Transient Receptor Potential Melastatin 5 (TRPM5) is an intracellular calcium-activated cation-selective ion channel expressed in a variety of cell types. Dysfunction of this channel has recently been implied in a range of disease states including diabetes, enteric infections, inflammatory responses, parasitic infection and other pathologies. However, to date, agonists and positive modulators of this channel with sufficient selectivity to enable target validation studies have not been described, limiting the evaluation of TRPM5 biology and its potential as a drug target. We developed a high-throughput assay using a fluorescent membrane potential dye and a medium- and high-throughput electrophysiology assay using QPatch HTX and SyncroPatch 384PE. By employing these assays, we conducted a primary screening campaign and identified hit compounds as TRPM5 channel positive modulators. An initial selectivity profile confirmed hit selectivity to TRPM5 and is presented here. These small molecule TRPM5 compounds have a high potential both as early tool compounds to enable pharmacological studies of TRPM5 and as starting points for the development of potent, selective TRPM5 openers or positive modulators as novel drugs targeting several pathological states.

Transient receptor potential cation channel subfamily M member 5 (TRPM5) is a nonselective monovalent cation channel activated by intracellular Ca2+ increase. Within the gastrointestinal system, TRPM5 is expressed in the stoma, small intestine, and colon. In the search for a selective agonist of TRPM5 possessing in vivo gastrointestinal prokinetic activity, a high-throughput screening was performed and compound 1 was identified as a promising hit. Hit validation and hit to lead activities led to the discovery of a series of benzo[d]isothiazole derivatives. Among these, compounds 61 and 64 showed nanomolar activity and excellent selectivity (>100-fold) versus related cation channels. The in vivo drug metabolism and pharmacokinetic profile of compound 64 was found to be ideal for a compound acting locally at the intestinal level, with minimal absorption into systemic circulation. Compound 64 was tested in vivo in a mouse motility assay at 100 mg/kg, and demonstrated increased prokinetic activity.

Genetic Engineering & Biotechnology NewsVol. 39, No. 2 Drug Discovery TutorialCompound Library Profiling of ImmunomodulatorsEvotec and Intellicyt describe multiplexed measurements of cell phenotype, viability, and cytokine secretion of human PBMCsSerge P. Parel, Laurent Brault, Tom Duensing, Zhaoping Liu, and Daniela BrodbeckSerge P. ParelSerge P. Parel, PhD, is director, chemistry and research informatics, Laurent Brault, PhD, serves as project leader assay development and hit discovery, and Daniela Brodbeck, PhD, works as director, biological sciences at Evotec. Website: www.evotec.com. Tom Duensing, PhD, is chief technology officer and Zhaoping Liu, PhD, is senior scientist at IntelliCyt. Website: www.intellicyt.com.Search for more papers by this author, Laurent BraultSerge P. Parel, PhD, is director, chemistry and research informatics, Laurent Brault, PhD, serves as project leader assay development and hit discovery, and Daniela Brodbeck, PhD, works as director, biological sciences at Evotec. Website: www.evotec.com. Tom Duensing, PhD, is chief technology officer and Zhaoping Liu, PhD, is senior scientist at IntelliCyt. Website: www.intellicyt.com.Search for more papers by this author, Tom DuensingSerge P. Parel, PhD, is director, chemistry and research informatics, Laurent Brault, PhD, serves as project leader assay development and hit discovery, and Daniela Brodbeck, PhD, works as director, biological sciences at Evotec. Website: www.evotec.com. Tom Duensing, PhD, is chief technology officer and Zhaoping Liu, PhD, is senior scientist at IntelliCyt. Website: www.intellicyt.com.Search for more papers by this author, Zhaoping LiuSerge P. Parel, PhD, is director, chemistry and research informatics, Laurent Brault, PhD, serves as project leader assay development and hit discovery, and Daniela Brodbeck, PhD, works as director, biological sciences at Evotec. Website: www.evotec.com. Tom Duensing, PhD, is chief technology officer and Zhaoping Liu, PhD, is senior scientist at IntelliCyt. Website: www.intellicyt.com.Search for more papers by this author, and Daniela BrodbeckSerge P. Parel, PhD, is director, chemistry and research informatics, Laurent Brault, PhD, serves as project leader assay development and hit discovery, and Daniela Brodbeck, PhD, works as director, biological sciences at Evotec. Website: www.evotec.com. Tom Duensing, PhD, is chief technology officer and Zhaoping Liu, PhD, is senior scientist at IntelliCyt. Website: www.intellicyt.com.Search for more papers by this authorPublished Online:31 Jan 2019https://doi.org/10.1089/gen.39.02.17AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetails Volume 39Issue 2Feb 2019 InformationCopyright © GEN PublishingTo cite this article:Serge P. Parel, Laurent Brault, Tom Duensing, Zhaoping Liu, and Daniela Brodbeck.Compound Library Profiling of Immunomodulators.Genetic Engineering & Biotechnology News.Feb 2019.54-55.http://doi.org/10.1089/gen.39.02.17Published in Volume: 39 Issue 2: January 31, 2019PDF download

Abstract The success of immunomodulatory approaches for the treatment of disease has generated tremendous interest in discovery of new immunotherapy based therapeutics. Immunotherapy has the potential of affecting - positively and negatively - the myriad interactions among cells and signaling molecules regulating the immune system, and projects should include high throughput screening campaigns aimed at profiling the effects of compounds on these complex interactions. We describe a no-wash, high throughput screen that provides a multiparameter activity profile for a structure activity relationship (SAR) library of immunomodulatory compounds. Seven known immunomodulatory compounds were used as templates to generate an SAR expansion library of 1438 compounds. The selection utilized a combination of various substructure and similarity search methods combined with the generation of virtual templates. Phytohemagglutinin (PHA) activated PBMCs were treated with the compounds at two doses, 10µM and 50µM. Immunophenotyping was done by α-CD3 and α-CD8 antibodies and differential toxic effects of compounds on the viability of individual cell subsets plus effects on the secretion of three cytokines were used to generate an immunological profile for each compound tested. We demonstrate the combination of a robust SAR expansion system with high throughput, multiplex screening on primary cells to generate activity profiles that can be used for identifying viable therapeutic candidates.

The mTOR pathway is a critical integrator of nutrient and growth factor signaling. Once activated, mTOR promotes cell growth and proliferation. Several components of the mTOR pathway are frequently deregulated in tumors, leading to constitutive activation of the pathway and thus contribute to uncontrolled cell growth. We performed a high-throughput screen with an isogenic cell line system to identify compounds specifically inhibiting proliferation of PTEN/mTOR-pathway addicted cells. We show here the characterization and mode of action of two such compound classes. One compound class inhibits components of the PTEN/mTOR signaling pathway, such as S6 ribosomal protein phosphorylation, and leads to cyclin D3 downregulation. These compounds are not adenosine triphosphate competitive inhibitors for kinases in the pathway, nor do they require FKBP12 for activity like rapamycin. The other compound class turned out to be a farnesylation inhibitor, blocking the activity of GTPases, as well as an inducer of oxidative stress. Our results demonstrate that an isogenic cell system with few specific mutations in oncogenes and tumor suppressor genes can identify different classes of compounds selectively inhibiting proliferation of PTEN/mTOR pathway–addicted isogenic clones. The identified mechanisms are in line with the known cellular signaling networks activated by the altered oncogenes and suppressor genes in the isogenic system.

Genetic Engineering & Biotechnology NewsVol. 34, No. 2 Drug Discovery TutorialPhenotypic Compound ProfilingApplying Multiplexing Capabilities to a Range of Drug Discovery ActivitiesKim Luu and Daniela BrodbeckKim LuuKim Luu, Ph.D. (E-mail Address: kluu@intellicyt.com), is manager, assay development at Intelli-Cyt. Website: www.intellicyt.com. Daniela Brodbeck works at Exquiron Biotech. Website: www.exquiron.com.Search for more papers by this author and Daniela BrodbeckSearch for more papers by this authorPublished Online:15 Jan 2014https://doi.org/10.1089/gen.34.02.09AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetails Volume 34Issue 2Jan 2014 Information© 2014 by GEN PublishingTo cite this article:Kim Luu and Daniela Brodbeck.Phenotypic Compound Profiling.Genetic Engineering & Biotechnology News.Jan 2014.16-17.http://doi.org/10.1089/gen.34.02.09Published in Volume: 34 Issue 2: January 15, 2014PDF download