Profiling a structure activity relationship library for immunological activity in human PBMCs: multiplexed measurements of cell phenotype, viability, and cytokine secretion from a single high throughput screen

Thomas Duensing, Zhaoping Liu, Daniela Brodbeck, Serge Parel and Kim Luu

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, 16.7µM and 83.3µM. 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.

J. Immunol., 2015, 194 (Suppl 1), 260.33, Poster presented at the Immunology 2015 meeting (Download)

An Isogenic Cell Panel Identifies Compounds That Inhibit Proliferation of mTOR-Pathway Addicted Cells by Different Mechanisms

Lorenza Wyder Peters, Klaus D. Molle, Anke Thiemeyer, Agnes Knopf, Marie Goxe, Philippe Guerry, Daniela Brodbeck, Marco Colombi, Michael N. Hall, Christoph Moroni, Urs Regenass

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.

J. Biomol. Screening, 2013, 19, pp 131-144 (full text)

Hit Expansion Approaches Using Multiple Similarity Methods and Virtualized Query Structures

Andreas Bergner, Serge P. Parel

ABSTRACT: Ligand-based virtual screening and computational hit expansion methods undoubtedly facilitate the finding of novel active chemical entities, utilizing already existing knowledge of active compounds. It as been demonstrated that the parallel execution of complementary similarity search methods enhances the performance of such virtual screening campaigns. In this article, we examine the use of virtualized emplate (query, seed) structures as an extension to common search methods, such as fingerprint and pharmacophore graph-based similarity searches. We demonstrate that template virtualization by bioisosteric enumeration and other rule-based methods, in combination with standard similarity search techniques, represents a powerful approach for hit expansion following high-throughput screening campaigns. The reliability of the methods is demonstrated by four different test data sets representing different target classes and two hit finding case studies on the epigenetic targets G9a and LSD1.

J. Chem. Inf. Model., 2013, 53(5), pp 1057–1066 (full text)