qHTS for small molecule inhibitors of Yes1 kinase: Primary Screen
The Src family of non-receptor protein tyrosine kinases contains nine members, including Yes1, Src, Fyn, Lyn, and Lyk. These kinases have important roles in a variety of cellular functions, such as cell proliferation, survival, and differentiation. Src, the most well characterized member of this family, has been previously identified as a proto-oncogene. Like Src, Yes1 kinase activity has been more ..
BioActive Compounds: 349
The Src family of non-receptor protein tyrosine kinases contains nine members, including Yes1, Src, Fyn, Lyn, and Lyk. These kinases have important roles in a variety of cellular functions, such as cell proliferation, survival, and differentiation. Src, the most well characterized member of this family, has been previously identified as a proto-oncogene. Like Src, Yes1 kinase activity has been shown to be activated in a variety of cancers, including colon carcinomas, melanoma, head and neck, renal, lung, and stomach cancers. In melanoma, it has been shown that Yes1, and not other family members, such as Src, is functionally involved in the malignant phenotype. Additionally, a recent study examining the downregulation of Yes1 by shRNA found significant effects on cell survival/growth for basal-like and Her2-positive breast cancers. Despite the potential for Yes1 to be a target for the above-described cancers, there are very few reports on the identification of potent and selective inhibitors of Yes1 kinase. Antagonists of Yes1 would further elucidate Yes1 biology and help to confirm whether Yes1 is a viable target for therapeutic intervention in a variety of cancers.
A compound that inhibits Yes1 could be important for the treatment of various cancers in which Yes1 is highly expressed and can also be used as a tool compound to help further elucidate Yes1 biology. To that end, we have developed a sensitive HTS biochemical assay for Yes1 activity. This assay was screened against several small compound libraries to look for modulators of activity. Inhibition is the desired output for this assay.
NIH Molecular Libraries Probe Production Centers Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
MLPCN Grant: MH084681
Two micoliter of Yes1 enzyme (columns 1-2, 5-48; Millipore, Billerica, MA) are dispensed into Greiner, white solid-bottom 1536-well assay plates; columns 3 and 4 receive buffer without Yes1 as a control for maximum inhibition. Compounds (23 nL) are then transferred via Kalypsys pin tool equipped with 1536-pin array (10 nL slotted pins, V&P Scientific, San Diego, CA). The library compounds are in columns 5-48 and columns 1-4 serve as controls with DMSO in columns 1, 3 and 4 and 10 mM 1:3 fold serial of dasatinib in column 2. Following addition of compound and a 20 minute incubation, 1 uL of substrate (ATP and poly-E4Y from Sigma-Aldrich, St. Louis, MO) is added, and the biochemical reaction is allowed to progress for 60 minutes at room temperature. The production of ADP is then quantified by the ADP-Glo Kit (Promega, Madison, WI). First, 2 uL of the first kit reagent is added, and the reaction is incubated for 45 minutes; then, 4 uL of the second kit reagent are added and the reaction is incubated for 30 minutes at which time the luminescence is read on a Viewlux plate reader (Perkin-Elmer, Waltham, MA). The final concentration of reagents in the reaction are 4 nM Yes1, 0.1 mM ATP, 100 uM EGTA, 10 mM MgCl2, 0.3 mg/mL poly(E4Y), 50 mM Tris pH 7.5, 1 mM DTT, 150 mM NaCl, 0.01% Brij-35, and 5% glycerol. Data are normalized to DMSO-treated control columns with (maximum signal) and without (minimum signal) enzyme.
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)