Page 91 - 2019_09-HaematologicaMondo-web

Thiazolidinones reduce iron overload
common worldwide in regions where malaria was histori- cally endemic, is a genetic erythrocyte disorder character- ized by ineffective erythropoiesis, anemia, and progressive iron overload.8 For both HH and β-thalassemia patients, long-term iron overload causes liver cirrhosis, cardiomy- opathy, and endocrinopathies.7 Iron excess is currently managed by phlebotomy in HH and chelation in iron-load- ing anemias,9 but both treatments have serious limitations, including suboptimal compliance and secondary suppres- sion of hepcidin, which results in a further increase of dietary iron uptake.7,10 Iron-chelating drugs can adversely affect ocular, auditory and renal functions11-13 and their administration can be burdensome, e.g., because of the short half-life of desferrioxamine.14,15 Other approaches, such as mini-hepcidin peptides, are still at the experimental stage.16,17 We therefore searched for hepcidin agonists with favorable characteristics for clinical applications.
The thiazolidinone scaffold can be engineered to target diverse pathologies, with derivatives that inhibit tumor growth, repress viral replication and diminish inflammato- ry responses.18,19 A thiazolidinone derivative [(Z)-5-(4- methoxybenzylidene) thiazolidine-2,4-dione] ameliorated liver injury and fibrogenesis,20 suggesting that this class of compounds could target hepatocytes. In the current study, we established a library of thiazolidinone derivatives to look for lead compounds that could increase hepcidin con- centration. We report here the identification of three novel compounds that ameliorated iron overload in HH and β- thalassemia mice by stimulating the hepatic production of hepcidin.
Methods
Synthesis and characterization of thiazolidinone compounds
Thiazolidinone compounds were synthesized using the combi- natorial library synthesis approach, and a previously described overall synthesis route.21 Briefly, primary thioureas (b) were con- structed by reacting aniline (a) with ammonium thiocyanate, in the presence of acid (Figure 1A). 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. Finally, approximately 95% of the product (d) was formed as a precipitate (Online Supplementary Figure S1).
Statistical analysis
The differences between individual treated groups relative to the untreated control were assessed using independent t-tests. The significance of mean differences for two or more treatment groups relative to the untreated control was determined by one-way analysis of variance. Data are shown as the mean ± standard devi- ation (SD). Statistical significance was accepted when P<0.05.
Other experimental details are provided in the Online Supplementary Data.
Results
Synthesis and characterization of the combinatorial thiazolidinone library
To search for hepcidin agonists, we designed a thiazo- lidinone compound library by incorporating diverse R1
and R2 groups on the thiazolidinone core.18 Following the procedure illustrated in Figure 1A, a combinatorial library of thiazolidinone compounds containing 210 members was synthesized (Online Supplementary Figure S1 and Online Supplementary Table S2), using protocols that we have previously reported.21 All compounds used for the animal experiments were then purified either by recrystal- lization or column chromatography to reach a purity ≥98% as measured by liquid chromatography with ultra- violet detection at 214 nm (LC/UV214), and their structures were characterized by 1H-nuclear magnetic resonance and high-resolution mass spectrometry (Online Supplementary Table S3).
Screening of thiazolidinone derivatives for hepcidin-stimulatory activity
We performed high-throughput screening of the thiazo- lidinone library using a dual luciferase reporter system developed in the laboratory.22 As shown in Online Supplementary Figure S2, no significant cytotoxicity was detected at 10 μM or 50 μM for these thiazolidinone com- pounds in SMMC-7721 cells, a hepatocyte cell line used for hepcidin screening. Hence, 10 μM was chosen as the test concentration. Following treatment with thiazolidi- none derivatives for 24 h, hepcidin-luciferase activity was measured. As shown in Figure 1B, of the 210 compounds tested, 42 compounds were identified to increase luciferase activity by >1.3 fold relative to the untreated control. Of these, 30 compounds increased luciferase activity by more than 1.5 fold: 12 (compounds 2, 15, 49, 53, 68, 93, 96, 139, 163, 165, 189 and 194) by approximate- ly 2 fold, two (compounds 22 and 23) by 2.5 fold, and one (compound 3) by 3.5 fold, relative to untreated cells (Figure 1B). The 30 compounds that were found to increase luciferase activity by more than 1.5 fold were subsequently rescreened by quantitative reverse transcrip- tase polymerase chain reaction (qRT-PCR) for stimulation of endogenous hepcidin expression in SMMC-7721 cells.
As the qRT-PCR results revealed (Figure 1C), ten of the 30 compounds increased hepcidin mRNA expression by more than 1.5 fold, compared to untreated cells, consistent with the luciferase reporter results (Figure 1B). Hepcidin mRNA expression was increased by nearly 6 fold after treatment with compounds 48 and 165 for 24 h, and com- pound 69 enhanced hepcidin expression by more than 3 fold, compared to untreated cells (P<0.001) (Figure 1C). Compound 93 stimulated hepcidin expression by approx- imately 2.5 fold (P<0.05) (Figure 1C) and compounds 49, 53, 139, 140, 142 and 156 increased hepcidin expression by approximately 2 fold (P<0.05) (Figure 1C), compared to untreated cells. By contrast, 13 compounds were not found to alter endogenous hepcidin expression, while compounds 2, 5, 11, 23 and 112 elicited inhibition of endogenous hepcidin transcription (P<0.05) (Figure 1C). Accordingly, compounds 48, 49, 53, 69, 93, 139, 140, 142, 156 and 165 were selected for further assessment.
To examine the hepcidin-stimulating activity of the ten potential agonists in vivo, we administered them by intraperitoneal injection to wildtype (Wt) Balb/C mice at a dose of 30 mg/kg body weight. As shown in Online Supplementary Figure S3A, compounds 93 and 156 signifi- cantly increased hepatic hepcidin mRNA expression by 1.8 fold, respectively, and 1.5 fold at 6 h following admin- istration of the compound, with a concomitant reduction of serum iron levels (P<0.05) (Online Supplementary Figure
haematologica | 2019; 104(9)
1769