Flow Cytometric HTS Screening for Inhibitors of Lytic Granule Exocytosis with MLPCN Compound Library
Target cell killing by cytotoxic T lymphocytes (CTLs), critical for the immune response to viruses and tumors, is involved in transplant rejection and contributes to autoimmune disease pathogenesis. A key mechanism CTLs use to kill target cells is secretion of cell-killing agents from specialized lysosomal lytic granules. Screening a small molecule library for compounds that inhibit lytic granule more ..
BioActive Compounds: 2400
University of New Mexico Assay Overview:
Assay Support: NIH R21 NS066462
A high-throughput screen of lytic granule exocytosis
PI: Adam Zweifach, Ph.D.
PI Affiliation: Department of Molecular and Cell Biology, University of Connecticut
Screening Center PI: Larry Sklar, Ph.D. / UNMCMD
Screening Lead: Mark Haynes, Ph.D.
Assay Implementation: Bruce Edwards, Ph.D., Matthew Garcia, Mark Carter, M.S.
UNM Cheminformatics: Oleg Ursu, Ph.D.
Chemistry: Vanderbilt Specialized Chemistry Center for Accelerated Probe Development
Vanderbilt Specialized Chemistry Center PI: Craig Lindsley, Ph.D.
Vanderbilt Chemistry Lead: Michael Wood, Ph.D.
Assay Background and Significance:
Target cell killing by cytotoxic T lymphocytes (CTLs), critical for the immune response to viruses and tumors, is involved in transplant rejection and contributes to autoimmune disease pathogenesis. A key mechanism CTLs use to kill target cells is secretion of cell-killing agents from specialized lysosomal lytic granules. Screening a small molecule library for compounds that inhibit lytic granule exocytosis would 1) serve as an assay for blockers of the known signals that control the process; 2) reveal the presence of and define chemical probes for as-yet-unknown signals and 3) lead to novel immunomodulatory drugs that could enhance our ability to transplant organs and stem cells and to treat autoimmune disorders.
CTL granule exocytosis can be monitored using fluorescently-labeled antibodies to detect the externalization of lysosome-associated membrane protein 1 (LAMP-1) from lytic granules to the plasma membrane in a simple no-wash assay protocol suitable for flow cytometry. We plan to capitalize on this simple assay together with the unique HTS flow cytometry capabilities at the University of New Mexico Center for Molecular Discovery (UNMCMD) to establish a phenotypic screen and to discover chemical probes that inhibit granulocyte exocytosis.
The purpose of this assay is to detect compounds that inhibit lytic granule exocytosis in TALL-104 cells. Lytic granule exocytosis is detected using a fluorescently-labeled antibody to LAMP-1, a lysosomal integral membrane protein that transfers to the plasma membrane as a consequence of exocytosis of lytic granules.
Briefly the protocol for HTS, 4 microL of medium will be added to columns 2-47 of a 1536-well microtitre plate followed by 0.1 microL of test compounds in DMSO (1milliM) added to columns 5-44. Columns 2, 46 and 47 are left empty for analysis purposes. Negative (columns 3 and 4) and positive (column 45) control wells will receive 0.1 microL of DMSO. Cells (5-6 x10^3) are added to columns 3 - 45 in a volume of 4 microL and the plates are mixed and incubated for 30 minutes at 37oC. The resulting mixture's final compound concentration is 10 microM with a DMSO concentration of ~ 1%. Following incubation with test compounds, columns 3 and 4 (unstimulated controls) will receive 2 microL of medium containing alexa647 conjugated anti-LAMP antibody (final dilution 1:500), while wells in columns 5-45 will receive 2 microL of a stimulation solution containing 1 milliM thapsigargin, 50 microM PMA, and alexa647 conjugated anti-LAMP antibody; this addition results in wells that contain 1 microM TG, 50 nanoM PMA and ~1/500 anti-lamp. Plates are incubated with rotation for 120 minutes. Individual wells are sampled using the HyperCyt HTFC platform at a rate of 1 sec/sip resulting in a delivery of >500 cells for analysis (~12 min/plate). Channel fluorescence parameters of individual samples is acquired and saved. Data analysis is accomplished using in-house HyperView software that parses the single data file, generating individual fluorescence values for each well.
Flow cytometeric analysis of the data includes forward and side light scatter that identifies the cell population of interest, alexa647 emission data from stimulated and unstimulated controls wells from each plate are used to create a region gate that delineates stimulated and unstimulated cells such that 95% of the stimulated population falls within the region gate.
Averages of % Positive value for the Negative control (NCntrl, stimulated) wells and the Positive control (PCntrl, unstimulated) wells are calculated and their range is used to calculate the % Inhibition of individual sample wells by the following equation:
%Inhibition =100 * (NCntrl%Pos - Sample%Pos)/(NCntrl%Pos - PCntrl%Pos)
A hit compound has a %Inhibition > 55%
Note, lack of sufficient sample size (less than 10 events) results in a well being labeled 'undetermined'. In total there were 767 undetermined wells or 'missing' compounds from a total set of 364202 compound library.
** Test Concentration.
Data Table (Concise)