qHTS for Activators of Integrin-Mediated Alleviation for Muscular Dystrophy
Integrins are a diverse family of transmembrane cell surface receptors that mediate interactions between cells and the extracellular matrix. These heterodimeric proteins are composed of alpha and beta subunits and the a7b1 interacts with laminin in the extracellular matrix and actin of the cell cytoskeleton. Both chains contribute to ligand binding, but the alpha chain mediates the specificity of more ..
BioActive Compounds: 222
Integrins are a diverse family of transmembrane cell surface receptors that mediate interactions between cells and the extracellular matrix. These heterodimeric proteins are composed of alpha and beta subunits and the a7b1 interacts with laminin in the extracellular matrix and actin of the cell cytoskeleton. Both chains contribute to ligand binding, but the alpha chain mediates the specificity of the interaction. The alpha7beta1 integrin is a major laminin receptor in skeletal, cardiac and vascular smooth muscle. Mutations in the alpha7 integrin gene are responsible for muscular dystrophy in both humans and mice. The alpha7beta1 integrin is also a major modifier of muscle disease progression in various muscle diseases including Duchenne muscular dystrophy, Fukuyama muscular dystrophy and merosin deficient congenital muscular dystrophy type 1A. In addition the alpha7beta1 integrin has been shown to be a major tumor suppressor gene in prostate cancer and plays a role in the progression of cardiovascular disease.
Transcriptional regulation of the alpha7 integrin gene is poorly understood and probably involves multiple transcription factors. The goal of identifying small molecules that enhance alpha7 integrin expression is two-fold: firstly, such molecules would help us gain insight into the complex transcriptional control of alpha7 integrin. It is known that alpha7 integrin expression is tightly controlled during various stages of development, disease and in response to external stimuli. The second goal of the project is to translate the above finding into a therapeutic strategy for muscular dystrophy. As increased alpha7 integrin levels have been shown to be beneficial in ameliorating muscular dystrophy in animal models, small molecules enhancing levels of this integrin are clearly useful and may be developed into therapeutic leads. Thus this project aims to discover small molecule probes that would not only help in gaining a better understanding of the complex regulatory mechanism of alpha7 integrin expression, but also lead to development of pharmacological agents to fight muscular dystrophy.
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]
MLPCN Grant: NS058429
Assay Submitter (PI): Dean Burkin, University of Nevada School of Medicine
A total of 250 alpha7betagal+/- myoblasts at passage 13 were dispensed using a mulitdrop from Thermo into black low base tissue culture treated microclear aurora plates in 6 ul media containing DMEM without phenol, 5% FBS, 1x GluMax and 1x Penicillin/Streptomycin. The plates were incubated 16-24 hr at 37C, 5% CO2, 95% humidity covered with low evaporation stainless steel lids from Kalypsys. Compounds were then dispensed using a Kalypsys pintool to deliver 23 nl/well compounds in DMSO (diluted into 6 uL resulting in a 1:260 dilution of compound). The control compound used was the cdk2 inhibitor SU9516 from Tocirs (found in the initial LOPAC screen). The plates were incubated for 48 hours at 37C, 5% CO2, 95% humidity using the same stainless steel lids. After incubation with compound, 5 ul of the media was aspirated using the Kalypsys washer/dispenser and 3 ul of the Mammalian Protein Extraction Reagent (MPER) lysis buffer (Thermo Fisher) was added. The plates were spun at 2000 rpms to remove bubbles and an initial capture was acquired on the Viewlux (Perkin Elmer) with exciation at 480 nm and emission at 540 nm for 25 seconds to omit any auto fluorescent compounds. The plates were then incubated for 10 minutes at room temperature. After incubation, 3 ul of 125 uM Fluorescein di-galactoside (FDG) (Marker Gene Technologies) diluted in PBS with 2 mM MgCl2 and 0.2% betamercaptoethanol (BME) was added, the plates were spun at 2000 rpms and incubated for 30 minutes at room temperature. The plates were again read on the Viewlux with exciation at 480 nm and emission at 540 nm for 25 seconds.
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 activators are ranked higher than compounds that showed apparent inhibition.
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.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)