Robust anti-obesity and metabolic effects of a dual GLP-1/glucagon receptor peptide agonist in rodents and non-human primates.

AIMS: To characterize the pharmacology of MEDI0382, a peptide dual agonist of glucagon-like peptide-1 (GLP-1) and glucagon receptors. MATERIALS AND METHODS: MEDI0382 was evaluated in vitro for its ability to stimulate cAMP accumulation in cell lines expressing transfected recombinant or endogenous GLP-1 or glucagon receptors, to potentiate glucose-stimulated insulin secretion (GSIS) in pancreatic ß-cell lines and stimulate hepatic glucose output (HGO) by primary hepatocytes. The ability of MEDI0382 to reduce body weight and improve energy balance (i.e. food intake and energy expenditure), as well as control blood glucose, was evaluated in mouse models of obesity and healthy cynomolgus monkeys following single and repeated daily subcutaneous administration for up to 2 months. RESULTS: MEDI0382 potently activated rodent, cynomolgus and human GLP-1 and glucagon receptors and exhibited a fivefold bias for activation of GLP-1 receptor versus the glucagon receptor. MEDI0382 produced superior weight loss and comparable glucose lowering to the GLP-1 peptide analogue liraglutide when administered daily at comparable doses in DIO mice. The additional fat mass reduction elicited by MEDI0382 probably results from a glucagon receptor-mediated increase in energy expenditure, whereas food intake suppression results from activation of the GLP-1 receptor. Notably, the significant weight loss elicited by MEDI0382 in DIO mice was recapitulated in cynomolgus monkeys. CONCLUSIONS: Repeated administration of MEDI0382 elicits profound weight loss in DIO mice and non-human primates, produces robust glucose control and reduces hepatic fat content and fasting insulin and glucose levels. The balance of activities at the GLP-1 and glucagon receptors is considered to be optimal for achieving weight and glucose control in overweight or obese Type 2 diabetic patients.

Genetic Diversity and Protective Efficacy of the RTS,S/AS01 Malaria Vaccine.

BACKGROUND: The RTS,S/AS01 vaccine targets the circumsporozoite protein of Plasmodium falciparum and has partial protective efficacy against clinical and severe malaria disease in infants and children. We investigated whether the vaccine efficacy was specific to certain parasite genotypes at the circumsporozoite protein locus. METHODS: We used polymerase chain reaction-based next-generation sequencing of DNA extracted from samples from 4985 participants to survey circumsporozoite protein polymorphisms. We evaluated the effect that polymorphic positions and haplotypic regions within the circumsporozoite protein had on vaccine efficacy against first episodes of clinical malaria within 1 year after vaccination. RESULTS: In the per-protocol group of 4577 RTS,S/AS01-vaccinated participants and 2335 control-vaccinated participants who were 5 to 17 months of age, the 1-year cumulative vaccine efficacy was 50.3% (95% confidence interval [CI], 34.6 to 62.3) against clinical malaria in which parasites matched the vaccine in the entire circumsporozoite protein C-terminal (139 infections), as compared with 33.4% (95% CI, 29.3 to 37.2) against mismatched malaria (1951 infections) (P=0.04 for differential vaccine efficacy). The vaccine efficacy based on the hazard ratio was 62.7% (95% CI, 51.6 to 71.3) against matched infections versus 54.2% (95% CI, 49.9 to 58.1) against mismatched infections (P=0.06). In the group of infants 6 to 12 weeks of age, there was no evidence of differential allele-specific vaccine efficacy. CONCLUSIONS: These results suggest that among children 5 to 17 months of age, the RTS,S vaccine has greater activity against malaria parasites with the matched circumsporozoite protein allele than against mismatched malaria. The overall vaccine efficacy in this age category will depend on the proportion of matched alleles in the local parasite population; in this trial, less than 10% of parasites had matched alleles. (Funded by the National Institutes of Health and others.).

Thermal stability of glucokinase (GK) as influenced by the substrate glucose, an allosteric glucokinase activator drug (GKA) and the osmolytes glycerol and urea.

We investigated how glycerol, urea, glucose and a GKA influence kinetics and stability of wild-type and mutant GK. Glycerol and glucose stabilized GK additively. Glycerol barely affected the TF spectra of all GKs but decreased k(cat), glucose S(0.5) and K(D) values and ATP K(M) while leaving cooperativity unchanged. Glycerol sensitized all GKs to GKA as shown by TF. Glucose increased TF of GKs without influence of glycerol on the effect. Glycerol and GKA affected kinetics and binding additively. The activation energies for thermal denaturation of GK were a function of glucose with K(D)s of 3 and 1mM without and with glycerol, respectively. High urea denatured wild type GK reversibly at 20 and 60°C and urea treatment of irreversibly heat denatured GK allowed refolding as demonstrated by TF including glucose response. We concluded: Glycerol stabilizes GK indirectly without changing the folding structure of the apoenzyme, by restructuring the surface water of the protein, whereas glucose stabilizes GK directly by binding to its substrate site and inducing a compact conformation. Glucose or glycerol (alone or combined) is unable to prevent irreversible heat denaturation above 40°C. However, urea denatures GK reversibly even at 60°C by binding to the protein backbone and directly interacting with hydrophobic side chains. It prevents irreversible aggregation allowing complete refolding when urea is removed. This study establishes the foundation for exploring numerous instability mutants among the more than 600 variant GKs causing diabetes in animals and humans.

Glucokinase activators for the potential treatment of type 2 diabetes.

The search for innovative and clinically-differentiated medicines for the treatment of type 2 diabetes is an active area of research for pharmaceutical companies. The discovery of allosteric Glucokinase (GK) activators in 2003 represents the first time a pharmaceutical agent was used to directly augment the actions of an enzyme by increasing its maximal velocity and substrate affinity. This discovery, coupled with translational medicine which has shown that inactivating and activating GK mutations cause glycemic diseases, has triggered an intensive medicinal chemistry effort in the field of glucokinase activators (GKAs). The antidiabetic effects of GK activators observed in animal models support the notion that these agents act to both augment insulin release from pancreatic beta-cells and suppress hepatic glucose production in the liver. This review describes the unprecedented task of optimizing small molecules in order to affect the appropriate changes in the kinetic parameters of an enzyme. In addition, a pharmacophore model for the various classes of glucokinase activators that have been described in the literature will be presented. Overall, the available data suggests that potent glucokinase activators with the desired effects on the kinetic properties of the enzyme can be designed to achieve strong and persistent antidiabetic effects. GK activators thus represent a promising new treatment for type 2 diabetes.

Glucokinase and glucose homeostasis: proven concepts and new ideas.

The enzyme GK (glucokinase), which phosphorylates glucose to form glucose 6-phosphate, serves as the glucose sensor of insulin-producing beta-cells. GK has thermodynamic, kinetic, regulatory and molecular genetic characteristics that are ideal for its glucose sensor function and allow it to control glycolytic flux of the beta-cells as indicated by control-, elasticity- and response-coefficients close to or larger than 1.0. GK operates in tandem with the K(+) and Ca(2+) channels of the beta-cell membrane, resulting in a threshold for glucose-stimulated insulin release of approx. 5 mM, which is the set point of glucose homoeostasis for most laboratory animals and humans. Point mutations of GK cause 'glucokinase disease' in humans, which includes hypo- and hyper-glycaemia syndromes resulting from activating or inactivating mutations respectively. GK is allosterically activated by pharmacological agents (called GK activators), which lower blood glucose in normal animals and animal models of T2DM. On the basis of crystallographic studies that identified a ligand-free 'super-open' and a liganded closed structure of GK, on thermostability studies using glucose or mannoheptulose as ligands and studies showing that mannoheptulose alone or combined with GK activators induces expression of GK in pancreatic islets and partially preserves insulin secretory competency, a new hypothesis was developed that GK may function as a metabolic switch per se without involvement of enhanced glucose metabolism. Current research has the goal to find molecular targets of this putative 'GK-switch'. The case of GK research illustrates how basic science may culminate in therapeutic advances of human medicine.

Clinical features and mutations in patients with dominant retinitis pigmentosa-1 (RP1).

PURPOSE: To survey patients with dominant retinitis pigmentosa (RP) for mutations in the RP1 gene to determine the spectrum of dominant mutations in this gene, to estimate the proportion of dominant RP caused by this gene, and to determine whether the clinical features of patients with RP1 mutations differ from features of those with rhodopsin mutations. METHODS: A set of 241 patients who did not have mutations in the rhodopsin gene (based on previous work) formed the basis for the study. Of these patients, 117 had also been previously evaluated and were found not to carry mutations in the RDS gene. The single-strand conformation polymorphism (SSCP) method was used to search for sequence variants, which were then directly sequenced. The relatives of selected patients were recruited for segregation analyses. Clinical evaluations of patients included a measurement of Snellen visual acuity, final dark adaptation thresholds, visual fields, and ERGs. Clinical data were compared with those obtained earlier from a study of 128 patients with dominant rhodopsin mutations. RESULTS: Of the 241 patients, all were screened for the most common RP1 mutation (Arg677Ter), and 10 patients were found to have this mutation. In addition, an evaluation of a subset of 189 patients in whom the entire coding sequence was evaluated revealed the following mutations: Gln679Ter (1 case), Gly723Ter (2 cases), Glu729(1-bp del) (1 case), Leu762(5-bp del) (2 cases), and Asn763(4-bp del) (1 case). All of these mutations cosegregated with RP in the families of the index patients. Nine missense mutations that were each found in six or fewer patients were encountered. The segregation of eight of these was evaluated in the respective patients' families, and only one segregated with dominant RP. This cosegregating missense change was in cis with the nonsense mutation Gln679Ter. Although patients with RP1 mutations had, on average, slightly better visual acuity than patients with rhodopsin mutations, there was no statistically significant difference in final dark-adaptation thresholds, visual field diameters, or cone electroretinogram (ERG) amplitudes. Comparably aged patients with RP1 mutations had visual function that varied by approximately two orders of magnitude, based on visual fields and ERG amplitudes. CONCLUSIONS: Dominant RP1 alleles typically have premature nonsense codons occurring in the last exon of the gene and would be expected to encode mutant proteins that are only approximately one third the size of the wild-type protein, suggesting that a dominant negative effect rather than haploinsufficiency is the mechanism leading to RP caused by RP1 mutations. On average, patients with RP1 mutations have slightly better visual acuity than patients with dominant rhodopsin mutations; otherwise, they have similarly severe disease. The wide range in severity among patients with RP1 mutations indicates that other genetic or environmental factors modulate the effect of the primary mutation.

Null RPGRIP1 alleles in patients with Leber congenital amaurosis.

We isolated and characterized the entire coding sequence of a human gene encoding a protein that interacts with RPGR, a protein that is absent or mutant in many cases of X-linked retinitis pigmentosa. The newly identified gene, called "RPGRIP1" for RPGR-interacting protein (MIM 605446), is located within 14q11, and it encodes a protein predicted to contain 1,259 amino acids. Previously published work showed that both proteins, RPGR and RPGRIP1, are present in the ciliary structure that connects the inner and outer segments of rod and cone photoreceptors. We surveyed 57 unrelated patients who had Leber congenital amaurosis for mutations in RPGRIP1 and found recessive mutations involving both RPGRIP1 alleles in 3 (6%) patients. The mutations all create premature termination codons and are likely to be null alleles. Patients with RPGRIP1 mutations have a degeneration of both rod and cone photoreceptors, and, early in life, they experience a severe loss of central acuity, which leads to nystagmus.

Glucokinase gene locus transgenic mice are resistant to the development of obesity-induced type 2 diabetes.

Transgenic mice that overexpress the entire glucokinase (GK) gene locus have been previously shown to be mildly hypoglycemic and to have improved tolerance to glucose. To determine whether increased GK might also prevent or diminish diabetes in diet-induced obese animals, we examined the effect of feeding these mice a high-fat high-simple carbohydrate low-fiber diet (HF diet) for 30 weeks. In response to this diet, both normal and transgenic mice became obese and had similar BMIs (5.3 +/- 0.1 and 5.0 +/- 0.1 kg/m2 in transgenic and non-transgenic mice, respectively). The blood glucose concentration of the control mice increased linearly with time and reached 17.0 +/- 1.3 mmol/l at the 30th week. In contrast, the blood glucose of GK transgenic mice rose to only 9.7 +/- 1.2 mmol/l at the 15th week, after which it returned to 7.6 +/- 1.0 mmol/l by the 30th week. The plasma insulin concentration was also lower in the GK transgenic animals (232 +/- 79 pmol/l) than in the controls (595 +/- 77 pmol/l), but there was no difference in plasma glucagon concentrations. Together, these data indicate that increased GK levels dramatically lessen the development of both hyperglycemia and hyperinsulinemia associated with the feeding of an HF diet.

Characterization of glucokinase regulatory protein-deficient mice.

The glucokinase regulatory protein (GKRP) inhibits glucokinase competitively with respect to glucose by forming a protein-protein complex with this enzyme. The physiological role of GKRP in controlling hepatic glucokinase activity was addressed using gene targeting to disrupt GKRP gene expression. Heterozygote and homozygote knockout mice have a substantial decrease in hepatic glucokinase expression and enzymatic activity as measured at saturating glucose concentrations when compared with wild-type mice, with no change in basal blood glucose levels. Interestingly, when assayed under conditions to promote the association between glucokinase and GKRP, liver glucokinase activity in wild-type and null mice displayed comparable glucose phosphorylation capacities at physiological glucose concentrations (5 mM). Thus, despite reduced hepatic glucokinase expression levels in the null mice, glucokinase activity in the liver homogenates was maintained at nearly normal levels due to the absence of the inhibitory effects of GKRP. However, following a glucose tolerance test, the homozygote knockout mice show impaired glucose clearance, indicating that they cannot recruit sufficient glucokinase due to the absence of a nuclear reserve. These data suggest both a regulatory and a stabilizing role for GKRP in maintaining adequate glucokinase in the liver. Furthermore, this study provides evidence for the important role GKRP plays in acutely regulating of hepatic glucose metabolism.