A high-throughput phenotypic screen of cytotoxic T lymphocyte lytic granule exocytosis reveals candidate immunosuppressants.
We screened the National Institutes of Health's Molecular Libraries Small Molecule Repository for inhibitors of cytotoxic T lymphocyte (CTL) lytic granule exocytosis by measuring binding of an antibody in the extracellular solution to a lysosomal membrane protein (LAMP-1) that is transferred to the plasma membrane by exocytosis. We used TALL-104 human leukemic CTLs stimulated with soluble chemicals. Using high-throughput cluster cytometry to screen 364,202 compounds in a 1536-well plate format, we identified 2404 initial hits: 161 were confirmed on retesting, and dose-response measurements were performed. Seventy-five of those compounds were obtained, and 48 were confirmed active. Experiments were conducted to determine the molecular mechanism of action (MMOA) of the active compounds. Fifteen blocked increases in intracellular calcium >50%. Seven blocked phosphorylation of extracellular signal-regulated kinase (ERK) by upstream mitogen-activated protein kinase kinases >50%. One completely blocked the activity of the calcium-dependent phosphatase calcineurin. None blocked ERK catalytic activity. Eight blocked more than one pathway. For 8 compounds, we were unable to determine an MMOA. The activity of 1 of these compounds was confirmed from powder resupply. We conclude that a screen based on antibody binding to CTLs is a good means of identifying novel candidate immunosuppressants with either known or unknown MMOAs.
Department of Molecular and Cell Biology, University of Connecticut at Storrs, Storrs, CT, USA.
Ziyan
Z
Zhao
University of New Mexico Center for Molecular Discovery, Albuquerque, NM, USA.
Mark K
MK
Haynes
University of New Mexico Center for Molecular Discovery, Albuquerque, NM, USA.
Oleg
O
Ursu
University of New Mexico Center for Molecular Discovery, Albuquerque, NM, USA.
Bruce S
BS
Edwards
University of New Mexico Center for Molecular Discovery, Albuquerque, NM, USA.
Larry A
LA
Sklar
Department of Molecular and Cell Biology, University of Connecticut at Storrs, Storrs, CT, USA adam.zweifach@uconn.edu.
Adam
A
Zweifach
D019703:Calcineurin / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D004353:Drug Evaluation, Preclinical; D004789:Enzyme Activation; D005089:Exocytosis / Q000187:drug effects* / Q000276:immunology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D057166:High-Throughput Screening Assays; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D015203:Reproducibility of Results; D015398:Signal Transduction / Q000187:drug effects; D054852:Small Molecule Libraries; D013602:T-Lymphocytes, Cytotoxic / Q000187:drug effects* / Q000276:immunology* / Q000378:metabolism
D007166:Immunosuppressive Agents; D054852:Small Molecule Libraries; D011493:Protein Kinase C; D048049:Extracellular Signal-Regulated MAP Kinases; D019703:Calcineurin; D002118:Calcium
chemical biology; high-throughput screening (HTS); immunosuppressor; lymphocyte; signal transduction