Thermodynamic Characterization of Allosteric Glycogen Phosphorylase Inhibitors

Abstract

Glycogen phosphorylase (GP) is a validated target for the treatment of type 2 diabetes. Here we describe highly potent GP inhibitors, AVE5688, AVE2865, and AVE9423. The first two compounds are optimized members of the acyl urea series. The latter represents a novel quinolone class of GP inhibitors, which is introduced in this study. In the enzyme assay, both inhibitor types compete with the physiological activator AMP and act synergistically with glucose. Isothermal titration calorimetry (ITC) shows that the compounds strongly bind to nonphosphorylated, inactive GP (GPb). Binding to phosphorylated, active GP (GPa) is substantially weaker, and the thermodynamic profile reflects a coupled transition to the inactive (tense) conformation. Crystal structures confirm that the three inhibitors bind to the AMP site of tense state GP. These data provide the first direct evidence that acyl urea and quinolone compounds are allosteric inhibitors that selectively bind to and stabilize the inactive conformation of the enzyme. Furthermore, ITC reveals markedly different thermodynamic contributions to inhibitor potency that can be related to the binding modes observed in the cocrystal structures. For AVE5688, which occupies only the lower part of the bifurcated AMP site, binding to GPb (Kd = 170 nM) is exclusively enthalpic (Delta H = -9.0 kcal/mol, TDelta S = 0.3 kcal/mol). The inhibitors AVE2865 (Kd = 9 nM, Delta H = -6.8 kcal/mol, TDelta S = 4.2 kcal/mol) and AVE9423 (Kd = 24 nM, Delta H = -5.9 kcal/mol, TDelta S = 4.6 kcal/mol) fully exploit the volume of the binding pocket. Their pronounced binding entropy can be attributed to the extensive displacement of solvent molecules as well as to ionic interactions with the phosphate recognition site.

Surf1, Associated with Leigh Syndrome in Humans, Is a Heme-binding Protein in Bacterial Oxidase Biogenesis

“…Protein samples were dialyzed twice for several hours at 4°C against measuring buffer (see Table 1) and centrifuged (20,000 ϫ g, 15 min, 4°C) to remove insoluble material. ITC titrations and data analyses were performed as described (28).…”

Biophysical Characterization of the Interaction between Hepatic Glucokinase and Its Regulatory Protein

“…In experiments, where the influence of effectors on the GK-GKRP interaction was studied, these compounds were added at equal concentrations to both cell and syringe solutions. ITC titrations and data analyses were performed as described (21). Unless otherwise stated, the titrations were performed at 25°C.…”

Understanding the Rate‐Limiting Step of Glycogenolysis by Using QM/MM Calculations on Human Glycogen Phosphorylase

“…There are three different isoenzymes depending on the tissue where they are expressed: liver, muscle, or brain. However, the liver GP is the one responsible for the glycemic demands of the body as a whole, and it is already a validated target for the treatment of DM‐2, as some specific inhibitors for GP decrease blood glucose levels in vivo . However, drugs against this target should exhibit selectivity over the liver isoform to prevent potential damage in other tissues, such as muscle cramping due to a buildup of glycogen in the tissue.…”

“…As shown in Figure 1, GP may exist in an active and relaxed state (R-GP) or in aless active tense state (T-GP). [6] Phosphorylation of Ser 14 by phosphorylase kinase (PKA) converts the inactive form (GPb) into the active one (GPa) and simultaneously promotes the Rstate (R-GPa,h igh activity andh igh substrate affinity),w hile dephosphorylationl eads to the opposite (T-GPb, low activity and low substrate affinity). [3b] As an allosteric enzyme,G Pf ollows the Monod-Wyman-Changeux model [7] for regulation and hast he typical multiple ligand bindings ites ( Figure 1a).…”