title: A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules.
abstract: High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a "target hypothesis", which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.
authors.affiliation: Department of Microbiology, Infectiology and Immunology, Université de Montréal.
authors.forename: Ahmed
authors.initials: A
authors.lastname: Fouda
authors.affiliation: Department of Pharmacology and Physiology, Université de Montréal.
authors.forename: Mahasti
authors.initials: M
authors.lastname: Tahsini
authors.affiliation: Department of Pharmacology and Physiology, Université de Montréal.
authors.forename: Fatemeh
authors.initials: F
authors.lastname: Khodayarian
authors.affiliation: Department of Pharmacology and Physiology, Université de Montréal.
authors.forename: Fatimah
authors.initials: F
authors.lastname: Al-Nafisah
authors.affiliation: Department of Microbiology, Infectiology and Immunology, Université de Montréal; Department of Pharmacology and Physiology, Université de Montréal; Molecular Biology Program, Université de Montréal; moutih.rafei.1@umontreal.ca.
authors.forename: Moutih
authors.initials: M
authors.lastname: Rafei
mesh_terms: D000818:Animals; D001681:Biological Assay; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005260:Female; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D007155:Immunologic Factors / Q000494:pharmacology; D008214:Lymphocytes / Q000187:drug effects* / Q000502:physiology; D008822:Mice, Transgenic; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1 / Q000235:genetics; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D054852:Small Molecule Libraries / Q000494:pharmacology*
chemical_list: D007155:Immunologic Factors; C539818:Nr4a1 protein, mouse; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D011948:Receptors, Antigen, T-Cell; D054852:Small Molecule Libraries; D049452:Green Fluorescent Proteins