[Show abstract][Hide abstract] ABSTRACT: M1 muscarinic receptors play an important role in cognition and memory, and are considered to be attractive targets for the development of novel medications to treat cognitive impairments seen in schizophrenia and Alzheimer's disease. Indeed, the M1 agonist xanomeline has been shown to produce beneficial cognitive effects in both Alzheimer's disease and schizophrenia patients. Unfortunately, the therapeutic utility of xanomeline was limited by "cholinergic" side effects (sweating, salivation, gastrointestinal distress) which are believed to result from nonselective activation of other muscarinic receptor subtypes such as M2 and M3. Therefore, drug discovery efforts targeting the M1 receptor have focused on the discovery of compounds with improved selectivity profiles. Recently, allosteric M1 receptor ligands have been described, which exhibit excellent selectivity for M1 over other muscarinic receptor subtypes. In the current study, three compounds (3-((1S,2S)-2-hydrocyclohexyl)-6-((6-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)methyl)benzo[h]quinazolin-4(3H)-one, 1-((4-cyano-4-(pyridin-2-yl)piperidin-1-yl)methyl)-4-oxo-4H-quinolizine-3-carboxylic acid, and (R)-ethyl 3-(2-methylbenzamido)-[1,4'-bipiperidine]-1'-carboxylate) with mixed agonist/positive allosteric modulator activities that are highly functionally selective for the M1 receptor were tested in rats, dogs, and cynomologous monkeys. Despite their selectivity for the M1 receptor, all three compounds elicited cholinergic side-effects such as salivation, diarrhea and emesis. These effects could not be explained by activity at other muscarinic receptor subtypes, or by activity at other receptors tested. Together, these results suggest that activation of M1 receptors alone is sufficient to produce unwanted cholinergic side effects such as those seen with xanomeline. This has important implications for the development of M1 receptor-targeted therapeutics, as it suggests that dose-limiting cholinergic side effects still reside in M1 receptor selective activating agents.
Full-text · Article · Nov 2015 · Journal of Pharmacology and Experimental Therapeutics
[Show abstract][Hide abstract] ABSTRACT: Hetero-oligomeric complexes of G protein-coupled receptors (GPCRs) may represent novel therapeutic targets exhibiting different pharmacology and tissue- or cell-specific site of action compared with receptor monomers or homo-oligomers. An ideal tool for validating this concept pharmacologically would be a hetero-oligomer selective ligand. We set out to develop and execute a 1536-well high-throughput screen of over 1 million compounds to detect potential hetero-oligomer selective ligands using a β-arrestin recruitment assay in U2OS cells coexpressing recombinant µ- and δ-opioid receptors. Hetero-oligomer selective ligands may bind to orthosteric or allosteric sites, and we might anticipate that the formation of hetero-oligomers may provide novel allosteric binding pockets for ligand binding. Therefore, our goal was to execute the screen in such a way as to identify positive allosteric modulators (PAMs) as well as agonists for µ, δ, and hetero-oligomeric receptors. While no hetero-oligomer selective ligands were identified (based on our selection criteria), this single screen did identify numerous µ- and δ-selective agonists and PAMs as well as nonselective agonists and PAMs. To our knowledge, these are the first µ- and δ-opioid receptor PAMs described in the literature.
[Show abstract][Hide abstract] ABSTRACT: G protein-coupled receptors (GPCRs) are one of the most popular and proven target classes for therapeutic intervention. The increased appreciation for allosteric modulation, receptor oligomerization, and biased agonism has led to the development of new assay platforms that seek to capitalize on these aspects of GPCR biology. High-content screening is particularly well suited for GPCR drug discovery given the ability to image and quantify changes in multiple cellular parameters, to resolve subcellular structures, and to monitor events within a physiologically relevant environment. Focusing on the sphingosine-1-phosphate (S1P1) receptor, we evaluated the utility of high-content approaches in hit identification efforts by developing and applying assays to monitor β-arrestin translocation, GPCR internalization, and GPCR recycling kinetics. Using these approaches in combination with more traditional GPCR screening assays, we identified compounds whose unique pharmacological profiles would have gone unnoticed if using a single platform. In addition, we identified a compound that induces an atypical pattern of β-arrestin translocation and GPCR recycling kinetics. Our results highlight the value of high-content imaging in GPCR drug discovery efforts and emphasize the value of a multiassay approach to study pharmacological properties of compounds of interest.
[Show abstract][Hide abstract] ABSTRACT: Recent genetic evidence suggests that the diacylglycerol lipase (DAGL-α) isoform is the major biosynthetic enzyme for the most abundant endocannabinoid, 2-arachidonoyl-glycerol (2-AG), in the central nervous system. Revelation of its essential role in regulating retrograde synaptic plasticity and adult neurogenesis has made it an attractive therapeutic target. Therefore, it has become apparent that selective inhibition of DAGL-α enzyme activity with a small molecule could be a strategy for the development of novel therapies for the treatment of disease indications such as depression, anxiety, pain, and cognition. In this report, the authors present the identification of small-molecule inhibitor chemotypes of DAGL-α, which were selective (≥10-fold) against two other lipases, pancreatic lipase and monoacylglycerol lipase, via high-throughput screening of a diverse compound collection. Seven chemotypes of interest from a list of 185 structural clusters, which included 132 singletons, were initially selected for evaluation and characterization. Selection was based on potency, selectivity, and chemical tractability. One of the chemotypes, the glycine sulfonamide series, was prioritized as an initial lead for further medicinal chemistry optimization.
Preview · Article · Nov 2013 · Journal of Biomolecular Screening
[Show abstract][Hide abstract] ABSTRACT: Transporter proteins are known to play a critical role in affecting the overall absorption, distribution, metabolism, and excretion characteristics of drug candidates. In addition to efflux transporters (P-gp, BCRP, MRP2, etc.) that limit absorption, there has been a renewed interest in influx transporters at the renal (OATs, OCTs) and hepatic (OATPs, BSEP, NTCP, etc.) organ level that can cause significant clinical drug-drug interactions (DDIs). Several of these transporters are also critical for hepatobiliary disposition of bilirubin and bile acid/salts, and their inhibition is directly implicated in hepatic toxicities. Regulatory agencies took action to address transporter-mediated DDI with the goal of ensuring drug safety in the clinic and on the market. To meet regulatory requirements, advanced bioassay technology and automation solutions were implemented for high-throughput transporter screening to provide structure-activity relationship within lead optimization. To enhance capacity, several functional assay formats were miniaturized to 384-well throughput including novel fluorescence-based uptake and efflux inhibition assays using high-content image analysis as well as cell-based radioactive uptake and vesicle-based efflux inhibition assays. This high-throughput capability enabled a paradigm shift from studying transporter-related issues in the development space to identifying and dialing out these concerns early on in discovery for enhanced mechanism-based efficacy while circumventing DDIs and transporter toxicities.
No preview · Article · Oct 2013 · Journal of Biomolecular Screening
[Show abstract][Hide abstract] ABSTRACT: μ-Opioid receptors are among the most studied G protein-coupled receptors because of the therapeutic value of agonists, such as morphine, that are used to treat chronic pain. However, these drugs have significant side effects, such as respiratory suppression, constipation, allodynia, tolerance, and dependence, as well as abuse potential. Efforts to fine tune pain control while alleviating the side effects of drugs, both physiological and psychological, have led to the development of a wide variety of structurally diverse agonist ligands for the μ-opioid receptor, as well as compounds that target κ- and δ-opioid receptors. In recent years, the identification of allosteric ligands for some G protein-coupled receptors has provided breakthroughs in obtaining receptor subtype-selectivity that can reduce the overall side effect profiles of a potential drug. However, positive allosteric modulators (PAMs) can also have the specific advantage of only modulating the activity of the receptor when the orthosteric agonist occupies the receptor, thus maintaining spatial and temporal control of receptor signaling in vivo. This second advantage of allosteric modulators may yield breakthroughs in opioid receptor research and could lead to drugs with improved side-effect profiles or fewer tolerance and dependence issues compared with orthosteric opioid receptor agonists. Here, we describe the discovery and characterization of μ-opioid receptor PAMs and silent allosteric modulators, identified from high-throughput screening using a β-arrestin-recruitment assay.
Full-text · Article · Jun 2013 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Abstract In recent years, the increased use of cell-based functional assays for G protein-coupled receptors in high-throughput screening has enabled the design of robust assays to identify allosteric modulators (AMs) in addition to the more traditional orthosteric agonists and antagonists. In this article, the authors describe a screening format able to identify all ligand types using a triple-add assay that measures changes in cytosolic calcium concentration with three separate additions and reads in the same assay plate. This triple-add assay captures more small molecule ligand types than previously described assay formats without a significant increase in screening cost. Finally, the customizability of the triple-add assay to suit the needs of various AM screening programs is demonstrated.
Full-text · Article · Jun 2012 · Assay and Drug Development Technologies
[Show abstract][Hide abstract] ABSTRACT: Protein tyrosine phosphatases (PTPs) catalyze the dephosphorylation of tyrosine residues, a process that involves a conserved tryptophan-proline-aspartate (WPD) loop in catalysis. In previously determined structures of PTPs, the WPD-loop has been observed in either an "open" conformation or a "closed" conformation. In the current work, X-ray structures of the catalytic domain of receptor-like protein tyrosine phosphatase γ (RPTPγ) revealed a ligand-induced "superopen" conformation not previously reported for PTPs. In the superopen conformation, the ligand acts as an apparent competitive inhibitor and binds in a small hydrophobic pocket adjacent to, but distinct from, the active site. In the open and closed WPD-loop conformations of RPTPγ, the side chain of Trp1026 partially occupies this pocket. In the superopen conformation, Trp1026 is displaced allowing a 3,4-dichlorobenzyl substituent to occupy this site. The bound ligand prevents closure of the WPD-loop over the active site and disrupts the catalytic cycle of the enzyme.
No preview · Article · Sep 2011 · Journal of Medicinal Chemistry
[Show abstract][Hide abstract] ABSTRACT: Protein tyrosine phosphatase-γ (PTP-γ) is a receptor-like PTP whose biological function is poorly understood. A recent mouse PTP-γ genetic deletion model associated the loss of PTP-γ gene expression with a potential antidepressant phenotype. This led the authors to screen a subset of the Bristol-Myers Squibb (BMS) compound collection to identify selective small-molecule inhibitors of receptor-like PTP-γ (RPTP-γ) for use in evaluating enzyme function in vivo. Here, they report the design of a high-throughput fluorescence resonance energy transfer (FRET) assay based on the Z'-LYTE technology to screen for inhibitors of RPTP-γ. A subset of the BMS diverse compound collection was screened and several compounds identified as RPTP-γ inhibitors in the assay. After chemical triage and clustering, compounds were assessed for potency and selectivity by IC(50) determination with RPTP-γ and two other phosphatases, PTP-1B and CD45. One hundred twenty-nine RPTP-γ selective (defined as IC(50) value greater than 5- to 10-fold over PTP-1B and CD45) inhibitors were identified and prioritized for evaluation. One of these hits, 3-(3, 4-dichlorobenzylthio) thiophene-2-carboxylic acid, was the primary chemotype for the initiation of a medicinal chemistry program.
Full-text · Article · Mar 2011 · Journal of Biomolecular Screening
[Show abstract][Hide abstract] ABSTRACT: Allosteric modulation of G protein-coupled receptors (GPCRs) is rapidly emerging as a therapeutic strategy. The functional assays that are currently in common use for highthroughput screening at GPCR targets can be used for the identification of both positive allosteric modulators (PAMs) and negative allosteric modulators (NAMs). However, these assays must be modified in order to maximize their potential to detect all allosteric chemotypes within a screening deck. This is particularly true in the case of dynamic Ca2+ flux assays. Specifically, because PAMs often exhibit direct agonist activity in addition to their modulator activity when tested in recombinant receptor systems, and because this agonist activity can desensitize the assay system, compounds must be tested in both agonist and PAM modes in order to avoid false negatives. Additionally, because extremely minor chemical modifications can change a PAM into a NAM and vice versa, the inclusion of an antagonist/NAM mode read is also necessary in order to facilitate the discovery of as many lead chemotypes as possible, as well as to provide the maximum amount of information regarding the structure-activity relationship for these lead chemotypes.
[Show abstract][Hide abstract] ABSTRACT: 2-Arylbenzoxazole 5 was identified as a hit from a fluorescence-based high-throughput screen for CETP inhibitors. The synthesis and SAR investigation employing array synthesis of the A- and B-rings are described.
No preview · Article · May 2008 · Bioorganic & medicinal chemistry letters
[Show abstract][Hide abstract] ABSTRACT: We review strategic approaches taken over an eight-year period at BMS to implement new high-throughput approaches to lead discovery. Investments in compound management infrastructure and chemistry library production capability allowed significant growth in the size, diversity and quality of the BMS compound collection. Screening platforms were upgraded with robust automated technology to support miniaturized assay formats, while workflows and information handling technologies were streamlined for improved performance. These technology changes drove the need for a supporting organization in which critical engineering, informatics and scientific skills were more strongly represented. Taken together, these investments led to significant improvements in speed and productivity as well a greater impact of screening campaigns on the initiation of new drug discovery programs.
No preview · Article · Feb 2008 · Drug Discovery Today
[Show abstract][Hide abstract] ABSTRACT: Cryopreserved, transiently transfected HepG2 cells were compared to freshly transfected HepG2 cells for use in a pregnane X receptor (PXR) transactivation assay. Assay performance was similar for both cell preparations; however, cryopreserved cells demonstrated less interassay variation. Validation with drugs of different PXR activation potencies and efficacies demonstrated an excellent correlation (r(2) > 0.95) between cryopreserved and fresh cells. Cryopreservation did not change the effect of known CYP3A4 inducers that have poor cell permeability, indicating that cryopreservation had little effect on membrane permeability. In addition, cryopreserved HepG2 cells did not exhibit enhanced susceptibility to cytotoxic compounds compared to transiently transfected control cells. The use of cryopreserved cells enables this assay to run with enhanced efficiency.
Full-text · Article · Mar 2007 · Journal of Biomolecular Screening
[Show abstract][Hide abstract] ABSTRACT: Introduction Evolution and Integration of HT Platforms into the Discovery Process HTS Automated Systems: Technology and Process Advances in High-Throughput Bioassay Technology References