AID	Name	Type	Probe	Comment
1458	qHTS Assay for Enhancers of SMN2 Splice Variant Expression	confirmatory
1474	Quantitative High-Throughput Screen for Enhancers of SMN2 Splice Variant Expression: Summary	summary	2
2515	Counterscreen Assay for Enhancers of SMN2 Splice Variant Expression: Interaction with Luciferase Reporter for Probe SAR	confirmatory
2513	Counterscreen Assay for Enhancers of SMN2 Splice Variant Expression: Modulation of SMN1 Expression for Probe SAR	confirmatory
2514	Confirmation Concentration-Response Assay for Enhancers of SMN2 Splice Variant Expression for Probe SAR	confirmatory

NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Production centers Network [MLPCN]

MLPCN Grant: R03 MH084179-01
Assay Submitter (PI): Elliot Androphy

NCGC Assay Overview:

Spinal muscular atrophy (SMA) is caused by insufficient levels of the survival motor neuron protein SMN. The SMN locus on chromosome 5q13 contains two inverted copies of SMN called SMN1 and SMN2 which are 99% identical at the amino acid level. SMN1 is a fully functional protein and SMN2 skips exon 7 90% of the time. Skipping of exon 7 produces non-functional protein product. 10% of the SMN2 protein includes exon 7 and is fully functional. In the SMA disease state, mutations in the SMN1 locus are the cause of the disease state. Because only 10% of SMN2 is of the fully functional form, it is not sufficient to overcome the deficiency produced by the loss of the SMN1 product. A therapy that either increases the amount of SMN2 product made increases the inclusion of exon 7 has been proposed for the treatment of SMA.

We have designed an assay to identify small molecules that can increase the amount of functional SMN2 product by appending a luciferase reporter gene after the native SMN2 gene, such that inclusion of exon 7 in the expressed product places the luciferase sequence in frame, thus generating functional luciferase enzyme.

One potential source of false positives in the primary screening assay is stabilization of the reporter luciferase via direct binding. This assay attempts to identify such compounds by screening directly for luciferase activity in the absence of the cellular system.

NCGC Assay Protocol Summary:

The primary screen utilizes a luciferase reporter gene assay, combining the promoter and splicing based cassettes in tandem with the major portion of the native SMN1 cDNA, and then transfected into HEK293 cells. Compounds that modulate luciferase signal presumably modulate expression of native SMN1 expression. In this assay, we utilize purified firefly luciferase in lieu of a cellular reporter to identify compounds interacting directly with luciferase in the primary screen.

Sequence, Parameter, Value, Description
(1) Reagent, 2.5 uL, Enzyme in buffer (note A)
(2) Compound, 23 nL, From stock solutions
(3) Time, 5 minutes, Room temp
(4) Reagent#2.5 ul, Luciferase detection reagent (note B)
(5) Detector, 1 sec integrate, Luminescent read on Viewlux (note C)

Sigma enzyme (1 mg) considered to be MW 61,000 diluted in 357 ul assay buffer to 50 uM stock.
A - Enzyme and buffer consists of 50 mM Tris-acetate pH 7.6, 10 mM Mg acetate 0.05% BSA, 0.01% Tween and 5 nM firefly luciferase
B - Detection reagent = britelite plus diluted 1:10 in PBS
C - Viewlux settings were 1 second integration medium sensitivity

Keywords: Spinal muscular atrophy, SMA, SMN2, high throughput screening, MLSMR, MLPCN, NIH Roadmap, qHTS and NCGC

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: MH084179-01