Old drugs with new skills: fenoprofen as an allosteric enhancer at melanocortin receptor 3

Abstract

The efficiency of drug research and development has paradoxically declined over the last decades despite major scientific and technological advances, promoting new cost-effective strategies such as drug repositioning by systematic screening for new actions of known drugs. Here, we performed a screening for positive allosteric modulators (PAMs) at melanocortin (MC) receptors. The non-steroidal anti-inflammatory drug fenoprofen, but not the similar compound ibuprofen, presented PAM activity at MC₃, MC₄, and MC₅ receptors. In a model of inflammatory arthritis, fenoprofen afforded potent inhibition while ibuprofen was nearly inactive. Fenoprofen presented anti-arthritic actions on cartilage integrity and synovitis, effects markedly attenuated in Mc3r-/- mice. Fenoprofen displayed pro-resolving properties promoting macrophage phagocytosis and efferocytosis, independently of cyclooxygenase inhibition. In conclusion, combining repositioning with advances in G-protein coupled receptor biology (allosterism) may lead to potential new therapeutics. In addition, MC₃ PAMs emerged as a viable approach to the development of innovative therapeutics for joint diseases.

Keywords: Arthritis; Drug repositioning; GPCR; Inflammation; Resolution of inflammation.

Fig. 1

Allosteric modulatory activity of fenoprofen at the human MC₃ receptor. cAMP production was studied in human MC₃ transfected CHO-K1 cells (GeneBLAzer^® beta-lactamase Reporter Technology). Fenoprofen was tested for agonistic activity (a, agonist mode) or in the presence of the agonist αMSH to address PAM activity in human and mouse receptors (b, PAM mode)

Fig. 2

Antiarthritic effect of fenoprofen in the K/BxN serum transfer model. Arthritis was induced on wild type (a–c) and Mc3r−/− (d–f) mice by two i.p. injections of arthritogenic serum on day 0 and 2. Pharmacological treatments (fenoprofen, Fen, and ibuprofen, Ibu: 10 mg/kg; vehicle: PBS) were administered i.p. twice daily from day 2. Non-arthritic mice were used as controls (Ctrl). Clinical score (a, d), paw volume (b,e) and disease incidence (c,f) were recorded over 8 days. Data are mean ± SEM of n = 5; *p < 0.05 ANOVA followed by Bonferroni multiple comparison test

Fig. 3

Gene expression and histological changes in arthritic joints. a Gene expression was analyzed in ankles from wild type (WT, black bars) and Mc3r−/− (white bars) mice as collected at day 8, and expressed as fold change with respect to non-arthritic control mouse joints. Groups correspond to vehicle (Veh), fenoprofen 10 mg/kg (Fen), and ibuprofen 10 mg/kg (Ibu). Data are mean ± SEM of n = 5; *p < 0.05 two-way ANOVA followed by Bonferroni multiple comparison test of veh vs. drugs, ^# p < 0.05 WT vs. Mc3r−/−. b Ankle sections (4 µm) were stained with hematoxylin and eosin (H&E) and fast green and safranin-O. Sections were graded from 0 (no disease) to 3 (severe) based on the degree of synovitis (purple staining in the H&E sections) and cartilage erosion (loss of red coloration in the safranin-O sections). C cell infiltrate, B bone, M muscle, F fat. The sum of H&E and safranin-O scores are represented in the left graph. Data are mean ± SEM of n = 5; p < 0.05 drug vs. Veh (*), or Veh vs. Ctrl (^#) ANOVA followed by Bonferroni multiple comparison test

Fig. 4

Production of endogenous melanocortin peptides in arthritic mouse joints and primary macrophages. a Wrists joints from K/BxN arthritic mice (wild type and Mc3r−/−) were collected at day 8 and protein extracts prepared on RIPA buffer. The melanocortin peptides αMSH, γMSH, and ACTH were determined by enzyme immunoassay following manufacturers’ protocol. b Biogel-elicited macrophages were collected from mice (wild type and Mc3r−/−) 4 days after an i.p. injection with 1 ml of 2% biogel. Cells were cultured in vitro during 24 h in 10% FCS-RPMI. Supernatants were analyzed for αMSH, γMSH, and ACTH levels by enzyme immunoassay. Data are mean ± SEM of n = 5; p < 0.05 ANOVA followed by Bonferroni multiple comparison test K/BxN vs. Ctrl (*) or WT vs. Mc3r−/− (^#)

Fig. 5

Pro-phagocytic actions of fenoprofen. Biogel-elicited peritoneal macrophages from wild type (WT) and Mc3r−/− mice were incubated with: a pHrodo™ Red E. coli for 20 min and analyzed by flow cytometry, or b apoptotic human neutrophils for 1 h and analyzed by myeloperoxidase (MPO) staining and cell counting. c Fenoprofen (Fen) and acetylsalicylic acid (ASA) were added 30 min prior addition of E. coli. d ASA was added 20 min before fenoprofen, and E. coli was added after 30 min incubation with fenoprofen. Images in e show that fluorescence develops specifically on ingested bacteria. Images in f show neutrophils (dark brown MPO staining) ingested by macrophages (efferocytosis). Data are mean ± SEM of n = 3; *p < 0.05 ANOVA followed by Bonferroni multiple comparison test