AID	Name	Type	Comment
2288	qHTS Assay for Modulators of miRNAs and/orActivators of miR-21	confirmatory	qHTS assay 1
2289	qHTS Assay for Modulators of miRNAs and/or Inhibitors of miR-21	confirmatory	qHTS assay 2
2507	Secondary Assay for General miRNAs Modulators and/or Inhibitors/Activators of miR-21: miR-30 Firefly Luciferase Assay	confirmatory	confirmation miR-30 assay
2508	Confirmatory Assay for General miRNAs Modulators and/or Inhibitors/Activators of miR-21	confirmatory	confirmation miR-21 assay
2598	qHTS Assay for General miRNAs Modulators and/or Inhibitors/Activators of miR-21: Summary	summary	summary assay

MLPCN Grant: 1 R21 NS059478-01

Assay Provider: Qihong Huang, The Wistar Institute

NCGC Assay Overview:

MicroRNAs (miRNAs) are endogenously encoded small (~20-25 nucleotides), nonprotein-coding RNAs that are involved in post-transcriptional repression of target messenger RNAs (mRNAs) (Le and Hannon, 2004). Estimated to be involved in the regulation of 30% of all protein-coding mRNAs (Filipowicz et al., 2008), miRNAs play a significant role in many biological processes including cellular differentiation and tissue development (Ghildiyal and Zamore, 2009). Not surprisingly, miRNAs have been found to be oncogenic or tumor suppressive, with altered miRNA expression levels associated with human cancers (Esquela-Kerscher and Slack, 2006). The miRNA miR-21 has been found to be expressed in most human tissues (Sonkoly et al., 2007), and its overexpression has been associated with a variety of human tumors (Tong and Nemunanitis, 2008). miR-21 has been shown to be anti-apoptotic: knock-down of miR-21 in a glioblastoma cell line was found to activate caspases and lead to apoptosis (Chan et al., 2005). Identification of small molecules that modulate miR-21 specifically, or miRNAs generally, would prove useful as tools to better understand miRNA biogenesis and mRNA regulation by miRNAs. Of special interest are compounds that specifically inhibit miR-21, and a cell-based firefly luciferase reporter gene assay optimized for 1536-well format quantitative HTS (qHTS) was developed and performed in an effort to identify such compounds.

Compounds active in the miR-21 FLuc assay were clustered using Leadscope and active chemical series were prioritized based on chemical properties, potential for medicinal chemistry optimization, and SAR. Within each chemical series, representative compounds were re-acquired to test for activity in a secondary assay based on the quality of their CRC, efficacy, and potency. This secondary assay to determine if compounds active in the miR-21 FLuc assay were just luciferase inhibitors. Compounds found active in both the miR-21 FLuc and purified FLuc assays are considered as false positives.

Keywords: NIH Roadmap, MLPCN, MLSMR, qHTS, miR-21, firefly luciferase, FLuc, miRNA.

NCGC Assay Protocol Summary:

Reagents: 50mM Tris acetate, pH 7.5; 10mM Mg acetate; 10uM D-luciferin (Sigma #L9504); 10uM ATP; 0.01% Tween-20; 0.05% BSA; 10nM P. pyralis luciferase (Sigma #L9506)
Control compounds used were two known firefly luciferase inhibitors (compounds (2) and (5) in Auld et al., 2010), and DMSO.

Assay Summary: Three microliters containing firefly luciferase substrates in buffer (final concentrations: 50mM Tris acetate, pH 7.5, 10mM Mg acetate, 0.01% Tween-20, 0.05% BSA, 10uM D-luciferin, and 10uM ATP) are dispensed into each well of a Greiner white, solid-bottom 1536-well format plate using a flying reagent dispenser (FRD). These assay plates were then treated with 23nL of compound or DMSO using a Kalypsys pin tool, which allows for delivery of a 6-point interplate titration of each compound to the assay plate (quantitative HTS), with a final compound concentrations ranging from approximately 60muM to 7pM. One microliter of firefly luciferase in 500mM Tris-acetate buffer was then delivered by FRD to each well for a final enzyme concentration of 10nM. Luciferase activity was then measured using a ViewLux CCD imager (PerkinElmer), with an average exposure time of 2-30 seconds (2X binning, medium/high gain).

Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.

Grant Number: 1 R21 NS059478-01