Parallel functional activity profiling reveals valvulopathogens are potent 5-HT2B receptor agonists: implications for drug safety assessment

Data Supplement

Files in this Data Supplement:

• Data Supplement - Supplemental Table 3. Expression of 5-HT2B receptors (Bmax) and radioligand affinity (KD) are similar for the cell lines assayed. See Methods and Supplemental Figure 4 for details.
• Data Supplement - Supplemental Figure 1. Agonist concentration-dependent stimulation of calcium flux in FlpIn HEK293 5-HT2B receptor-expressing cells is sensitive to the 5-HT2B receptor antagonist SB 206553. Maximal intracellular calcium mobilization to each concentration of agonist, measured by Calcium Plus dye fluorescence, is expressed as fold increase over baseline. Each panel shows isotherms for one of the drugs listed in Table 1 either in the absence or presence of the 5-HT2B receptor antagonist 2B 206553.
• Data Supplement - Supplemental Figure 2. Agonist-mediated proliferation responses in parental FlpIn HEK293 cells. XTT-based proliferation assay of agonist-treated FlpIn HEK293 cells. Cells were stimulated for 48 hours with the indicated drug at the indicated concentration (see legend). Four hours prior to the end of the drug treatment phase, XTT reagent was added. Then, the 490-nm absorbance (ODmax of the XTT formazan metabolite) was read. OD490 was proportional to cell number (data not shown). *: p < 0.05 compared with vehicle (two-way ANOVA followed by Bonferroni post-test).
• Data Supplement - Supplemental Figure 3. Homologous radioligand competition binding assay of 5-HT2B receptor-expressing cell lines does not reveal large differences in receptor expression (Bmax). Membrane fractions from 5-HT2B receptor-expressing cell lines were prepared as described in the Methods. For each cell line, radioligand (3Hmesulergine at 2 nM and 4 nM) competition (with unlabeled mesulergine) isotherms were obtained and the data were fit simulataneously to equation 1 (for 2 nM radioligand) and 2 (for 4 nM radioligand), with Bmax and KD values shared (i.e., not dependent on radioligand concentration). Best-fit Bmax values (in dpm) were adjusted for protein content per sample (in mg) and radioligand specific activity to obtain Bmax values in fmol/mg (see Supplementary Table 3).
• Data Supplement - Supplemental Figure 4. Chemical structures of the drugs in Table 1.
• Data Supplement - Supplemental Figure 4. Chemical structures of the drugs in Table 1.
• Data Supplement - Supplemental Figure 4. Chemical structures of the drugs in Table 1.
• Data Supplement - Supplemental Figure 4. Chemical structures of the drugs in Table 1.
• Data Supplement - Supplemental Figure 4. Chemical structures of the drugs in Table 1.
• Data Supplement - Supplemental Figure 4. Chemical structures of the drugs in Table 1.
• Data Supplement - Supplemental Table 1. Results from the preliminary screen of several libraries for modulators of human 5-HT2B receptor-mediated calcium flux. For the Prestwick library, the average test concentration of the compounds was 3 micromolar; for the other libraries, the test concentration was 10 micromolar for each compound. �First Add� values correspond to the calcium flux response evoked by the test compound (expressed as percent of the average calcium response elicited by an EC90 of 5-HT); �Second Add� values correspond to the calcium flux response to an EC90 of 5-HT approximately six minutes after the addition of the test compound. Thus, a large �First Add� value suggests efficacious agonism. Compounds having �First Add� values >15% were considered putative agonists.
• Data Supplement - Supplemental Table 2. Results from the confirmatory screen of �hits� from the preliminary screen (Supplemental Table 1). Values are as indicated in Supplemental Table 1. Compounds are annotated such that the rationale for inclusion in/exclusion from further study is clear.