Fmo induction as a tool to screen for pro-longevity drugs

Abstract

Dietary restriction (DR) and hypoxia (low oxygen) extend lifespan in Caenorhabditis elegans through the induction of a convergent downstream longevity gene, fmo-2. Flavin-containing monooxygenases (FMOs) are highly conserved xenobiotic-metabolizing enzymes with a clear role in promoting longevity in nematodes and a plausible similar role in mammals. This makes them an attractive potential target of small molecule drugs to stimulate the health-promoting effects of longevity pathways. Here, we utilize an fmo-2 fluorescent transcriptional reporter in C. elegans to screen a set of 80 compounds previously shown to improve stress resistance in mouse fibroblasts. Our data show that 19 compounds significantly induce fmo-2, and 10 of the compounds induce fmo-2 more than twofold. Interestingly, 9 of the 10 high fmo-2 inducers also extend lifespan in C. elegans. Two of these drugs, mitochondrial respiration chain complex inhibitors, interact with the hypoxia pathway to induce fmo-2, whereas two dopamine receptor type 2 (DRD2) antagonists interact with the DR pathway to induce fmo-2, indicating that dopamine signaling is involved in DR-mediated fmo-2 induction. Together, our data identify nine drugs that each (1) increase stress resistance in mouse fibroblasts, (2) induce fmo-2 in C. elegans, and (3) extend nematode lifespan, some through known longevity pathways. These results define fmo-2 induction as a viable approach to identifying and understanding mechanisms of putative longevity compounds.

Keywords: C. elegans; Fmo-2 induction; Dietary restriction; Drugs; Flavin-containing monooxygenases; Hypoxia; Longevity.

Fig. 1

Drugs that increase stress resistance in mouse fibroblasts also increase Fmo4 and Fmo5 transcript levels. A–D Changes in expression of Fmo4 and Fmo5 mRNA in mouse fibroblasts from UM-HET3 mice treated with 4 μM of the indicated drugs compared to DMSO control. Fmo4 or Fmo5 mRNA levels were measured by qRT-PCR. Horizontal lines represent the mean, and error bars represent SD. Drugs that increase both Fmo4 and Fmo5 mRNA levels are shown in red in A and C. The x-axis values, shown on a log2 scale in B and D, represent the change of mRNA levels. The y-axis values, shown on a − log10 scale in B and D, represent the P-values. Drugs that affect the mRNA levels of Fmo4 or Fmo5 significantly are shown in red in B and D. E Drugs that co-regulate mRNA levels of Fmo4 and Fmo5 are shown by a scatter plot on a log2 scale fold change. Drugs that significantly increase both Fmo4 and Fmo5 mRNA levels are shown in red in the second quadrant. F Diagram showing that bioactive compounds increase stress resistance and induce Fmo4 and Fmo5 levels in mouse fibroblasts and will be tested for fmo-2 induction and lifespan extension in C. elegans. All drugs shown have P < 0.05 when compared to control (Welch two-sample t-test, two-sided)

Fig. 2

Ten strong and nine weak C. elegans fmo-2 inducers are identified from the 80 primary hits that increase stress resistance in mouse fibroblasts. Images and quantification of fmo-2p::mCherry after drug treatments. Strong hits (A–B, red) are defined as drugs that induce fmo-2 more than mianserin (as a cut-off threshold shown in green). Weak hits (C–D, blue) are those agents that induce fmo-2 more than 1.5-fold but less than mianserin dose (green). Dietary restriction (DR) condition is a positive control that induces fmo-2 (purple). Fluorescence intensity in A cannot be compared to intensity in C, since they were performed and analyzed in different experiments with different exposure times. Worms were treated with 1-μM rotenone, 100-μM deguelin, 100-μM pinaverium bromide (pinaverium), 100-μM dihydrorotenone, 30-μM chlorhexidine, 100-μM trifluoperazine, 500-μM diphenyleneiodonium (DPI), 500-μM butaclamol, 100-μM methylbenzethonium, 100-μM thioridazine for strong hits; 100-μM mianserin (~ twofold induction) as a cut-off threshold between strong hits (> twofold induction) and weak hits (< twofold but > 1.5-fold); 1-mM astemizole, 1-mM flunarizine, 1-mM MG-101, 1-mM furosemide, 0.5-mM curcumin, 1-mM vinblastine, 0.5-mM promethazine hydrochloride, 1-mM picropodophyllin, or 1-mM AEG3482 for weak hits. Scale bar, 1 mm. ****Indicates P < 0.0001 when compared to control worms (Welch two-sample t-test, two-sided). Each symbol represents 1 worm, horizontal lines represent means, and error bars represent standard deviation. Each experiment was repeated at least three times. Single dose selected from toxicity tests (Supplementary Table 4) of each drug was used in the experiment

Fig. 3

Nine of ten strong fmo-2 inducers increase lifespan in C. elegans. Survival curves of wild-type C. elegans after drug treatments. Lifespans were significantly (P < 0.05 by log-rank) extended by the indicated fmo-2 inducers under the specified concentration. Wild-type C. elegans (N2 Bristol) were synchronized to L4 stages, treated with indicated drugs, and then lifespan was measured. ****indicates P < 0.0001; ***indicates P < 0.001; **indicates P < 0.01 when compared to control treated worms (log-rank test). P-values (log-rank test), lifespan replicates, and lifespan statistics are listed in Supplementary Table 3. Each lifespan was performed at least two times with multiple doses. The best dose was performed at least four times, with one replicate shown here

Fig. 4

fmo-2 is required for fmo-2 inducers to increase lifespan in C. elegans. Survival curves of wild-type (N2 Bristol) or fmo-2 (ok2147) knockout animals after drug treatment. Lifespan extension by seven of nine of these drugs is significantly dependent on fmo-2. N2 and fmo-2 knockout strains were synchronized at L4 stages, treated with indicated drugs, and then lifespan was measured. ***indicates P < 0.001; **indicates P < 0.01 when drug induced lifespan extension of wild-type strain compared to fmo-2 strain (cox regression). P-values (log-rank test) and Cox regression lifespan analyses are listed in Supplementary Tables 3 and 5

Fig. 5

Mitochondrial complex I inhibitors induce fmo-2 and extend lifespan within the hypoxia pathway. A–B Images and quantification of fmo-2p::mCherry by strong drug hits in the egl-9 mutant background. Red denotes drugs that do not further induce fmo-2, while gray denotes significant additional induction of fmo-2 in the egl-9 mutant. Dietary restriction (DR) condition is a positive control that induces fmo-2 (purple). In all experiments, fmo-2p::mCherry;egl-9(sa307) animals were synchronized to L4 stage before treatment with the indicated drugs. Worms were treated with 1-μM rotenone, 100-μM deguelin, 100-μM pinaverium bromide, 100-μM dihydrorotenone, 30-μM chlorhexidine, 100-μM trifluoperazine, 500-μM diphenyleneiodonium (DPI), 500-μM butaclamol, 100-μM methylbenzethonium, 100-μM thioridazine, and 100-μM mianserin as a positive control. Scale bar, 1 mm. C–D Survival curves of vhl-1 knockout strains treated with 1-μM rotenone or 5-μM dihydrorotenone from L4 stage. Rotenone and dihydrorotenone do not significantly extend lifespan of vhl-1(ok161) worms. ****indicates P < 0.0001 when compared to control (Welch two-sample t-test, two-sided) in B. ***indicates P < 0.001 when drug induced lifespan extension of wild-type strain compared to vhl-1 strain (Cox regression) in C and D. P-values (log-rank test) and Cox regression analyses of lifespan statistics are listed in Supplementary Tables 3 and 5

Fig. 6

Dopamine D2 receptor antagonists and rotenone induce and extend lifespan in the DR pathway. A–B Images and quantification of fmo-2p::mCherry induction by strong drug hits under DR condition. Drugs that do not further induce fmo-2 are in red, while drugs that further induce are in gray. The DR condition (without drug treatment) is in purple. All fmo-2p::mCherry animals were synchronized to L4 stage and treated with indicated drugs under DR condition for 18 h. Worms were treated with 1-μM rotenone, 100-μM deguelin, 100-μM pinaverium bromide, 100-μM dihydrorotenone, 30-μM chlorhexidine, 100-μM trifluoperazine, 500-μM diphenyleneiodonium (DPI), 500-μM butaclamol, 100-μM methylbenzethonium, 100-μM thioridazine, and 100-μM mianserin as a control. Scale bar, 1 mm. C–F Survival curves of wild-type worms after drug treatments under DR condition. Rotenone and three DRD2 antagonists thioridazine, trifluoperazine, and butaclamol do not further extend lifespan under DR. Wild-type worms were synchronized to L4 stage, treated with 1-μM rotenone, 25-μM thioridazine, 0.1-μM trifluoperazine, or 5-μM butaclamol under DR condition, and lifespan was then measured. ***indicates P < 0.001; *indicates P < 0.05 when drug induced lifespan extension of worms under DR condition compared to fed condition (Cox regression) in C–F. P-values (log-rank test) and Cox regression analyses of lifespan curves comparisons were listed in Supplementary Table 3 by log-rank test. ****indicates P < 0.0001; **indicates P < 0.01; *indicates P < 0.05 when compared to control (Welch two-sample t-test, two-sided) in B