[Show abstract][Hide abstract] ABSTRACT: NADPH oxidase 1 (NOX1) is highly expressed in colon epithelial cells, where it generates reactive oxygen species (ROS) to interact with normal and pathogenic bacteria. Excessive reactive ROS production is associated with damage to the intestinal mucosa, particularly in mucosal lesions of inflammatory bowel disease (IBD). Studies have shown that NOX1 levels are increased in human prostate cancer, and might also play a role in angiogenesis, cell growth, and tumor pathogenesis. The identification of potent, selective inhibitors of NOX1 may lead to potential therapeutic candidates for excess cell proliferation, cancer, and IBD. This project demonstrated that the molecular probe ML090 (CID-616479) is neither a hydrogen peroxide scavenger, nor a general cell toxin on the time scale of cellular NOX inhibition assays. The specificity of the probe for NOX1 over NOX2, 3 and 4 in a 293 assay system suggests that a target specific to the NOX1 system is the molecular target. ML090 should serve as a useful probe for cellular systems where inhibition of NOX1, and not other members of the NOX family, is desired. This compound provides a significant improvement over the previously existing non-selective NOX inhibitor, diphenylene iodium.
[Show abstract][Hide abstract] ABSTRACT: The retinoblastoma (RB) tumor suppressor protein controls cell cycle progression by regulating the activity of the transcription factor E2F, which in turn activates genes essential for DNA replication. Thus, factors that bind and regulate RB activity provide for valuable targets for preventing tumorigenesis. The enzyme, RB binding protein 9 (RBBP9), is widely expressed in a number of different tissues and is upregulated in certain tumors. As a result, the identification of compounds that selectively inhibit RBBP9 activity would serve as potentially valuable probes for the study of apoptosis, cell cycle, and tumorigenesis. The probe ML081 (CID- 6603320; emetine hydrochloride) represents the first non-covalent, selective RBBP9 inhibitor, and will be useful for exploring the enzymatic functions of RBBP9 in biological systems. Moreover, the tight structure-activity relationship of the emetine-RBBP9 interaction suggests that only minor modifications to the emetine structure will improve its activity. As a result, future studies will involve semi-synthetic addition of small moieties to the emetine and cephaeline scaffolds.
[Show abstract][Hide abstract] ABSTRACT: The highly conserved p97 ATPase functions in endoplasmic reticulum-associated degradation (ERAD) by hydrolyzing ATP needed to export ubiquitinated substrates to the cytosol for degradation by the proteasome. Inhibition of p97 leads to unfolded protein accumulation, and ultimately, cell death. The discovery of p97 missense mutations in a genetic form of human dementia, in addition to the localization of p97 in ubiquitylated inclusions in affected neurons of amyotrophic lateral sclerosis and Parkinson's disease, and the overproduction of p97 in multiple cancers suggests that p97 has diverse and essential cellular roles. Thus, the identification of probes that selectively target p97 activity would be useful for providing insights into the biological roles of P97. The probe ML080 (CID-25110544) is the first cell active, reversible inhibitor of P97. This compound in the ATP-depletion-based assay, is cell penetrable as demonstrated by activity in the cell-based ubiquitin-GFP turnover assay.
[Show abstract][Hide abstract] ABSTRACT: Recent findings have identified protein phosphatase methylesterase-1 (PME-1) as a protector of sustained ERK pathway activity in malignant gliomas. PME-1 is a protein methylesterase that functions in the regulation of protein phosphatase 2A (PP2A) by reversible methylation. Biochemical elucidation of PME-1 would thus greatly benefit from the development of potent and selective chemical inhibitors. The probe compound ML136 (CID-44607965), containing a sulfonyl acrylonitrile core, represents the first potent, selective inhibitor of PME-1. Moreover, the probe does not appear to exhibit cytotoxicity. Thus, ML136 should serve as a useful tool for in vitro and in situ research assays in which it is desirable to specifically block PME-1 activity.
[Show abstract][Hide abstract] ABSTRACT: The retinoblastoma (RB) tumor suppressor protein controls cell cycle progression by regulating the activity of the transcription factor E2F, which activates genes essential for DNA replication. Thus, factors that bind and regulate RB activity are considered valuable targets for preventing tumorigenesis. The enzyme RB binding protein 9 (RBBP9) is widely expressed in different tissues and upregulated in certain tumors. As a result, the identification of compounds that selectively inhibit RBBP9 activity would serve as potentially valuable probes for the study of apoptosis, cell cycle, and tumorigenesis. We previously reported a modestly potent, RBBP9 reversible inhibitor, ML081 (CID-6603320). However, ML081 exhibits high cytotoxicity. We, therefore, have now identified a newer probe, ML114 (CID-5934766), which is 10-fold more potent than ML081, exhibits no cytotoxicity, and is from an entirely different structural and mechanistic class of compounds that covalently inhibit RBBP9. This new probe will be useful for in vitro assays in which it is desirable to specifically block RBBP9 activity for primary research purposes.
[Show abstract][Hide abstract] ABSTRACT: 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 ensure ultra-high-throughput screening (uHTS) compatibility, the assay was scaled to a 1536-well plate format and cells were transfected in bulk and cryopreserved. This miniaturized homogeneous assay demonstrated robust performance, with a calculated Z′ factor of 0.65 +/− 0.05. The assay was screened against a publicly available library of approximately 218,000 compounds to identify Wee1 stabilizers. Nonselective, cytotoxic, and promiscuous compounds were rapidly triaged through the use of a similarly formatted counterscreen that measured stabilization of an 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 4 unrelated cell-permeable small molecules that showed selective Wee1-luciferase stabilization with micromolar potency. One of these compounds, SID-4243143(ML118), was shown to inhibit cell cycle progression, underscoring the importance of Wee1 degradation to the cell cycle. This probe was found to be inactive in a whole-cell assay against its anti-target, cyclin B. In contrast, the current state-of-the-art probe, MG132, inhibits degradation of both Wee1 and cyclin B in the same assays. More importantly, flow-cytometry assays confirm that the probe is able to induce an increase in the G2/M population after cell treatment, without increasing the sub-G1 population, suggesting the probe is not toxic to cells. These 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.