An allosteric activator of glucokinase impairs the interaction of glucokinase and glucokinase regulatory protein and regulates glucose metabolism

Glucokinase (GK) plays a key role in the control of blood glucose homeostasis. We identified a small molecule GK activator, compound A, that increased the glucose affinity and maximal velocity (V(max)) of GK. Compound A augmented insulin secretion from isolated rat islets and enhanced glucose utilization in primary cultured rat hepatocytes. In rat oral glucose tolerance tests, orally administrated compound A lowered plasma glucose elevation with a concomitant increase in plasma insulin and hepatic glycogen. In liver, GK activity is acutely controlled by its association to the glucokinase regulatory protein (GKRP). In order to decipher the molecular aspects of how GK activator affects the shuttling of GK between nucleus and cytoplasm, the effect of compound A on GK-GKRP interaction was further investigated. Compound A increased the level of cytoplasmic GK in both isolated rat primary hepatocytes and the liver tissues from rats. Experiments in a cell-free system revealed that compound A interacted with glucose-bound free GK, thereby impairing the association of GK and GKRP. On the other hand, compound A did not bind to glucose-unbound GK or GKRP-associated GK. Furthermore, we found that glucose-dependent GK-GKRP interaction also required ATP. Given the combined prominent role of GK on insulin secretion and hepatic glucose metabolism where the GK-GKRP mechanism is involved, activation of GK has a new therapeutic potential in the treatment of type 2 diabetes.     

Modulation of glucokinase by glucose, small-molecule activator and glucokinase regulatory protein: steady-state kinetic and cell-based analysis

GK (glucokinase) is an enzyme central to glucose metabolism that displays positive co-operativity to substrate glucose. Small-molecule GKAs (GK activators) modulate GK catalytic activity and glucose affinity and are currently being pursued as a treatment for Type 2 diabetes. GK progress curves monitoring product formation are linear up to 1 mM glucose, but biphasic at 5 mM, with the transition from the lower initial velocity to the higher steady-state velocity being described by the rate constant kact. In the presence of a liver-specific GKA (compound A), progress curves at 1 mM glucose are similar to those at 5 mM, reflecting activation of GK by compound A. We show that GKRP (GK regulatory protein) is a slow tight-binding inhibitor of GK. Analysis of progress curves indicate that this inhibition is time dependent, with apparent initial and final Ki values being 113 and 12.8 nM respectively. When GK is pre-incubated with glucose and compound A, the inhibition observed by GKRP is time dependent, but independent of GKRP concentration, reflecting the GKA-controlled transition between closed and open GK conformations. These data are supported by cell-based imaging data from primary rat hepatocytes. This work characterizes the modulation of GK by a novel GKA that may enable the design of new and improved GKAs.     

Discovery and preclinical development of AR453588 as an anti-diabetic glucokinase activator

Glucose flux through glucokinase (GK) controls insulin release from the pancreas in response to high levels of glucose. Flux through GK is also responsible for reducing hepatic glucose output. Since many individuals with type 2 diabetes appear to have an inadequacy or defect in one or both of these processes, identifying compounds that can activate GK could provide a therapeutic benefit. Herein we report the further structure activity studies of a novel series of glucokinase activators (GKA). These studies led to the identification of pyridine 72 as a potent GKA that lowered post-prandial glucose in normal C57BL/6J mice, and after 14d dosing in ob/ob mice.     

Salmonella regrowth in compost as influenced by substrate (salmonella regrowth in compost)

Composting can eliminate pathogenic organisms, including salmonellae, from sewage sludge. However, if salmonellae are present in the compost at undetectable levels or are inoculated into the compost by infected animals or from other sources, they may regrow presenting a health hazard for certain uses of compost. In this study, we examined dilute mineral-salt extracts of three composts from widely separate composting sites in the United States and found that they supported growth ofSalmonella typhimurium. From kinetic studies of the growth of the organism on these extracts, we concluded that each compost produced on extraction a single water-soluble substrate and that the substrates from the different composts were very similar, if not identical.We thank J. Robert Burge, Statistical Consulting and Analysis, ARS, USDA for invaluable help with the statistical analyses, and Alice V. Gibson of our laboratory for technical help.     

A substrate in pieces: allosteric activation of glycerol 3-phosphate dehydrogenase (NAD+) by phosphite dianion

The ratio of the second-order rate constants for reduction of dihydroxyacetone phosphate (DHAP) and of the neutral truncated substrate glycolaldehyde (GLY) by glycerol 3-phosphate dehydrogenase (NAD (+), GPDH) saturated with NADH is (1.0 x 10 (6) M (-1) s (-1))/(8.7 x 10 (-3) M (-1) s (-1)) = 1.1 x 10 (8), which was used to calculate an intrinsic phosphate binding energy of at least 11.0 kcal/mol. Phosphite dianion binds very weakly to GPDH ( K d > 0.1 M), but the bound dianion strongly activates GLY toward enzyme-catalyzed reduction by NADH. Thus, the large intrinsic phosphite binding energy is expressed only at the transition state for the GPDH-catalyzed reaction. The ratio of rate constants for the phosphite-activated and the unactivated GPDH-catalyzed reduction of GLY by NADH is (4300 M (-2) s (-1))/(8.7 x 10 (-3) M (-1) s (-1)) = 5 x 10 (5) M (-1), which was used to calculate an intrinsic phosphite binding energy of -7.7 kcal/mol for the association of phosphite dianion with the transition state complex for the GPDH-catalyzed reduction of GLY. Phosphite dianion has now been shown to activate bound substrates for enzyme-catalyzed proton transfer, decarboxylation, hydride transfer, and phosphoryl transfer reactions. Structural data provide strong evidence that enzymic activation by the binding of phosphite dianion occurs at a modular active site featuring (1) a binding pocket complementary to the reactive substrate fragment which contains all the active site residues needed to catalyze the reaction of the substrate piece or of the whole substrate and (2) a phosphate/phosphite dianion binding pocket that is completed by the movement of flexible protein loop(s) to surround the nonreacting oxydianion. We propose that loop motion and associated protein conformational changes that accompany the binding of phosphite dianion and/or phosphodianion substrates lead to encapsulation of the substrate and/or its pieces in the protein interior, and to placement of the active site residues in positions where they provide optimal stabilization of the transition state for the catalyzed reaction.     

Rate of hydrolysis of urea as influenced by thiourea and pellet size

Summary Two incubation experiments and a number of field experiments were conducted to determine the effect of soil moisture tension, pellet size and addition of thiourea to urea on the rate of urea hydrolysis. In the incubation experiments at 20°C, the rate of hydrolysis of urea increased from 15 bar to 1/3 bar soil moisture tension, with the largest change (doubling) occurring from 15 bar to 7 bar moisture tension. Increasing pellet size reduced the rate of urea hydrolysis by about 12% with urea pellets weighing 0.21 g as compared to 0.01 g urea pellets after 114h. When thiourea (a metabolic inhibitor) was pelleted with urea in a ratio of two parts urea and one part thiourea, the rate of hydrolysis was halved.In a field experiment, the addition of thiourea to urea and increasing pellet size suppressed the rate of urea hydrolysis considerably for 8 days. The amount of urea hydrolyzed with urea+thiourea (21) pellets weighing 2.51 g was one-fourth of the amount of urea hydrolyzed with 0.01 g pellets of urea alone. In the other six field experiments which were set out in October, only 22% to 39% of urea +thiourea (21) was hydrolyzed at two weeks after application, while almost all of the urea was hydrolyzed when it was mixed into the soil without an inhibitor.Unter our field conditions, we would estimate that the hydrolysis of urea can be inhibited for at least one week. The inhibition of urea hydrolysis appears to be great enough that the problems encountered from the rapid hydrolysis of urea, wherever these occur, may be reduced by combined use of thiourea and either increased pellet size or band placement.     

Discovery of 3-(4-methanesulfonylphenoxy)-N-[1-(2-methoxy-ethoxymethyl)-1H-pyrazol-3-yl]-5-(3-methylpyridin-2-yl)-benzamide as a novel glucokinase activator (GKA) for the treatment of type 2 diabetes mellitus

Novel heteroaryl-containing benzamide derivatives were synthesized and screened using an in vitro assay measuring increases in glucose uptake and glucokinase activity stimulated by 10 mM glucose in rat hepatocytes. From a library of synthesized compounds, 3-(4-methanesulfonylphenoxy)-N-[1-(2-methoxy-ethoxymethyl)-1H-pyrazol-3-yl]-5-(3-methyl pyridin-2-yl)-benzamide (19e) was identified as a potent glucokinase activator with assays demonstrating an EC₅₀ of 315 nM and the induction of a 2.23 fold increase in glucose uptake. Compound 19e exhibited a glucose AUC reduction of 32% (50 mg/kg) in an OGTT study with C57BL/6J mice compared to 28% for metformin (300 mg/kg). Single treatment of the compound in C57BL/J6 and ob/ob mice elicited basal glucose lowering activity, while in a two-week repeated dose study with ob/ob mice, the compound significantly decreased blood glucose levels with no evidence of hypoglycemia risk. In addition, 19e exhibited favorable pharmacokinetic parameters in mice and rats and excellent safety margins in liver and testicular toxicity studies. Compound 19e was therefore selected as a development candidate for the potential treatment of type 2 diabetes.     

Probing the allosteric activation of pyruvate carboxylase using 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

2′,3′-O-(2,4,6-Trinitrophenyl) adenosine 5′-triphosphate (TNP-ATP) is a fluorescent analogue of ATP. MgTNP-ATP was found to be an allosteric activator of pyruvate carboxylase that exhibits competition with acetyl CoA in activating the enzyme. There is no evidence that MgTNP-ATP binds to the MgATP substrate binding site of the enzyme. At concentrations above saturating, MgATP activates bicarbonate-dependent ATP cleavage, but inhibits the overall reaction. The fluorescence of MgTNP-ATP increases by about 2.5-fold upon binding to the enzyme and decreases on addition of saturating acetyl CoA. However, not all the MgTNP-ATP is displaced by acetyl CoA, or with a combination of saturating concentrations of MgATP and acetyl CoA. The kinetics of the binding of MgTNP-ATP to pyruvate carboxylase have been measured and shown to be triphasic, with the two fastest phases having pseudo first-order rate constants that are dependent on the concentration of MgTNP-ATP. The kinetics of displacement from the enzyme by acetyl CoA have been measured and also shown to be triphasic. A model of the binding process is proposed that links the kinetics of MgTNP-ATP binding to the allosteric activation of the enzyme.     

Notochordal development as influenced by the insecticide dicrotophos (Bidrin)

White Leghorn chicken embryos were treated at different ages with the insecticide dicrotophos to determine the time period of maximum effect upon notochordal development. Doses of insecticide ranging from 250 micrograms to 2.0 mg were injected into eggs at 8, 16, 24, 32, 40, 48, 72, or 96 hr of incubation and the eggs allowed to incubate for an additional 48 hr. Dicrotophos treatment caused dorsoventral and lateral folding of the notochord, with the cervical region being most severely affected. Although there was no apparent difference in dose responsiveness at any one age, there was an obvious age relationship. Notochordal responsiveness, expressed as both the number and severity of folds, was low among the 8- and 16-hr treated embryos, increased to a maximum in the 48-hr treatment group, and then declined among the older embryos. The time of maximum effect correlates closely with the time of sheath deposition and vacuolization of the notochord, but not to initial formation of the notochord from the mesoblast or later extracellular matrix production by sclerotome cells. It is proposed that dicrotophos interferes with some aspect of sheath formation. The pressure exerted by the vacuolization upon a structurally weakened sheath is thought to cause the observed folding.     

The design and optimization of a series of 2-(pyridin-2-yl)-1H-benzimidazole compounds as allosteric glucokinase activators

The optimization of a series of benzimidazole glucokinase activators is described. We identified a novel and potent achiral benzimidazole derivative as an allosteric GK activator. This activator was designed and synthesized via removal of the chiral center of the lead compound, 6-(N-acylpyrrolidin-2-yl)benzimidazole. The activator exhibited good PK profiles in rats and dogs, and significant hypoglycemic efficacy at 1 mg/kg po dosing in a rat OGTT model. The binding site and binding mode of the benzimidazole class of GKA with GK protein was confirmed by X-ray crystallographic analysis.     

Affinity labeling of the allosteric activator site(s) of spinach leaf ADP-glucose pyrophosphorylase

Pyridoxal-P has been shown to be an activator of the spinach leaf ADP-glucose pyrophosphorylase. It has a higher apparent affinity than the physiological activator 3-phosphoglycerate but only activates the enzyme activity 6-fold whereas 3-phosphoglycerate gives a 25-fold activation. Reductive phosphopyridoxylation of the spinach leaf enzyme results in enzyme having less dependence on the presence of activator for activity. Labeled pyridoxal-P is incorporated into both the 54- and 51-kilodalton subunits of the spinach leaf enzyme. The incorporation is inhibited by the presence of either 3-phosphoglycerate or the allosteric inhibitor, inorganic phosphate, thus suggesting that pyridoxal phosphate is covalently bound to the allosteric activator site. The pyridoxal phosphate is bound to an epsilon-amino group of a lysine residue. The phosphopyridoxylated enzyme is more resistant to phosphate inhibition than the unmodified form. The modified 51-kDa subunit has been digested with trypsin, and the peptide containing the labeled pyridoxal phosphate has been purified via high performance liquid chromatography and sequenced. Comparison of this sequence with the deduced amino acid sequence of a rice endosperm cDNA clone indicates that the putative allosteric site of the 51-kDa subunit is close to the carboxyl-terminal. This is in contrast to what had been demonstrated for the position of the activator site of the Escherichia coli ADP-glucose pyrophosphorylase which was shown to be close to the amino-terminal of the subunit.     

Biotoxicity of mercury as influenced by mercury(II) speciation

Integration of physicochemical procedures for studying mercury(II) speciation with microbiological procedures for studying the effects of mercury on bacterial growth allows evaluation of ionic factors (e.g., pH and ligand species and concentration) which affect biotoxicity. A Pseudomonas fluorescens strain capable of methylating inorganic Hg(II) was isolated from sediment samples collected at Buffalo Pound Lake in Saskatchewan, Canada. The effect of pH and ligand species on the toxic response (i.e., 50% inhibitory concentration [IC50]) of the P. fluorescens isolated to mercury were determined and related to the aqueous speciation of Hg(II). It was determined that the toxicities of different mercury salts were influenced by the nature of the co-ion. At a given pH level, mercuric acetate and mercuric nitrate yielded essentially the same IC50s; mercuric chloride, on the other hand, always produced lower IC50s. For each Hg salt, toxicity was greatest at pH 6.0 and decreased significantly (P = 0.05) at pH 7.0. Increasing the pH to 8.0 had no effect on the toxicity of mercuric acetate or mercuric nitrate but significantly (P = 0.05) reduced the toxicity of mercuric chloride. The aqueous speciation of Hg(II) in the synthetic growth medium M-IIY (a minimal salts medium amended to contain 0.1% yeast extract and 0.1% glycerol) was calculated by using the computer program GEOCHEM-PC with a modified data base. Results of the speciation calculations indicated that complexes of Hg(II) with histidine [Hg(H-HIS)HIS+ and Hg(H-HIS)2(2+)], chloride (HgCl+, HgCl2(0), HgClOH0, and HgCl3-), phosphate (HgHPO4(0), ammonia (HgNH3(2+), glycine [Hg(GLY)+], alanine [Hg(ALA)+], and hydroxyl ion (HgOH+) were the Hg species primarily responsible for toxicity in the M-IIY medium. The toxicity of mercuric nitrate at pH 8.0 was unaffected by the addition of citrate, enhanced by the addition of chloride, and reduced by the addition of cysteine. In the chloride-amended system, HgCl+, HgCl2(0), and HgClOH0 were the species primarily responsible for observed increases in toxicity. In the cysteine-amended system, formation of Hg(CYS)2(2-) was responsible for detoxification effects that were observed. The formation of Hg-citrate complexes was insignificant and had no effect on Hg toxicity.     

The regulatory design of an allosteric feedback loop: the effect of saturation by pathway substrate

Aspects of metabolic regulation can be fruitfully studied with a combination of generic modelling, control analysis and graphical analysis using rate characteristics. This paper analyses a prototypical supply-demand system consisting of a biosynthetic subsystem subject to allosteric inhibition by its product and a demand process that consumes this product. The effect of changes in affinity of the committing supply enzyme for the pathway substrate on the regulatory properties of the supply subsystem is compared for the Monod-Wyman-Changeux and the reversible Hill allosteric enzyme models. We found that the Hill model has a distinct advantage in that the steady-state concentration at which it maintains the product is set by the half-saturating product concentration and is independent of changes in the degree of saturation for substrate. In contrast, with the Monod-Wyman-Changeux model this set point varies with affinity for substrate. Explicitly incorporating reversibility in all rate equations made it possible to distinguish between kinetic and thermodynamic aspects of regulation. Combining the supply and demand rate characteristics allows us to explore both the control distribution at steady state and the regulatory performance of the system over a wide range of demand activities.