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

Abstract

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.

Fig. 1.

Agonist concentration-dependent stimulation of calcium flux in FlpIn HEK293 5-HT_2B receptor-expressing cells. Maximal intracellular calcium response to each concentration of agonist, measured by Calcium Plus dye fluorescence, is expressed as a percentage of the 5-HT E_max. Each panel shows isotherms for two of the drugs listed in Table 1 and for 5-HT. Similar experiments were performed for all drugs listed in Table 1, and the data were analyzed as described under Materials and Methods to obtain potency (pEC₅₀) and efficacy (E_max) estimates. Parental untransfected FlpIn HEK293 cells did not exhibit calcium flux responses to any of the tested drugs at any concentration (data not shown).

Fig. 2.

Agonist concentration-dependent stimulation of a 5-HT_2B-mediated NFAT-β-lactamase reporter. The FRET ratio observed for each concentration of agonist is expressed as a percentage of the 5-HT E_max. Each panel shows isotherms for two of the drugs listed in Table 1 and for 5-HT. Similar experiments were performed for all drugs listed in Table 1, and the data were analyzed as described under Materials and Methods to obtain potency (pEC₅₀) and efficacy (E_max) estimates.

Fig. 3.

Agonist concentration-dependent stimulation of ERK2 activation (phosphorylation) in U2OS 5-HT_2B ERK2-GFP cells. Time-resolved FRET between ERK2-GFP and a terbium-labeled anti-phospho-ERK2 antibody is measured after 1) a 5-min agonist challenge and 2) a 2-h lysis/antibody incubation step. The time-resolved FRET observed for each concentration of agonist is expressed as a percentage of the 5-HT E_max. Each panel shows isotherms for two of the drugs listed in Table 1 and for 5-HT. Similar experiments were performed for all drugs listed in Table 1, and the data were analyzed as described under Materials and Methods to obtain potency (pEC₅₀) and efficacy (E_max) estimates.

Fig. 4.

Immunoblot analysis of agonist-mediated ERK1/2 phosphorylation in FlpIn HEK293 5-HT_2B receptor-expressing cells. A, representative immunoblots probed with anti-phospho-ERK1/2 or anti-total-ERK1/2 antibodies as indicated. B, image densitometry of immunoblot scans in A to quantify phospho-ERK1/2 content in cell lysates. Immunoreactivity for phospho-ERK1/2 (i.e., mean pixel intensity per region of interest that contained the p42 and p44 bands, minus the background) was measured for each sample. Blots were then stripped and reprobed for total-ERK1/2 and analyzed as before. The phospho-ERK1/2 immunoreactivity for each sample was normalized to its total-ERK1/2 immunoreactivity. The normalized phospho-ERK1/2 immunoractivity elicited by 5-HT (measured on each gel) was set to 100%. SB, SB-206,553 pretreatment.

Fig. 5.

Agonist concentration-dependent stimulation of β-arrestin translocation. The FRET ratio observed for each concentration of agonist is expressed as a percentage of the 5-HT E_max. Each panel shows isotherms for two of the drugs listed in Table 1 and for 5-HT. Similar experiments were performed for all drugs listed in Table 1, and the data were analyzed as described under Materials and Methods to obtain potency (pEC₅₀) and efficacy (E_max) estimates.

Fig. 6.

Agonist concentration-dependent stimulation of InsP accumulation in FlpIn HEK293 5-HT_2B receptor-expressing cells. The InsP accumulation observed for each concentration of agonist is expressed as a percentage of the 5-HT E_max. Each panel shows isotherms for two drugs listed in Table 1 and for 5-HT. Similar experiments were performed for all drugs listed in Table 1, and the data were analyzed as described under Materials and Methods to obtain potency (pEC₅₀) and efficacy (E_max) estimates. Note: InsP accumulation stimulated by all tested drugs (at 1 μM) was completely blocked by pretreatment with 10 μM SB-206593 (data not shown).

Fig. 7.

Agonist-mediated proliferation responses in FlpIn HEK293 5-HT_2B receptor-expressing cells. XTT-based proliferation assay of agonist-treated FlpIn HEK293 5-HT_2B cells. Cells were stimulated for 48 h with the indicated drug at 1 nM, 30 nM, or 1 μM. Four hours before the end of the drug treatment phase, XTT reagent was added. Then, the 490-nm absorbance (OD_max of the XTT formazan metabolite) was read. OD₄₉₀ was proportional to cell number (data not shown). *, p < 0.05 compared with vehicle (two-way analysis of variance followed by Bonferroni post test). A, VHD-associated drugs were all potent at 30 nM; ropinirole was distinct from the valvulopathogens in that is was inactive at 30 nM. B, of the five FDA-approved medications we identified, only guanfacine and xylometazoline were active at 30 nM.

Fig. 8.

Hierarchical clustering analysis reveals a separation between ropinirole and valvulopathogens. Shown is an image generated by TreeView ver. 1.1.3 of pEC₅₀ data derived from Table 1 (see Materials and Methods for details). The x-axis represents “Assays,” whereas the y-axis represents “Drugs tested.” Data are colored for activity: <5, black; >9, yellow.