A Reversible and Highly Selective Inhibitor of the Proteasomal Ubiquitin Receptor Rpn13 Is Toxic to Multiple Myeloma Cells

The proteasome is a multisubunit complex responsible for most nonlysosomal turnover of proteins in eukaryotic cells. Proteasome inhibitors are of great interest clinically, particularly for the treatment of multiple myeloma (MM). Unfortunately, resistance arises almost inevitably to these active site-targeted drugs. One strategy to overcome this resistance is to inhibit other steps in the protein turnover cascade mediated by the proteasome. Previously, Anchoori et al. identified Rpn13 as the target of an electrophilic compound (RA-190) that was selectively toxic to MM cells (<i>Cancer Cell</i> <b>2013</b>, <i>24</i>, 791–805), suggesting that this subunit of the proteasome is also a viable cancer drug target. Here we describe the discovery of the first highly selective, reversible Rpn13 ligands and show that they are also selectively toxic to MM cells. These data strongly support the hypothesis that Rpn13 is a viable target for the development of drugs to treat MM and other cancers

Discovery of Native Autoantigens via Antigen Surrogate Technology: Application to Type 1 Diabetes

A fundamental goal in understanding the mechanisms of autoimmune disease is the characterization of autoantigens that are targeted by autoreactive antibodies and T cells. Unfortunately, the identification of autoantigens is a difficult problem. We have begun to explore a novel route to the discovery of autoantibody/autoantigen pairs that involves comparative screening of combinatorial libraries of unnatural, synthetic molecules for compounds that bind antibodies present at much higher levels in the serum of individuals with a given autoimmune disease than in the serum of control individuals. We have shown that this approach can yield “antigen surrogates” capable of capturing disease-specific autoantibodies from serum. In this report, we demonstrate that the synthetic antigen surrogates can be used to affinity purify the autoantibodies from serum and that these antibodies can then be used to identify their cognate autoantigen in an appropriate tissue lysate. Specifically, we report the discovery of a peptoid able to bind autoantibodies present in about one-third of nonobese diabetic (NOD) mice. The peptoid-binding autoantibodies were highly enriched through peptoid affinity chromatography and employed to probe mouse pancreatic and brain lysates. This resulted in identification of murine GAD65 as the native autoantigen. GAD65 is a known humoral autoantigen in human type 1 diabetes mellitus (T1DM), but its existence in mice had been controversial. This study demonstrates the potential of this chemical approach for the unbiased identification of autoantigen/autoantibody complexes

Screening of cell cycle fusion proteins to identify kinase signaling networks

<p>Kinase signaling networks are well-established mediators of cell cycle transitions. However, how kinases interact with the ubiquitin proteasome system (UPS) to elicit protein turnover is not fully understood. We sought a means of identifying kinase-substrate interactions to better understand signaling pathways controlling protein degradation. Our prior studies used a luciferase fusion protein to uncover kinase networks controlling protein turnover. In this study, we utilized a similar approach to identify pathways controlling the cell cycle protein p27^Kip1. We generated a p27^Kip1-luciferase fusion and expressed it in cells incubated with compounds from a library of pharmacologically active compounds. We then compared the relative effects of the compounds on p27^Kip1-luciferase fusion stabilization. This was combined with <i>in silico</i> kinome profiling to identify potential kinases inhibited by each compound. This approach effectively uncovered known kinases regulating p27^Kip1 turnover. Collectively, our studies suggest that this parallel screening approach is robust and can be applied to fully understand kinase-ubiquitin pathway interactions.</p

Utility of Redundant Combinatorial Libraries in Distinguishing High and Low Quality Screening Hits

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

A cell based screening approach for identifying protein degradation regulators

<p>Cellular transitions are achieved by the concerted actions of regulated degradation pathways. In the case of the cell cycle, ubiquitin mediated degradation ensures unidirectional transition from one phase to another. For instance, turnover of the cell cycle regulator cyclin B1 occurs after metaphase to induce mitotic exit. To better understand pathways controlling cyclin B1 turnover, the N-terminal domain of cyclin B1 was fused to luciferase to generate an N-cyclin B1-luciferase protein that can be used as a reporter for protein turnover. Prior studies demonstrated that cell-based screens using this reporter identified small molecules inhibiting the ubiquitin ligase controlling cyclin B1-turnover. Our group adapted this approach for the G2-M regulator Wee1 where a Wee1-luciferase construct was used to identify selective small molecules inhibiting an upstream kinase that controls Wee1 turnover. In the present study we present a screening approach where cell cycle regulators are fused to luciferase and overexpressed with cDNAs to identify specific regulators of protein turnover. We overexpressed approximately 14,000 cDNAs with the N-cyclin B1-luciferase fusion protein and determined its steady-state level relative to other luciferase fusion proteins. We identified the known APC/C regulator Cdh1 and the F-box protein Fbxl15 as specific modulators of N-cyclin B1-luciferase steady-state levels and turnover. Collectively, our studies suggest that analyzing the steady-state levels of luciferase fusion proteins in parallel facilitates identification of specific regulators of protein turnover.</p