Research Spotlight: The University of Kansas High-Throughput Screening Laboratory. Part II: Enabling Collaborative Drug-Discovery Partnerships Through Cutting-Edge Screening Technology

McDonald, Peter R.; Roy, Anuradha; Chaguturu, Rathnam

doi:10.4155/fmc.11.84

Cited by 5 publications

(2 citation statements)

References 5 publications

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“…The chemical library consisted of a 100,000-compound library (ChemBridge), the 1,280-member Library of Pharmacologically Active Compounds (LOPAC; Sigma-Aldrich), 100,000 compounds acquired from the University of Kansas High-Throughput Screening Laboratory [44] , focused natural product libraries and also 6,700 peptide and peptidomimetic compounds. Additional compounds were obtained from the MicroSource Spectrum Collection (Discovery Systems), the Prestwick Chemical collection, the NIH Clinical Collection, Tocris chemical standard libraries (Tocris Bioscience), in-house and miscellaneous commercial compounds.…”

Section: Methodsmentioning

confidence: 99%

A Cell-Free Fluorometric High-Throughput Screen for Inhibitors of Rtt109-Catalyzed Histone Acetylation

et al. 2013

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The lysine acetyltransferase (KAT) Rtt109 forms a complex with Vps75 and catalyzes the acetylation of histone H3 lysine 56 (H3K56ac) in the Asf1-H3-H4 complex. Rtt109 and H3K56ac are vital for replication-coupled nucleosome assembly and genotoxic resistance in yeast and pathogenic fungal species such as Candida albicans. Remarkably, sequence homologs of Rtt109 are absent in humans. Therefore, inhibitors of Rtt109 are hypothesized as potential and minimally toxic antifungal agents. Herein, we report the development and optimization of a cell-free fluorometric high-throughput screen (HTS) for small-molecule inhibitors of Rtt109-catalyzed histone acetylation. The KAT component of the assay consists of the yeast Rtt109-Vps75 complex, while the histone substrate complex consists of full-length Drosophila histone H3-H4 bound to yeast Asf1. Duplicated assay runs of the LOPAC demonstrated day-to-day and plate-to-plate reproducibility. Approximately 225,000 compounds were assayed in a 384-well plate format with an average Z' factor of 0.71. Based on a 3σ cut-off criterion, 1,587 actives (0.7%) were identified in the primary screen. The assay method is capable of identifying previously reported KAT inhibitors such as garcinol. We also observed several prominent active classes of pan-assay interference compounds such as Mannich bases, catechols and p-hydroxyarylsulfonamides. The majority of the primary active compounds showed assay signal interference, though most assay artifacts can be efficiently removed by a series of straightforward counter-screens and orthogonal assays. Post-HTS triage demonstrated a comparatively small number of confirmed actives with IC50 values in the low micromolar range. This assay, which utilizes five label-free proteins involved in H3K56 acetylation in vivo, can in principle identify compounds that inhibit Rtt109-catalyzed H3K56 acetylation via different mechanisms. Compounds discovered via this assay or adaptations thereof could serve as chemical probes or leads for a new class of antifungals targeting an epigenetic enzyme.

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Section: Methodsmentioning

confidence: 99%

A Cell-Free Fluorometric High-Throughput Screen for Inhibitors of Rtt109-Catalyzed Histone Acetylation

et al. 2013

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“…The KU-HTS team has extensive background in assay formats compatible with high throughput and high content screening with siRNAs and compounds. The KU-HTS laboratory routinely performs both biochemical and cell-based assays in homogeneous as well as heterogeneous formats [3]. A vast majority of assays are homogeneous and involve one or two steps based on a wide variety of assay platforms: absorbance, fluorescence (fluorescence intensity, FRET, fluorescence polarization and time-resolved FRET), luminescence, and AlphaScreen.…”

Section: Ku-hts Capabilities: Assay Formatsmentioning

confidence: 99%

High Throughput Screening Operations at the University of Kansas

Roy¹

2014

CCHTS

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The High Throughput Screening Laboratory at University of Kansas plays a critical role in advancing academic interest in the identification of chemical probes as tools to better understand the biological and biochemical basis of new therapeutic targets. The HTS laboratory has an open service policy and collaborates with internal and external academia as well as for-profit organizations to execute projects requiring HTS-compatible assay development and screening of chemical libraries for target validation, probe selection, hit identification and lead optimization.

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Experiences in Academic and Industry Partnerships – Forging a Path to Translational Drug Discovery

Dolle

Barrault²,

Carragher³

et al. 2021

Burger's Medicinal Chemistry and Drug Discovery

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Research Spotlight: The University of Kansas High-Throughput Screening Laboratory. Part II: Enabling Collaborative Drug-Discovery Partnerships Through Cutting-Edge Screening Technology

Cited by 5 publications

References 5 publications

A Cell-Free Fluorometric High-Throughput Screen for Inhibitors of Rtt109-Catalyzed Histone Acetylation

A Cell-Free Fluorometric High-Throughput Screen for Inhibitors of Rtt109-Catalyzed Histone Acetylation

High Throughput Screening Operations at the University of Kansas

Experiences in Academic and Industry Partnerships – Forging a Path to Translational Drug Discovery

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