Aaron D. Showalter

Novel Small Molecule Glucagon-like Peptide-1 Receptor Agonist Stimulates Insulin Secretion in Rodents and from Human Islets

OBJECTIVE The clinical effectiveness of parenterally-administered glucagon-like peptide-1 (GLP-1) mimetics to improve glucose control in patients suffering from type 2 diabetes strongly supports discovery pursuits aimed at identifying and developing orally active, small molecule GLP-1 receptor agonists. The purpose of these studies was to identify and characterize novel nonpeptide agonists of the GLP-1 receptor. RESEARCH DESIGN AND METHODS Screening using cells expressing the GLP-1 receptor and insulin secretion assays with rodent and human islets were used to identify novel molecules. The intravenous glucose tolerance test (IVGTT) and hyperglycemic clamp characterized the insulinotropic effects of compounds in vivo. RESULTS Novel low molecular weight pyrimidine-based compounds that activate the GLP-1 receptor and stimulate glucose-dependent insulin secretion are described. These molecules induce GLP-1 receptor-mediated cAMP signaling in HEK293 cells expressing the GLP-1 receptor and increase insulin secretion from rodent islets in a dose-dependent manner. The compounds activate GLP-1 receptor signaling, both alone or in an additive fashion when combined with the endogenous GLP-1 peptide; however, these agonists do not compete with radiolabeled GLP-1 in receptor-binding assays. In vivo studies using the IVGTT and the hyperglycemic clamp in Sprague Dawley rats demonstrate increased insulin secretion in compound-treated animals. Further, perifusion assays with human islets isolated from a donor with type 2 diabetes show near-normalization of insulin secretion upon compound treatment. CONCLUSIONS These studies characterize the insulinotropic effects of an early-stage, small molecule GLP-1 receptor agonist and provide compelling evidence to support pharmaceutical optimization.

Allosteric Modulation of Endogenous Metabolites as an Avenue for Drug Discovery

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors and a key drug target class. Recently, allosteric drugs that can cobind with and modulate the activity of the endogenous ligand(s) for the receptor have become a major focus of the pharmaceutical and biotechnology industry for the development of novel GPCR therapeutic agents. This class of drugs has distinct properties compared with drugs targeting the endogenous (orthosteric) ligand-binding site that include the ability to sculpt cellular signaling and to respond differently in the presence of discrete orthosteric ligands, a behavior termed “probe dependence.” Here, using cell signaling assays combined with ex vivo and in vivo studies of insulin secretion, we demonstrate that allosteric ligands can cause marked potentiation of previously “inert” metabolic products of neurotransmitters and peptide hormones, a novel consequence of the phenomenon of probe dependence. Indeed, at the muscarinic M2 receptor and glucagon-like peptide 1 (GLP-1) receptor, allosteric potentiation of the metabolites, choline and GLP-1(9–36)NH2, respectively, was ∼100-fold and up to 200-fold greater than that seen with the physiological signaling molecules acetylcholine and GLP-1(7–36)NH2. Modulation of GLP-1(9–36)NH2 was also demonstrated in ex vivo and in vivo assays of insulin secretion. This work opens up new avenues for allosteric drug discovery by directly targeting modulation of metabolites, but it also identifies a behavior that could contribute to unexpected clinical outcomes if interaction of allosteric drugs with metabolites is not part of their preclinical assessment.

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.

Targeted suppression of an amyloidogenic transthyretin with antisense oligonucleotides

Transthyretin (TTR) amyloidosis, the most common form of hereditary systemic amyloidosis, is characterized clinically by adult‐onset axonal neuropathy and restrictive cardiomyopathy. More than 85 mutations in transthyretin have been found to cause this hereditary disease. Since essentially all circulating TTR is of hepatic origin, orthotopic liver transplantation has been used as the only specific form of therapy. Unfortunately, in many patients amyloid deposition continues after orthotopic liver transplantation, indicating that mutant TTR is no longer required for progression of the disease after tissue deposits have been initiated. As a first step toward medical treatment of this disease, we have employed antisense oligonucleotides (ASOs) to inhibit hepatic expression of TTR. A transgenic mouse model carrying the human TTR Ile84Ser mutation was created and shown to express high levels of human mutant transthyretin. TTR ASOs suppressed hepatic TTR mRNA levels and serum TTR levels by as much as 80%. Suppression of hepatic synthesis of transthyretin may offer a medical treatment for transthyretin systemic amyloidosis. Muscle Nerve, 2006

Genetic defects in the development and function of the anterior pituitary gland

Genetic defects affecting the hypothalamic-pituitary-target organ axes can cause a variety of diseases involving restricted or broad disruptions of human development and physiology. At the level of the anterior pituitary gland, mutations in the genes encoding key transcription factors, hypothalamic releasing and inhibiting hormone receptors, and the pituitary hormones themselves, can all result in the loss of action of one or more of the specialized hormone-secreting cell types. This article focuses on the effects of inherited and sporadic mutations on the development and function of the anterior pituitary. Mutations in the genes encoding the HESX1, PITX2, LHX3, LHX4, PROP1, PIT1, SF1, and TPIT developmental transcription factors are associated with combined pituitary hormone deficiency diseases. By contrast, deleterious alterations in the genes that encode hypothalamic releasing hormone receptors or pituitary hormones, such as the growth hormone releasing hormone receptor or growth hormone genes, usually result in phenotypes that reflect specific defects in the hormonesecreting capacities of individual anterior pituitary cell types.

Analysis of the human LHX3 neuroendocrine transcription factor gene and mapping to the subtelomeric region of chromosome 9

The Lhx3 LIM homeodomain transcription factor is critical to pituitary organogenesis and motor neuron development. We determined the genomic structure and chromosomal localization of human LHX3. The gene contains seven coding exons and six introns that span 8.7 kilobases in length. The LHX3 gene codes for two functionally distinct isoforms that differ in their amino termini but share common LIM domains and a homeodomain. The functional domains of the LHX3 proteins are encoded by distinct exons. The alternate amino termini and LIM domains lie within individual exons, and the homeodomain is coded by two exons interrupted by a small intron. Human LHX3 maps to the subtelomeric region of chromosome 9 at band 9q34.3, within a region noted for chromosomal translocation and insertion events. Characterization of the genomic organization and chromosomal localization of LHX3 will enable molecular evaluation and genetic diagnoses of pituitary diseases and central nervous system developmental disorders in humans.

Suppression of choroid plexus transthyretin levels by antisense oligonucleotide treatment

Leptomeningeal amyloidosis associated with mutations in transthyretin (TTR) is a rare but fatal form of amyloidosis. Dementia and intracerebral haemorrhage are prominent features of this disease for which no specific therapy is known. In previous studies, we have shown that antisense oligonucleotides (ASOs) specific for human TTR could inhibit hepatic synthesis of TTR in mice transgenic for a human amyloid-associated TTR and may offer a medical means of treating systemic TTR amyloidosis. Parenteral administration of TTR-specific ASO, however, had no effect on the expression of TTR by the choroid plexus, which is believed to be the source of the amyloid protein in patients who have leptomeningeal amyloidosis. In the present study, mice transgenic for the human TTR amyloid-associated mutation Ile84Ser were treated by administration of TTR-specific ASO (50 μg or 75 μg per day) via an osmotic pump into the cerebral ventricular system over a 4-week period. Intraventricular administration of TTR-specific ASO significantly reduced choroid human TTR mRNA levels, and these findings correlated with decreased TTR in choroid plexus epithelial cells as demonstrated by immunohistochemistry. Suppression of choroid TTR expression by intraventricular administered ASO may offer a medical means of treating leptomeningeal amyloidosis.

Differential conservation of transcriptional domains of mammalian Prophet of Pit-1 proteins revealed by structural studies of the bovine gene and comparative functional analysis of the protein

The Prophet of Pit-1 (PROP1) gene encodes a paired class homeodomain transcription factor that is exclusively expressed in the developing mammalian pituitary gland. PROP1 function is essential for anterior pituitary organogenesis, and heritable mutations in the gene are associated with combined pituitary hormone deficiency in human patients and animals. By cloning the bovine PROP1 gene and by comparative analysis, we demonstrate that the homeodomains and carboxyl termini of mammalian PROP1 proteins are highly conserved while the amino termini are diverged. Whereas the carboxyl termini of the human and bovine PROP1 proteins contain potent transcriptional activation domains, the amino termini and homeodomains have repressive activities. The bovine PROP1 gene has four exons and three introns and maps to a region of chromosome seven carrying a quantitative trait locus affecting ovulation rate. Two alleles of the bovine gene were found that encode distinct protein products with different DNA binding and transcriptional activities. These experiments demonstrate that mammalian PROP1 genes encode proteins with complex regulatory capacities and that modest changes in protein sequence can significantly alter the activity of this pituitary developmental transcription factor.

Absence of Glucagon and Insulin Action Reveals a Role for the GLP-1 Receptor in Endogenous Glucose Production

The absence of insulin results in oscillating hyperglycemia and ketoacidosis in type 1 diabetes. Remarkably, mice genetically deficient in the glucagon receptor (Gcgr) are refractory to the pathophysiological symptoms of insulin deficiency, and therefore, studies interrogating this unique model may uncover metabolic regulatory mechanisms that are independent of insulin. A significant feature of Gcgr-null mice is the high circulating concentrations of GLP-1. Hence, the objective of this report was to investigate potential noninsulinotropic roles of GLP-1 in mice where GCGR signaling is inactivated. For these studies, pancreatic β-cells were chemically destroyed by streptozotocin (STZ) in Gcgr−/−:Glp-1r−/− mice and in Glp-1r−/− animals that were subsequently treated with a high-affinity GCGR antagonist antibody that recapitulates the physiological state of Gcgr ablation. Loss of GLP-1 action substantially worsened nonfasting glucose concentrations and glucose tolerance in mice deficient in, and undergoing pharmacological inhibition of, the GCGR. Further, lack of the Glp-1r in STZ-treated Gcgr−/− mice elevated rates of endogenous glucose production, likely accounting for the differences in glucose homeostasis. These results support the emerging hypothesis that non–β-cell actions of GLP-1 analogs may improve metabolic control in patients with insulinopenic diabetes.

The identification of novel proteins that interact with the GLP-1 receptor and restrain its activity.

Glucagon-like peptide 1 receptor (GLP-1R) controls diverse physiological functions in tissues including the pancreatic islets, brain, and heart. To understand the mechanisms that control glucagon-like peptide 1 (GLP-1) signaling better, we sought to identify proteins that interact with the GLP-1R using a membrane-based split ubiquitin yeast two-hybrid (MYTH) assay. A screen of a human fetal brain cDNA prey library with an unliganded human GLP-1R as bait in yeast revealed 38 novel interactor protein candidates. These interactions were confirmed in mammalian Chinese hamster ovarian cells by coimmunoprecipitation. Immunofluorescence was used to show subcellular colocalization of the interactors with GLP-1R. Cluster analysis revealed that the interactors were primarily associated with signal transduction, metabolism, and cell development. When coexpressed with the GLP-1R in Chinese hamster ovarian cells, 15 interactors significantly altered GLP-1-induced cAMP accumulation. Surprisingly, all 15 proteins inhibited GLP-1-activated cAMP. Given GLP-1s prominent role as an incretin, we then focused on 3 novel interactors, SLC15A4, APLP1, and AP2M1, because they are highly expressed and localized to the membrane in mouse insulinoma β-cells. Small interfering RNA-mediated knockdown of each candidate gene significantly enhanced GLP-1-induced insulin secretion. In conclusion, we have generated a novel GLP-1R-protein interactome, identifying several interactors that suppress GLP-1R signaling. We suggest that the inhibition of these interactors may serve as a novel strategy to enhance GLP-1R activity.