Design and synthesis of water soluble ß-aminosulfone analogues of SCH 900229 as γ-secretase inhibitors.

In this paper we describe our strategy to improve the aqueous solubility of SCH 900229, a potent PS1-selective γ-secretase inhibitor for the treatment of Alzheimer's disease. Incorporation of ionizable amino groups into the side chain terminal generates water soluble ß-aminosulfone analogues of SCH 900229 that maintain robust in vitro potency and in vivo efficacy.

Structure activity relationship studies of tricyclic bispyran sulfone γ-secretase inhibitors.

An investigation is detailed of the structure activity relationships (SAR) of two sulfone side chains of compound (-)-1a (SCH 900229), a potent, PS1-selective γ-secretase inhibitor and clinical candidate for the treatment of Alzheimer's disease. Specifically, 4-CF(3) and 4-Br substituted arylsulfone analogs, (-)-1b and (-)-1c, are equipotent to compound (-)-1a. On the right hand side chain, linker size and terminal substituents of the pendant sulfone group are also investigated.

Discovery of amide and heteroaryl isosteres as carbamate replacements in a series of orally active gamma-secretase inhibitors.

The design of amide and heteroaryl amide isosteres as replacements for the carbamate substructure in previously disclosed 2,6-disubstituted piperidine N-arylsulfonamides is described. In several cases, amides lessened CYP liabilities in this class of gamma-secretase inhibitors. Selected compounds showed significant reduction of Abeta levels upon oral dosing in a transgenic murine model of Alzheimer's disease.

Effects of a selective melanin-concentrating hormone 1 receptor antagonist on food intake and energy homeostasis in diet-induced obese mice.

Melanin concentrating hormone (MCH) is a cyclic neuropeptide expressed in the lateral hypothalamus that plays an important role in energy homeostasis. To investigate the pharmacological consequences of inhibiting MCH signaling in murine obesity models, we examined the effect of acute and chronic administration of a selective MCH1 receptor antagonist (SCH-A) in diet-induced obese (DIO) and Lep(ob/ob) mice. Oral administration of SCH-A for 5 consecutive days (30 mg/kg q.d.) produced hypophagia, a loss of body weight and adiposity, and decreased plasma leptin levels in DIO mice, and hypophagia and reduced weight gain in Lep(ob/ob) mice. Chronic administration of SCH-A to DIO mice decreased food intake, body weight and adiposity, and plasma leptin and free fatty acids. These effects were accompanied by increases in several hypothalamic neuropeptides. Acute administration of SCH-A (30 mg/kg) prevented the decrease in energy expenditure associated with food restriction. These results indicate that MCH1 receptor antagonists may be effective in the treatment of obesity.

A genomic perspective on protein tyrosine phosphatases: gene structure, pseudogenes, and genetic disease linkage.

The protein tyrosine phosphatases (PTPs) are now recognized as critical regulators of signal transduction under normal and pathophysiological conditions. In this analysis we have explored the sequence of the human genome to define the composition of the PTP family. Using public and proprietary sequence databases, we discovered one novel human PTP gene and defined chromosomal loci and exon structure of the additional 37 genes encoding known PTP transcripts. Direct orthologs were present in the mouse genome for all 38 human PTP genes. In addition, we identified 12 PTP pseudogenes unique to humans that have probably contaminated previous bioinformatics analysis of this gene family. PCR amplification and transcript sequencing indicate that some PTP pseudogenes are expressed, but their function (if any) is unknown. Furthermore, we analyzed the enhanced diversity generated by alternative splicing and provide predicted amino acid sequences for four human PTPs that are currently defined by fragments only. Finally, we correlated each PTP locus with genetic disease markers and identified 4 PTPs that map to known susceptibility loci for type 2 diabetes and 19 PTPs that map to regions frequently deleted in human cancers. We have made our analysis available at http://ptp.cshl.edu or http://science.novonordisk.com/ptp and we hope this resource will facilitate the functional characterization of these key enzymes.

Increased auditory startle response and reduced prepulse inhibition of startle in transgenic mice expressing a double mutant form of amyloid precursor protein.

Prepulse inhibition (PPI), a form of sensorimotor gating, occurs when an auditory startle response is markedly inhibited by a preceding sub-threshold stimulus (prepulse). Deficits in PPI have been demonstrated in patients with certain psychiatric disorders, such as schizophrenia, and in laboratory animals following specific pharmacological manipulations. Patients with Alzheimer's disease (AD) have not been tested in PPI, but have been shown to have abnormal sensory gating in another paradigm. Transgenic (Tg) CRND8 mice, which model Alzheimer's disease, carry the Swedish and Indiana familial Alzheimer's disease mutations of the human amyloid precursor protein gene and show age-related increases in beta-amyloid (Abeta) production, as well as plaque deposition. The present experiment investigated auditory startle threshold and PPI in TgCRND8 mice at various ages. In two longitudinal studies, PPI was examined in male TgCRND8 mice and non-transgenic (non-Tg) controls at 6-8 weeks of age (pre-plaque), and every 2 weeks thereafter until all mice were at least 16 weeks old (post-plaque). In a cross-sectional study, three different age sets of nai;ve TgCRND8 and non-Tg mice were tested: 10-12, 12-14, and 15-17 weeks old. In all three studies, TgCRND8 mice consistently and robustly demonstrated an enhanced response to a range of auditory startle stimuli compared to non-Tg mice. In addition, the TgCRND8 mice exhibited modest reductions in PPI, compared to non-Tg controls. These PPI deficits were present at pre- and post-plaque time points and did not appear to intensify with age; thus, they do not seem to correlate with the known neuropathology of TgCRND8 mice.

Increased seizure threshold and severity in young transgenic CRND8 mice.

Reports suggest that Alzheimer's disease (AD) patients show a high life-time prevalence of seizure-like disorders. The transgenic CRND8 (TgCRDN8) is a mouse model of AD-like amyloid pathogenesis that expresses a double-mutant form of human amyloid precursor protein 695 (K670N/M671L and V717F). We have previously reported that post-plaque TgCRND8 mice exhibited a lower threshold to seizure with a more severe seizure type when challenged with pentylenetetrazole (PTZ) intravenously. Here, we now report that pre-plaque TgCRND8 mice also demonstrate an increased sensitivity to PTZ-induced seizures with a more severe seizure type over age-matched littermate controls. A lower threshold and more severe seizure type in TgCRND8 mice prior to and after plaque deposition suggest that this genotype difference may be due to beta-amyloid (Abeta) toxicity rather than plaque formation. Thus, the TgCRND8 mice are not only a model for Abeta production and plaque deposition, but may also be useful for AD associated seizure.

In Vivo Characterization of a Novel γ-Secretase Inhibitor SCH 697466 in Rodents and Investigation of Strategies for Managing Notch-Related Side Effects.

Substantial evidence implicates ß-amyloid (Aß) peptides in the etiology of Alzheimer's disease (AD). Aß is produced by the proteolytic cleavage of the amyloid precursor protein by ß- and γ-secretase suggesting that γ-secretase inhibition may provide therapeutic benefit for AD. Although many γ-secretase inhibitors have been shown to be potent at lowering Aß, some have also been shown to have side effects following repeated administration. All of these side effects can be attributed to altered Notch signaling, another γ-secretase substrate. Here we describe the in vivo characterization of the novel γ-secretase inhibitor SCH 697466 in rodents. Although SCH 697466 was effective at lowering Aß, Notch-related side effects in the intestine and thymus were observed following subchronic administration at doses that provided sustained and complete lowering of Aß. However, additional studies revealed that both partial but sustained lowering of Aßand complete but less sustained lowering of Aß were successful approaches for managing Notch-related side effects. Further, changes in several Notch-related biomarkers paralleled the side effect observations. Taken together, these studies demonstrated that, by carefully varying the extent and duration of Aß lowering by γ-secretase inhibitors, it is possible to obtain robust and sustained lowering of Aß without evidence of Notch-related side effects.

Discovery of SCH 900229, a Potent Presenilin 1 Selective γ-Secretase Inhibitor for the Treatment of Alzheimer's Disease.

An exploration of the SAR of the side chain of a novel tricyclic series of γ-secretase inhibitors led to the identification of compound (-)-16 (SCH 900229), which is a potent and PS1 selective inhibitor of γ-secretase (Aß40 IC50 = 1.3 nM). Compound (-)-16 demonstrated excellent lowering of Aß after oral administration in preclinical animal models and was advanced to human clinical trials for further development as a therapeutic agent for the treatment of Alzheimer's disease.

The anxiolytic-like profile of the nociceptin receptor agonist, endo-8-[bis(2-chlorophenyl)methyl]-3-phenyl-8-azabicyclo[3.2.1]octane-3-carboxamide (SCH 655842): comparison of efficacy and side effects across rodent species.

The endogenous opioid-like peptide, nociceptin, produces anxiolytic-like effects that are mediated via the nociceptin (NOP) receptor. Similarly, synthetic, non-peptide NOP agonists produce robust anxiolytic-like effects although these effects are limited by marked side effects. In the present studies, the effects of a novel NOP receptor agonist, SCH 655842, were examined in rodent models sensitive to anxiolytic drugs and tests measuring potential adverse affects. Oral administration of SCH 655842 produced robust, anxiolytic-like effects in three species, i.e., rat, guinea pig, and mouse. Specifically, SCH 655842 was effective in rat conditioned lick suppression (3-10 mg/kg) and fear-potentiated startle (3-10 mg/kg) tests, a guinea pig pup vocalization test (1-3 mg/kg), as well as in mouse Geller-Seifter (30 mg/kg) and marble burying (30 mg/kg) tests. The anxiolytic-like effect of SCH 655842 in the conditioned lick suppression test was attenuated by the NOP antagonist, J-113397. In mice, SCH 655842 reduced locomotor activity and body temperature at doses similar to the anxiolytic-like dose and these effects were absent in NOP receptor knockout mice. In rats, SCH 655842 did not produce adverse behavioral effects up to doses of 70-100 mg/kg. Pharmacokinetic studies in the rat confirmed dose-related increases in plasma and brain levels of SCH 655842 across a wide oral dose range. Taken together, SCH 655842 may represent a NOP receptor agonist with improved tolerability compared to other members of this class although further studies are necessary to establish whether this extends to higher species.

GPR119 is required for physiological regulation of glucagon-like peptide-1 secretion but not for metabolic homeostasis.

G protein-coupled receptor 119 (GPR119) is expressed in pancreatic islets and intestine, and is involved in insulin and incretin hormone release. GPR119-knockout (Gpr119(-/-)) mice were reported to have normal islet morphology and normal size, body weight (BW), and fed/fasted glucose levels. However, the physiological function of GPR119 and its role in maintaining glucose homeostasis under metabolic stress remain unknown. Here, we report the phenotypes of an independently generated line of Gpr119(-/-) mice under basal and high-fat diet (HFD)-induced obesity. Under low-fat diet feeding, Gpr119(-/-) mice show normal plasma glucose and lipids, but have lower BWs and lower post-prandial levels of active glucagon-like peptide 1 (GLP-1). Nutrient-stimulated GLP-1 release is attenuated in Gpr119(-/-) mice, suggesting that GPR119 plays a role in physiological regulation of GLP-1 secretion. Under HFD-feeding, both Gpr119(+)(/)(+) and Gpr119(-/-) mice gain weight similarly, develop hyperinsulinemia and hyperleptinemia, but not hyperglycemia or dyslipidemia. Glucose and insulin tolerance tests did not reveal a genotypic difference. These data show that GPR119 is not essential for the maintenance of glucose homeostasis. Moreover, we found that oleoylethanolamide (OEA), reported as a ligand for GPR119, was able to suppress food intake in both Gpr119(+)(/)(+) and Gpr119(-/-) mice, indicating that GPR119 is not required for the hypophagic effect of OEA. Our results demonstrate that GPR119 is important for incretin and insulin secretion, but not for appetite suppression.

Automated evaluation of sensitivity to foot shock in mice: inbred strain differences and pharmacological validation.

Assessing foot shock sensitivity in rodents can be useful in identifying analgesic or hyperalgesic drugs, and phenotyping inbred or genetically altered mice. Furthermore, as foot shock is an integral part of several rodent behavioral models, sensitivity should also be assessed to accurately interpret behavioral measures from these models. To eliminate variability and increase the efficiency of manually scored shock sensitivity paradigms, we utilized a startle reflex system to automatically quantify responses to varying levels of foot shock. Eight inbred mouse strains were tested for reactivity to foot shock in this system, as well as inherent startle response activity to loud noise bursts. Strain rank order for shock reactivity differed from that for acoustic startle, suggesting that pathways activated in response to each differed. Analgesic doses of morphine and acetaminophen specifically reduced foot shock responses without affecting motor reflexive responses to loud noises in each strain tested. We also tested diazepam and scopolamine, which are often used to disrupt behavior in shock-related paradigms to illustrate the usefulness of this assay. Overall, these results demonstrate that our automated method is a quick and simple way to accurately assess potential foot shock sensitivity differences owing to strain, genotype or drug treatments.

The conserved immunoglobulin domain controls the subcellular localization of the homophilic adhesion receptor protein-tyrosine phosphatase mu.

The receptor protein-tyrosine phosphatase mu (PTPmu) is a homophilic adhesion protein thought to regulate cell-cell adhesion in the vascular endothelium through dephosphorylation of cell junction proteins. In subconfluent cell cultures, PTPmu resides in an intracellular membrane pool; however, as culture density increases and cell contacts form, the phosphatase localizes to sites of cell-cell contact, and its expression level increases. These characteristics of PTPmu, which are consistent with a role in cell-cell adhesion, suggest that control of subcellular localization is an important mechanism to regulate the function of this phosphatase. To gain a better understanding of how PTPmu is regulated, we examined the importance of the conserved immunoglobulin domain, containing the homophilic binding site, in control of the localization of the enzyme. Deletion of the immunoglobulin domain impaired localization of PTPmu to the cell-cell contacts in endothelial and epithelial cells. In addition, deletion of the immunoglobulin domain affected the distribution of PTPmu in subconfluent endothelial cells when homophilic binding to another PTPmu molecule on an apposing cell was not possible, resulting in an accumulation of the mutant phosphatase at the cell surface with a concentration at the cell periphery in the region occupied by focal adhesions. This aberrant localization correlated with reduced survival and alterations in normal focal adhesion and cytoskeleton morphology. This study therefore illustrates the critical role of the immunoglobulin domain in regulation of the localization of PTPmu and the importance of such control for the maintenance of normal cell physiology.

Computational analysis of protein tyrosine phosphatases: practical guide to bioinformatics and data resources.

The exponential growth of sequence data has become a challenge to database curators and end-users alike and biologists seeking to utilize the data effectively are faced with numerous analysis methods. Here, with practical examples from our bioinformatics analysis of the protein tyrosine phosphatases (PTPs), we show how computational analysis can be exploited to fuel hypothesis-driven experimental research through the exploration of online databases. We cover the following elements: (i) similarity searches and strategies to collect a non-redundant database of tyrosine-specific PTP domains; (ii) utilization of this database to classify human, fly, and worm PTPs (based on alignments and phylogenetic analysis); (iii) three-dimensional structural analysis to identify conserved regions (structure-function) and non-conserved selectivity-determining regions (substrate specificity); and (iv) genomic analysis, including mapping of exon structure, identification of pseudogenes, and exploration of disease databases. We discuss the importance of manual curation, illustrating examples in which pseudogenes give rise to predicted proteins in GenBank and note that domain servers, such as PFAM and SMART, erroneously include dual-specificity and lipid phosphatases in their collection of tyrosine-specific PTPs. To capitalize on our annotated set of 402 PTP domains (from 47 species and five phyla), we identify sequence conservation across taxonomic categories and explore structure-function relationships among tandem domain receptor-like PTPs. We define three Src homology 2 domain-containing PTP genes in stingray, zebrafish, and fugu and speculate on their evolutionary relationship with human pseudogenes. Our annotated sequences, along with a web service for phylogenetic classification of PTP domains, are available online (http://ptp.cshl.edu and http://science.novonordisk.com/ptp).

Receptor protein tyrosine phosphatase micro regulates the paracellular pathway in human lung microvascular endothelia.

The pulmonary vascular endothelial paracellular pathway and zonula adherens (ZA) integrity are regulated, in part, through protein tyrosine phosphorylation. ZA-associated protein tyrosine phosphatase (PTP)s are thought to counterregulate tyrosine phosphorylation events within the ZA multiprotein complex. One such receptor PTP, PTPmu, is highly expressed in lung tissue and is almost exclusively restricted to the endothelium. We therefore studied whether PTPmu, in pulmonary vascular endothelia, associates with and/or regulates both the tyrosine phosphorylation state of vascular endothelial (VE)-cadherin and the paracellular pathway. PTPmu was expressed in postconfluent human pulmonary artery and lung microvascular endothelial cells (ECs) where it was almost exclusively restricted to EC-EC boundaries. In human lung microvascular ECs, knockdown of PTPmu through RNA interference dramatically impaired barrier function. In immortalized human microvascular ECs, overexpression of wild-type PTPmu enhanced barrier function. PTPmu-VE-cadherin interactions were demonstrated through reciprocal co-immunoprecipitation assays and co-localization with double-label fluorescence microscopy. When glutathione S-transferase-PTPmu was incubated with purified recombinant VE-cadherin, and when glutathione S-transferase-VE-cadherin was incubated with purified recombinant PTPmu, PTPmu directly bound to VE-cadherin. Overexpression of wild-type PTPmu decreased tyrosine phosphorylation of VE-cadherin. Therefore, PTPmu is expressed in human pulmonary vascular endothelia where it directly binds to VE-cadherin and regulates both the tyrosine phosphorylation state of VE-cadherin and barrier integrity.

Long-term potentiation is increased in the CA1 area of the hippocampus of APP(swe/ind) CRND8 mice.

The present study reports changes in synaptic function and plasticity [long-term potentiation (LTP)] in a recently developed mouse model of Alzheimer's disease (CRND8 line) harboring a double amyloid precursor protein mutation (APP(swe/ind)). In 9-week-old preplaque transgenic (Tg) mice brain slices, basal synaptic function in the hippocampal CA1 area was unchanged. Only one of three different LTP induction protocols revealed early influence of genotype on synaptic plasticity. By 20 weeks of age, there were numerous plaques in the hippocampus from Tg mice associated with more robust evidence for genotype-related effects in synaptic function. Field potential maximum slope was consistently decreased and LTP was increased, irrespective of the stimulation protocol used. In addition, there was clear evidence of increased synaptic excitability in Tg mice. Furthermore, the maximum amplitude of evoked IPSCs was decreased whereas the maximum amplitude of evoked EPSCs was increased in 20-week-old Tg mice. Collectively, these results suggest a number of APP genotype-related changes in the fine-tuning of the CA1 area circuitry, some of which are likely to contribute to the pathology-dependent effects on LTP observed in CRND8 mice.