Synthesis and SAR studies of novel triazolobenzazepinones as gamma secretase modulators (GSMs) are presented in this communication. Starting from our azepinone leads, optimization studies toward improving central lowering of Aβ42 led to the discovery of novel benzo-fused azepinones. Several benzazepinones were profiled in vivo and found to lower brain Aβ42 levels in Sprague Dawley rats and transgenic APP-YAC mice in a dose-dependent manner after a single oral dose. Compound 34 was further progressed into a pilot study in our cisterna-magna-ported rhesus monkey model, where we observed robust lowering of CSF Aβ42 levels.

ADVERTISEMENT RETURN TO ISSUEPerspectiveNEXTModern Phenotypic Drug Discovery Is a Viable, Neoclassic Pharma StrategyJonathan A. Lee*†, Mark T. Uhlik‡, Christopher M. Moxham⊥, Dirk Tomandl§, and Daniel J. Sall∥View Author Information∥ Departments of †Quantitative Biology, ‡Cancer-Angiogenesis, §Discovery Informatics, and ∥Discovery Chemistry Research and Technologies, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana⊥ ImClone Systems, a Wholly Owned Subsidiary of Eli Lilly and Company, New York, New York*Phone: 317-277-8123. Fax: 317-276-6009. E-mail: [email protected]Cite this: J. Med. Chem. 2012, 55, 10, 4527–4538Publication Date (Web):March 12, 2012Publication History Received6 December 2011Published online23 March 2012Published inissue 24 May 2012https://pubs.acs.org/doi/10.1021/jm201649shttps://doi.org/10.1021/jm201649sreview-articleACS PublicationsCopyright © 2012 American Chemical SocietyRequest reuse permissionsArticle Views6957Altmetric-Citations160LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose SUBJECTS:Angiogenesis,Assays,Inhibition,Mathematical methods,Pharmaceuticals Get e-Alerts

Synthesis and SAR studies of novel aryl triazoles as gamma secretase modulators (GSMs) are presented in this communication. Starting from our aryl triazole leads, optimization studies were continued and the series progressed towards novel amides and lactams. Triazole 57 was identified as the most potent analog in this series, displaying single-digit nanomolar Aβ42 IC(50) in cell-based assays and reduced affinity for the hERG channel.

Phenotypic lead generation strategies seek to identify compounds that modulate complex, physiologically relevant systems, an approach that is complementary to traditional, target-directed strategies. Unlike gene-specific assays, phenotypic assays interrogate multiple molecular targets and signaling pathways in a target “agnostic” fashion, which may reveal novel functions for well-studied proteins and discover new pathways of therapeutic value. Significantly, existing compound libraries may not have sufficient chemical diversity to fully leverage a phenotypic strategy. To address this issue, Eli Lilly and Company launched the Phenotypic Drug Discovery Initiative (PD2), a model of open innovation whereby external research groups can submit compounds for testing in a panel of Lilly phenotypic assays. This communication describes the statistical validation, operations, and initial screening results from the first PD2 assay panel. Analysis of PD2 submissions indicates that chemical diversity from open source collaborations complements internal sources. Screening results for the first 4691 compounds submitted to PD2 have confirmed hit rates from 1.6% to 10%, with the majority of active compounds exhibiting acceptable potency and selectivity. Phenotypic lead generation strategies, in conjunction with novel chemical diversity obtained via open-source initiatives such as PD2, may provide a means to identify compounds that modulate biology by novel mechanisms and expand the innovation potential of drug discovery.

The development of a novel series of purines as gamma-secretase modulators for potential use in the treatment of Alzheimer's disease is disclosed herein. Optimization of a previously disclosed pyrimidine series afforded a series of potent purine-based gamma-secretase modulators with 300- to 2000-fold in vitro selectivity over inhibition of Notch cleavage and that selectively reduces Alphabeta42 in an APP-YAC transgenic mouse model.

c-Met is a receptor tyrosine kinase (RTK) with a critical role in many fundamental cellular processes, including cell proliferation and differentiation. Deregulated c-Met signaling has been implicated in both the initiation and progression of human cancers and therefore represents an attractive target for anticancer therapy. Monitoring the phosphorylation status of relevant tyrosine residues provides an important method of assessing c-Met kinase activity. This report describes a novel assay to monitor c-Met phosphorylation in cells using Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen) technology. Using AlphaScreen, the authors were able to detect both global and site-specific phosphorylation of c-Met in transformed cell lines. Data obtained from the AlphaScreen assay were compared to data obtained from a high-content imaging (HCI) method developed in parallel to monitor c-Met phosphorylation at the single cell level. The AlphaScreen assay was miniaturized to a 384-well format with acceptable signal-to-background ratio (S/B) and Z' statistics and was employed to measure c-Met kinase activity in situ after treatment with potent c-Met-specific kinase inhibitors. The authors discuss the utility of quantifying endogenous cellular c-Met phosphorylation in lead optimization and how the modular design of the AlphaScreen assay allows its adaptation to measure cellular activity of other kinases.

Insulin resistance in the liver and peripheral tissues, together with a pancreatic cell defect, are the common causes of Type 2 diabetes. It is now appreciated that insulin resistance can result from a defect in the insulin receptor signaling system, at a site post binding of insulin to its receptor. Protein tyrosine phosphatases (PTPases) have been shown to be negative regulators of the insulin receptor. Inhibition of PTPases may be an effective method in the treatment of Type 2 diabetes. We have identified two novel series of benzofuran/benzothiophene biphenyl oxo-acetic acids and sulfonyl-salicylic acids as potent inhibitors of PTP1B with good oral antihyperglycemic activity. To assist in the design of these inhibitors, crystallographic studies have attempted to identify enzyme inhibitor interactions. Resolution of crystal complexes has suggested that the inhibitors bind to the enzyme active site and are held in place through hydrogen bonding and van der Waals interactions formed within two hydrophobic pockets. In the oxo-acetic acid series, hydrophobic substitutents at position-2 of the benzofuran/benzothiophene biphenyl framework interacted with Phe182 of the catalytic site and were very critical to the intrinsic activity of the molecule. The hydrophobic region of the catalytic-site pocket was exploited and taken advantage by hydrophobic substituents at either the alpha-carbon or the ortho aromatic positions of the oxo-acetic acid moiety. Similar ortho aromatic substitutions on the salicylic acid-type inhibitors had no effect, primarily due to the different orientation of these inhibitors in the catalytic site. The most active inhibitors of both series inhibited recombinant human PTP1B with phosphotyrosyl dodecapeptide TRDI(P)YETD(P)Y(P)YRK as the source of the substrate with IC(50) values in the range of 20-50 nM. Compound 68 was one of the most active compounds in vivo, normalizing plasma glucose levels at the 25 mg/kg dose (po) and the 1 mg/kg dose (ip). Compound 68 was also selective against several other PTPases.

ADVERTISEMENT RETURN TO ISSUEPREVLetterNEXTPTP1B Inhibition and Antihyperglycemic Activity in the ob/ob Mouse Model of Novel 11-Arylbenzo[b]naphtho[2,3-d]furans and 11-Arylbenzo[b]naphtho[2,3-d]thiophenesJay Wrobel, Janet Sredy, Christopher Moxham, Arlene Dietrich, Zenan Li, Diane R. Sawicki, Laura Seestaller, Li Wu, Alan Katz, Donald Sullivan, Cesario Tio, and Zhong-Yin ZhangView Author Information Wyeth-Ayerst Research, Inc., CN 8000, Princeton, New Jersey 08543-8000, and Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461 Cite this: J. Med. Chem. 1999, 42, 17, 3199–3202Publication Date (Web):August 10, 1999Publication History Received25 May 1999Published online10 August 1999Published inissue 1 August 1999https://pubs.acs.org/doi/10.1021/jm990260vhttps://doi.org/10.1021/jm990260vrapid-communicationACS PublicationsCopyright © 1999 American Chemical SocietyRequest reuse permissionsArticle Views1493Altmetric-Citations105LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-AlertscloseSupporting Info (1)»Supporting Information Supporting Information SUBJECTS:Carbohydrates,Inhibitors,Peptides and proteins,Post-translational modification,Rodent models Get e-Alerts

This chapter introduces a novel approach to the study of G-protein function in vivo in which transgenic mice are created that harbor α conditional, tissue-specific expression vector that can produce RNA antisense to target mRNA(s). The central role of G proteins in transmembrane signaling from the superfamily of G-protein-linked receptors (GPLRs) to a less populous class of effector molecules that includes adenyl cyclase, phospholipase C (PLC), and various ion channels needs little explanation (see earlier review articles 1–10). Much less obvious is the pivotal role G proteins play in more complex biological processes, such as growth and development. Infectious diseases such as cholera and whooping cough, for example, express elements of their pathology via covalent modification (mono-adenosine diphosphate [ADP]-ribosylation) of G-protein targets. In endocrine tissues, mutations of specific G-protein subunits have been shown to induce tumor growth (11). Finally, genetic mutations of G proteins have been linked to pseudo-hypoparathyroidism, McCune-Albright syndrome (MAS), and Albright's hereditary osteodystrophy in humans (12).

Deficiency of the G protein subunit G alpha i2 that is known to mediate the inhibitory control of adenylylcyclase impairs insulin action [11]. Using the promoter for the phosphoenolpyruvate carboxykinase gene, conditional tissue-specific expression of the constitutively active mutant form (Q205L) of G alpha i2 was achieved in mice harboring the transgene. Expression of Q205L G alpha i2 was detected in liver and adipose tissue of transgenic mice. Whereas the G alpha i2 deficient mice displayed blunted glucose tolerance, the Q205L G alpha i2 expressing mice displayed enhanced glucose tolerance. Hexose transport and the recruitment of GLUT4, but not GLUT1, transporters to the membrane were elevated in adipocytes from Q205L G alpha i2 expressing mice in the absence of insulin. Additionally, hepatic glycogen synthase was found to be activated in Q205L G alpha i2 expressing mice, in the absence of the administration of insulin. Serum insulin levels in transgenic mice fasted overnight were equivalent to those of their control littermates. These data demonstrate that much as G alpha i2 deficiency leads to insulin resistance, expression of Q205L constitutively active G alpha i2 mimics insulin action in vivo, reflecting a permissive role of G alpha i2 in signaling via this growth factor receptor tyrosine kinase linked pathway.

Antisense RNA technology provides for selective suppression of proteins of interest and thus a new strategy with which to probe the emerging complexity of the various regulatory networks of signal transduction pathways. Considering sheer economics, the use of antisense DNA oligodeoxynucleotides is practical for studies requiring small-scale culture of cells, pilot studies seeking to test the antisense DNA strategy, and in systems in which single-cell assays can be performed (e.g., patch-clamping or histochemical analysis). Vector-driven antisense RNA expression, both constitutive and inducible, in cell culture allows for large-scale cell growth capacities enabling biochemical analyses. Expanding the antisense RNA approach to transgenic mice provides the means to generate unique animal models with which to explore the role of transmembrane signaling elements in complex biological processes in vivo. In our studies, suppression of Gsα with antisense DNA oligodeoxynucleotides provided exciting information concerning the role(s) of this G protein in adipogenesis (16). Similarly, the role of Giα2 in early mouse development has been addressed in F9 embryonic stem cells stably expressing antisense RNA (10). Finally, investigation of the role of Giα2 in adipose tissue and liver function as well as its role in whole-body metabolism, growth, and development have been made possible only through the hybrid PEPCK gene construct employed in our laboratory (11). Using a variety of antisense DNA/RNA-based approaches, investigators are now able to explore the roles of signaling elements at several distinctly different levels, selectively targeting the expression of a protein of interest in vitro or in tissues in vivo. Our knowledge of the role of transmembrane signaling elements in disease is growing rapidly. Our success with antisense DNA/RNA-based approaches in vitro and in vivo highlights the potential applications of this technology for use in gene therapy to treat pathological disorders. Expression of antisense RNA in a global set of tissues by retroviral infection or expression in a tissue-specific manner using selective promoters has implications not only for our basic understanding of how signal transduction pathways impinge on these complex events, but also for the development of new therapeutic agents with which to treat human disease.

The ability to selectively suppress the expression of specific signaling elements has provided a new strategy with which to probe the complex regulatory networks of signal transduction pathways. From an economical perspective, the use of antisense DNA oligodeoxynucleotides is practical for studies which require relatively small-scale culture of cells, for pilot studies seeking to test the antisense DNA strategy, and for cell systems amenable to single-cell assays (i.e., patch clamping or histochemical analysis). Vector-driven antisense RNA expression, both constitutive and inducible, in cell culture allows for large-scale cell growth capacities enabling biochemical analyses. Expanding the antisense RNA approach to transgenic animals provides the means to generate unique mouse models with which to explore the role of transmembrane signaling elements in complex biological processes in vivo . In our studies, the use of antisense oligodeoxynucleotides for suppression of G sα or for suppression of specific protein kinases provided powerful insights into the roles of these proteins in differentiation and receptor desensitization, respectively. Similarly the role of G iα2 in stem cell differentiation and the role of PKC in receptor desensitization have been addressed in cells stably expressing antisense RNA. Finally, investigation of the role of G iα2 in adipose tissue and liver function as well as its role in whole-body metabolism, growth, and development has been made possible only through the hybrid PEPCK gene construct employed in our laboratory. Using a panel of different antisense DNA/RNA-based approaches, one can explore the roles of signaling elements at several distinctly different levels by selectively suppressing either a single target or a family of targets in cells in vitro or in tissues in vivo . Our knowledge of the role of transmembrane signaling elements in disease is growing rapidly. Our success with antisense DNA/RNA-based approaches in vitro and in vivo highlights the potential applications of this technology for use in gene therapy to treat pathological disorders. The delivery of anti-sense DNA oligodeoxynucleotides or retroviruses harboring antisense RNA sequences to tissues as well as the ability to express antisense RNA in a narrowly defined and specific set of tissues has great implications not only for our basic understanding of how signal transduction pathways impinge on these complex events but also for the treatment of human disease.

Guanosine triphosphate-binding regulatory proteins (G proteins) are key elements in transmembrane signaling and have been implicated as regulators of more complex biological processes such as differentiation and development. The G protein Gα i2 is capable of mediating the inhibitory control of adenylylcyclase and regulates stem cell differentiation to primitive endoderm. Here an antisense RNA to Gα i2 was expressed in a hybrid RNA construct whose expression was both tissue-specific and induced at birth. Transgenic mice in which the antisense construct was expressed displayed a lack of normal development in targeted organs that correlated with the absence of Gα i2 . The loss of Gα i2 expression in adipose tissue of the transgenic mice was correlated with a rise in basal levels of adenosine 3′,5′-monophosphate (cAMP) and the loss of receptor-mediated inhibition of adenylylcyclase. These data expand our understanding of G protein function in vivo and demonstrate the necessity for Gα i2 in the development of liver and fat.