Asli N. Goktug

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.

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/.

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

N-terminal acetylation is an abundant modification influencing protein functions. Since ≈80% of mammalian cytosolic proteins are N-terminally acetylated, this potentially represents 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, suggesting it 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, aka UBC12) and DCN1 (aka 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 the anchorage-independent growth of a cell line harboring DCN1 amplification. Overall, the data demonstrate that N-terminal acetyl-dependent protein interactions are druggable targets, and provide insights into targeting multiprotein E2–E3 ligases.

High-throughput screening reveals alsterpaullone, 2-cyanoethyl as a potent p27Kip1 transcriptional inhibitor.

p27Kip1 is a cell cycle inhibitor that prevents cyclin dependent kinase (CDK)/cyclin complexes from phosphorylating their targets. p27Kip1 is a known tumor suppressor, as the germline loss of p27Kip1 results in sporadic pituitary formation in aged rodents, and its presence in human cancers is indicative of a poor prognosis. In addition to its role in cancer, loss of p27Kip1 results in regenerative phenotypes in some tissues and maintenance of stem cell pluripotency, suggesting that p27Kip1 inhibitors could be beneficial for tissue regeneration. Because p27Kip1 is an intrinsically disordered protein, identifying direct inhibitors of the p27Kip1 protein is difficult. Therefore, we pursued a high-throughput screening strategy to identify novel p27Kip1 transcriptional inhibitors. We utilized a luciferase reporter plasmid driven by the p27Kip1 promoter to transiently transfect HeLa cells and used cyclohexamide as a positive control for non-specific inhibition. We screened a “bioactive” library consisting of 8,904 (4,359 unique) compounds, of which 830 are Food and Drug Administration (FDA) approved. From this screen, we successfully identified 111 primary hits with inhibitory effect against the promoter of p27Kip1. These hits were further refined using a battery of secondary screens. Here we report four novel p27Kip1 transcriptional inhibitors, and further demonstrate that our most potent hit compound (IC50 = 200 nM) Alsterpaullone 2-cyanoethyl, inhibits p27Kip1 transcription by preventing FoxO3a from binding to the p27Kip1 promoter. This screen represents one of the first attempts to identify inhibitors of p27Kip1 and may prove useful for future tissue regeneration studies.

A high-throughput screen reveals new small-molecule activators and inhibitors of pantothenate kinases.

Pantothenate kinase (PanK) is a regulatory enzyme that controls coenzyme A (CoA) biosynthesis. The association of PanK with neurodegeneration and diabetes suggests that chemical modifiers of PanK activity may be useful therapeutics. We performed a high throughput screen of >520000 compounds from the St. Jude compound library and identified new potent PanK inhibitors and activators with chemically tractable scaffolds. The HTS identified PanK inhibitors exemplified by the detailed characterization of a tricyclic compound (7) and a preliminary SAR. Biophysical studies reveal that the PanK inhibitor acts by binding to the ATP–enzyme complex.

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.

Data Analysis Approaches in High Throughput Screening

With the advances in biotechnology, identification of new therapeutic targets, and better un‐ derstanding of human diseases, pharmaceutical companies and academic institutions have accelerated their efforts in drug discovery. The pipeline to obtain therapeutics often involves target identification and validation, lead discovery and optimization, pre-clinical animal studies, and eventually clinical trials to test the safety and effectiveness of the new drugs. In most cases, screening using genome-scale RNA interference (RNAi) technology or diverse compound libraries comprises the first step of the drug discovery initiatives. Small interfer‐ ing RNA (siRNA, a class of double-stranded RNA molecules 20-25 nucleotides in length ca‐ pable of interfering with the expression of specific genes with complementary nucleotide sequence) screen is an effective tool to identify upstream or downstream regulators of a spe‐ cific target gene, which may also potentially serve as drug targets for a more efficient and successful treatment. On the other hand, screening of diverse small molecule libraries against a known target or disease-relevant pathway facilitates the discovery of chemical tools as candidates for further development.

Practical Considerations of Liquid Handling Devices in Drug Discovery

Automated liquid handling has become an indispensable tool in drug discovery, particular‐ ly in screening campaigns ranging millions of compounds. Intense innovations of these de‐ vices go hand in hand with the progression towards assay miniaturization, accelerating dramatically the discovery of drug candidates and chemical probes for querying biological systems. The advancement in this technology is driven in large part by much impetus in cost reduction and efficiency. In addition to increased throughput, streamlining screening opera‐ tions using automated fluid devices ensures consistency and reliability while avoiding hu‐ man error.

Alteration of RNA Splicing by Small-Molecule Inhibitors of the Interaction between NHP2L1 and U4:

Splicing is an important eukaryotic mechanism for expanding the transcriptome and proteome, influencing a number of biological processes. Understanding its regulation and identifying small molecules that modulate this process remain a challenge. We developed an assay based on time-resolved fluorescence resonance energy transfer (TR-FRET) to detect the interaction between the protein NHP2L1 and U4 RNA, which are two key components of the spliceosome. We used this assay to identify small molecules that interfere with this interaction in a high-throughput screening (HTS) campaign. Topotecan and other camptothecin derivatives were among the top hits. We confirmed that topotecan disrupts the interaction between NHP2L1 and U4 by binding to U4 and inhibits RNA splicing. Our data reveal new functions of known drugs that could facilitate the development of therapeutic strategies to modify splicing and alter gene function.

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.