A Genome-Wide RNAi Screen Reveals Common Host-Virus Gene Signatures: Implication for Dengue Antiviral Drug Discovery.

Dengue is the most common mosquito-borne viral disease that in recent years has become a major international public health concern. Dengue is a tropical neglected disease with increasing global incidences, affecting millions of people worldwide, and without the availability of specific treatments to combat it. The identification of host-target genes essential for the virus life cycle, for which effective modulators may already exist, would provide an alternative path to a rapid drug development of the much needed antidengue agents. For this purpose, we performed the first genome-wide RNAi screen, combining two high-content readouts for dengue virus infection (DENV E infection intensity) and host cell toxicity (host cell stained nuclei), against an arrayed lentiviral-based short hairpin RNA library covering 16,000 genes with a redundancy of at least 5 hairpins per gene. The screen identified 1924 gene candidates in total; of which, 1730 gene candidates abrogated dengue infection, whereas 194 gene candidates were found to enhance its infectivity in HEK293 cells. A first pass clustering analysis of hits revealed a well-orchestrated gene-network dependency on host cell homeostasis and physiology triggering distinct cellular pathways for infectivity, replication, trafficking, and egress; a second analysis revealed a comprehensive gene signature of 331 genes common to hits identified in 28 published RNAi host-viral interaction screens. Taken together, our findings provide novel antiviral molecular targets with the potential for drug discovery and development.

Translational Strategies for Repotrectinib in Neuroblastoma.

Limited clinical data are available regarding the utility of multikinase inhibition in neuroblastoma. Repotrectinib (TPX-0005) is a multikinase inhibitor that targets ALK, TRK, JAK2/STAT, and Src/FAK, which have all been implicated in the pathogenesis of neuroblastoma. We evaluated the preclinical activity of repotrectinib monotherapy and in combination with chemotherapy as a potential therapeutic approach for relapsed/refractory neuroblastoma. In vitro sensitivity to repotrectinib, ensartinib, and cytotoxic chemotherapy was evaluated in neuroblastoma cell lines. In vivo antitumor effect of repotrectinib monotherapy, and in combination with chemotherapy, was evaluated using a genotypically diverse cohort of patient-derived xenograft (PDX) models of neuroblastoma. Repotrectinib had comparable cytotoxic activity across cell lines irrespective of ALK mutational status. Combination with chemotherapy demonstrated increased antiproliferative activity across several cell lines. Repotrectinib monotherapy had notable antitumor activity and prolonged event-free survival compared with vehicle and ensartinib in PDX models (P < 0.05). Repotrectinib plus chemotherapy was superior to chemotherapy alone in ALK-mutant and ALK wild-type PDX models. These results demonstrate that repotrectinib has antitumor activity in genotypically diverse neuroblastoma models, and that combination of a multikinase inhibitor with chemotherapy may be a promising treatment paradigm for translation to the clinic.

Evaluation of Compound Optical Interference in High-Content Screening.

Compound optical interference remains an inherent problem in chemical screening and has been well documented for biochemical assays and less so for automated microscopy-based assays. It has also been the assumption that the latter should not suffer from such interference because of the washing steps involved in the process, thus eliminating the residual nonspecific compound effects. Instead, these compounds may have no relevance to the actual target, and as such, compound optical interference contributes to a number of false-positives, resulting in a high attrition rate during subsequent follow-up studies. In this report, we analyze the outcome of a high-content screen using enhanced green fluorescent protein as a reporter in a gain-of-function cell-based assay in search of modulators of the micro RNA (miRNA) biogenesis pathway. Using a previously validated image-based biosensor, we screened a diverse library collection of ~315,000 compounds covering natural and synthetic derivatives in which 1130 positives were identified to enhance green fluorescence expression. Lateral confirmation and dose-response studies revealed that all of these compounds were the result of optical interference and not specific inhibition of miRNA biogenesis. Here, we highlight the chemical classes that are susceptible to compound optical interference and discuss their implications in automated microscopy-based assays.

Chemical & RNAi screening at MSKCC: a collaborative platform to discover & repurpose drugs to fight disease.

Memorial Sloan Kettering Cancer Center (MSKCC) has implemented the creation of a full service state-of-the-art High-throughput Screening Core Facility (HTSCF) equipped with modern robotics and custom-built screening data management resources to rapidly store and query chemical and RNAi screening data outputs. The mission of the facility is to provide oncology clinicians and researchers alike with access to cost-effective HTS solutions for both chemical and RNAi screening, with an ultimate goal of novel target identification and drug discovery. HTSCF was established in 2003 to support the institution's commitment to growth in molecular pharmacology and in the realm of therapeutic agents to fight chronic diseases such as cancer. This endeavor required broad range of expertise in technology development to establish robust and innovative assays, large collections of diverse chemical and RNAi duplexes to probe specific cellular events, sophisticated compound and data handling capabilities, and a profound knowledge in assay development, hit validation, and characterization. Our goal has been to strive for constant innovation, and we strongly believe in shifting the paradigm from traditional drug discovery towards translational research now, making allowance for unmet clinical needs in patients. Our efforts towards repurposing FDA-approved drugs fructified when digoxin, identified through primary HTS, was administered in the clinic for treatment of stage Vb retinoblastoma. In summary, the overall aim of our facility is to identify novel chemical probes, to study cellular processes relevant to investigator's research interest in chemical biology and functional genomics, and to be instrumental in accelerating the process of drug discovery in academia.

A high content assay to assess cellular fitness.

A universal process in experimental biology is the use of engineered cells; more often, stably or transiently transfected cells are generated for the purpose. Therefore, it is important that cell health assessment is conducted to check for stress mediated by induction of heat shock proteins (Hsps). For this purpose, we have developed an integrated platform that would enable a direct assessment of transfection efficiency (TE) combined with cellular toxicity and stress response. We make use of automated microscopy and high content analysis to extract from the same well a multiplexed readout to assess and determine optimal chemical transfection conditions. As a proof of concept, we investigated seven commercial reagents, in a matrix of dose and time, to study transfection of an EGFP DNA plasmid into HeLa cells and their consequences on health and fitness; where we scored for cellular proliferation, EGFP positive cells, and induction of Hsp10 and Hsp70 as makers of stress responses. FuGENE HD emerged as the most optimal reagent with no apparent side effects suitable for performing microtiter based miniaturized transfection for both chemical and RNAi screening. In summary, we report on a high content assay method to assess cellular overall fitness upon chemical transfection.

An arrayed RNA interference genome-wide screen identifies candidate genes involved in the MicroRNA 21 biogenesis pathway.

MicroRNAs (miRNAs) are evolutionary conserved noncoding molecules that regulate gene expression. They influence a number of diverse biological functions, such as development and differentiation. However, their dysregulation has been shown to be associated with disease states, such as cancer. Genes and pathways regulating their biogenesis remain unknown and are highly sought after. For this purpose, we have validated a multiplexed high-content assay strategy to screen for such modulators. Here, we describe its implementation that makes use of a cell-based gain-of-function reporter assay monitoring enhanced green fluorescent protein expression under the control of miRNA 21 (miR-21); combined with measures of both cell metabolic activities through the use of Alamar Blue and cell death through imaged Hoechst-stained nuclei. The strategy was validated using a panel of known genes and enabled us to successfully progress to and complete an arrayed genome-wide short interfering RNA (siRNA) screen against the Ambion Silencer Select v4.0 library containing 64,755 siRNA duplexes covering 21,565 genes. We applied a high-stringency hit analysis method, referred to as the Bhinder-Djaballah analysis method, leading to the nomination of 1,273 genes as candidate inhibitors of the miR-21 biogenesis pathway; after several iterations eliminating those genes with only one active duplex and those enriched in seed sequence mediated off-target effects. Biological classifications revealed four major control junctions among them vesicular transport via clathrin-mediated endocytosis. Altogether, our screen has uncovered a number of novel candidate regulators that are potentially good druggable targets allowing for the discovery and development of small molecules for regulating miRNA function.

A high-content biosensor-based screen identifies cell-permeable activators and inhibitors of EGFR function: implications in drug discovery.

Early success of kinase inhibitors has validated their use as drugs. However, discovery efforts have also suffered from high attrition rates due to lack of cellular activity. We reasoned that screening for such candidates in live cells would identify novel cell-permeable modulators for development. For this purpose, we have used our recently optimized epidermal growth factor receptor (EGFR) biosensor assay to screen for modulators of EGFR activity. Here, we report on its validation under high-throughput screening (HTS) conditions displaying a signal-to-noise ratio of 21 and a Z' value of 0.56-attributes of a robust cell-based assay. We performed a pilot screen against a library of 6912 compounds demonstrating good reproducibility and identifying 82 inhibitors and 66 activators with initial hit rates of 1.2% and 0.95%, respectively. Follow-up dose-response studies revealed that 12 of the 13 known EGFR inhibitors in the library were confirmed as hits. ZM-306416, a vascular endothelial growth factor receptor (VEGFR) antagonist, was identified as a potent inhibitor of EGFR function. Flurandrenolide, beclomethasone, and ebastine were confirmed as activators of EGFR function. Taken together, our results validate this novel approach and demonstrate its utility in the discovery of novel kinase modulators with potential use in the clinic.

An image-based biosensor assay strategy to screen for modulators of the microRNA 21 biogenesis pathway.

microRNAs (miRNAs) are evolutionary conserved, small endogenous non-coding, RNA molecules. Although their mode of action has been extensively studied, little is known about their biogenesis. As their altered expression has been implicated in many diseases, small molecules that would modulate their expression are sought after. They are generated through the concerted action of several complexes which promote their transcription, maturation, export, trafficking, and loading of mature miRNA into silencing complexes. An increasing number of studies have suggested that each of these steps serves as a regulatory junction in the process, and therefore provides an intervention point. For this purpose, we have developed a simple image-based assay strategy to screen for such modulators. Here, we describe its successful implementation which combines the use of a microRNA 21 (miR-21) synthetic mimic together with an EGFP based reporter cell line, where its expression is under the control of miR-21, to monitor EGFP expression in a format suitable for HTS. The strategy was further validated using a small panel of known gene modulators of the miRNA pathway. A screen was performed in duplicate against a library of 6,912 compounds and identified 48 initial positives exhibiting enhanced EGFP fluorescence intensity. 42 compounds were found to be inherently fluorescent in the green channel leaving the remaining 6 as potential inhibitors and with a positive rate of 0.09%. Taken together, this validated strategy offers the opportunity to discover novel and specific inhibitors of the pathway through the screening of diverse chemical libraries.

Domain-based biosensor assay to screen for epidermal growth factor receptor modulators in live cells.

Traditional drug discovery efforts have resulted in the approval of a handful of receptor tyrosine kinase (RTK) inhibitors; however, their discovery relied solely on screening recombinant kinases, often with poor cellular activity outcome. The ability to screen RTKs in their natural environment is sought as an alternative approach. We have adapted a novel strategy utilizing a green fluorescent protein-labeled SRC homology 2 domain-based biosensor as a surrogate reporter of endogenous epidermal growth factor receptor (EGFR) activity in A549 cells. Upon activation of the receptor, EGFR function in live cells is measured by the number of green granules that form. Here we describe assay miniaturization and demonstrate specificity for EGFR through its chemical inhibition and RNAi-dependent knockdown resulting in complete abrogation of granule formation. Gefitinib and PD 153035 were identified as hits in a pilot screen. This approach allows for the identification of novel EGFR modulators in high-throughput formats for screening chemical and RNAi libraries.

Comparison of luminescence ADP production assay and radiometric scintillation proximity assay for Cdc7 kinase.

Several assay technologies have been successfully adapted and used in HTS to screen for protein kinase inhibitors; however, emerging comparative analysis studies report very low hit overlap between the different technologies, which challenges the working assumption that hit identification is not dependent on the assay method of choice. To help address this issue, we performed two screens on the cancer target, Cdc7-Dbf4 heterodimeric protein kinase, using a direct assay detection method measuring [(33)P]-phosphate incorporation into the substrate and an indirect method measuring residual ADP production using luminescence. We conducted the two screens under similar conditions, where in one, we measured [(33)P]-phosphate incorporation using scintillation proximity assay (SPA), and in the other, we detected luminescence signal of the ATP-dependent luciferase after regenerating ATP from residual ADP (LUM). Surprisingly, little or no correlation were observed between the positives identified by the two methods; at a threshold of 30% inhibition, 25 positives were identified in the LUM screen whereas the SPA screen only identified two positives, Tannic acid and Gentian violet, with Tannic acid being common to both. We tested 20 out of the 25 positive compounds in secondary confirmatory study and confirmed 12 compounds including Tannic acid as Cdc7-Dbf4 kinase inhibitors. Gentian violet, which was only positive in the SPA screen, inhibited luminescence detection and categorized as a false positive. This report demonstrates the strong impact in detection format on the success of a screening campaign and the importance of carefully designed confirmatory assays to eliminate those compounds that target the detection part of the assay.

Structure-activity relationships of 6-(2,6-dichlorophenyl)-8-methyl-2-(phenylamino)pyrido[2,3-d]pyrimidin-7-ones: toward selective Abl inhibitors.

We report the design, synthesis, and structure-activity relationship (SAR) of a series of novel pyrido[2,3-d]pyrimidin-7-one compounds as potent Abl kinase inhibitors. We evaluate their specificity profile against a panel of human recombinant kinases, as well as their biological profile toward a panel of well-characterized cancer cell lines. Our study reveals that substitutions in the 3- and 4-positions of the phenylamino moiety lead to improved potency and improved selectivity both in target-based and cell-based assays. Altogether, our results provide an insight into the SAR of pyrido[2,3-d]pyrimidin-7-ones for the development of drug candidates with improved potency and selectivity for the targeted treatment of CML.