A novel mechanism-based mammalian cell assay for the identification of SH2-domain-specific protein-protein inhibitors.
BACKGROUND
Many intracellular signal-transduction pathways are regulated by specific protein-protein interactions. These interactions are mediated by structural domains within signaling proteins that modulate a protein's cellular location, stability or activity. For example, Src-homology 2 (SH2) domains mediate protein-protein interactions through short contiguous amino acid motifs containing phosphotyrosine. As SH2 domains have been recognized as key regulatory molecules in a variety of cellular processes, they have become attractive drug targets.


RESULTS
We have developed a novel mechanism-based cellular assay to monitor specific SH2-domain-dependent protein-protein interactions. The assay is based on a two-hybrid system adapted to function in mammalian cells where the SH2 domain ligand is phosphorylated, and binding to a specific SH2 domain can be induced and easily monitored. As examples, we have generated a series of mammalian cell lines that can be used to monitor SH2-domain-dependent activity of the signaling proteins ZAP-70 and Src. We are utilizing these cell lines to screen for immunosuppressive and anti-osteoclastic compounds, respectively, and demonstrate here the utility of this system for the identification of small-molecule, cell-permeant SH2 domain inhibitors.


CONCLUSIONS
A mechanism-based mammalian cell assay has been developed to identify inhibitors of SH2-domain-dependent protein-protein interactions. Mechanism-based assays similar to that described here might have general use as screens for cell-permeant, nontoxic inhibitors of protein-protein interactions.
Rickles
R J
RJ
Ariad Pharmaceuticals, Inc., Cambridge, MA 02139, USA. ricklesr@ariad.com
Henry
P A
PA
Guan
W
W
Azimioara
M
M
Shakespeare
W C
WC
Violette
S
S
Zoller
M J
MJ
D000818:Animals; D006801:Humans; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011506:Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D051746:ZAP-70 Protein-Tyrosine Kinase; D018909:src Homology Domains
D011506:Proteins; D011505:Protein-Tyrosine Kinases; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human