Discovery of Small-Molecule Glucokinase Regulatory Protein Modulators That Restore Glucokinase Activity

Abstract

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. Keywords glucokinase (GK), glucokinase regulatory protein (GKRP), luminescence assay, NADPH-coupled fluorescence assay, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), differential scanning fluorimetry (DSF)

Discovery and Structure-Guided Optimization of Diarylmethanesulfonamide Disrupters of Glucokinase–Glucokinase Regulatory Protein (GK–GKRP) Binding: Strategic Use of a N → S (nN → σ*S–X) Interaction for Conformational Constraint

“…A second HTS campaign of a separate small-molecule library of over 126 000 compounds identified a single potent and selective hit, N -(1-benzofuran-2-yl­(phenyl)­methyl)-3,4-dihydro-2 H -1,5-benzodioxepine-7-sulfonamide ( 6 , Figure ). In these luciferase-based luminescence assays measuring ATP depletion, 6 was found to increase GK activity in the presence of GKRP (hGK–hGKRP ATP EC 50 = 0.26 μM) but to have no effect on GK activity in the absence of GKRP (hGK ATP EC 50 > 100 μM) .…”

“…A second HTS campaign of a separate small-molecule library of over 126 000 compounds identified a single potent and selective hit, N -(1-benzofuran-2-yl­(phenyl)­methyl)-3,4-dihydro-2 H -1,5-benzodioxepine-7-sulfonamide ( 6 , Figure ). In these luciferase-based luminescence assays measuring ATP depletion, 6 was found to increase GK activity in the presence of GKRP (hGK–hGKRP ATP EC 50 = 0.26 μM) but to have no effect on GK activity in the absence of GKRP (hGK ATP EC 50 > 100 μM) . Subsequently, HTS hit 6 was demonstrated in surface plasmon resonance assays to selectively bind GKRP (hGKRP K d = 1.1 μM; hGK K d > 20 μM) and was shown in a bead-based proximity assay (AlphaScreen) to disrupt the GK–GKRP binding interaction (hGK–hGKRP IC 50 = 1.2 μM) .…”

Non-stoichiometric inhibition in integrated lead finding – a literature review

“…However, protocols to immobilize even challenging protein species such as GPCRs [112] or the monomeric, oligomeric and fibrillar conformational variants of the Abeta protein fragment [113] have recently been published, indicating that the approach is now quite universally applicable. [100,[114][115][116] The behavior of four different types of non-stoichiometric inhibitors, i.e. chelating compounds, aggregating compounds, redox cycling compounds and compounds acting through redox-mediated covalent modification in DSF and SPR, has been experimentally analyzed and compared with the effect expected for stoichiometric inhibitors.…”

“…orthogonal and selectivity assays or in silico analysis. [100] Because hit compounds validated by experimental proof of stoichiometric target interaction can be directly passed on to medicinal chemists, binding assays should be applied as early as and whenever logistically possible. For example, AS-MS (affinity selection-mass spectrometry) provides evidence of compound-target interaction through separation of targetbound and free compound by size exclusion chromatography and subsequent identification of the ligand based on its molecular mass.…”

Glucokinase and glucokinase regulatory proteins as molecular targets for novel antidiabetic drugs

“…An analysis of the spatial structures made it possible to localize the fructose phosphate binding sites in the GKRP mole cule and to characterize in detail the structural changes occurring in the course of the GK-GKRP interaction. At the same time, the development of high throughput methods of quantitative analysis enabled massive screening of small molecule libraries in order to identify the compounds capable of modu lating the GK-GKRP interaction [44].…”