Transcriptional-based screens for pathway-specific, high-throughput target discovery in endothelial cells.
With the sequence of the human genome at hand, target discovery strategies are needed that can rapidly identify novel gene products involved in human disease pathways. In this article, the authors describe a cell-based, high-throughput assay that can identify gene products capable of modulating the vascular endothelial growth factor (VEGF) and tumor necrosis factor alpha (TNFalpha) signaling pathways in human endothelial cells. The assay uses real-time PCR technology to measure downstream reporter mRNA transcripts induced upon cytokine stimulation in a 96-well plate format and has been adapted for use with recombinant adenoviruses. The authors specifically demonstrate modulation of cytokine-driven reporter transcripts using drug inhibitors and through adenoviral-mediated expression of known signaling intermediates of the respective pathways. In addition, they have used an arrayed library of 350 recombinant adenoviruses to screen for novel modulators of the VEGF and TNFalpha pathways. The high-throughput screening capacity and sensitivity of this system make it a useful tool for new drug target identification.
Yauch
Robert L
RL
SUGEN, Inc., South San Francisco, CA, USA. bobyauch@gene.com
Kadel
Edward E
EE
Nicholas
Cory
C
Tetangco
Selwyna
S
Clary
Douglas O
DO
D000256:Adenoviridae / Q000235:genetics; D001681:Biological Assay; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D005786:Gene Expression Regulation; D015723:Gene Library; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D023281:Genomics / Q000379:methods*; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors; D016133:Polymerase Chain Reaction; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013925:Thromboplastin / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology* / Q000502:physiology; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology* / Q000502:physiology
D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D042461:Vascular Endothelial Growth Factor A; D018799:Intercellular Adhesion Molecule-1; D013925:Thromboplastin; D020928:Mitogen-Activated Protein Kinases