Jorge Alsina-Fernandez

Small molecule allosteric modulation of the glucagon-like Peptide-1 receptor enhances the insulinotropic effect of oxyntomodulin.

Identifying novel mechanisms to enhance glucagon-like peptide-1 (GLP-1) receptor signaling may enable nascent medicinal chemistry strategies with the aim of developing new orally available therapeutic agents for the treatment of type 2 diabetes mellitus. Therefore, we tested the hypothesis that selectively modulating the low-affinity GLP-1 receptor agonist, oxyntomodulin, would improve the insulin secretory properties of this naturally occurring hormone to provide a rationale for pursuing an unexplored therapeutic approach. Signal transduction and competition binding studies were used to investigate oxyntomodulin activity on the GLP-1 receptor in the presence of the small molecule GLP-1 receptor modulator, 4-(3-benzyloxyphenyl)-2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine (BETP). In vivo, the intravenous glucose tolerance test characterized oxyntomodulin-induced insulin secretion in animals administered the small molecule. BETP increased oxyntomodulin binding affinity for the GLP-1 receptor and enhanced oxyntomodulin-mediated GLP-1 receptor signaling as measured by activation of the α subunit of heterotrimeric G protein and cAMP accumulation. In addition, oxyntomodulin-induced insulin secretion was enhanced in the presence of the compound. BETP was pharmacologically characterized to induce biased signaling by oxyntomodulin. These studies demonstrate that small molecules targeting the GLP-1 receptor can increase binding and receptor activation of the endogenous peptide oxyntomodulin. The biased signaling engendered by BETP suggests that GLP-1 receptor mobilization of cAMP is the critical insulinotropic signaling event. Because of the unique metabolic properties of oxyntomodulin, identifying molecules that enhance its activity should be pursued to assess the efficacy and safety of this novel mechanism.

Central injection of the stable somatostatin analog ODT8-SST induces a somatostatin2 receptor-mediated orexigenic effect: role of neuropeptide Y and opioid signaling pathways in rats.

Somatostatin and octreotide injected into the brain have been reported to modulate food intake. However, little is known regarding the underlying mechanisms. The stable oligosomatostatin analog, des-AA(1,2,4,5,12,13)-[DTrp(8)]-somatostatin (ODT8-SST), like somatostatin, binds to all five somatostatin receptors (sst(1-5)). We characterized the effects of ODT8-SST injected intracerebroventricularly (i.c.v.) on food consumption and related mechanisms of action in freely fed rats. ODT8-SST (0.3 and 1 microg per rat, i.c.v.) injected during the light or dark phase induced an early onset (within 1 h) and long-lasting (4 h) increase in food intake in nonfasted rats. By contrast, i.p. injection (0.3-3 mg/kg) or i.c.v. injection of selective sst(1) or sst(4) agonists (1 microg per rat) had no effect. The 2 h food intake response during the light phase was blocked by i.c.v. injection of a sst(2) antagonist, the neuropeptide Y (NPY) Y(1) receptor antagonist, BIBP-3226, and ip injection of the mu-opioid receptor antagonist, naloxone, and not associated with changes in plasma ghrelin levels. ODT8-SST (1 microg per rat, i.c.v.) stimulated gastric emptying of a solid meal which was also blocked by naloxone. The increased food intake was accompanied by a sustained increase in respiratory quotient, energy expenditure, and drinking as well as mu-opioid receptor-independent grooming behavior and hyperthermia, while ambulatory movements were not altered after ODT8-SST (1 microg per rat, i.c.v.). These data show that ODT8-SST acts primarily through brain sst(2) receptors to induce a long-lasting orexigenic effect that involves the activation of Y(1) and opiate-receptors, accompanied by enhanced gastric transit and energy expenditure suggesting a modulation of NPYergic and opioidergic orexigenic systems by brain sst(2) receptors.

Pattern of Fos expression in the brain induced by selective activation of somatostatin receptor 2 in rats.

Central activation of somatostatin (sst) receptors by oligosomatostatin analogs inhibits growth hormone and stress-related rise in catecholamine plasma levels while stimulating grooming, feeding behaviors, gastric transit and acid secretion, which can be mimicked by selective sst(2) receptor agonist. To evaluate the pattern of neuronal activation induced by peptide sst receptor agonists, we assessed Fos-expression in rat brain after intracerebroventricular (i.c.v.) injection of a newly developed selective sst(2) agonist compared to the oligosomatostatin ODT8-SST, a pan-sst(1-5) agonist. Ninety min after injection of vehicle (10 microl) or previously established maximal orexigenic dose of peptides (1 microg=1 nmol/rat), brains were assessed for Fos-immunohistochemistry and doublelabeling. Food and water were removed after injection. The sst(2) agonist and ODT8-SST induced a similar Fos distribution pattern except in the arcuate nucleus where only the sst(2) agonist increased Fos. Compared to ODT8-SST, the sst(2) agonist induced higher Fos-expression by 3.7-times in the basolateral amygdaloid nucleus, 1.2-times in the supraoptic nucleus (SON), 1.6-times in the magnocellular paraventricular hypothalamic nucleus (mPVN), 4.1-times in the external lateral parabrachial nucleus, and 2.6-times in both the inferior olivary nucleus and superficial layer of the caudal spinal trigeminal nucleus. Doublelabeling in the hypothalamus showed that ODT8-SST activates 36% of oxytocin, 63% of vasopressin and 79% of sst(2) immunoreactive neurons in the mPVN and 28%, 55% and 25% in the SON, respectively. Selective activation of sst(2) receptor results in a more robust neuronal activation than the pan-sst(1-5) agonist in various brain regions that may have relevance in sst(2) mediated alterations of behavioral, autonomic and endocrine functions.

Chronic injection of pansomatostatin agonist ODT8-SST differentially modulates food intake and decreases body weight gain in lean and diet-induced obese rats.

The aim of this study was to investigate the central actions of the stable pansomatostatin peptide agonist, ODT8-SST on body weight. ODT8-SST or vehicle was acutely (1μg/rat) injected or chronically infused (5μg/rat/d, 14d) intracerebroventricularly and daily food intake, body weight and composition were monitored. In lean rats, neither acute nor chronic ODT8-SST influenced daily food intake while body weight was reduced by 2.2% after acute injection and there was a 14g reduction of body weight gain after 14d compared to vehicle (p<0.01). In diet-induced obese (DIO) rats, chronic ODT8-SST increased cumulative 2-week food intake compared to vehicle (+14%, p<0.05) and also blunted body weight change (-11g, p<0.05). ODT8-SST for 14d reduced lean mass (-22g and -25g respectively, p<0.001) and total water (-19g and -22g respectively, p<0.001) in lean and DIO rats and increased fat mass in DIO (+16g, p<0.001) but not lean rats (+1g, p>0.05) compared to vehicle. In DIO rats, ODT8-SST reduced ambulatory (-27%/24h, p<0.05) and fine movements (-38%, p<0.01) which was associated with an increased positive energy balance compared to vehicle (+50g, p<0.01). Chronic central somatostatin receptor activation in lean rats reduces body weight gain and lean mass independently of food intake which is likely related to growth hormone inhibition. In DIO rats, ODT8-SST reduces lean mass but promotes food intake and fat mass, indicating differential responsiveness to somatostatin under obese conditions.

Glucose dysregulation and response to common anti-diabetic agents in the FATZO/Pco mouse

The FATZO/Pco mouse is the result of a cross of the C57BL/6J and AKR/J strains. The crossing of these two strains and the selective inbreeding for obesity, insulin resistance and hyperglycemia has resulted in an inbred strain exhibiting obesity in the presumed presence of an intact leptin pathway. Routinely used rodent models for obesity and diabetes research have a monogenic defect in leptin signaling that initiates obesity. Given that obesity and its sequelae in humans are polygenic in nature and not associated with leptin signaling defects, the FATZO mouse may represent a more translatable rodent model for study of obesity and its associated metabolic disturbances. The FATZO mouse develops obesity spontaneously when fed a normal chow diet. Glucose intolerance with increased insulin levels are apparent in FATZO mice as young as 6 weeks of age. These progress to hyperglycemia/pre-diabetes and frank diabetes with decreasing insulin levels as they age. The disease in these mice is multi-faceted, similar to the metabolic syndrome apparent in obese individuals, and thus provides a long pre-diabetic state for determining the preventive value of new interventions. We have assessed the utility of this new model for the pre-clinical screening of agents to stop or slow progression of the metabolic syndrome to severe diabetes. Our assessment included: 1) characterization of the spontaneous development of disease, 2) comparison of metabolic disturbances of FATZO mice to control mice and 3) validation of the model with regard to the effectiveness of current and emerging anti-diabetic agents; rosiglitazone, metformin and semaglutide. Conclusion: Male FATZO mice spontaneously develop significant metabolic disease when compared to normal controls while maintaining hyperglycemia in the presence of high leptin levels and hyperinsulinemia. The disease condition responds to commonly used antidiabetic agents.

Thioredoxin-mimetic peptides (TXM) inhibit inflammatory pathways associated with high-glucose and oxidative stress

Impaired insulin signaling and the associated insulin-resistance in liver, adipose tissue, and skeletal muscle, represents a hallmark of the pathogenesis of type 2-diabetes-mellitus. Here we show that in the liver of db/db mice, a murine model of obesity, type 2 diabetes, and dyslipidemia, the elevated activities of mitogen-activated protein kinases (MAPK; ERK1/2 and p38MAPK), and Akt/PKB are abolished by rosiglitazone-treatment, which normalizes blood glucose in db/db mice. This is unequivocal evidence of a functional link between the activation of the MAPK specific inflammatory-pathway and high-blood sugar. A similar reduction in ERK1/2, p38MAPK, and Akt activities but without affecting blood-glucose was observed in the liver of db/db mice treated with a molecule that mimics the action of thioredoxin, called thioredoxin-mimetic peptide (TXM). N-Acetyl-Cys-Pro-Cys-amide (TXM-CB3) is a free radical scavenger, a reducing and denitrosylating reagent that protects the cells from early death induced by inflammatory pathways. TXM-CB3 also lowered MAPK signaling activated by the disruption of the thioredoxin-reductase-thioredoxin (Trx-TrxR) redox-system and restored Akt activity in rat hepatoma FAO cells. Similarly, two other TXM-peptides, N-Acetyl-Cys-Met-Lys-Cys-amide (TXM-CB13; DY70), and N-Acetyl-Cys-γGlu-Cys-Cys-amide (TXM-CB16; DY71), lowered insulin- and oxidative stress-induced ERK1/2 activation, and rescued HepG2 cells from cell death. The potential impact of TXM-peptides on inhibiting inflammatory pathways associated with high-glucose could be effective in reversing low-grade inflammation. TXM-peptides might also have the potential to improve insulin resistance by protecting from posttranslational modifications like nitrosylation.

LY3298176, a novel dual GIP and GLP-1 receptor agonist for the treatment of type 2 diabetes mellitus: From discovery to clinical proof of concept

Objective A novel dual GIP and GLP-1 receptor agonist, LY3298176, was developed to determine whether the metabolic action of GIP adds to the established clinical benefits of selective GLP-1 receptor agonists in type 2 diabetes mellitus (T2DM). Methods LY3298176 is a fatty acid modified peptide with dual GIP and GLP-1 receptor agonist activity designed for once-weekly subcutaneous administration. LY3298176 was characterised in vitro, using signaling and functional assays in cell lines expressing recombinant or endogenous incretin receptors, and in vivo using body weight, food intake, insulin secretion and glycemic profiles in mice. A Phase 1, randomised, placebo-controlled, double-blind study was comprised of three parts: a single-ascending dose (SAD; doses 0.25–8 mg) and 4-week multiple-ascending dose (MAD; doses 0.5–10 mg) studies in healthy subjects (HS), followed by a 4-week multiple-dose Phase 1 b proof-of-concept (POC; doses 0.5–15 mg) in patients with T2DM (ClinicalTrials.gov no. NCT02759107). Doses higher than 5 mg were attained by titration, dulaglutide (DU) was used as a positive control. The primary objective was to investigate safety and tolerability of LY3298176. Results LY3298176 activated both GIP and GLP-1 receptor signaling in vitro and showed glucose-dependent insulin secretion and improved glucose tolerance by acting on both GIP and GLP-1 receptors in mice. With chronic administration to mice, LY3298176 potently decreased body weight and food intake; these effects were significantly greater than the effects of a GLP-1 receptor agonist. A total of 142 human subjects received at least 1 dose of LY3298176, dulaglutide, or placebo. The PK profile of LY3298176 was investigated over a wide dose range (0.25–15 mg) and supports once-weekly administration. In the Phase 1 b trial of diabetic subjects, LY3298176 doses of 10 mg and 15 mg significantly reduced fasting serum glucose compared to placebo (least square mean [LSM] difference [95% CI]: −49.12 mg/dL [−78.14, −20.12] and −43.15 mg/dL [−73.06, −13.21], respectively). Reductions in body weight were significantly greater with the LY3298176 1.5 mg, 4.5 mg and 10 mg doses versus placebo in MAD HS (LSM difference [95% CI]: −1.75 kg [−3.38, −0.12], −5.09 kg [−6.72, −3.46] and −4.61 kg [−6.21, −3.01], respectively) and doses of 10 mg and 15 mg had a relevant effect in T2DM patients (LSM difference [95% CI]: −2.62 kg [−3.79, −1.45] and −2.07 kg [−3.25, −0.88], respectively. The most frequent side effects reported with LY3298176 were gastrointestinal (vomiting, nausea, decreased appetite, diarrhoea, and abdominal distension) in both HS and patients with T2DM; all were dose-dependent and considered mild to moderate in severity. Conclusions Based on these results, the pharmacology of LY3298176 translates from preclinical to clinical studies. LY3298176 has the potential to deliver clinically meaningful improvement in glycaemic control and body weight. The data warrant further clinical evaluation of LY3298176 for the treatment of T2DM and potentially obesity.

Deciphering binding interactions of IL-23R with HDX-MS: Mapping protein and macrocyclic dodecapeptide ligands

Molecular characterization of the binding epitope of IL-23R and its cognate cytokine IL-23 is paramount to understand the role in autoimmune diseases and to support the discovery of new inhibitors of this protein-protein interaction. Our results revealed that HDX-MS was able to identify the binding epitope of IL-23R:IL-23, which opened the way to evaluate a peptide macrocycle described in the literature as disrupter of this autoimmune target. Thus, the characterization of the interactions of this chemotype by HDX-MS in combination with computational approaches was achieved. To our knowledge, this is the first reported structural evidence regarding the site where a small compound binds to IL-23R.