New thiazolidinones reduce iron overload in mouse models of hereditary hemochromatosis and β-thalassemia

Abstract

Genetic iron-overload disorders, mainly hereditary hemochromatosis and untransfused β-thalassemia, affect a large population worldwide. The primary etiology of iron overload in these diseases is insufficient production of hepcidin by the liver, leading to excessive intestinal iron absorption and iron efflux from macrophages. Hepcidin agonists would therefore be expected to ameliorate iron overload in hereditary hemochromatosis and β-thalassemia. In the current study, we screened our synthetic library of 210 thiazolidinone compounds and identified three thiazolidinone compounds, 93, 156 and 165, which stimulated hepatic hepcidin production. In a hemochromatosis mouse model with hemochromatosis deficiency, the three compounds prevented the development of iron overload and elicited iron redistribution from the liver to the spleen. Moreover, these compounds also greatly ameliorated iron overload and mitigated ineffective erythropoiesis in β-thalassemic mice. Compounds 93, 156 and 165 acted by promoting SMAD1/5/8 signaling through differentially repressing ERK1/2 phosphorylation and decreasing transmembrane protease serine 6 activity. Additionally, compounds 93, 156 and 165 targeted erythroid regulators to strengthen hepcidin expression. Therefore, our hepcidin agonists induced hepcidin expression synergistically through a direct action on hepatocytes via SMAD1/5/8 signaling and an indirect action via eythroid cells. By increasing hepcidin production, thiazolidinone compounds may provide a useful alternative for the treatment of iron-overload disorders.

Figure 1.

Screening of the thiazolidinone library for compounds stimulating hepcidin expression. (A) Synthesis of the thiazolidinone library. A total of 18 anilines and 20 aromatic aldehydes were used as reactants, and 210 thiazolidinone compounds were obtained with a purity greater than 95%, as determined by liquid chromatography/mass spectrometry. Reagents and conditions: (i) R₁-NH₂, NH SCN, H⁺², H₂O, 80°C; (ii) ethyl chloroacetate, NaOAC, EtOH, 60°C; (iii) aldehydes, piperdine, EtOH, 60°C. The bold letters (a d) delineate the synthesis procedure, as described in the Methods section: primary thioureas (b) were constructed by reacting aniline (a) with ammonium thiocyanate, in the presence of acid; thioureas (b) reacted with ethyl 2-chloroacetate to generate thiazolidinones (c) as a precipitate, which was filtered and washed with absolute ethanol to obtain the product; the final step of the reaction was carried out in piperidine and absolute ethanol at 60°C, and approximately 95% of the product (d) was formed as precipitate. (B) A heatmap diagram showing the average fold changes of hepcidin-promoter luciferase activity relative to that of untreated cells (n=4). (C) Endogenous hepcidin mRNA expression in SMMC-7721 cells upon administration of compounds at a concentration of 10 μM for 24 h (n=4). *P<0.05, #P<0.001, compared to untreated control (Ctrl).

Figure 2.

Testing thiazolidinone derivatives for their effects on hepatic hepcidin in wildtype mice. (A) Diagram of the experimental design. (B) Hepatic hepcidin mRNA, (C) serum hepcidin, (D) serum iron and (E) splenic iron in 8-week old Balb/C wildtype (Wt) mice treated with compounds 93, 156 and 165 at a dose of 30 mg/kg body weight at various time points (n=4-6). (F) Splenic iron shown by Perls Prussian blue staining (blue areas evidenced by arrows) of mice treated with compounds 93, 156 and 165 at a dose of 30 mg/kg body weight for 24 h and 12 days. Original magnification, ×200. *P<0.05; ^#P<0.001, compared to untreated control (Ctrl).

Figure 3.

Compounds 93, 156 and 165 targeted SMAD1/5/8 signaling. (A) P-SMAD1/5/8, P-ERK1/2 and TMPRSS6 levels determined by western blot analysis of liver specimens from 8-week old Balb/C mice 24 h after administration of compounds 93, 156 and 165 at a dose of 30 mg/kg body weight. (B) Changes of TMPRSS6 and downstream target genes of P-SMAD1/5/8 signaling: Id1 and Smad7 determined by quantitative reverse transcriptase polymerase chain reaction analysis (qRT-PCR) (n=4-6) in liver specimens of these mice. (C) Changes of hepcidin mRNA in Hepa 1-6 cells at the indicated times after treatment with compounds 93, 156 and 165 at a concentration of 10 μM (n=4-6). (D) Changes of Id1 and Samd7 were determined by qRT-PCR analysis (n=4) of Hepa 1-6 cells 24 h after treatment with compounds 93, 156 and 165 (10 μM). (E) Variations of P-SAMD1/5/8, SMAD1, P-ERK1/2, ERK1/2 and TMPRSS6 levels analyzed by western blot in Hepa 1-6 cells 24 h after treatment with compounds 93, 156 and 165 at a concentration of 10 μM. *P<0.05; ^#P<0.001, relative to untreated control (Ctrl).

Figure 4.

Compounds 93, 156 and 165 regulated Gdf15, Twsg1 and Erfe in bone marrow cells. (A) Changes of Gdf15, Twsg1 and Erfe mRNA expression in bone marrow cells from wildtype (Wt) Balb/C mice upon administration of the various compounds at a dose of 30 mg/kg for 24 h. (B) The variations of Gdf15, Twsg1 and Erfe mRNA expression in bone marrow cells from Hbb^th3/+ mice treated with the various compounds at a dose of 30 mg/kg body weight for 24 h. *P<0.05; ^#P<0.001, relative to untreated control (Ctrl).

Figure 5.

Treatment with compounds 93, 156 and 165 redistributed iron in Hfe^−/− mice. (A) Serum hepcidin in wildtype (Wt) 129S and Hfe^−/− mice at different ages. (B) The experimental scheme. Changes of (C) serum hepcidin, (D) serum iron and (E) hepatic iron in Hfe^−/− mice after treatment with compounds 93, 156 and 165 at a dose of 10 mg/kg body weight for 2 weeks (n=4-6). (F) Perls Prussian blue staining of liver and spleen (in blue, indicated by arrows) and 3′-diaminobenzidine-enhanced Perls Prussion staining of duodenum (in brown) of 5-week-old Hfe^−/− mice treated with compounds 93, 156 and 165 at a dose of 10 mg/kg body weight every other day for 2 weeks. Original magnification, ×200 for spleen sections; ×400 for liver and duodenum sections. *P<0.05; ^#P<0.001, compared to the untreated, control (Ctrl) Hfe^−/− mice.

Figure 6.

Compound administration to iron-depleted Hfe^−/− mice. (A) The experimental design of treatment of iron-depleted Hfe^−/− mice with compounds 93, 156 and 165. Changes in (B) serum hepcidin, (C) serum iron, (D) splenic iron and (E) hepatic iron of 9-week old Hfe^−/− mice with iron depletion for 3 weeks prior to the administration of compounds 93, 156 and 165 at a dose of 30 mg/kg body weight for another 2 weeks (n=4-6). (F) Tissue iron staining of liver and spleen sections with Perls Prussian blue (in blue, indicated by arrows) and duodenal sections with 3′-diaminobenzidine-enhanced Perls stain (in brown). Original magnification, ×200 for spleen, and ×400 for liver and duodenum. *P<0.05; ^#P<0.001, relative to untreated control (Ctrl) mice.

Figure 7.

Compounds 93, 156 and 165 alleviated iron overload in Hbb^th3/+ mice. (A) Serum hepcidin, (B) serum iron, (C) serum ferritin (D) hepatic iron, (E) splenic iron and (F) 3′-diaminobenzidine-enhanced Perls iron staining of liver sections (in brown) and Perls Prussion staining of spleen sections (in blue, indicated by arrows) after administration of compounds 93, 156 and 165 to Hbb^th3/+ mice at a dose of 10 mg/kg body weight every other day for 2 weeks (n=4-6). Original magnification, ×400. *P<0.05, relative to untreated, control (Ctrl) mice.

Figure 8.

Compounds 93, 156 and 165 improved ineffective erythropoiesis in Hbb^th3/+ mice. (A) Hemoglobin (HGB) content and (B) red blood cell (RBC) count in peripheral blood samples from 4-week old Hbb^th3/+ mice following administration of compounds 93, 156 and 165 at a dose of 10 mg/kg body weight every other day for 2 weeks (n=3-4). (C) Blood smears (original magnification, ×1,000) with damaged or deformed erythrocytes indicated by arrows and (D) representative erythropoiesis profiles of bone marrow cells from these mice (n=3-4). *P<0.05, compared to untreated, control (Ctrl) Hbb^th3/+ mice.