AID	Name	Type	Comment
1474	Quantitative High-Throughput Screen for Enhancers of SMN2 Splice Variant Expression: Summary	summary

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.

As a counterscreen for nonspecific modulators of expression, we have run compounds through a similar assay that measures expression of an SMN1-luciferase chimera.

NCGC Assay Protocol Summary:

This screen utilizes a luciferase reporter gene assay, combining the promoter with the major portion of the native SMN1 cDNA (exons 1 -7, intron 8 , exon 8 fused to luciferase) stably expressed in HEK293 cells. Compounds that increase luciferase signal presumably enhance expression of the functional SMN1 splice variant.

Passaging media contained DMEM w/ glutamax (+phenol red) 10% FCS, 1x pen/strep, 200 ug/ml hygro, 1x sodium pyruvate.
Assay media contained DMEM w/ glutamax (-phenol red) 10% FCS, 1x pen strep, 1x pyruvate

Sequence, Parameter, Value, Description
(1) Cells, 5 uL, 2000 cells/well, 1536 TC treated White solid bottom plate
(2) Time, 10-12 hours, 37C 5% CO2
(3) Compound, 23 nl, MLSMR Library
(4) Control Compound, 23 nL, Sodium butyrate st 4.5mM (final) conc
(5) Time, 30-36 hours, 37C 5% CO2
(6) Reagent, 3 ul, One Glo (TM) from Promega
(7) Time, 5-15 minutes, Room temp
(8) Detector, Viewlux: luminescent read, 60 second integration, high speed 2x binning

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