Discovery of Novel Fetal Hemoglobin Inducers Through Small Chemical Library Screening

Home , Fetal hemoglobin, Globin, HBG1, HBG2, Sickle cell disease

International Journal of Molecular Sciences

Article Discovery of Novel Fetal Hemoglobin Inducers through Small Chemical Library Screening

Giulia Breveglieri 1, Salvatore Paciﬁco 2 , Cristina Zuccato 1, Lucia Carmela Cosenza 1, Shaiq Sultan 1, Elisabetta D’Aversa 1, Roberto Gambari 1, Delia Preti 2, Claudio Trapella 2, Remo Guerrini 2,* and Monica Borgatti 1,3,*

1 Department of Life Sciences and Biotechnology, University of Ferrara, Via Fossato di Mortara 74, 44121 Ferrara, Italy; [email protected] (G.B.); [email protected] (C.Z.); [email protected] (L.C.C.); [email protected] (S.S.); [email protected] (E.D.); [email protected] (R.G.) 2 Department of Chemical and Pharmaceutical Sciences, University of Ferrara, Via Luigi Borsari 46, 44121 Ferrara, Italy; salvatore.paciﬁ[email protected] (S.P.); [email protected] (D.P.); [email protected] (C.T.) 3 Center of Biotechnology, University of Ferrara, Via Fossato di Mortara 64b, 44121 Ferrara, Italy * Correspondence: [email protected] (R.G.); [email protected] (M.B.)

Received: 4 June 2020; Accepted: 2 October 2020; Published: 8 October 2020

Abstract: The screening of chemical libraries based on cellular biosensors is a useful approach to identify new hits for novel therapeutic targets involved in rare genetic pathologies, such as β-thalassemia and sickle cell disease. In particular, pharmacologically mediated stimulation of human γ-globin gene expression, and increase of fetal hemoglobin (HbF) production, have been suggested as potential therapeutic strategies for these hemoglobinopathies. In this article, we screened a small chemical library, constituted of 150 compounds, using the cellular biosensor K562.GR, carrying enhanced green fluorescence protein (EGFP) and red fluorescence protein (RFP) genes under the control of the human γ-globin and β-globin gene promoters, respectively. Then the identified compounds were analyzed as HbF inducers on primary cell cultures, obtained from β-thalassemia patients, confirming their activity as HbF inducers, and suggesting these molecules as lead compounds for further chemical and biological investigations.

Keywords: hemoglobinopathies; β-thalassemia; sickle cell disease; fetal hemoglobin; chemical screening; compound library; cellular biosensors

1. Introduction The screening of chemical libraries is a useful approach for drug discovery and identification of new therapeutic molecules, by using in vitro assays based on enzymatic activity, fluorescence signaling, calcium or ionic efflux, and cellular biosensors. In this context, these tools can be important in identifying new hits for novel therapeutic targets involved in rare genetic pathologies, such as β-thalassemia and sickle cell disease (SCD). The β-thalassemias are inherited hematological genetic disorders, caused by more than 300 mutations of the human β-globin gene, causing reduced or absent synthesis of adult β-globin, and a consequent excess of α-globins, which induces ineffective erythropoiesis and severe anemia [1]. The annual incidence at birth of symptomatic cases is estimated at 1/100,000 worldwide, with a great prevalence in the developing world [1]. The β-thalassemias can be grouped in different classes, depending on the β-globin gene mutations (β0 and β+-thalassemias) and on the transfusion dependency (transfusion-dependent and transfusion independent β-thalassemias). In the case of the most severe forms of β-thalassemia, regular blood transfusions and chelating therapies are the

Int. J. Mol. Sci. 2020, 21, 7426; doi:10.3390/ijms21197426 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 7426 2 of 12 conventional therapeutic approaches [2–5]. While bone marrow transplantation is currently the only resolving treatment of the disease [6], several potentially curative strategies are under clinical validation, including gene therapy [7], gene editing [8], and treatment with novel agents potentiating hematopoiesis (i.e., luspatercept) [9]. SCD is a rare monogenic, hematological, and multi-organ disorder, associated with acute and chronic illness, and progressive organ damage [10]. The disease is due to a single point mutation (Glu6Val) on the HBB gene, causing polymerization of the mutant hemoglobin (Hb) S, and resulting in sickling of erythrocytes [11]. The clinical severity for these two hemoglobinopathies could be reduced by reactivating the expression of γ-globin, absent after birth. In this case, even a small increase in the percentage of fetal hemoglobin (HbF), replacing the deficient adult hemoglobin (HbA), could ameliorate the clinical picture, reducing anemia and transfusion burden in β-thalassemia, and inhibiting polymerization of sickle hemoglobin (HbS) in SCD [12–14]. This has been demonstrated by several findings, including the fact that hereditary persistence of fetal hemoglobin (HPFH) is associated with high HbF production in adults, causing clinically mild phenotypes, even in severe forms of β-thalassemia and SCD [15–17]. Accordingly, genome editing to create the HPFH genotype in erythroid precursor cells has been proposed as a useful approach for treating SCD and β-thalassemia [17]. In addition, several investigations have been instigated to identify effective HbF inducers [18–22]. Currently, hydroxyurea (HU) is the only FDA-approved therapeutic treatment for the induction of HbF in SCD patients [13,23]. With respect to β-thalassemia, in spite of its verified benefits in certain cases, demonstrated by the reach of a transfusion-independent phenotype in patients with β-thalassemia intermedia [24,25], specific approvals for β-thalassemia are lacking. On the other hand, adverse effects and potential toxicity in the case of long-term treatment are expected, and have been described for HU [26,27]. Furthermore, not all patients respond to HU treatment, or can become insensitive after repeated administrations [13,28]; therefore, the discovery and investigation of new HbF inducers is certainly needed. In this work, we have screened a small chemical library constituted of 150 compounds, randomly selected, and exhibiting heterogeneous chemotypes, using our cellular biosensor K562.GR carrying enhanced green fluorescence protein (EGFP) and red fluorescence protein (RFP) genes under the control of the human γ-globin and β-globin gene promoters, respectively [29]. This system is suitable for identifying the induction ability on the transcription of γ-globin and β-globin genes in erythroid cells in an efficient and reproducible way [29,30]. This investigation resulted in the identification of a subset of compounds that were ultimately analyzed as Hb inducers on primary cell cultures obtained from β-thalassemia patients. This assay has been widely used to validate the HbF-inducing potential of molecules identified using large or middle-scale screening approaches [31–33].

2. Results

2.1. Screening of the Chemical Library Using the K562.GR Cellular Biosensor In order to discover novel fetal hemoglobin (HbF) inducers from the collected chemical library, we performed a first screening based on the K562.GR reporter system. The clone K562.GR contains a reporter construct with EGFP (enhanced green fluorescent protein) and RFP (red fluorescent protein) genes under the control of the human γ-globin or β-globin gene promoters, respectively, and it has been validated as a high throughput screening (HTS) system in previous publications [29,30]. With this system, a potential HbF inducer can be easily identified by fluorescence-activated cell sorting (FACS) detection of the specific fluorescent protein, in response to the activation of the corresponding promoter. On the contrary, if the tested compound does not induce either the γ-globin or the β-globin promoter, no fluorescence is detected. Despite the fact that in this study we focused on HbF inducers, it should be underlined that the system would also allow the identification of transcription activators of the β-globin promoter, in the case RFP is increased instead of EGFP [29,30]. Int. J. J. Mol. Mol. Sci. Sci. 20202020,, 2121,, x 7426 3 of 14 12

For each compound of the chemical library, four concentrations (10 μM, 1 μM, 100 nM, and 10 nM) wereFor each administered compound to of K562.GR the chemical cells library, for 5 days, four and concentrations then FACS (10 analysisµM, 1 µwasM, 100performed. nM, and 10 nM) wereFigure administered 1 shows to a K562.GR representative cells for example 5 days, andof the then FACS FACS analysis, analysis obtained was performed. during the screening of theFigure 150 compounds1 shows a representative of the library, example dissolved of in the dimethylsulfoxide FACS analysis, obtained (DMSO), during plotting the the screening curves generatedof the 150 by compounds cells in the of presence, the library, and dissolved in the absence, in dimethylsulfoxide of the treatment. (DMSO), plotting the curves generatedHydroxyurea by cells in(HU) the presence,was selected and as in a the positive absence, control of the (Figure treatment. 1A), considering the fact that it has beenHydroxyurea firmly established (HU) was selectedto be able as to a positiveinduce controlγ-globin (Figure gene 1expression.A), considering For this the factreason, that HU it has is usedbeen firmlyin clinical established applications to be ablefor β to-thalassemia induce γ-globin and SCA gene treatment expression. [1 For0]. As this expected, reason, HU when is used EGFP in fluorescence,clinical applications dependent for β -thalassemiaon the activation and SCAof the treatment γ-globin gene [10]. Aspromoter, expected, was when measured EGFP fluorescence,(Figure 1A), adependent clear shift on to thethe activationright of the of curve the γ-globin for the geneHU tr promoter,eated cells was (solid measured line), compared (Figure1A), to athe clear untreated shift to cellsthe right (bold of solid the curve line), forwas the obtained. HU treated On the cells contra (solidry, line), no shift compared was observed to the untreated when RFP cells fluorescence (bold solid forline), β-globin was obtained. gene promoter On the contrary,activation no was shift analyzed was observed (Figure when 1B). RFPAfter fluorescence a qualitative for analysisβ-globin of gene the promoterhistograms activation obtained was from analyzed all the (Figurecompounds1B). After of the a qualitative library, most analysis of the of molecules the histograms did not obtained show detectablefrom all the fluorescence compounds increases, of the library, similarly most to of compound the molecules 1 (Figure did not 1E,F); show the detectable gaussian curves fluorescence of the treatedincreases, samples similarly were to compoundperfectly superimposed 1 (Figure1E,F); on the those gaussian of the curves untreated of the control, treated samplesindicating were the perfectlyabsence superimposedof an induction on thoseeffect of on the both untreated promot control,ers. On indicating the contrary, the absence some of anof inductionthe analyzed effect compoundson both promoters. produced On thea shift contrary, to higher some oflevels the analyzed of EGFP compounds fluorescence, produced similar a shiftto that to higher found levels for compoundof EGFP fluorescence, 63 (Figure similar1C,D). toInterestingly, that found forcompou compoundnd 63 63(as (Figure HU) was1C,D). also Interestingly, unable to induce compound RFP, confirming63 (as HU) wasthat alsoits effects unable are to inducerestricted RFP, toconfirming the γ-globin that gene its epromoterffects are (associated restricted to to the a clearγ-globin increase gene promoterof EGFP production). (associated to a clear increase of EGFP production).

Figure 1. Representative examples examples of of FACS FACS analysisanalysis of K562.GR cells, untreated (bold solid lines) or µ µ µ treated (solid(solid lines),lines), with with 150 150 MμM HU HU (A (,A,BB), 10), 10M μ compoundM compound 63 (63C,D (C,D), and), and 10 M10 compoundμM compound1 (E,F 1), (respectively.E,F), respectively. The CheLiFe The CheLiFe compounds compounds were dissolved were dissolved in DMSO. in 30,000DMSO. cells 30,000 were cells analyzed were analyzed after five days of treatment, by detecting both enhanced green fluorescence protein (EGFP) (A,C,E) and RFP after five days of treatment, by detecting both enhanced green fluorescence protein (EGFP) (A,C,E) (B,D,F) fluorescence. and RFP (B,D,F) fluorescence. In order to identify the compounds able to induce the γ-globin gene, we selected nine potentially In order to identify the compounds able to induce the γ-globin gene, we selected nine active molecules, with values of median fold over control, for EGFP signal, greater than 1.35 in at least potentially active molecules, with values of median fold over control, for EGFP signal, greater than one of the four tested concentrations (Table1 and Table S1 of Supplementary Materials). 1.35 in at least one of the four tested concentrations (Table 1 and Table S1 of Supplementary Interestingly, while most of the compounds were active only at the highest concentration (10 µM), Materials). showing no induction activity at the other concentrations, some of them, such as 56, 58, and 59, Interestingly, while most of the compounds were active only at the highest concentration (10 also showed a relevant activity at low concentrations (1 µM). The 1.35 value was identified as a μM), showing no induction activity at the other concentrations, some of them, such as 56, 58, and 59, also showed a relevant activity at low concentrations (1 μM). The 1.35 value was identified as a 3 Int. J. Mol. Sci. 2020, 21, 7426 4 of 12 minimum cut-off in previous articles, where the same K562.GR cellular biosensor was validated using well-known fetal hemoglobin inducers, such as 4,40,6-Trimethylangelicin (TMA) and HU [29,30,34]. In addition, the experiments shown in Figure1, Table1, and Table S1 (Supplementary Materials) were repeated using the same compounds dissolved in a methanol solution containing 5% DMSO. This was done considering that DMSO decreased the number of K562.GR cells (data not shown) when used as a negative control. Moreover, as reported in the literature, DMSO reduces the production of hemoglobin and represses the erythroid phenotype in K562 cells [35]. On the contrary, we demonstrated that methanol/DMSO does not affect the erythroid differentiation after treatment of immortalized K562.GRInt. J. Mol. Sci. clones, 2020, 21 showing, x no changes in relative EGFP (Figure S1A,C of Supplementary Materials)5 of 14 and Int. J. Mol. Sci. 2020, 21, x 5 of 14 RFP (Figure S1B,D of Supplementary Materials) fluorescence, detectable by FACS analysis. Int. J. Mol. Sci. 2020, 21, x 5 of 14 Compound Structure 10 nM 100 nM 1 µM 10 µM Int.Int. J. Table J.Mol. Mol. Sci. Sci. 1.Compound 2020 2020Values, 21, 21, x, x of median fold Structure over control for EGFP 10 nM signals, 100 expressed nM 1 µM as mean 10 µM value 5standard 5of of 14 14 ± Int.deviation, J. Mol. Sci. 2020 obtained, 21, x from three diNCfferent H treatments of K562.GR cells with each compound at5 of 10 14 nM, Int. J. Mol. Sci. 2020, 21, x N 5 of 14 100 nM,Compound 1 µM6 and 10 µM concentration. StructureNC H The CheLiFe compounds101.15 nM 1001.05 were nM dissolved 11.00 µM 10in1.57 DMSO.µM Compound N N CompoundCompound Structure Structure CF 10 10 nM nM 100 100 nM nM 1 1µM µM 10 10 µM µM 56 resulted toxic6 at 10 µM( ). * Structure non disclosable3 1.15 for patenting1.05 reasons.1.00 1.57 Compound − N StructureNC H 10 nM 100 nM 1 µM 10 µM N CF3 Compound6 StructureNCNC H H 101.15 nM 1001.05 nM 11.00 µM 101.57 µM CompoundN Structure NN 10 nM 100 nM 1 µM 10 µM NC H CF 6 6 N 3 1.151.15 1.051.05 1.001.00 1.571.57 NNNC H 6 N CFCF3 3 1.15 1.05 1.00 1.57 N 66 CF 1.151.15 1.05 1.051.00 1.001.57 1.57 N 3 17 CF3 0.90 0.94 0.99 1.48 17 0.90 0.94 0.99 1.48

17 0.90 0.94 0.99 1.48 1717 0.900.90 0.940.94 0.990.99 1.481.48 1717 0.90 0.94 0.94 0.99 0.99 1.48 1.48 17 0.90 0.94 0.99 1.48 22 0.94 0.92 1.02 1.53 22 0.94 0.92 1.02 1.53

2256 ND * 0.941.02 0.921.05 1.021.59 1.53 - 2256 ND * 0.941.02 0.921.05 1.021.59 1.53 - 572222 ND * 1.100.940.94 1.000.92 0.92 1.071.02 1.021.361.53 1.53 5722 ND * 1.100.94 1.000.92 1.071.02 1.361.53

2256 ND * 0.941.02 0.921.05 1.021.59 1.53 - 56 ND * 1.02 1.05 1.59 - 57585656 ND ND ** * 1.100.991.02 1.02 1.001.131.05 1.05 1.071.551.59 1.591.361.62 - - 575756 NDNDND * ** 1.101.101.02 1.001.001.05 1.071.071.59 1.361.36 - 585657 ND ND * 0.991.02 1.10 1.131.05 1.001.551.59 1.071.62 - 1.36 57 ND * 1.10 1.00 1.07 1.36 57 ND * 1.10 1.00 1.07 1.36 58 0.99 1.13 1.55 1.62

585858 O 0.990.990.99 1.131.13 1.13 1.551.55 1.551.621.62 1.62 O 58 N 0.99 1.13 1.55 1.62 58 N H 0.99 1.13 1.55 1.62 59 N 1.03 1.20 2.00 2.10 N O 59 N H 1.03 1.20 2.00 2.10 O N NO 59 N 1.03 1.20 2.00 2.10 59 H O 1.03 1.20 2.00 2.10 NN NN OH H 5959 N 1.031.03 1.201.20 2.002.00 2.102.10 N H 59 NN N 1.03 1.20 2.00 2.10 59 N H 1.03 1.20 2.00 2.10 62 N 1.01 0.96 0.98 1.35 N 6262 1.011.01 0.96 0.960.98 0.981.35 1.35

62 1.01 0.96 0.98 1.35

6262 1.011.01 0.960.96 0.980.98 1.351.35

62 1.01 0.96 0.98 1.35 62 1.01 0.96 0.98 1.35

6363 1.021.02 1.01 1.01 1.04 1.041.78 1.78 63 1.02 1.01 1.04 1.78

63 1.02 1.01 1.04 1.78

* Structure non disclosable for patenting reasons. 6363 * Structure non disclosable for patenting1.021.02 reasons.1.011.01 1.041.04 1.781.78 63 * Structure non disclosable for patenting1.02 reasons.1.01 1.04 1.78 63 1.02 1.01 1.04 1.78 Table 2. Values of median fold over control for EGFP signals, expressed as mean value ± standard

Tabledeviation, 2. Values obtained of median from three* Structurefold diff overerent noncontrol treatments disclosable for EGFP of for K562.GR signals, patenting expressedcells, reasons. with aseach mean compound value ± atstandard 10 nM, * *Structure Structure non non disclosable disclosable for for patenting patenting reasons. reasons. deviation,100 nM, 1 obtainedμM, and from10 μM three concentration. different treatments The CheLiFe of K562.GR compounds cells, were with dissolved each compound in methanol at 10 nM,+5% Table 2. Values of median *fold Structure over control non disclosable for EGFP for signals, patenting expressed reasons. as mean value ± standard 100DMSO. nM, Compounds1 μM, and 10 56 μ andM concentration. 59 resulted toxic The at CheLiFe 10 μM (compounds−). were dissolved in methanol +5% Tabledeviation, 2. Values obtained of median from three* Structurefold diff overerent noncontrol treatments disclosable for EGFP of for K562.GR signals, patenting expressedcells, reasons. with aseach mean compound value ± atstandard 10 nM, DMSO.Table 2.Compounds Values of median56 and 59fold resulted over control toxic at for 10 EGFPμM (− ).signals, expressed as mean value ± standard deviation,100deviation,Table nM, 2.1 obtainedμValues obtainedM, and of 10from medianfrom μM three threeconcentration. fold diff diff overerenterent control treatments treatmentsThe CheLiFe for EGFP of of K562.GR compoundsK562.GR signals, cells, cells,expressed were with with dissolved each eachas meancompound compound in value methanol at± at standard10 10 nM,+5% nM, Table 2. Values ofCompd median fold 10 over nM control 100for EGFPnM signals, 1 µM expressed 10as µMmean value ± standard 100DMSO.100deviation, nM, nM, Compounds1 1μ μ M,obtainedM, and and 10 1056 from μ μandMM concentration.three concentration.59 resulted different toxic Thetreatments The at CheLiFe CheLiFe10 μM of (compounds− K562.GRcompounds). cells, were were with dissolved dissolved each compound in in methanol methanol at 10 +5% +5% nM, Compd6 1.09 10 nM± 0.08 1.11 100 ±nM 0.07 1.14 1 µM ± 0.06 1.47 10 µM± 0.27 DMSO.deviation,100 nM, Compounds 1obtained μM, and from 56 10 and μ threeM 59concentration. resulteddifferent toxic treatments The at 10CheLiFe μ ofM K562.GR(−). compounds cells, withwere each dissolved compound in methanol at 10 nM, +5% DMSO. Compounds6 56 and 1.0959 resulted ± 0.08 toxic 1.11 at ± 10 0.07 μM (− 1.14). ± 0.06 1.47 ± 0.27 100DMSO. nM, 1 Compounds μM, andCompd 10 μ56M and concentration. 59 10 resulted nM toxicThe 100 CheLiFe at nM10 μ M compounds (−). 1 µM were 10dissolved µM in methanol +5% 5 DMSO. CompoundsCompdCompd 566 and 59 1.09 resulted 10 10 nM± nM0.08 toxic 1.11 100at 100 10 ±nM 0.07nMμM ( −). 1.14 1 1µM ±µM 0.06 1.47 10 10 µM± µM 0.27 5 Compd 10 nM 100 nM 1 µM 10 µM 6 6 1.09 1.09 ± ±0.08 0.08 1.11 1.11 ± ±0.07 0.07 1.14 1.14 ± ±0.06 0.06 1.47 1.47 ± ±0.27 0.27 5 Compd6 1.0910 nM ± 0.08 100 1.11 nM ± 0.07 1.14 1 µM ± 0.06 1.4710 µM ± 0.27 6 1.09 ± 0.08 1.11 ± 0.07 1.14 ± 0.06 1.47 ± 0.27 55 5 5 Int. J. Mol. Sci. 2020, 21, 7426 5 of 12

Using methanol/DMSO, three independent experiments on K562.GR clones were performed (Table2), in order to confirm the increase in EGFP levels, detected by FACS analysis in the previous experiments performed in DMSO (Table1). The obtained median folds over control for EGFP analysis were greater than 1 and, in general, higher than the values measured during the first screening. In any case, we were able to confirm that the effects of the selected compounds were independent from the employed vehicle (DMSO versus methanol/DMSO).

Table 2. Values of median fold over control for EGFP signals, expressed as mean value standard ± deviation, obtained from three diﬀerent treatments of K562.GR cells, with each compound at 10 nM, 100 nM, 1 µM, and 10 µM concentration. The CheLiFe compounds were dissolved in methanol +5% DMSO. Compounds 56 and 59 resulted toxic at 10 µM( ). − Compd 10 nM 100 nM 1 µM 10 µM 6 1.09 0.08 1.11 0.07 1.14 0.06 1.47 0.27 ± ± ± ± 17 1.13 0.07 1.17 0.05 1.18 0.04 1.42 0.13 ± ± ± ± 22 1.11 0.08 1.19 0.06 1.29 0.05 1.89 0.27 ± ± ± ± 56 1.11 0.05 1.19 0.12 1.47 0.20 - ± ± ± 57 1.13 0.12 1.13 0.09 1.21 0.10 1.60 0.30 ± ± ± ± 58 1.13 0.07 1.20 0.11 1.53 0.31 1.54 0.20 ± ± ± ± 59 1.14 0.06 1.38 0.24 1.77 0.15 - ± ± ± 62 1.15 0.11 1.18 0.05 1.24 0.09 1.61 0.28 ± ± ± ± 63 1.14 0.09 1.17 0.07 1.20 0.08 1.63 0.28 ± ± ± ±

2.2. Analysis of HbF Induction in a Human Erythroleukemic K562 Cell Line The specificity of the induction activity of the nine compounds was determined by performing the same treatments on wild type K562 cells without the reporter construct. As reported in Table3, the values of median fold over control were close to or less than 1, confirming for all nine compounds the absence of increased cellular fluorescence, and indicating that any fluorescence detection after the K562.GR treatment was presumably due to a specific induction of the γ-globin promoter, and not to an autofluorescence phenomenon of the employed compounds.

Table 3. Values of median fold over control for EGFP signals, expressed as mean value standard ± deviation, obtained from three diﬀerent treatments of K562 cells, with each compound at 10 nM, 100 nM, 1 µM, and 10 µM concentration. The CheLiFe compounds were dissolved in methanol +5% DMSO. Compounds 56 and 59 resulted toxic at 10 µM.

Compound 10 nM 100 nM 1 µM 10 µM 6 0.76 0.17 0.67 0.15 0.72 0.17 0.57 0.02 ± ± ± ± 17 0.70 0.19 0.62 0.16 0.58 0.12 0.50 0.03 ± ± ± ± 22 0.70 0.23 0.62 0.15 0.61 0.13 0.49 0.04 ± ± ± ± 56 0.75 0.15 0.69 0.14 0.72 0.24 - ± ± ± 57 0.75 0.25 0.63 0.22 0.65 0.21 0.63 0.22 ± ± ± ± 58 0.73 0.18 0.67 0.18 1.07 0.43 0.79 0.24 ± ± ± ± 59 0.76 0.20 0.66 0.18 0.83 0.43 - ± ± ± 62 0.69 0.25 0.60 0.24 0.61 0.22 0.60 0.23 ± ± ± ± 63 0.74 0.27 0.63 0.21 0.61 0.23 0.58 0.19 ± ± ± ± Int. J. Mol. Sci. 2020, 21, 7426 6 of 12

After this first screening, the effects of the selected compounds were investigated on cell growth, and on hemoglobin production by the K562 cell line. This cell line is a very useful experimental model system, since it synthesizes hemoglobin at very low levels when untreated, but significantly increases the expression of embryo-fetal globins after treatment with inducers of erythroid differentiation [36]. Table4 reports the IC 50 values (relative to the effects on cell proliferation) and the percentages of benzidine-positive cells calculated after 5 days of treatment. The analyzed compounds can be divided into three classes based on the relative IC50. The first one comprises 56, 58, and 59, showing the lowest values: 0.85 µM, 0.69 µM, and 0.10 µM, respectively. The second class includes compounds 6, 17, and 22, exhibiting intermediate values, between 1.2 and 5. Finally, for the other three molecules it was not possible to determine the IC50 value, since in our experimental conditions we did not detect significant antiproliferative effects at the maximum tested concentration (10 µM).

Table 4. Eﬀects of the nine compounds from the CheLiFe library, selected after screening with the

K562.GR reporter system, on K562 cell proliferation and diﬀerentiation. The IC50 value, determined, when possible, by counting K562 cells after 5 days from administration, is reported for each compound, together with the percentage of benzidine-positive cells after treatment with diﬀerent concentrations of compounds for 7 days.

Benzidine Assay Compound IC50 (µM) Concentration Benzidine-Positive Cells (%) 1 µM 4 6 5.00 5 µM 9 10 µM 15 1 µM 6 17 3.60 5 µM 10 10 µM 11 1 µM 4 22 1.20 5 µM 7 10 µM 18 0.5 µM 1 56 0.85 1 µM 2 5 µM 1 1 µM 5 57 >10.00 5 µM 6 10 µM 8 0.5 µM 6 58 0.69 1 µM 5 5 µM 3 0.5 µM 2 59 0.10 1 µM 3 5 µM 2 1 µM 3 62 >10.00 5 µM 4 10 µM 8 1 µM 2 63 >10.00 5 µM 3 10 µM 6 Int. J. Mol. Sci. 2020, 21, 7426 7 of 12

As far as the hemoglobin production analyzed using a benzidine test (Table4), the same compounds showed an induction of effects on erythroid differentiation of K562 cells at different concentrations, and most of them in a dose-dependent manner. During this analysis, we noted that compounds with higher (>1 µM) or not determined IC50 (because it was greater than 10 µM) generally showed differentiation values dependent on the administered dose, even reaching quite high percentages of positive cells (for example, 15% for compound 6, 11% for compound 17, or 18% for compound 22, at 10 µM). In contrast, the three compounds with lower IC50 showed lower differentiation values, between 1 and 6%.

2.3. HbF Induction of Erythroid Precursors from β-Thalassemia Patients The results obtained in K562 cell lines, prompted us to determine whether the nine molecules were able to induce the expression of γ-globin genes in primary cultures of erythroid precursor cells (ErPCs) obtained from β-thalassemia patients, evaluating the HbF production by HPLC. In total seven β-thalassemia patients were recruited, and their ErPCs isolated and treated as described elsewhere [37]. A representative example of HPLC analysis after 5-day treatment of these isolated ErPCs is reported in Figure2. The analysis of the Hb content of these cultures showed an increase of the HbF peak after the treatment with compound 63 at 10 µM (Figure1B), relative to untreated cells (Figure1A), Int. J. Mol. Sci. 2020, 21, x 8 of 14 while no relevant increase of the peaks corresponding to HbA and HbA2 was detected.

FigureFigure 2. 2. RepresentativeRepresentative example example of of HPLC HPLC analysis analysis after after erythroid erythroid precursor precursor cells cells treatment. treatment. ChromatogramsChromatograms obtained obtained from from HPLC HPLC assay assay of of erythroid erythroid precursor precursor cells cells of of patient patient 6 6 (a (a ββ-thalassemia-thalassemia patient),patient), untreated ((AA)) oror afterafter 5 5 days days of of treatment treatment with with 10 10µM μM compound compound 63 (63B). ( TheB). The positions positions of fetal of (HbF) and adult (HbA ed HbA ) hemoglobin peaks are indicated by arrows. fetal (HbF) and adult (HbA ed HbA2 2) hemoglobin peaks are indicated by arrows.

For each compound, we compared the HbF fold induction over untreated control values to those For each compound, we compared the HbF fold induction over untreated control values to obtained after HU treatment (used as positive control) (Table5). those obtained after HU treatment (used as positive control) (Table 5).

8 Int. J. Mol.Int. J.Sci. Mol. 2020 Sci., 21 2020, x , 21, x 9 of 149 of 14

TableTable 5. Graphic 5. Graphic representation representation of fold of induction fold induction over controlover control values values for HbF for production,HbF production, obtained obtained Int. J.Int. Mol. Int.J. Mol.Sci. J. Mol. 2020 Sci. Sci. 2020, 21 2020, x, 21 , 21x , x 9 of 149 of 9 14 of 14 Int. J. Mol. Sci. 2020, 21, x after treatmentafter treatment of erythroid of erythroid precursor precursor cells ofcells seven of seven different different 8β of-thalassemic 12 β-thalassemic patients patients with thewith nine the nine compoundscompounds under under investigation. investigation. TableTable 5.Table Graphic 5. Graphic5. GraphicInt. representation J. Mol.representation representation Sci. 2020 of, 21 fold ,of 7426 foldofinduction fold induction induction over over control over control control values values valuesfor HbFfor forHbF production, HbF production, production, obtained obtained obtained 8 of 12

afterafter treatmentafter treatment treatment of erythroid of erythroidof erythroid precursor precursor precursor cells cells of cells seven of sevenof differentseven different different β-thalassemic β-thalassemic β-thalassemic patients patients patients with with the with ninethe the nine nine PatientPatient No. No. compoundscompoundscompounds under under investigation.under investigation. investigation. CompoundCompound Table 5.5. GraphicGraphic representation of fold induction over control values for HbF production,1 2 1 3 obtained 2 4 3 5 4 6 5 7 6 7 after treatment of erythroid precursorPatientPatientPatient cellsNo. No. of No.seven different β-thalassemic6 6patients with the nine after treatmentCompoundCompoundCompound of erythroid precursor cells of seven diﬀerent β-thalassemic patients with the nine compounds under investigation.1 12 123 234 345 456 567 67 7 17 17 6 6 6 Patient No. 22 22 Compound 17 17 17 1 2 3 4 5 6 7 56 56 22 22 22 6 57 57 56 56 56 17 58 58 57 57 57 22 59 59 58 58 58 56 62 62 57 59 59 59 63 63 58 62 62 62 59 63 63 63 62 not treated; not analyzable; fold induction <50% HU; 63 not treated; not analyzable; fold induction <50% HU;

not treated;notnot notnot treated; treated; treated; treated; not not not analyzabnot analyzable; not analyzable;analyzab analyzable; le; le; fold fold fold induction fold induction induct fold induction inductionHU. >HU. induction >HU. fold induction >HU.

In 50% detail, 50% HU 50% compounds HUHU. induction >HU. >HU. values greater than that of HU in all treatmentsIn detail, of erythroid compounds precursor 6, 17, and cells 63 of showed different HbFβ-thalassemia fold induction patients, values while greater the other than compoundsthat of HU In detail,In detail, compounds compounds 6, 17, 6, and 17, 63and showed 63 showed HbF foHbFld inductionfold induction values values greater greater than thanthat ofthat HU of HU reportedin all treatments fold induction of erythroid values precursor greater than cells that of ofdifferent HU in oneβ-thalassemia or two patient patients, cultures. while This the resultother in allin treatments all treatments of erythroid of erythroid precursor precursor cells cellsof different of different β-thalassemia β-thalassemia patients, patients, while while the other the other confirmedcompounds the reported erythroid fold di inductionfferentiation values ability greater of compounds than that of 6 andHU in 17 one demonstrated or two patient in K562 cultures. cells compoundscompounds reported reported fold inductionfold induction values values greater greater than ththatan ofthat HU of inHU one in or one two or patienttwo patient cultures. cultures. In detail,In Indetail, detail,compounds compounds(TableThis compounds result4 ),6, suggesting 17, 6,confirmed and17,6, 17, and 63 and their showed63 the 63showed potentialerythroid showed HbF HbF fo application HbFdifferentiationld fo induction ldfo ldinduction induction as chemicalvalues ability values values greater of inducers greater compounds greater than ofthan that HbFthan that 6of synthesis.andthat HU of 17HU of HUdemonstrated in in allin intreatmentsall alltreatments treatments ofK562 erythroidof of erythroidcells erythroid (Table precursor precursor 4), precursor suggesting cells cells of cells different oftheirThis ofdifferent potentialdifferent resultThis β-thalassemia result βconfirmed-thalassemia appβ-thalassemia confirmedlication patients,the as patients,erythroid thechemicalpatients, erythroidwhile while differentiation inducerswhilethe differentiationtheother the other of otherHbF ability synthesis. ability of compounds of compounds 6 and 6 17and demonstrated 17 demonstrated in in compoundscompoundscompounds reported reported reported3. Discussionfold fold induction fold induction induction values values values greater greater greater thK562an th that K562ancellsth anthat of (Tablecellsthat HU of (TableofHU in 4), HU one insuggesting 4),in oneor suggestingone two or ortwo patient their two patient theirpotentialpatient cultures. potentialcultures. cultures. app lication app lication as chemical as chemical inducers inducers of HbF of synthesis.HbF synthesis. ThisThis resultThis result resultconfirmed confirmed confirmed3. Discussion the theerythroid the erythroid erythroid differentiation differentiation differentiation ability ability ability of compounds of ofcompounds compounds 6 and 6 and 617 and demonstrated17 17demonstrated demonstrated in in in Inherited hemoglobin disorders (such as sickle-cell disease and thalassemias) are among the most K562K562 cellsK562 cells (Table cells (Table (Table4), suggesting 4), 4),suggesting suggesting their their potential their potential potential app application 3.application Discussionlication3. as Discussion chemical as chemicalas chemical inducers inducers inducers of HbF of HbFof synthesis. HbF synthesis. synthesis. frequentInherited monogenic hemoglobin diseases, disorders originally (such characteristic as sickle-cell of the disease tropics andand subtropics,thalassemias) but noware among commonly the InheritedInherited hemoglobin hemoglobin disorders disorders (such (such as sickle-cell as sickle-cell disease disease and thalassemias)and thalassemias) are among are among the the distributedmost frequent worldwide, monogenic mainly diseases, due originally to migration characteristic [38]. Hydroxyurea of the tropics (HU) and is subtropics, currently thebut onlynow 3. Discussion3. Discussion3. Discussion most mostfrequent frequent monogenic monogenic diseases, diseases, originally originally characteristic characteristic of the of tropics the tropics and subtropics,and subtropics, but now but now FDA-approvedcommonly distributed drug for worldwide, the induction mainly of HbF due in to SCD migration patients, [38]. but itHydroxyurea is effective in (HU) only 70%is currently of patients, the commonlycommonly distributed distributed worldwide, worldwide, mainly mainly due to due migration to migration [38]. Hydroxyurea[38]. Hydroxyurea (HU) (HU) is currently is currently the the InheritedInheritedInherited hemoglobin hemoglobinandonly hemoglobin aFDA-approved solution disorders disorders disorders is required (such drug (such as(such for sickle-cellas the assickle-cell induction 30%sickle-cell disease non-responders disease of disease HbFand and thalassemias)in and SCD thalassemias) [39 thalassemias)], patients, while are regarding but areamong areit among is amongeffective βthe-thalassemia the the in only patients, 70% of only FDA-approvedonly FDA-approved drug drugfor the for induction the induction of HbF of inHbF SCD in SCDpatients, patients, but it but is effective it is effective in only in 70%only of70% of mostmost frequentmost frequent frequent monogenic monogenicinpatients, monogenic spite diseases, of and itsdiseases, sustainedadiseases, solution originally originally benefitsoriginally is required characteristic characteristic for characteristic some for th untransfusedeof 30% the of ofthenon-responderstropics the tropics patientstropics and and subtropics, and with subtropics,[39], subtropics,β -thalassemiawhile but butregardingnow but now intermedianow β -thalassemia [24,25], patients,patients, and aand solution a solution is required is required for th fore 30% the non-responders30% non-responders [39], while[39], while regarding regarding β-thalassemia β-thalassemia commonlycommonlycommonly distributed distributed distributedspecificpatients, worldwide, worldwide, approvals inworldwide, spite mainly for ofmainly βmainlyits -thalassemiadue sustained due to due migration to migrationto aremigrationbenefi lacking. [38].ts [38]. Hydroxyureafor [38]. Therefore, Hydroxyureasome Hydroxyurea untransfused HbF(HU) (HU) induction is (HU) currently is patients currentlyis currently might the with the be the of β great-thalassemia interest patients,patients, in spite in spiteof its of sustained its sustained benefi benefits forts somefor some untransfused untransfused patients patients with withβ-thalassemia β-thalassemia onlyonly FDA-approvedonly FDA-approved FDA-approvedforintermedia drug the drug developmentfor drug thefor [24,25], for theinduction the induction specific induction of therapeutic of HbFapprovals of HbFof in HbF SCD protocolsin forSCDin patients, SCDβ-thalassemia patients, for patients, thesebut butit hemoglobinopathies is butare iteffective is itlacking. effectiveis effective in Therefore, only in onlyin 70% [only13 70%, 23ofHbF 70% ,38 of ].induction of In this context, might intermediaintermedia [24,25], [24,25], specific specific approvals approvals for β -thalassemiafor β-thalassemia are lacking. are lacking. Therefore, Therefore, HbF inductionHbF induction might might patients,patients,patients, and and a solutionand a solutionchemicalbe a solutionof is great required is library requiredis interest required for screening thforfore for th30% the eth using30% enon-respondersdevelopment 30% non-responders cellular non-responders biosensors of [39], th erapeutic[39], while [39], canwhile regardingwhile be protocols regarding considered regarding β-thalassemia for β-thalassemia atheseβ potential-thalassemia hemoglobinopathies strategy to help be ofbe great of great interest interest for the for developmentthe development of th oferapeutic therapeutic protocols protocols for these for these hemoglobinopathies hemoglobinopathies patients,patients,patients, in spitein inspite ofthe[13,23,38].spite itsof pharmaceutical ofsustainedits its sustainedIn sustainedthis beneficontext, developmentbenefi tsbenefi chemicalforts tsforsome for andsome librarysomeuntransfused identification untransfused untransfusedscreening patients of using drugspatients patients cellularwith for with their βwith -thalassemiabiosenso therapeuticβ-thalassemia β-thalassemiars can treatment. be considered In this a [13,23,38].[13,23,38]. In this In context,this context, chemical chemical library library screen screening usinging using cellular cellular biosenso biosensors canrs be can considered be considered a a intermediaintermediaintermedia [24,25], [24,25], [24,25], articlepotentialspecific specific specific we approvals strategy screened approvals approvals forto a small helpβfor-thalassemia for β -thalassemia chemicalthe β-thalassemia pharmaceutical are library arelacking. are lacking. composed lacking. deTherefore,velopment Therefore, Therefore, of 150 HbF hits,and HbF induction HbF andidentification induction induction we identifiedmight might mightof drugs two interesting for their potentialpotential strategy strategy to help to helpthe pharmaceuticalthe pharmaceutical development development and identificationand identification of drugs of drugs for their for their be ofbe begreatof ofgreat interestgreat interest interestleadtherapeutic for moleculesforthe for thedevelopment treatment.the development (compoundsdevelopment In of this thof 6erapeutic article ofth and erapeuticth 17),erapeutic we protocols confirmingscreened protocols protocols afor small their forthese for HbFchemicalthese hemoglobinopathiesthese induction hemoglobinopathies hemoglobinopathieslibrary in composed different cellularof 150 hits, models and therapeutictherapeutic treatment. treatment. In this In article this article we screened we screened a small a small chemical chemical library library composed composed of 150 of hits, 150 andhits, and [13,23,38].[13,23,38].[13,23,38]. In this In Inthis context, (cellularwethis context, identified context, chemical biosensor, chemical chemicaltwo library interesting immortalized library library screen screen lead screening erythroleukemic ingmoleculesusinging using usingcellular cellular(com cellular biosensopounds cell biosenso line,biosenso rs6 andcanrs primarycanrs17),be can considered beconfirming beconsidered cell considered cultures atheir a HbF obtaineda induction from we identifiedwe identified two interesting two interesting lead moleculeslead molecules (com pounds(compounds 6 and 6 17), and confirming 17), confirming their theirHbF inductionHbF induction potentialpotentialpotential strategy strategy strategy toβin-thalassemia differenthelpto tohelp thehelp thecellularpharmaceutical patients)the pharmaceutical pharmaceutical models without (cellular development cytotoxicityde velopmentde biosensor,velopment and eff ects.and immortalizedidentification and Inidentification addition,identification erythroleukemicof compound drugsof ofdrugs drugsfor 63 fortheir deserves forcell their theirline, consideration and primary in differentin different cellular cellular models models (cellular (cellular biosensor, biosensor, immortalized immortalized erythroleukemic erythroleukemic cell line, cell andline, primary and primary therapeutictherapeutictherapeutic treatment. treatment. treatment.incell future Incultures this In experiments,Inthis article this articleobtained articlewe screenedwe focusing we screenedfrom screened aβ onsmall-thalassemia a itssmall a mechanismchemicalsmall chemical chemical patients) library for library activitylibrary composed without composed composed as a HbFofcytotoxicity 150 of inducer 150ofhits, 150 hits, and hits, (seeeffects. and Figuresand In 1addition, and2). cell culturescell cultures obtained obtained from fromβ-thalassemia β-thalassemia patients) patients) without without cytotoxicity cytotoxicity effects. effects. In addition, In addition, we identifiedwewe identified identified two two interestingcompound two interesting Theseinteresting lead data63 lead deservesmolecules representlead molecules molecules consideration the(com first(compounds (com steppoundspounds ofin 6 aandfuture process6 and 17),6 and experiments, 17),confirming of 17), drugconfirming confirming discovery, theirfocusing their HbF their that HbF oninduction involvesHbF its induction mechanisminduction the identification for activity of compoundcompound 63 deserves 63 deserves consideration consideration in future in future experiments, experiments, focusing focusing on its on mechanism its mechanism for activity for activity in differentin differentin different cellular cellular cellular novelasmodels a modelsHbF hits models (cellular inducer (considered (cellular (cellular biosensor,(see biosensor, inFigures biosensor, this immortalized study 1 immortalizedand byimmortalized 2). the screening erythroleukemic erythroleukemic erythroleukemic of a small cell library cellline, cell ofline, and randomlyline, and primary and primary primary selected compounds), as a HbFas a inducerHbF inducer (see Figures (see Figures 1 and 1 2). and 2). cell cellculturescell cultures cultures obtained obtained andobtained theThesefrom developmentfrom fromβdata-thalassemia β -thalassemiarepresentβ-thalassemia of a programpatients) the patients) patients)first of without medicinalstep without withoutof cytotoxicity chemistry,a cytotoxicityproc cytotoxicityess ineffects.of order drugeffects. effects. toIn producediscovery, addition,In Inaddition, addition, analogues, that involves to increase the TheseThese data datarepresent represent the firstthe firststep stepof a ofproc a essproc ofess drug of drugdiscovery, discovery, that thatinvolves involves the the compoundcompoundcompound 63 deserves63 63deservestheidentification deserves a considerationffinity, consideration consideration selectivity, of novel in future inhits effi futureincacy (considered futureexperiments,/potency, experiments, experiments, in metabolic thisfocusing focusing study focusing stability,on by itson the mechanismonits screening and itsmechanism mechanism oral bioavailability forof a activityfor small for activity activitylibrary of the of randomly identified identificationidentification of novel of novel hits (considered hits (considered in this in study this study by the by screening the screening of a small of a small library library of randomly of randomly as aas HbF aas HbF ainducer HbF inducer inducer (seeleadselected (see Figures (see compounds. Figures Figurescompounds), 1 and 1 and 2).1 Inand 2). addition, 2).and the these development data represent of a the program starting of point medicinal for the futurechemistry, structure–activity in order to selectedselected compounds), compounds), and theand developmentthe development of a ofprogram a program of medicinal of medicinal chemistry, chemistry, in order in order to to TheseThese Thesedata data representdatarelationshipproduce represent represent analogues,the (SAR), thefirst the first instep firstto order step increase ofstep to aof analyze procof athe procaess theaffinity,proc essof chemicaless drugof selectivity,ofdrug structuredrugdiscovery, discovery, discovery, efficacy/potency,/biological that that involvesthat activity involves involves metabolic correlation,the the the stability, and identify and produceproduce analogues, analogues, to increase to increase the affinity,the affinity, selectivity, selectivity, efficacy/potency, efficacy/potency, metabolic metabolic stability, stability, and and identificationidentificationidentification of novel oftheoral novelof hitsnovel specific bioavailability hits (considered hits (considered chemical (considered ofin group thethis in identifiedthis instudy responsible this study studyby theleadby for bythescreening compounds. HbFthe screening screening increase of a of Insmall in aof addition, oursmall a librarysmall cellular library librarythese of models. randomly of data randomlyof randomly Therepresent SAR investigation the starting selectedselectedselected compounds), compounds), compounds),willpoint and permit forand the andthe synthetizing thedevelopment thefuture development development structure–activity new of derivatives aof program ofa programa program of relationof the medicinalof bioactive ofmedicinalship medicinal (SAR), chemistry, compounds chemistry, chemistry,in order in 6, order in17,to inorder and analyze orderto 63, to modifying to the chemical their 9 9 produceproduceproduce analogues, analogues, analogues,chemicalstructure/biological to increaseto toincrease structure increase the theaffinity, to theactivity improveaffinity, affinity, selectivity, correlation, theirselectivity, selectivity, biological efficacy/potency, andefficacy/potency, efficacy/potency, eidenffecttify and the potencymetabolic specificmetabolic metabolic with stability,chemical lowstability, stability, toxicity. andgroup and and responsible for 9 9 9 8 Int. J. Mol. Sci. 2020, 21, 7426 9 of 12

Further experimental efforts on ErPCs isolated from β-thalassemia patients with different mutations of the β-globin gene, different HbF-related polymorphisms, and different starting levels of endogenously produced HbF are necessary to verify the therapeutic potential of the identified compounds. In addition, since SCD is expected to greatly benefit from production of anti-sickling HbF, ErPCs from SCD patients should be considered for experimental validation. Furthermore, combined treatments using compounds identified in the present study administered together with HbF-inducers employed in clinical trials (i.e., sirolimus, see clinical trials NCT03877809 and NCT04247750) might help to verify whether synergisms using different molecules can occur in inducing HbF. Finally, treatment with HbF inducers (including those described in this study) might also be combined with other strategies aimed at augmentation of HbF, for instance (a) inhibiting the activity of γ-globin gene transcriptional repressors (such as BCL11A, MYB, and ZBTB7A/LRF) [40–43], and (b) gene editing aimed at the genomic sequences negatively regulating γ-globin genes [44–46].

4. Materials and Methods

4.1. Chemical Library The chemical library collects 150 chemical compounds developed at the University of Ferrara. Each compound was dissolved in dimethylsulfoxide (DMSO) at a concentration of 10 mM, with a minimum degree of purity of 90%, evaluated by HPLC or mass analysis. In the ﬁrst screening of K562.GR clone, the compounds were tested in DMSO, while for the following experimental cultures they were diluted in methanol + 5% DMSO. Analytical data (1HNMR spectra and found (M+H) + ESI mass) of the reference compounds of the manuscript are reported in Table S2, Supplementary Materials.

4.2. Cell Lines and Culture Conditions The human erythroid leukemia K562 cells [47] and the derived cellular biosensor K562.GR [29,30] were cultured in RPMI 1640 medium (Sigma Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (FBS; Biowest, Nuaillé, France) with addition of 100 units/mL penicillin and 100 µg/mL streptomycin (Pen-Strep; Lonza Group, Basel, Switzerland). Experimental cultures were carried out by adding the appropriate drug concentrations to the cells usually seeded at 8000 cells/mL in 24-well plates. Cell growth was determined as cell number per ml using a Z2 Coulter (Beckman Coulter, Brea, CA, USA). The number of K562 cells containing hemoglobin was assayed by benzidine staining as elsewhere described [37].

4.3. FACS Analysis For the determination of fluorescence intensity of treated K562 and K562.GR clones, BD FACSCanto™ II instrument and BD FACSDiva™ Software (Becton Dickinson, Franklin Lakes, NJ, USA) were used. After a PBS washing step, 30,000 cells were analyzed by the fl1 channel to detect green fluorescence. The results were expressed as median fold over control, that is the ratio between the median of fluorescence intensity values obtained by cells in the presence, and in the absence, of treatment.

4.4. Erythroid Precursor Cultures Obtained from Blood of Thalassemia Patients Blood samples were collected from the Thalassemic Day Hospital (DHT) of the University Hospital “Sant’Anna” (Cona, Ferrara, Italy). Written informed consent was obtained from each patient, and peripheral blood samples were collected just before the blood transfusion. The primary cell cultures were obtained using a two-phase liquid culture procedure, as previously described [37,48]. Brieﬂy, mononuclear cells were isolated from blood by density gradient centrifugation, and cultured in α-MEM (Sigma Aldrich, St. Louis, MO, USA), 10% FBS, 1 µg/mL cyclosporine A (Sigma Aldrich, St. Louis, MO, USA), 10% conditioned medium from the 5637 bladder carcinoma cell line and 10 ng/mL stem cell Int. J. Mol. Sci. 2020, 21, 7426 10 of 12

factor (SCF; Life Technologies, Carlsbad, CA, USA). After seven days incubation at 37 ◦C and in an atmosphere of 5% CO2, the non-adherent cells were harvested, washed, and cultured in fresh medium composed of α-MEM, 30% FBS, 1% deionized bovine serum albumin (BSA; Sigma Aldrich, St. Louis, 5 MO, USA), 10− M β-mercaptoethanol (Sigma Aldrich, St. Louis, MO, USA), 2 mM L-glutamine (Sigma 6 Aldrich, St. Louis, MO, USA), 10− M dexamethasone (Sigma Aldrich, St. Louis, MO, USA), 1 U/mL human recombinant erythropoietin (Tebu-bio, Magenta, MI, Italy), and 10 ng/mL SCF. After seven days of incubation, the cells were seeded at 1 106/well in a 12-well plate, and treated with compounds × for five days before the HPLC analysis to determine the proportion of HbF (as percentage of total Hb). In particular, a Beckman Coulter instrument System Gold 126 Solvent Module-166 Detector was used, in combination with a Syncropak CCM 103/25 column eluting the samples in a solvent gradient using aqueous sodium acetate-BisTris-KCN buffers, as previously reported [37,48]. The calculated percentages were used to evaluate HbF induction after the treatment, taking into consideration the starting hemoglobin levels of each individual patient, by applying the following algorithm: % induction = ((% HbF after treatment—% HbF before treatment)/(100—% HbF before treatment)) 100. × Supplementary Materials: The following are available online at http://www.mdpi.com/1422-0067/21/19/7426/s1, Figure S1: Representative examples of FACS analysis of K562.GR cells, Table S1: Values of median fold over control obtained after treatment of K562.GR cells, Table S2: Analytical data (1HNMR spectra and found [M + H] + ESI mass) of the reference compounds of the manuscript. Author Contributions: Conceptualization: R.G. (Remo Guerrini), D.P., C.T., R.G. (Roberto Gambari) and M.B.; Data curation: G.B., R.G. (Remo Guerrini), D.P., C.T., R.G. (Roberto Gambari) and M.B.; Funding acquisition: R.G. (Remo Guerrini) and R.G.(Roberto Gambari); Investigation: G.B., S.P., C.Z., L.C.C., S.S. and E.D.; Methodology: G.B., S.P., C.Z., L.C.C., S.S., E.D., C.T. and M.B.; Validation: G.B., D.P. and M.B.; Writing—original draft: G.B. and M.B.; Writing—review and editing: R.G. (Remo Guerrini), D.P., C.T. and R.G. (Roberto Gambari) All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by Bando per il finanziamento di Progetti di Ricerca Interdisciplinari di Ateneo (PRIA)-2014 to Remo Guerrini. Roberto Gambari was supported by: UE THALAMOSS Project (Thalassemia Modular Stratification System for Personalized Therapy of β Thalassemia; n. 306201-FP7-HEALTH-2012- INNOVATION-1); Wellcome Trust, United Kingdom (Innovator Award 208872/Z/17/Z); AIFA, Italy (AIFA- 2016-02364887). Conflicts of Interest: The authors declare no conflict of interest.

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