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ACS Combinatorial Science (1)
Journal of biomolecular screening (1)
AYAD, NAGI G. (1)
CHASE, PETER (1)
Dean, Sonja (1)
Doran, Todd M. (1)
Gao, Yu (1)
HODDER, PETER (1)
Kodadek, Thomas (1)
MADOUX, FRANCK (1)
MISHRA, JITENDRA K. (1)
Mendes, Kimberly (1)
ROUSH, WILLIAM R. (1)
SIMANSKI, SCOTT (1)
Simanski, Scott (1)
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Utility of Redundant Combinatorial Libraries in Distinguishing High and Low Quality Screening Hits
Doran, Todd M.
ACS Combinatorial Science
Large one-bead one-compound (OBOC) combinatorial libraries can be constructed relatively easily by solid-phase split and pool synthesis. The use of resins with hydrophilic surfaces, such as TentaGel, allows the beads to be used directly in screens for compounds that bind selectively to labeled proteins, nucleic acids, or other biomolecules. However, we have found that this method, while useful, has a high false positive rate. In other words, beads that are scored as hits often display compounds that prove to be poor ligands for the target of interest when they are resynthesized and carried through validation trials. This results in a significant waste of time and resources in cases where putative hits cannot be validated without resynthesis. Here, we report that this problem can be largely eliminated through the use of redundant OBOC libraries, where more than one bead displaying the same compound is present in the screen. We show that compounds isolated more than once are likely to be high quality ligands for the target of interest, whereas compounds isolated only once have a much higher likelihood of being poor ligands. While the use of redundant libraries does limit the number of unique compounds that can be screened at one time in this format, the overall savings in time, effort, and materials makes this a more efficient route to the isolation of useful ligands for biomolecules.
OBOC library; peptoids; redundant library; nonspecific binding; antibody screen; antigen surrogate; serum screen
An Ultra-High Throughput Cell-Based Screen for Wee1 Degradation Inhibitors
MISHRA, JITENDRA K.
ROUSH, WILLIAM R.
AYAD, NAGI G.
Journal of biomolecular screening
The tyrosine kinase Wee1 is part of a key cellular sensing mechanism that signals completion of DNA replication, ensuring proper timing of entry into mitosis. Wee1 acts as an inhibitor of mitotic entry by phosphorylating cyclin-dependent kinase CDK1. Wee1 activity is mainly regulated at the protein level through its phosphorylation and subsequent degradation by the ubiquitin proteasome pathway. To facilitate identification of small molecules preventing Wee1 degradation, a homogeneous cell-based assay was developed using HeLa cells transiently transfected with a Wee1-Luciferase fusion protein. To insure uHTS compatibility, the assay was scaled to 1,536-well plate format and cells were transfected in bulk and cryopreserved. This miniaturized homogenous assay demonstrated robust performance, with a calculated Z′ factor of 0.65±0.05. The assay was screened against a publicly available library of ~218,000 compounds in order to identify Wee1 stabilizers. Nonselective, cytotoxic and promiscuous compounds were rapidly triaged through the use of a similarly formatted counterscreen that measured stabilization of a N-cyclin B-Luciferase fusion protein, as well as execution of viability assessment in the parental HeLa cell line. This screening campaign led to the discovery of four unrelated cell-permeable small molecules that showed selective Wee1-Luciferase stabilization with micromolar potency. One of these compounds, SID4243143, was shown to inhibit cell cycle progression, underscoring the importance of Wee1 degradation to the cell cycle. Our results suggest that this uHTS approach is suitable for identifying selective chemical probes that prevent Wee1 degradation, and generally applicable to discovering inhibitors of the ubiquitin proteasome pathway.
Wee1; degradation; stabilizer; reporter assay; transient transfection; cryopreserved cells; ubiquitin; proteasome
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