Blocking an N-terminal acetylation-dependent protein interaction inhibits an E3 ligase.

N-terminal acetylation is an abundant modification influencing protein functions. Because ∼80% of mammalian cytosolic proteins are N-terminally acetylated, this modification is potentially an untapped target for chemical control of their functions. Structural studies have revealed that, like lysine acetylation, N-terminal acetylation converts a positively charged amine into a hydrophobic handle that mediates protein interactions; hence, this modification may be a druggable target. We report the development of chemical probes targeting the N-terminal acetylation-dependent interaction between an E2 conjugating enzyme (UBE2M or UBC12) and DCN1 (DCUN1D1), a subunit of a multiprotein E3 ligase for the ubiquitin-like protein NEDD8. The inhibitors are highly selective with respect to other protein acetyl-amide-binding sites, inhibit NEDD8 ligation in vitro and in cells, and suppress anchorage-independent growth of a cell line with DCN1 amplification. Overall, our data demonstrate that N-terminal acetyl-dependent protein interactions are druggable targets and provide insights into targeting multiprotein E2-E3 ligases.

Stability of the human pregnane X receptor is regulated by E3 ligase UBR5 and serine/threonine kinase DYRK2.

The hPXR (human pregnane X receptor), a major chemical toxin sensor, is a ligand-induced transcription factor activated by various xenobiotics and toxins, resulting in the transcriptional up-regulation of detoxifying enzymes. To date, little is known about the upstream regulation of hPXR. Using MS analysis and a kinome-wide siRNA screen, we report that the E3 ligase UBR5 (ubiquitin protein ligase E3 component n-recognin 5) and DYRK2 (dual-specificity tyrosine-phosphorylation-regulated kinase 2) regulate hPXR stability. UBR5 knockdown resulted in accumulation of cellular hPXR and a concomitant increase in hPXR activity, whereas the rescue of UBR5 knockdown decreased the cellular hPXR level and activity. Importantly, UBR5 exerted its effect in concert with the serine/threonine kinase DYRK2, as the knockdown of DYRK2 phenocopied UBR5 knockdown. hPXR was shown to be a substrate for DYRK2, and DYRK2-dependent phosphorylation of hPXR facilitated its subsequent ubiquitination by UBR5. This is the first report of the post-translational regulation of hPXR via phosphorylation-facilitated ubiquitination by DYRK2 and UBR5. The results of the present study reveal the role of the ubiquitin-proteasomal pathway in modulating hPXR activity and indicate that pharmacological inhibitors of the ubiquitin-proteasomal pathway that regulate hPXR stability may negatively affect treatment outcome from unintended hPXR-mediated drug-drug interactions.

Development of Second-Generation CDK2 Inhibitors for the Prevention of Cisplatin-Induced Hearing Loss.

There are currently no FDA-approved therapies to prevent the hearing loss associated with the usage of cisplatin in chemotherapeutic regimens. We recently demonstrated that the pharmacologic inhibition with kenpaullone or genetic deletion of CDK2 preserved hearing function in animal models treated with cisplatin, which suggests that CDK2 is a promising therapeutic target to prevent cisplatin-induced ototoxicity. In this study, we identified two lead compounds, AT7519 and AZD5438, from a focused library screen of 187 CDK2 inhibitors, performed in an immortalized cell line derived from neonatal mouse cochleae treated with cisplatin. Moreover, we screened 36 analogues of AT7519 and identified analogue 7, which exhibited an improved therapeutic index. When delivered locally, analogue 7 and AZD5438 both provided significant protection against cisplatin-induced ototoxicity in mice. Thus, we have identified two additional compounds that prevent cisplatin-induced ototoxicity in vivo and provided further evidence that CDK2 is a druggable target for treating cisplatin-induced ototoxicity.

CDK2 inhibitors as candidate therapeutics for cisplatin- and noise-induced hearing loss.

Hearing loss caused by aging, noise, cisplatin toxicity, or other insults affects 360 million people worldwide, but there are no Food and Drug Administration-approved drugs to prevent or treat it. We screened 4,385 small molecules in a cochlear cell line and identified 10 compounds that protected against cisplatin toxicity in mouse cochlear explants. Among them, kenpaullone, an inhibitor of multiple kinases, including cyclin-dependent kinase 2 (CDK2), protected zebrafish lateral-line neuromasts from cisplatin toxicity and, when delivered locally, protected adult mice and rats against cisplatin- and noise-induced hearing loss. CDK2-deficient mice displayed enhanced resistance to cisplatin toxicity in cochlear explants and to cisplatin- and noise-induced hearing loss in vivo. Mechanistically, we showed that kenpaullone directly inhibits CDK2 kinase activity and reduces cisplatin-induced mitochondrial production of reactive oxygen species, thereby enhancing cell survival. Our experiments have revealed the proapoptotic function of CDK2 in postmitotic cochlear cells and have identified promising therapeutics for preventing hearing loss.

High-Throughput Screening Identifies 1,4,5-Substituted 1,2,3-Triazole Analogs as Potent and Specific Antagonists of Pregnane X Receptor.

Human pregnane X receptor (hPXR) is a nuclear receptor that regulates the expression of phase I and phase II drug-metabolism enzymes, as well as that of drug transporters. hPXR is a "xenobiotics sensor" and can be activated by structurally diverse compounds. The activation of hPXR by its agonists increases the clearance of xenobiotics by increasing the expression of drug-metabolism enzymes and drug transporters, possibly leading to drug toxicity, drug resistance, and other adverse drug reactions. Therefore, hPXR antagonists might attenuate agonist-mediated activation of hPXR and reduce the risk of adverse drug reactions. Several hPXR antagonists have been reported, but none of them is specific for hPXR. In this study, we present the first large-scale, unbiased, cell-based high-throughput screen to identify specific hPXR antagonists. Among the 132,975 compounds screened, we identified the 1,4,5-substituted 1,2,3-triazole analogs as potent and specific hPXR antagonists by sequentially performing primary screening, retesting, and dose-response analysis using cell-based hPXR gene reporter and receptor binding assays, as well as receptor and promoter specificity assays. The compound SJ000076745-1 is the most potent and specific hPXR antagonist in the 1,4,5-substituted 1,2,3-triazole chemical class, having a cell-based hPXR antagonist 50% inhibitory concentration (IC50) value of 377 ± 16 nM and an hPXR binding inhibitory IC50 value of 563 ± 40 nM.

Development of Cell-Based High-Throughput Chemical Screens for Protection Against Cisplatin-Induced Ototoxicity.

Various compounds have been tested in recent years for protection against cisplatin-induced hearing loss, but no compound has yet been FDA approved for clinical use in patients. Towards this goal, we developed an unbiased, high-throughput, mammalian cochlear cell-based chemical screen that allowed quantification of the protection ability of bioactive compounds and ranked them for future testing ex vivo in cochlear explant cultures and in vivo in animal models. In our primary screens, protection in the HEI-OC1 organ of Corti immortalized cell line was measured by the ability of each compound to inhibit caspase-3/7 activity triggered by cisplatin treatment (50 µM cisplatin for 22 h). A total of 4385 unique bioactive compounds were tested in a single dose of 8 µM and promising compounds were validated by dose response curves covering ten, 1:3 serial diluted concentrations. Primary hits were defined as having more than 60 % inhibition of the caspase-3/7 activity. Toxicity of the top compounds was measured by a CellTiter-Glo (CTG) assay that measured the viability of the cells in the presence of compound alone in similar dose responsive analysis. A combination of the caspase-3/7 inhibition activity assay (as measured by IC50) and the CTG viability assay (as determined by LD50) identified the top protective compounds in the HEI-OC1 cells. In the future, the top hits in our screens will be tested for their protective ability ex vivo in mouse cochlear explants and in vivo in animal models. Our mammalian cochlear cell-based, high-throughput chemical screening assays described here can be further modified and represent an initial successful step towards therapeutic intervention of hearing disorders, an unmet medical need of our society.

A timetable organizer for the planning and implementation of screenings in manual or semi-automation mode.

We have designed an Excel spreadsheet to facilitate the planning and execution of screenings performed manually or in semi-automation mode, following a sequential set of events. Many assays involve multiple steps, often including time-sensitive stages, thus complicating the proper implementation to ensure that all plates are treated equally to achieve reliable outcomes. The spreadsheet macro presented in this study analyzes and breaks down the timings for all tasks, calculates the limitation in the number of plates that suit the desired parameters, and allows for optimization based on tolerance of time delay and equal treatment of plates when possible. The generated Gantt charts allow for visual inspection of the screening process and provide timings in a tabulated form to assist the user to conduct the experiments as projected by the software. The program can be downloaded from http://sourceforge.net/projects/sams-hts/.

GUItars: a GUI tool for analysis of high-throughput RNA interference screening data.

BACKGROUND: High-throughput RNA interference (RNAi) screening has become a widely used approach to elucidating gene functions. However, analysis and annotation of large data sets generated from these screens has been a challenge for researchers without a programming background. Over the years, numerous data analysis methods were produced for plate quality control and hit selection and implemented by a few open-access software packages. Recently, strictly standardized mean difference (SSMD) has become a widely used method for RNAi screening analysis mainly due to its better control of false negative and false positive rates and its ability to quantify RNAi effects with a statistical basis. We have developed GUItars to enable researchers without a programming background to use SSMD as both a plate quality and a hit selection metric to analyze large data sets. RESULTS: The software is accompanied by an intuitive graphical user interface for easy and rapid analysis workflow. SSMD analysis methods have been provided to the users along with traditionally-used z-score, normalized percent activity, and t-test methods for hit selection. GUItars is capable of analyzing large-scale data sets from screens with or without replicates. The software is designed to automatically generate and save numerous graphical outputs known to be among the most informative high-throughput data visualization tools capturing plate-wise and screen-wise performances. Graphical outputs are also written in HTML format for easy access, and a comprehensive summary of screening results is written into tab-delimited output files. CONCLUSION: With GUItars, we demonstrated robust SSMD-based analysis workflow on a 3840-gene small interfering RNA (siRNA) library and identified 200 siRNAs that increased and 150 siRNAs that decreased the assay activities with moderate to stronger effects. GUItars enables rapid analysis and illustration of data from large- or small-scale RNAi screens using SSMD and other traditional analysis methods. The software is freely available at http://sourceforge.net/projects/guitars/.