Despite huge advancements in the process of synthesizing small molecules as part of one-bead-one-compound (OBOC) libraries, progress lags in the ability to screen these libraries against proteins of interest. Recently, we developed a method to screen OBOC libraries in which a target protein is labeled with a near-infrared (NIR) range fluorophore. The labeled protein incubates with beads of a library in a 96-well plate, then the plate is imaged for fluorescence. Fluorescence intensities produced by the labeled protein binding the bead can be quantitated and provide a basis to rank hits. Here, we present an application of this technique by screening the oncoprotein gankyrin against a 343-member peptoid library. The library was composed of four positions occupied by one of seven amines. In the third position, an amine that facilitates covalent binding via a sulfonyl fluoride moiety was incorporated. After screening for gankyrin binders twice, ten structures showed overlap in the types of amines present at each position. These initial hits were validated with an in-gel fluorescence assay in which the labeled ligands covalently interacted with purified gankyrin. Excitingly, one peptoid was validated from this analysis. This hit was also shown to bind gankyrin in the presence of HEK 293T lysate. Results from this study demonstrate successful use of our screening method to quickly identify quality binders to a target protein of interest.

A series of oleic acid amide derivatives were synthesized based on our previous and continuing endeavors towards stimulation of the 20S core particle of the proteasome (20S CP) with the goal of increasing the protein degradation rate via the ubiquitin-independent pathway. The designed compounds were tested in a variety of biochemical and cell-based assays to assess their ability to increase the rate of hydrolysis of the 20S CP, and compared to a known fatty acid amide stimulator of the 20S CP, AM-404. AM-404 was previously described to stimulate the activity of the 20S CP, however, it does negatively affect viability of cells after prolonged dosing. Here we report the development of several small molecules with a similar ability to enhance the activity of the 20S CP as AM-404. While one molecule (17) was just as potent as AM-404, it still caused significant unwanted cytotoxicity. Molecules such as these are compatible with biochemical assays and short-term cell-based proteasome activity assays, but their unwanted toxicity limits their use in prolonged cell assays or in vivo studies.