Francis S. Willard

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.

G-protein signaling : back to the future

Abstract.Heterotrimeric G-proteins are intracellular partners of G-protein-coupled receptors (GPCRs). GPCRs act on inactive Gα·GDP/Gβγ heterotrimers to promote GDP release and GTP binding, resulting in liberation of Gα from Gβγ. Gα·GTP and Gβγ target effectors including adenylyl cyclases, phospholipases and ion channels. Signaling is terminated by intrinsic GTPase activity of Gα and heterotrimer reformation — a cycle accelerated by ‘regulators of G-protein signaling’ (RGS proteins). Recent studies have identified several unconventional G-protein signaling pathways that diverge from this standard model. Whereas phospholipase C (PLC) β is activated by Gαq and Gβγ, novel PLC isoforms are regulated by both heterotrimeric and Ras-superfamily G-proteins. An Arabidopsis protein has been discovered containing both GPCR and RGS domains within the same protein. Most surprisingly, a receptor-independent Gα nucleotide cycle that regulates cell division has been delineated in both Caenorhabditis elegans and Drosophila melanogaster. Here, we revisit classical heterotrimeric G-protein signaling and explore these new, non-canonical G-protein signaling pathways.

RIC-8 Is Required for GPR-1/2-Dependent Gα Function during Asymmetric Division of C. elegans Embryos

Heterotrimeric G proteins are crucial for asymmetric cell division, but the mechanisms of signal activation remain poorly understood. Here, we establish that the evolutionarily conserved protein RIC-8 is required for proper asymmetric division of one-cell stage C. elegans embryos. Spindle severing experiments demonstrate that RIC-8 is required for generation of substantial pulling forces on astral microtubules. RIC-8 physically interacts with GOA-1 and GPA-16, two Galpha subunits that act in a partially redundant manner in one-cell stage embryos. RIC-8 preferentially binds to GDP bound GOA-1 and is a guanine nucleotide exchange factor (GEF) for GOA-1. Our analysis suggests that RIC-8 acts before the GoLoco protein GPR-1/2 in the sequence of events leading to Galpha activation. Furthermore, coimmunoprecipitation and in vivo epistasis demonstrate that inactivation of the Gbeta subunit GPB-1 alleviates the need for RIC-8 in one-cell stage embryos. Our findings suggest a mechanism in which RIC-8 favors generation of Galpha free from Gbetagamma and enables GPR-1/2 to mediate asymmetric cell division.

GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling

Heterotrimeric G protein signaling is important for cell-proliferative and glucose-sensing signal transduction pathways in the model plant organism Arabidopsis thaliana. AtRGS1 is a seven-transmembrane, RGS domain-containing protein that is a putative membrane receptor for d-glucose. Here we show, by using FRET, that d-glucose alters the interaction between the AtGPA1 and AtRGS1 in vivo. AtGPA1 is a unique heterotrimeric G protein α subunit that is constitutively GTP-bound given its high spontaneous nucleotide exchange coupled with slow GTP hydrolysis. Analysis of a point mutation in AtRGS1 that abrogates GTPase-accelerating activity demonstrates that the regulation of AtGPA1 GTP hydrolysis mediates sugar signal transduction during Arabidopsis development, in contrast to animals where nucleotide exchange is the limiting step in the heterotrimeric G protein nucleotide cycle.

Crystal structure of the human PRMT5:MEP50 complex

Protein arginine methyltransferases (PRMTs) play important roles in several cellular processes, including signaling, gene regulation, and transport of proteins and nucleic acids, to impact growth, differentiation, proliferation, and development. PRMT5 symmetrically di-methylates the two-terminal ω-guanidino nitrogens of arginine residues on substrate proteins. PRMT5 acts as part of a multimeric complex in concert with a variety of partner proteins that regulate its function and specificity. A core component of these complexes is the WD40 protein MEP50/WDR77/p44, which mediates interactions with binding partners and substrates. We have determined the crystal structure of human PRMT5 in complex with MEP50 (methylosome protein 50), bound to an S-adenosylmethionine analog and a peptide substrate derived from histone H4. The structure of the surprising hetero-octameric complex reveals the close interaction between the seven-bladed β-propeller MEP50 and the N-terminal domain of PRMT5, and delineates the structural elements of substrate recognition.

Structural diversity in the RGS domain and its interaction with heterotrimeric G protein alpha-subunits.

Regulator of G protein signaling (RGS) proteins accelerate GTP hydrolysis by Gα subunits and thus facilitate termination of signaling initiated by G protein-coupled receptors (GPCRs). RGS proteins hold great promise as disease intervention points, given their signature role as negative regulators of GPCRs—receptors to which the largest fraction of approved medications are currently directed. RGS proteins share a hallmark RGS domain that interacts most avidly with Gα when in its transition state for GTP hydrolysis; by binding and stabilizing switch regions I and II of Gα, RGS domain binding consequently accelerates Gα-mediated GTP hydrolysis. The human genome encodes more than three dozen RGS domain-containing proteins with varied Gα substrate specificities. To facilitate their exploitation as drug-discovery targets, we have taken a systematic structural biology approach toward cataloging the structural diversity present among RGS domains and identifying molecular determinants of their differential Gα selectivities. Here, we determined 14 structures derived from NMR and x-ray crystallography of members of the R4, R7, R12, and RZ subfamilies of RGS proteins, including 10 uncomplexed RGS domains and 4 RGS domain/Gα complexes. Heterogeneity observed in the structural architecture of the RGS domain, as well as in engagement of switch III and the all-helical domain of the Gα substrate, suggests that unique structural determinants specific to particular RGS protein/Gα pairings exist and could be used to achieve selective inhibition by small molecules.

Rgs1 regulates multiple Gα subunits in Magnaporthe pathogenesis, asexual growth and thigmotropism

Regulators of G‐protein signaling (RGS proteins) negatively regulate heterotrimeric G‐protein cascades that enable eukaryotic cells to perceive and respond to external stimuli. The rice‐blast fungus Magnaporthe grisea forms specialized infection structures called appressoria in response to inductive surface cues. We isolated Magnaporthe RGS1 in a screen for mutants that form precocious appressoria on non‐inductive surfaces. We report that a thigmotropic cue is necessary for initiating appressoria and for accumulating cAMP. Similar to an RGS1‐deletion strain, magAG187S (RGS‐insensitive Gαs) and magAQ208L (GTPase‐dead) mutants accumulated excessive cAMP and elaborated appressoria on non‐inductive surfaces, suggesting that Rgs1 regulates MagA during pathogenesis. Rgs1 was also found to negatively regulate the Gαi subunit MagB during asexual development. Deficiency of MAGB suppressed the hyper‐conidiation defect in RGS1‐deletion strain, whereas magBG183S and magBQ204L mutants produced more conidia, similar to the RGS1‐deletion strain. Rgs1 physically interacted with GDP·AlF4−‐activated forms of MagA, MagB and MagC (a GαII subunit). Thus, Rgs1 serves as a negative regulator of all Gα subunits in Magnaporthe and controls important developmental events during asexual and pathogenic development.

Chronic Olanzapine Treatment Causes Differential Expression of Genes in Frontal Cortex of Rats as Revealed by DNA Microarray Technique

Recent emerging biochemical data indicate that several important neuroregulatory genes and proteins may be involved in the etiology of schizophrenia and bipolar disorder. Additionally, the same genes appear to be targets of several psychotropic medications that are used to treat these disorders. Recent DNA microarray studies show that genes involved in synaptic neurotransmission, signal transduction, and glutamate/GABA regulation may be differentially regulated in brains of subjects with schizophrenia. We hypothesized that chronic administration of olanzapine to rats would alter expression of various genes that may be involved in the etiology of schizophrenia and mood disorders. Rats were administered olanzapine (N=20, 2 mg/kg/day) or sterile saline intraperitoneally (N=20) daily for 21 days. Control and olanzapine-treated frontal cortices were analyzed using cDNA microarray technology. The results showed significant downregulation of 31 genes and upregulation of 38 genes by greater than two-fold in the drug-treated brains vs controls. Our results provide evidence for altered regulation of genes involved with signal transduction and cell communication, metabolism and energy pathways, transport, immune response, nucleic acid metabolism, and neuronal growth factors. Real-time quantitative RT-PCR analysis verified the direction and magnitude of change in six genes of interest: calbindin 3, homer 1, regulator of G-protein signaling (RGS) 2, pyruvate kinase, Reelin and insulin 2. Western blotting showed significant upregulation in protein products for Reelin 410 and Reelin 180 kDa and downregulation for NMDA3B and RGS2. Our results show for the first time that olanzapine causes changes in levels of several important genes that may be involved in the etiology and treatment of schizophrenia and other psychiatric disorders.