Parallel functional activity profiling reveals valvulopathogens are potent 5-hydroxytryptamine(2B) receptor agonists: implications for drug safety assessment.

Drug-induced valvular heart disease (VHD) is a serious side effect of a few medications, including some that are on the market. Pharmacological studies of VHD-associated medications (e.g., fenfluramine, pergolide, methysergide, and cabergoline) have revealed that they and/or their metabolites are potent 5-hydroxytryptamine(2B) (5-HT(2B)) receptor agonists. We have shown that activation of 5-HT(2B) receptors on human heart valve interstitial cells in vitro induces a proliferative response reminiscent of the fibrosis that typifies VHD. To identify current or future drugs that might induce VHD, we screened approximately 2200 U.S. Food and Drug Administration (FDA)-approved or investigational medications to identify 5-HT(2B) receptor agonists, using calcium-based high-throughput screening. Of these 2200 compounds, 27 were 5-HT(2B) receptor agonists (hits); 14 of these had previously been identified as 5-HT(2B) receptor agonists, including seven bona fide valvulopathogens. Six of the hits (guanfacine, quinidine, xylometazoline, oxymetazoline, fenoldopam, and ropinirole) are approved medications. Twenty-three of the hits were then "functionally profiled" (i.e., assayed in parallel for 5-HT(2B) receptor agonism using multiple readouts to test for functional selectivity). In these assays, the known valvulopathogens were efficacious at concentrations as low as 30 nM, whereas the other compounds were less so. Hierarchical clustering analysis of the pEC(50) data revealed that ropinirole (which is not associated with valvulopathy) was clearly segregated from known valvulopathogens. Taken together, our data demonstrate that patterns of 5-HT(2B) receptor functional selectivity might be useful for identifying compounds likely to induce valvular heart disease.

Fluorescent labeling of proteins in living cells using the FKBP12 (F36V) tag.

Over the past decade live cell imaging has become a key technology to monitor and understand the dynamic behavior of proteins in the physiological context of living cells. The visualization of a protein of interest is most commonly achieved by genetically fusing it to green fluorescent protein (GFP) or one of it variants. Considerable effort has been made to develop alternative methods of protein labeling to overcome the intrinsic limitations of fluorescent proteins. In this report we show the optimization of a live cell labeling technology based on the use of a mutant form of FKBP12 (FKBP12(F36V)) in combination with a synthetic high affinity ligand (SLF') that specifically binds to this mutant. It had been previously shown that the use of a fluorescein-conjugated form of SLF' (5'-fluorescein-SLF') allowed the labeling of proteins genetically fused to FKBP-F36V in living cells. Here we describe the identification of novel fluorescent SLF'dye conjugates that allow specific labeling of FKBP12(F36V) fusion proteins in living cells. To further increase the versatility of this technology we developed a number of technical improvements. We implemented the use of pluronics during the labeling process to facilitate the uptake of the SLF'-dye conjugates and the use suppression dyes to reduce background signal. Furthermore, the time and dose dependency of labeling was investigated in order to determine optimal labeling conditions. Finally, the specificity of the FKBP12(F36V) labeling technology was extensively validated by morphological analysis using a diverse set of FKBP12(F36V) fusions proteins. In addition we show a number of different application examples, such as translocation assays, the generation of biosensors, and multiplex labeling in combination with different labeling technologies, such as FlAsH or GFP. In summary we show that the FKBP12(F36V)/SLF' labeling technology has a broad range of applications and should prove useful for the study of protein function in living cells.

Protein labeling with FlAsH and ReAsH.

The ability to image biochemical and phenotypical changes in living cells has become crucial for the investigation and understanding of the molecular mechanisms that govern all physiological cellular functions in health and disease. Genetically encoded reporters derived from fluorescent proteins (FPs) have proved to be extremely useful for localization and interaction studies in living cells. However, the large size and spectral properties of FP impose certain limitations for their use. The recently developed Fluorescein Arsenical Hairpin (FlAsH/tetracysteine) binder technology emerged as a promising alternative to FP for protein labeling and cellular localization studies. The combination of a small genetically encoded peptide tag with a small molecule detection reagent makes this technology particularly suitable for the investigation of biochemical changes in living cells that are difficult to approach with fluorescent proteins as molecular tags. We describe the practical application of this technology to image protein dynamics in living cells.

Mutational analysis of ThiH, a member of the radical S-adenosylmethionine (AdoMet) protein superfamily.

Thiamine pyrophosphate (TPP) is an essential cofactor for all forms of life. In Salmonella enterica, the thiH gene product is required for the synthesis of the 4-methyl-5-beta hydroxyethyl-thiazole monophosphate moiety of TPP. ThiH is a member of the radical S-adenosylmethionine (AdoMet) superfamily of proteins that is characterized by the presence of oxygen labile [Fe-S] clusters. Lack of an in vitro activity assay for ThiH has hampered the analysis of this interesting enzyme. We circumvented this problem by using an in vivo activity assay for ThiH. Random and directed mutagenesis of the thiH gene was performed. Analysis of auxotrophic thiH mutants defined two classes, those that required thiazole to make TPP (null mutants) and those with thiamine auxotrophy that was corrected by either L-tyrosine or thiazole (ThiH* mutants). Increased levels of AdoMet also corrected the thiamine requirement of members of the latter class. Residues required for in vivo function were identified and are discussed in the context of structures available for AdoMet enzymes.

A one-arm homologous recombination approach for developing nuclear receptor assays in somatic cells.

Drug discovery is in need of technologies that enable investigators to develop cell-based assays that accurately reflect the functional consequence of small molecule intervention on biological processes. Here, we describe a strategy that uses both one-arm homologous recombination and the beta-lactamase (BLA) reporter system, a sensitive and robust transcriptional reporter for gene activation. We demonstrate that this powerful approach can be utilized for developing cell-based assays for the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) in HEK293 somatic cells. Specifically, one-arm homologous recombination was used to introduce the GAL4 DNA-binding domain (GAL4DBD) in the GR and MR genomic loci such that a chimeric GAL4DBD-GR (ligand-binding domain) [GAL4DBD-GR(LBD)] and GAL4DBD-MR(LBD) transcript is produced from the strong CMV promoter in HEK293 cells previously stably transfected with the UAS(GAL4)-BLA reporter construct. Dexamethasone- and aldosterone-responding BLA-positive cells were isolated by fluorescence-activated cell sorting, and then further expanded into separate cell lines. The sensitivity and robustness of the resulting GR and MR assays are demonstrated by the fact that the addition of dexamethasone and aldosterone to the two transgenic clonal cell lines for 16 h results in high Z' values (>0.8) and EC(50) values of 1 and 0.3 nM, respectively. These assays illustrate the flexibility of this technology to generate high-performance cellular assays for nuclear receptor targets without the need for target-specific cDNA.