qHTS for Inhibitors of the vitamin D receptor (VDR): Hit Validation using a Beta-Lactamase Assay
The vitamin D receptor (VDR) is a ligand-activated transcription factor and is partially responsible for the regulation of the endocrine system. This includes the transcriptional regulation of genes involved in the production of the parathyroid hormone (PTH) and the regulation of the blood calcium level. High levels of PHT can cause hypercalcemia in the case of primary and tertiary more ..
BioActive Compounds: 187
The vitamin D receptor (VDR) is a ligand-activated transcription factor and is partially responsible for the regulation of the endocrine system. This includes the transcriptional regulation of genes involved in the production of the parathyroid hormone (PTH) and the regulation of the blood calcium level. High levels of PHT can cause hypercalcemia in the case of primary and tertiary hyperparathyroidism. The VDR-mediated gene regulation, activated by its ligand 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), is governed by the recruitment of coactivators. Our hypothesis is that small molecules, with the ability to inhibit the interaction between VDR and certain coactivators, are able to selectively modulate VDR-mediated transcription. Our long term objective is the application these VDR-coactivator inhibitors to study the role of VDR-coactivator binding for specific VDR regulated genes. Ultimately, these molecules can be developed into new treatments for hypercalcemic hyperparathyroidism. No small molecule or non-peptide inhibitors are known for the VDR-coactivator interaction.
In a collaboration between the University of Wisconsin, Milwaukee and the NIH Chemical Genomics Center (NCGC) a HTS-compatible enzymatic assay was developed. The assay uses fluorescence polarization as its read-out to measure the inhibition between VDR and coregulator peptide SRC2-3 exerted by small molecules. This assay was used to screen the NIH Molecular Libraries Small Molecule Repository (MLSMR). The cell-based VDR transcription assay was carried out with commercially available stably transfected HEK293 cells. The cell line expresses a fusion protein of VDR-GAL4 DNA binding domain, which is activated by agonist LG190178 and induces transcription of a beta-lactamase reporter gene under control of an UAS response element. Successful activation is detected by a decrease in time-resolved fluorescence resonance energy transfer (TR-FRET) caused by enzymatic cleavage of fluorescent betalactam substrate added after 24h incubation. The cleaved substrate concentration was quantified by measuring the fluorescence emission at 447 nm.
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]
MLPCN Grant: DA031090
Assay Submitter (PI): Alexander Arnold, University of Wilwaukee
Plate: 384 black clear bottom, tissue culture treated
Step 1: Plate 20,000 cell per well (32 uL)
Step 2: 4 hours incubatio
Step 3: Add screening compounds
Step 4: Add 300 nM LG190178
Step 5: 18 hour incubation
Step 6: Add FRET reagent (6 uL)
Step 7: 2 hour incubation
Step 8: Read at ex409, em460, em530 nM
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.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)