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

Transcriptional regulators lacking enzymatic activity or binding pockets with targetable molecular features have typically been considered “undruggable,” and a reductionist approach based on identification of their molecular targets has largely failed. We have demonstrated that the Ewing sarcoma chimeric transcription factor, EWSR1-FLI1, maintains accessible chromatin at disease-specific regions. We adapted formaldehyde-assisted isolation of regulatory elements (FAIRE), an assay for accessible chromatin, to screen an epigenetically targeted small molecule library for compounds that reverse the disease-associated signature. This approach can be applied broadly for discovery of chromatin-based developmental therapeutics and offers significant advantages because it does not require the selection of a single molecular target. Using this approach, we identified a specific class of compounds with therapeutic potential

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

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

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

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

<div><p>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.</p></div

Nanodroplets persisted in solution longer than microbubbles.

<p><b>A)</b> Flow chart outlining method for production of nanodroplets. <b>B)</b> A persistence study was performed in the ultrasonic bath to compare nanodroplets and microbubbles. An Accusizer particle sizing system (Particle Sizing Systems, Port Richey, FL) was used to measure the microbubble and nanodroplet concentrations at specific time points between 0 and 300 seconds (5 minutes). Nanodroplets maintained between 10–20% of their initial concentration as far out as 3 minutes into the sonication treatment, while the microbubble concentration dropped to 10% after 1 second.</p

DNA fragmentation in an ultrasonic water bath compared to a commercially available device.

<p><b>(A)</b> Flow chart outlining method for comparing DNA fragmentation methods.</p

<i>Saccharomyces cerevisiae</i> gDNA (BY4741) fragmented with nanodroplets in an ultrasonic bath was comparable in quality to DNA fragmented using a commercial method.

<p><b>(A)</b> Agilent D1000 ScreenTape system traces for DNA samples in microTUBES with the rod (left panel) or microTUBES with nanodroplets (right panel) that were subjected to sequencing. Average size is indicated in base pairs (bp). DNA size markers are denoted by Upper and Lower. <b>(B)</b> Traces showing similar size distribution of DNA after sequencing library preparation. Average size is indicated in base pairs (bp). DNA size markers are denoted by Upper and Lower. <b>(C)</b> Mapping sequencing reads to the <i>Saccharomyces cerevisiae</i> (S288c) reference genome is comparable in detection of single nucleotide variations, insertions, and deletions. Abundance and profile of relative errors in sequencing reads does not indicate a difference in the presence of error bias in the data.</p

<i>Saccharomyces cerevisiae</i> gDNA (BY4741) fragmented with nanodroplets in an ultrasonic water bath was comparable in quality to DNA fragmented in a commercially available device.

<p><b>(A)</b> Agilent D1000 ScreenTape data showing size distribution of DNA fragmented in tubes without (left panel) or tubes with nanodroplets (right panel). Average size is indicated in base pairs (bp). DNA size markers are denoted by Upper and Lower. <b>(B)</b> False gel picture indicating that DNA fragmented without nanodroplets had an average fragment size >1,500 bp. Purple bars indicate the upper (1,500 bp) molecular weight marker and green bars indicate the lower (25 bp) molecular weight marker in each lane. <b>(C)</b> Size distribution of DNA after sequencing library preparation. Average size is shown in base pairs (bp). DNA size markers are denoted by Upper and Lower. <b>(D)</b> Mapping sequencing reads to the <i>Saccharomyces cerevisiae</i> (S288c) reference genome is comparable in detection of single nucleotide variations and indels in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133014#pone.0133014.g004" target="_blank">Fig 4C</a>. Abundance and profile of relative errors in sequencing reads does not indicate a difference in the presence of error bias in the data compared to data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133014#pone.0133014.g004" target="_blank">Fig 4C</a>.</p

Nanodroplet-mediated DNA fragmentation compared to a commercial method.

<p><b>(A)</b> Flow chart outlining method for comparing DNA fragmentation methods. <b>(B)</b> False gel picture from Agilent D1000 ScreenTape system showing DNA fragment size distribution in base pairs for samples fragmented in the Covaris E110 sonicator. Purple bars indicate the upper (1,500 bp) molecular weight marker and green bars indicate the lower (25 bp) molecular weight marker in each lane.</p