Sequence: lamin A/C isoform 3 [Homo sapiens]

Gene:LMNA     Conserved Domain     Related Protein 3D Structures

NCGC Assay Overview:

Hutchinson-Gilford Progeria Syndrome (HGPS) is a pediatric premature aging disease caused by a spontaneous mutation in the lamin A/C (LMNA) gene. The mutation activates a cryptic splice site in the LMNA pre-mRNA which results in production of a pre-lamin A protein that cannot be processed properly. The mutant protein accumulates in the nucleus and negatively affects numerous cellular functions. Correction of the splicing defect in HGPS patient cells using a targeted oligonucleotide (exo11) leads to reversal of the cellular disease phenotypes. To identify small molecule modulators of aberrant LMNA splicing, a homogenous assay was constructed in HeLa cells that used a GFP containing minigene to report on correction of aberrant splicing in lamin A and a RFP (DsRed2) to report on cell viability, uniformity and nonspecific effects on the assay signal. This assay was adapted to laser-scanning microplate cytometry (Acumen, TTP Labtech) where miniaturization of the assay to a 1,536-well plate format was possible.

References:
Bowen, W. P.; Wylie, P. G. Application of Laser-Scanning Fluorescence Microplate Cytometry in High Content Screening. Assay Drug Dev Technol 2006;4:209-221.
Scaffidi P, Misteli T. Reversal of the cellular phenotype in the premature aging disease Hutchinson-Gilford progeria syndrome. Nat. Med. 2005;11:440-445.

NCGC Assay Protocol Summary:

Reagents/Controls: Stable clones expressing DsRed2 and lamin A-EGFP were generated by Cell and Molecular Technologies in S3 HeLa cells.
HeLa cells were prepared for the assay when they were 70-90% confluent. The cells were counted and resuspended in Opti-MEM I (no phenol red; Gibco), 2% FBS, 1% penicillin/ streptomycin (Gibco) at concentration of 120 cells/uL concentration. The suspension was passed through a Falcon basket filter with pore size 40um (Beckton Dickson).
Control titrations: column of the 1536-well plate contained a sixteen point titration (1:2 dilutions) in duplicate of ancitabine (Sigma: A8598) beginning at 40 uM final concentration. Column 2 contained a sixteen point titration (1:2 dilutions) of cycloheximide beginning at 1.4 mM final concentration. Column 3 contained ancitabine at 40 uM and column 4 contained was the vehicle control (0.5% DMSO final concentration).

Assay Steps:
5 uL of HeLa cells were plated in Aurora LoBase black clear bottom (cyclic-olefin polymer) 1536-well plates at a final density of 600 cells/well. Controls and library compounds were added with a 1536-well pin array using the Kalypsys pin-tool. The plates were then incubated for 48 hrs in a cell-culture incubator (37oC; 5% CO2, 98% humidity). Following incubation the plates were read on the Acumen Explorer using the 488 nm laser. Data was collected on objects between 10 and 100 um in size. Cell objects were defined by a 15 um width x 10 um depth filter. The ratio of the total object intensity in PMT1 (500-530 nm emission; GFP) over PMT3 (575-640 nm emission; RFP) was calculated for analysis.

Keywords: NIH Roadmap, MLSCN, MLI, MLSMR, qHTS, NCGC, lamin A, Progeria, RNA splicing.

Compound Ranking:
1. Compounds are first classified as being probable inhibitors (compounds decreasing ratio signal), activators (compounds increasing ratio signal), fluorescent (artifactual results), cytotoxic (artifactual results), inactive, or inconclusive based on the ratio and component channel readouts.
2. Compounds are then classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Ratio-Curve Description".
3. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active inhibitor 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. Ratio-Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.

Grant Number: 1R03MH084827-01