fluorimetry (DSF); glucokinase (GK); glucokinase regulatory protein (GKRP); isothermal titration calorimetry (ITC); luminescence assay; surface plasmon resonance (SPR)