Cornelia Hertel

A rationally designed monomeric peptide triagonist corrects obesity and diabetes in rodents

We report the discovery of a new monomeric peptide that reduces body weight and diabetic complications in rodent models of obesity by acting as an agonist at three key metabolically-related peptide hormone receptors: glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and glucagon receptors. This triple agonist demonstrates supraphysiological potency and equally aligned constituent activities at each receptor, all without cross-reactivity at other related receptors. Such balanced unimolecular triple agonism proved superior to any existing dual coagonists and best-in-class monoagonists to reduce body weight, enhance glycemic control and reverse hepatic steatosis in relevant rodent models. Various loss-of-function models, including genetic knockout, pharmacological blockade and selective chemical knockout, confirmed contributions of each constituent activity in vivo. We demonstrate that these individual constituent activities harmonize to govern the overall metabolic efficacy, which predominantly results from synergistic glucagon action to increase energy expenditure, GLP-1 action to reduce caloric intake and improve glucose control, and GIP action to potentiate the incretin effect and buffer against the diabetogenic effect of inherent glucagon activity. These preclinical studies suggest that, so far, this unimolecular, polypharmaceutical strategy has potential to be the most effective pharmacological approach to reversing obesity and related metabolic disorders.

β‐amyloid binds to p75NTR and activates NFκB in human neuroblastoma cells

Amyloid β peptide (Aβ), a proteolytic fragment of the amyloid precursor protein (APP), is a major component of the plaques found in the brain of Alzheimers disease (AD) patients. These plaques are thought to cause the observed loss of cholinergic neurons in the basal forebrain of AD patients. In these neurons, particularly those of the nucleus basalis of Meynert, an up‐regulation of 75kD‐neurotrophin receptor (p75NTR), a nonselective neurotrophin receptor belonging to the death receptor family, has been reported. p75NTR expression has been described to correlate with β‐amyloid sensitivity in vivo and in vitro, suggesting a possible role for p75NTR as a receptor for Aβ. Here we used a human neuroblastoma cell line to investigate the involvement of p75NTR in Aβ‐induced cell death. Aβ peptides were found to bind to p75NTR resulting in activation of NFκB in a time‐ and dose‐dependent manner. Blocking the interaction of Aβ with p75NTR using NGF or inhibition of NFκB activation by curcumin or NFκB SN50 attenuated or abolished Aβ‐induced apoptotic cell death. The present results suggest that p75NTR might be a death receptor for Aβ, thus being a possible drug target for treatment of AD. J. Neurosci. Res. 54:798–804, 1998.

Phytoestrogen kaempferol (3,4′,5,7-tetrahydroxyflavone) protects PC12 and T47D cells from β-amyloid–induced toxicity

In clinical studies, it has been shown that estrogen replacement therapy in menopause is strongly correlated with a reduced risk of the development of Alzheimers disease (AD). In in vitro experiments, it was demonstrated that estradiol protects cells against the toxic effects of β‐amyloid, the major component of plaques in brains of AD patients. Therefore, estrogens have become interesting candidates for a possible treatment of neurodegeneration. In plants, a class of compounds has been identified that bind to human estrogen receptor, so‐called phytoestrogens, which are part of our daily diet. Here, we compared the effects of α‐ and β‐estradiol with plant‐derived kaempferol on β‐amyloid peptide–induced toxicity in PC12 neuroblastoma and T47D human breast cancer cells. The present results demonstrate a protective effect of kaempferol comparable to that observed with estradiol. The effects of the weak estrogen receptor agonists α‐estradiol and kaempferol were found to be similar to the effects of the strong estrogen receptor agonist β‐estradiol, suggesting a mode of action independent from the nuclear estrogen receptor. J. Neurosci. Res. 57:399–404, 1999.

β‐Amyloid‐Induced Cell Toxicity: Enhancement of 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐Diphenyltetrazolium Bromide‐Dependent Cell Death

Abstract: In an attempt to understand the cause of neurodegeneration in Alzheimers disease, the toxic effects of β‐amyloid (Aβ) peptides have been widely studied. At high micromolar concentrations Aβ peptides have been demonstrated to be acutely toxic to various cell types. At submicromolar concentrations, Aβ peptides have been suggested to inhibit cellular metabolic activity, due to their inhibition of the ability of cells to metabolize the oxidoreductase substrate 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT). Here we show, first, that MTT reduction surprisingly leads to a breakdown in PC12 cell membrane integrity and cell death, presumably through the formation of a crystalline formazan product, and, second, that pretreatment of PC12 cells with nanomolar concentrations of Aβ peptide, rather than inhibiting their metabolic activity, increases the susceptibility of these cells to the secondary toxic effect of formazan crystal formation. These results suggest that low nanomolar concentrations of Aβ render membranes more susceptible to damage by a secondary insult, in this case, MTT reduction. It is plausible that such an effect, when combined with additional risk factors, could contribute to the neurodegeneration that occurs in Alzheimers disease.

5-Hydroxyindole-2-carboxylic Acid Amides: Novel Histamine-3 Receptor Inverse Agonists for the Treatment of Obesity

Obesity is a major risk factor in the development of conditions such as hypertension, hyperglycemia, dyslipidemia, coronary artery disease, and cancer. Several pieces of evidence across different species, including primates, underscore the implication of the histamine 3 receptor (H(3)R) in the regulation of food intake and body weight and the potential therapeutic effect of H(3)R inverse agonists. A pharmacophore model, based on public information and validated by previous investigations, was used to design several potential scaffolds. Out of these scaffolds, the 5-hydroxyindole-2-carboxylic acid amide appeared to be of great potential as a novel series of H(3)R inverse agonist. Extensive structure-activity relationships revealed the interconnectivity of microsomal clearance and hERG (human ether-a-go-go-related gene) affinity with lipophilicity, artificial membrane permeation, and basicity. This effort led to the identification of compounds reversing the (R)-alpha-methylhistamine-induced water intake increase in Wistar rats and, further, reducing food intake in diet-induced obese Sprague-Dawley rats. Of these, the biochemical, pharmacokinetic, and pharmacodynamic characteristics of (4,4-difluoropiperidin-1-yl)[1-isopropyl-5-(1-isopropylpiperidin-4-yloxy)-1H-indol-2-yl]methanone 36 are detailed.

Impedance measurement: a new method to detect ligand-biased receptor signaling.

Ligand-biased receptor signaling has been proposed for several G-protein coupled receptors including the niacin receptor GPR109A. Coupling to the G(i/o) pathway has been shown to be responsible for the well described triglyceride lowering effect of nicotinic acid in mice, while activation of the β-arrestin pathway has been suggested to be responsible for its peripheral vasodilatory effect that causes cutaneous flushing. Several ligands have been described to selectively induce triglyceride lowering without inducing flushing. Cellular impedance has been demonstrated to determine G-protein coupled receptors activation in a G-protein specific manner. Agonists, which induce triglyceride lowering, but not flushing show a profile in cellular impedance that is distinct from the one induced by niacin and those compounds that induce triglyceride lowering as well as flushing. The strength of the signal correlates with the activation of β-arrestin.

Nerve Growth Factor-Induced Differentiation in Neuroblastoma Cells Expressing TrkA but Lacking p75NGFR

Abstract: Nerve growth factor (NGF) binds to two distinct cell surface receptors, TrkA, which is a receptor tyrosine kinase, and p75NGFR, whose role in NGF‐induced signal transduction remains unclear. We have found that human neuroblastoma IMR‐32 cells express TrkA, but p75NGFR expression was not detectable in these cells by northern blot analysis, immunoblotting, or chemical crosslinking experiments. Despite the lack of p75NGFR expression, subnanomolar concentrations of recombinant human NGF induced neurite outgrowth, tyrosine phosphorylation, and immediate early gene expression in these cells. These results strongly suggest that NGF‐induced neuronal differentiation in IMR‐32 cells is initiated through TrkA in the absence of p75NGFR. Thus, IMR‐32 cells may provide a model for studying neurotrophic effects of NGF on adult striatal cholinergic neurons, which also lack p75NGFR expression.

Pyrido pyrimidinones as selective agonists of the high affinity niacin receptor GPR109A: optimization of in vitro activity.

Pyrido pyrimidinones are selective agonists of the human high affinity niacin receptor GPR109A (HM74A). They show no activity on the highly homologous low affinity receptor GPR109B (HM74). Starting from a high throughput screening hit the in vitro activity of the pyrido pyrimidinones was significantly improved providing lead compounds suitable for further optimization.

G protein-coupled receptor transmembrane binding pockets and their applications in GPCR research and drug discovery: a survey.

G protein-coupled receptors (GPCRs) share a common architecture consisting of seven transmembrane (TM) domains. Various lines of evidence suggest that this fold provides a generic binding pocket within the TM region for hosting agonists, antagonists, and allosteric modulators. Hence, an automated method was developed that allows a fast analysis and comparison of these generic ligand binding pockets across the entire GPCR family by providing the relevant information for all GPCRs in the same format. This methodology compiles amino acids lining the TM binding pocket including parts of the ECL2 loop in a so-called 1D ligand binding pocket vector and translates these 1D vectors in a second step into 3D receptor pharmacophore models. It aims to support various aspects of GPCR drug discovery in the pharmaceutical industry. Applications of pharmacophore similarity analysis of these 1D LPVs include definition of receptor subfamilies, prediction of species differences within subfamilies in regard to in vitro pharmacology and identification of nearest neighbors for GPCRs of interest to generate starting points for GPCR lead identification programs. These aspects of GPCR research are exemplified in the field of melanopsins, trace amine-associated receptors and somatostatin receptor subtype 5. In addition, it is demonstrated how 3D pharmacophore models of the LPVs can support the prediction of amino acids involved in ligand recognition, the understanding of mutational data in a 3D context and the elucidation of binding modes for GPCR ligands and their evaluation. Furthermore, guidance through 3D receptor pharmacophore modeling for the synthesis of subtype-specific GPCR ligands will be reported. Illustrative examples are taken from the GPCR family class C, metabotropic glutamate receptors 1 and 5 and sweet taste receptors, and from the GPCR class A, e.g. nicotinic acid and 5-hydroxytryptamine 5A receptor.

Protein Kinase Inhibitor H-7 Differentially Affects Early and Delayed Nerve Growth Factor Responses in PC12 Cells

Abstract: The effects of the protein kinase inhibitor H‐7 on early and delayed responses to nerve growth factor (NGF) were investigated in PC12 cells. H‐7 reduced the NGF‐induced expression of c‐Fos in a dose‐dependent manner without affecting the time course of c‐Fos appearance. Conversely, H‐7 potentiated delayed NGF effects, i.e., neurite outgrowth and Ca2+/phospholipid‐dependent protein kinase (PKC) induction, but not choline acetyltransferase induction. Long‐term treatment with NGF resulted in an increase of at least four tyrosine‐phosphorylated protein bands with molecular masses between 39 and 48 kDa, which was also potentiated by H‐7. In the absence of NGF, H‐7 had no significant effect on c‐Fos expression, tyrosine phosphorylation of the 45 kDa protein, or choline acetyltransferase activity. However, 4 days of exposure to H‐7 alone induced PKC activity and tyrosine phosphorylation of the 39‐kDa protein. The action of H‐7 derivatives on neurite outgrowth did not correlate with their inhibition profile of cyclic nucleotide‐dependent protein kinases. Down‐regulation of PKC activity by prolonged exposure to phorbol ester did not completely abolish the effects of NGF and H‐7 on induction of c‐Fos, choline acetyltransferase activity, and neurite outgrowth, indicating that PKC‐independent pathways contribute to these actions. These results suggest that additional pathway(s) sensitive to H‐7 may exist, which induce immediate early gene expression and suppress neuronal differentiation of PC12 cells.