10 research outputs found
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A Bioinformatics Method Identifies Prominent Off-targeted Transcripts in RNAi Screens
Because off-target effects hamper interpretation and validation of RNAi screens, we developed a bioinformatics method, Genome-wide Enrichment of Seed Sequence matches (GESS), to identify candidate off-targeted transcripts from direct analysis of primary screening data. GESS identified a prominent off-targeted transcript in several screens, including MAD2 in a screen for components of the spindle assembly checkpoint. We demonstrate how incorporation of the results of GESS analysis can enhance the validation rate in RNAi screens
Pharmacologic Inhibition of the Anaphase-Promoting Complex Induces A Spindle Checkpoint-Dependent Mitotic Arrest in the Absence of Spindle Damage
SummaryMicrotubule inhibitors are important cancer drugs that induce mitotic arrest by activating the spindle assembly checkpoint (SAC), which, in turn, inhibits the ubiquitin ligase activity of the anaphase-promoting complex (APC). Here, we report a small molecule, tosyl-L-arginine methyl ester (TAME), which binds to the APC and prevents its activation by Cdc20 and Cdh1. A prodrug of TAME arrests cells in metaphase without perturbing the spindle, but nonetheless the arrest is dependent on the SAC. Metaphase arrest induced by a proteasome inhibitor is also SAC dependent, suggesting that APC-dependent proteolysis is required to inactivate the SAC. We propose that mutual antagonism between the APC and the SAC yields a positive feedback loop that amplifies the ability of TAME to induce mitotic arrest
A Time-Series Method for Automated Measurement of Changes in Mitotic and Interphase Duration from Time-Lapse Movies
Automated time-lapse microscopy can visualize proliferation of large numbers of individual cells, enabling accurate measurement of the frequency of cell division and the duration of interphase and mitosis. However, extraction of quantitative information by manual inspection of time-lapse movies is too time-consuming to be useful for analysis of large experiments.Here we present an automated time-series approach that can measure changes in the duration of mitosis and interphase in individual cells expressing fluorescent histone 2B. The approach requires analysis of only 2 features, nuclear area and average intensity. Compared to supervised learning approaches, this method reduces processing time and does not require generation of training data sets. We demonstrate that this method is as sensitive as manual analysis in identifying small changes in interphase or mitotic duration induced by drug or siRNA treatment.This approach should facilitate automated analysis of high-throughput time-lapse data sets to identify small molecules or gene products that influence timing of cell division
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A genome-wide homologous recombination screen identifies the RNA-binding protein RBMX as a component of the DNA-damage response
Repair of DNA double-strand breaks is critical to genomic stability and the prevention of developmental disorders and cancer. A central pathway for this repair is homologous recombination (HR). Most knowledge of HR is derived from work in prokaryotic and eukaryotic model organisms. We carried out a genome-wide siRNA-based screen in human cells. Among positive regulators of HR we identified networks of DNA-damage-response and pre-mRNA-processing proteins, and among negative regulators we identified a phosphatase network. Three candidate proteins localized to DNA lesions, including RBMX, a heterogeneous nuclear ribonucleoprotein that has a role in alternative splicing. RBMX accumulated at DNA lesions through multiple domains in a poly(ADP-ribose) polymerase 1-dependent manner and promoted HR by facilitating proper BRCA2 expression. Our screen also revealed that off-target depletion of RAD51 is a common source of RNAi false positives, raising a cautionary note for siRNA screens and RNAi-based studies of HR
New insights from old data - Hunting for compounds with novel mechanisms using cellular high-throughput screening profiles with Grey Chemical Matter
Identifying high quality chemical starting points is a critical and challenging step in drug discovery, which typically involves screening large compound libraries or repurposing of compounds with known mechanisms of actions (MoAs). Here we introduce a novel cheminformatics approach that mines existing large-scale, phenotypic high throughput screening (HTS) data. Our method aims to identify bioactive compounds with distinct and specific MoAs, serving as a valuable complement to existing focused library collections. This approach identifies chemotypes with selectivity across multiple cell-based assays and characterized by persistent and broad structure activity relationships (SAR). We prospectively demonstrate the validity of the approach in broad cellular profiling assays (cell painting, DRUG-seq, Promotor Signature Profiling) and chemical proteomics experiments where the compounds behave similarly to known chemogenetic libraries, but with a bias towards novel protein targets and required no synthetic effort to improve compound properties. A public set of such compounds is provided based on the PubChem BioAssay dataset for use by the scientific community
Pharmacologic Inhibition of the Anaphase-Promoting Complex Induces A Spindle Checkpoint-Dependent Mitotic Arrest in the Absence of Spindle Damage
Adhesion G protein-coupled receptors: opportunities for drug discovery
The seminal discovery of the novel activation mechanism of Adhesion GPCRs (aGPCRs)1,2, together with their strong and growing links to disease from human genetics and pre-clinical research, has prompted a rapid reconsideration of this unique family of receptors for classical drug discovery. However, while acknowledged as a sub-family of GPCRs by the IUPHAR3, these receptors are anything but classical with their complex gene structures, large multi-domain N-termini, autocatalytic cleavage and tethered ligands. Initially thought to have a purely structural role, the increasing functional complexity of this GPCR sub-family and the many, potentially unique mechanisms of modulation challenges the way we have perceived this protein class until now. Significantly, if 50% of non-sensory GPCRs are unexploited as drug targets4, this figure reaches 100% for aGPCRs so the potential to develop novel therapies could be substantial5. Here, we discuss the unique opportunities and challenges brought by aGPCRs in the context of drug discovery programs naturally starting with target identification then extending to target validation, assay building and safety considerations