Nociceptin/orphanin FQ opioid receptor (NOP) selective ligand MCOPPB links anxiolytic and senolytic effects

Abstract

Accumulation of senescent cells may drive age-associated alterations and pathologies. Senolytics are promising therapeutics that can preferentially eliminate senescent cells. Here, we performed a high-throughput automatized screening (HTS) of the commercial LOPAC®Pfizer library on aphidicolin-induced senescent human fibroblasts, to identify novel senolytics. We discovered the nociceptin receptor FQ opioid receptor (NOP) selective ligand 1-[1-(1-methylcyclooctyl)-4-piperidinyl]-2-[(3R)-3-piperidinyl]-1H-benzimidazole (MCOPPB, a compound previously studied as potential anxiolytic) as the best scoring hit. The ability of MCOPPB to eliminate senescent cells in in vitro models was further tested in mice and in C. elegans. MCOPPB reduced the senescence cell burden in peripheral tissues but not in the central nervous system. Mice and worms exposed to MCOPPB also exhibited locomotion and lipid storage changes. Mechanistically, MCOPPB treatment activated transcriptional networks involved in the immune responses to external stressors, implicating Toll-like receptors (TLRs). Our study uncovers MCOPPB as a NOP ligand that, apart from anxiolytic effects, also shows tissue-specific senolytic effects.

Keywords: Aging; NOP; Senescence; Senolytic.

Fig. 1

Screening for senolytic compounds. A Setup for obtaining a large quantity of senescent MRC5 cells via prolonged exposition to low dose (0.2 μM) of replication stress inducer — aphidicolin. The images below are depicting the evolution of SA-β-gal staining (blue signal) during the treatment procedure. B Screening performed with LOPAC®Pfizer library on MRC5 cells expressed in the dot chart depicting the toxicity (percentage of inhibition, PI) of individual compounds towards normal proliferating cells (y-axis) and senescent cells (x-axis). The chart is accompanied by visual microscopic validation of the strongest hit MCOPPB. C Dose–response of MCOPPB on cell viability. Cells were incubated for 24 h with increasing concentrations (0–125-250–500-750 nM, 1–1.5–2-2.5–3-4–5 μM) of the drug, before viability assay (N = 6). Results are presented as mean ± SD. D CTL; MCOPPB; DOX; and DOX + MCOOPB-treated HepG2 or Huh-7 cells were incubated with C12FDG (N = 3), and positive cells were detected by flow cytometer. Results are presented as mean ± SEM. *p < 0.05 compared to CTL; ***p < 0.01 compared to CTL; # p < 0.05 compared to DOX

Fig. 2

Scheme of the experimental design of MCOPPB study. A Mice C57JBL6 20 weeks old fed chow diet were treated for 21 days with either 5 mg/kg of the drug or the vehicle with 2 days of wash-out every 5 days and then were divided into 2 groups (N = 12 each). Two groups received additional 2 days of wash-out and then were used for the PhenoMaster® experiment (Group 1) or sacrificed for Histological analysis at the 28th week (Group 2). B Effects of repeated treatment with MCOPPB or vehicle (CTL) on weight gain. Data are presented as mean ± SEM of n = 12 mice/group. *p < 0.05 vs CTL-treated group

Fig. 3

Effects of repeated treatment with MCOPPB or vehicle (CTL) on A–D locomotor activity in the open-field test (OFT), E mobility time in the forced swim test (FST), F cognitive performance, and in the Y-maze test of mice. Data are presented as mean ± SEM of n = 12 mice/group. *p < 0.05 and ***p < 0.01 vs CTL group

Fig. 4

MCOPPB decreases cellular senescence and increases fat accumulation in WAT adipocytes. A Representative SA-β-Gal staining and B representative H&E images of white adipose tissue from control and MCOPPB treated mice. The SA-β-Gal positive and lipid droplets area were calculated in 10 random high-power fields (HPF) at 200 × magnification and expressed as means. ***p < 0.01; compared to CTL

Fig. 5

MCOOPB decreases cellular senescence in mice livers. A Representative SA-β-Gal staining images from livers of control and MCOPPB treated mice. The SA-β-Gal positive areas were calculated in 10 random high-power fields (HPF) at 200 × magnification and expressed as means. MCOOPB induces mild hepatic stress in mice. B Representative liver H&E and PAS images from CTL and MCOPPB treated mice. C Steatosis, ballooning, and inflammation score of CTL and MCOPPB groups. The steatosis score for CTL group is zero. *p < 0.05; compared to CTL. ***p < 0.01; compared to CTL

Fig. 6

MCOOPB regulates senescence, immune, and pro-oxidative pathways in mice liver. A Heatmap showing differences in mRNA expression levels, B principal component analysis of the compared groups showing samples clustering on the first two principal components, C Bar plot reporting Z-score of biological pathways in crescent statistical significance order (bar length), and D STRING diagram displaying the protein interaction among the more representative pathways between control and MCOPPB-treated mice (n = 3–5 per group)

Fig. 7

MCOPPB decreases the number of macrophages and senescent cells in the mouse liver. A Immunofluorescence images of F4-80/B-gal double-positive cells. Frequency of B positive cells for F4-80, C positive cells for B-gal, D macrophages over B-gal + cells, and E B-gal + macrophages over total macrophages. Indicated in %, ***p < 0.01, ****p < 0.0001

Fig. 8

MCOPPB treatment increases lipid levels but decreases motility and is inconsequential to lifespan in C. elegans. A Quantification of lipid content in worms at L4 stage stained with Nile red, B activity measurement, C lifespan, and D qPCR of SKN-1 and DAF-16 target genes of worms treated with control or MCOPPB. ***p < 0.01, ****p < 0.0001