High-throughput small molecule screen identifies inhibitors of aberrant chromatin accessibility.

Mutations in chromatin-modifying proteins and transcription factors are commonly associated with a wide variety of cancers. Through gain- or loss-of-function, these mutations may result in characteristic alterations of accessible chromatin, indicative of shifts in the landscape of regulatory elements genome-wide. The identification of compounds that reverse a specific chromatin signature could lead to chemical probes or potential therapies. To explore whether chromatin accessibility could serve as a platform for small molecule screening, we adapted formaldehyde-assisted isolation of regulatory elements (FAIRE), a chemical method to enrich for nucleosome-depleted genomic regions, as a high-throughput, automated assay. After demonstrating the validity and robustness of this approach, we applied this method to screen an epigenetically targeted small molecule library by evaluating regions of aberrant nucleosome depletion mediated by EWSR1-FLI1, the chimeric transcription factor critical for the bone and soft tissue tumor Ewing sarcoma. As a class, histone deacetylase inhibitors were greatly overrepresented among active compounds. These compounds resulted in diminished accessibility at targeted sites by disrupting transcription of EWSR1-FLI1. Capitalizing on precise differences in chromatin accessibility for drug discovery efforts offers significant advantages because it does not depend on the a priori selection of a single molecular target and may detect novel biologically relevant pathways.

Establishing and Maintaining a Robust Sample Management System.

This paper has been written by the SLAS Sample Management Special Interest Group to serve as a guide to the best practices and methods in establishing and maintaining a high-quality sample management system. The topics covered are applicable to sample types ranging from small molecules to biologics to tissue samples. It has been put together using the collective experience of the authors in start-up companies, small pharma, agricultural research, IT, academia, biorepositories, and large pharma companies. Our hope is that sharing our experience will streamline the process of setting up a new sample management system and help others avoid some of the problems that we have encountered.

Instrument Quality Control.

Well-defined quality control (QC) processes are used to determine whether a certain procedure or action conforms to a widely accepted standard and/or set of guidelines, and are important components of any laboratory quality assurance program (Popa-Burke et al., J Biomol Screen 14: 1017-1030, 2009). In this chapter, we describe QC procedures useful for monitoring the accuracy and precision of laboratory instrumentation, most notably automated liquid dispensers. Two techniques, gravimetric QC and photometric QC, are highlighted in this chapter. When used together, these simple techniques provide a robust process for evaluating liquid handler accuracy and precision, and critically underpin high-quality research programs.

Cavitation Enhancing Nanodroplets Mediate Efficient DNA Fragmentation in a Bench Top Ultrasonic Water Bath.

A perfluorocarbon nanodroplet formulation is shown to be an effective cavitation enhancement agent, enabling rapid and consistent fragmentation of genomic DNA in a standard ultrasonic water bath. This nanodroplet-enhanced method produces genomic DNA libraries and next-generation sequencing results indistinguishable from DNA samples fragmented in dedicated commercial acoustic sonication equipment, and with higher throughput. This technique thus enables widespread access to fast bench-top genomic DNA fragmentation.

UNC1062, a new and potent Mer inhibitor.

Abnormal activation of Mer kinase has been implicated in the oncogenesis of many human cancers including acute lymphoblastic and myeloid leukemia, non-small cell lung cancer, and glioblastoma. We have discovered a new family of small molecule Mer inhibitors, pyrazolopyrimidine sulfonamides, that potently inhibit the kinase activity of Mer. Importantly, these compounds do not demonstrate significant hERG activity in the PatchXpress assay. Through structure-activity relationship studies, 35 (UNC1062) was identified as a potent (IC50 = 1.1 nM) and selective Mer inhibitor. When applied to live tumor cells, UNC1062 inhibited Mer phosphorylation and colony formation in soft agar. Given the potential of Mer as a therapeutic target, UNC1062 is a promising candidate for further drug development.