Baicalein, 7,8-Dihydroxyflavone and Myricetin As Potent Inhibitors Of

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Article Baicalein, 7,8-Dihydroxyﬂavone and Myricetin as Potent Inhibitors of Human Ornithine Decarboxylase

1, 1,2, 1, 1 2 Yun-Chin Liu †, Yi-Liang Liu †, Ju-Yi Hsieh †, Chang-Hsu Wang , Chi-Li Lin , Guang-Yaw Liu 2,3,* and Hui-Chih Hung 1,4,5,*

1 Department of Life Sciences, National Chung Hsing University, Taichung 402, Taiwan; [email protected] (Y.-C.L.); [email protected] (Y.-L.L.); [email protected] (J.-Y.H.); [email protected] (C.-H.W.) 2 Institute of Medicine, College of Medicine, Chung Shan Medical University, Taichung 402, Taiwan; [email protected] 3 Division of Allergy, Immunology, and Rheumatology, Chung Shan Medical University Hospital, Taichung 402, Taiwan 4 Institute of Genomics and Bioinformatics, National Chung Hsing University, Taichung 402, Taiwan 5 iEGG and Animal Biotechnology Center, National Chung Hsing University, Taichung 402, Taiwan * Correspondence: [email protected] (G.-Y.L.); [email protected] (H.-C.H.); Tel.: +886-4-2284-0416 (G.-Y.L.); +886-4-2473-0022 (H.-C.H.) These authors contributed equally to this work. † Received: 26 October 2020; Accepted: 15 December 2020; Published: 17 December 2020

Abstract: Background: Human ornithine decarboxylase (ODC) is a well-known oncogene, and the discovery of ODC enzyme inhibitors is a beneficial strategy for cancer therapy and prevention. Methods: We examined the inhibitory effects of a variety of flavone and flavonol derivatives on ODC enzymatic activity, and performed in silico molecular docking of baicalein, 7,8-dihydroxyflavone and myricetin to the whole dimer of human ODC to investigate the possible binding site of these compounds on ODC. We also examined the cytotoxic effects of these compounds with cell-based studies. Results: Baicalein, 7,8-dihydroxyflavone and myricetin exhibited significant ODC suppression activity with IC50 values of 0.88 µM, 2.54 µM, and 7.3 µM, respectively, which were much lower than that of the active-site irreversible inhibitor α-DL-difluoromethylornithine (IC50, the half maximal inhibitory concentration, of approximately 100 µM). Kinetic studies and molecular docking simulations suggested that baicalein, and 7,8-dihydroxyflavone act as noncompetitive inhibitors that are hydrogen-bonded to the region near the active site pocket in the dimer interface of the enzyme. Baicalein and myricetin suppress cell growth and induce cellular apoptosis, and both of these compounds suppress the ODC-evoked anti-apoptosis of cells. Conclusions: Therefore, we suggest that the flavone or flavonol derivatives baicalein, 7,8-dihydroxyflavone, and myricetin are potent chemopreventive and chemotherapeutic agents that target ODC.

Keywords: ornithine decarboxylase; chemoprevention; molecular docking simulations: baicalein; 7,8-dihydroxyﬂavone; myricetin

1. Introduction Ornithine decarboxylase (ODC; EC 4.1.1.17) is the ﬁrst and rate-limiting enzyme in the biosynthesis of polyamines (putrescine, spermidine, and spermine). The biological functions of ODC and polyamines, such as embryonic development, cell diﬀerentiation, cell growth, cancer proliferation, cell transformation and aging, have been extensively studied [1–6]. The cellular levels of ODC are tightly regulated by a variety of physiological events [7].

Nutrients 2020, 12, 3867; doi:10.3390/nu12123867 www.mdpi.com/journal/nutrients Nutrients 2020, 12, 3867 2 of 18

Cellular levels of ODC and polyamines are frequently dysregulated in cancer and are associated with a critical role in cell proliferation [8–12]. In addition, ODC is able to reduce the accumulation of intracellular reactive oxygen species (ROS), protecting cancer cells against cellular apoptosis and therefore maintaining cancer cell survival [4,5]. ODC is considered an oncogene because its enzymatic activity leads to cancer initiation and proliferation [2,13]. Targeting oncogenic ODC could manipulate cell cycle checkpoints and protect against tumorigenesis. Therefore, ODC is a potential cancer target, and the discovery of ODC enzyme inhibitors is a beneficial strategy for cancer therapy and prevention [14,15]. An active-site irreversible inhibitor of ODC, α-DL-difluoromethylornithine (DFMO; also called eflornithine), has been approved by the FDA as a therapeutic agent for cancer treatment. DFMO has also been successfully used against other diseases, such as African sleeping sickness and a number of tropical diseases [16]. Additionally, it is widely used against hirsutism in women [17]. ODC is a short-lived protein that is rapidly degraded by the 26S proteasome within 30 min. ODC activity and degradation are delicately controlled by antizyme (AZ), which is a natural ornithine decarboxylase inhibitor that binds, inhibits, and promotes the protein degradation of ODC by the 26S proteasome [18,19]. High polyamine concentrations trigger a +1 ribosomal frameshifting of AZ mRNA to generate mature full-length AZ proteins [20–22]. In addition, c-Myc is the gene upstream of ODC, suggesting a strong association between the oncogene myc and the overexpression of ODC [23]. ODC is functional as a homodimer with its two active sites at the dimer interface that includes residues from each subunit [24]. ODC is inactivated by AZ binding, causing dissociation of the ODC dimer into monomers to form an inactive AZ-ODC heterodimer, finally promoting ODC degradation through a ubiquitin-independent proteasomal pathway [1,20,25]. Another regulatory protein of ODC, antizyme inhibitor (AZI), has a similar structure to that of ODC but lacks decarboxylase activity [26,27]. AZI binds with a greater affinity to AZ than ODC, thus rescuing ODC activity by liberating the ODC monomer from the inactive ODC-AZ heterodimer to reform active ODC dimers [25,28,29]. Although DFMO is an approved FDA drug against ODC, high doses of DFMO lead to hearing loss [30]. Therefore, discovering a new ornithine decarboxylase inhibitor with fewer side-effects would be quite valuable for chemoprevention or cancer therapy. The discovery of agents from natural products that prevent cancer is a vital issue. A number of flavonoids have been investigated for their anticancer activities in a variety of cancer cell lines and animal models. The flavonol herbacetin has been found to be an allosteric inhibitor of ODC with anticancer activity [31]. By targeting ODC, herbacetin exerts activity against colon cancer and melanoma [31,32]. Baicalein (5,6,7-trihydroxyflavone) is a flavone that was originally isolated from the roots of Scutellaria baicalensis and Scutellaria lateriflora. 7,8-Dihydroxyflavone (7,8-DHF) is another natural flavone found in Godmania aesculifolia, Tridax procumbens, and primula tree leaves. Myricetin is easy to obtain from dietary sources, including vegetables, nuts, berries, oranges, grapes, and tea. Baicalein, 7,8-DHF and myricetin demonstrate diverse biological activities, including inhibiting cell proliferation, angiogenesis, and tumor metastasis; attenuating the neoplastic transformation of cancer cells; inducing cell cycle arrest, autophagy and apoptosis; targeting the mitochondria; and regulating enzymatic activity [33–35]. Additionally, these flavonoids protect against several human diseases, such as Alzheimer’s, Parkinson’s, cardiovascular pathologies, bone-related disorders, eye disorders and aging, and also have analgesic and antihypertensive activity [36–38]. In the present study, we provide evidence suggesting that baicalein, 7,8-DHF, and myricetin are potent inhibitors of human ODC, either in in vitro or in cell-based studies. This article also reveals the structure-activity relationships between ODC and these flavonoids through molecular docking simulations. Baicalein, 7,8-DHF, and myricetin are thus convincing candidates for the development of chemopreventive or chemotherapeutic agents. Nutrients 2020, 12, 3867 3 of 18

2. Materials and Methods

2.1. Materials Flavone, 7,8-DHF, 6,7-DHF, chrysin, pinocembrin, 6-hydroxyflavone, 7-hydroxyflavone, 7,8-dimethoxyflavone, flavonol, luteolin, and kaempferol were purchased from ChromaDex (Irvine, CA, USA). Baicalein, myricetin, quercetin, morin, galangin, apigenin, biochanin, rutin, naringenin, pyridoxamine, 4-pyridoxic acid, and 4-deoxypyridoxine were purchased from Sigma-Aldrich (St. Louis, MO, USA).

2.2. Cell Culture The human promyelocytic leukemia HL-60 and human acute Jurkat T cell leukemia cell lines were grown in 90% RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) at a temperature of 37 ◦C under a humidiﬁed 5% CO2 atmosphere. These cell lines were approved by short tandem repeat proﬁling, and the cells were passaged for no more than 2 months.

2.3. Cell Viability and Acridine Orange Staining Assay Cells (2 106/mL) were seeded into 60-mm Petri dishes and incubated at 37 C. The cells were × ◦ harvested by treatment with 5 µM rottlerin. Viable cells were counted using the trypan blue exclusion method. The cell suspension was mixed on a slide with an equal volume of acridine orange solution (10 mg/mL). Green ﬂuorescence was detected between 500 and 525 nm with a ﬂuorescence microscope (Zeiss, Oberkochen, Germany).

2.4. Transfection The ODC-empty vector (pcDNA3-ODC-empty) and ODC-overexpressing vector (pcDNA3- ODC-WT) were transfected into parental HL-60 cells according to calcium phosphate-mediated transfection. Stably transfected cells were selected with the antibiotic G418 (400 µg/mL). After three weeks, the isolated G418-resistant clones were individually selected to analyze the protein expression of ODC in the cells.

2.5. DNA Fragmentation Assay Cells were harvested and lysed overnight in digestion buﬀer (0.5% sarkosyl, 0.5 mg/mL proteinase K, 50 mM Tris-HCl, pH 8.0 and 10 mM ethylenediamine tetraacetic acid (EDTA) at 55 ◦C). Subsequently, cells were treated with 0.5 µg/mL RNase A for 2 h. Genomic DNA was extracted by phenol/chloroform/isoamyl alcohol extraction and analyzed by gel electrophoresis using 2% agarose.

2.6. Immunoblotting To purify the total proteins, cells were harvested and lysed in cold lysis buffer (10% v/v glycerol, 1% v/v Triton X-100, 1 mM sodium orthovanadate, 1 mM ethylene glycol tetraacetic acid (EGTA), 10 mM NaF, 1 mM sodium pyrophosphate, 20 mM Tris, pH 7.9, 100 µM β-glycerophosphate, 137 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/mL aprotinin, and 10 µg/mL leupeptin), homogenized, and centrifuged, and then the supernatant was boiled in loading buffer with an aliquot corresponding to 50 µg of protein. The samples were then separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes for immunoblotting using anti-ODC (sc-390366, Santa Cruz Biotechnology Inc., Dallas, TX, USA) or actin (mouse monoclonal, Santa Cruz Biotechnology Inc., Dallas, TX, USA) antibodies for 6 h, followed by a 1 h incubation with a horseradish peroxidase-labeled secondary antibody. Nutrients 2020, 12, 3867 4 of 18

2.7. Expression and Purification of Human ODC The human ODC gene was subcloned into the pQE30 vector, which carries an N-terminal His-tag sequence (Qiagen, Hilden, Germany). The protein expression and purification protocols have been reported in our previous papers [39]. Briefly, the expression vector with the ODC gene was transformed into the JM109 strain of Escherichia coli, and ODC protein overexpression was induced by 1 mM isopropyl-1-thio-b-D-galactoside (IPTG). The cells were grown at 25 ◦C, and the proteins were purified using Ni-NTA agarose (Sigma-Aldrich, St. Louis, MO, USA). Finally, the ODC protein was eluted with elution buffer (30 mM Tris-HCl, pH 7.6, 250–500 mM NaCl, 2 mM β-mercaptoethanol, and 250 mM imidazole), and the purified protein was dialyzed against dialysis buffer (50 mM Tris-HCl, pH 7.6, 0 or 500 mM NaCl, and 2 mM β-mercaptoethanol).

2.8. Enzyme Activity Assay and Inhibition Study of ODC

The ODC enzyme activity was measured at 37 ◦C using the CO2-L3K assay kit (DCL, Charlottetown, Canada) [39]. The enzyme assay couples the decarboxylation of ornithine to the carboxylation of phosphoenolpyruvate (PEP) to form oxaloacetate (OAA), which becomes malate following nicotinamide adenine dinucleotide (NADH) oxidation. The standard reaction mixture for the ODC spectrophotometric assay contained 30 mM Tris-HCl, pH 7.8, 10 mM ornithine, 0.2 mM pyridoxal phosphate (PLP), and 0.4 mL of CO2-L3K assay buffer containing 12.5 mM PEP, 0.4 units/mL microbial PEPC, 4.1 units/mL malate dehydrogenase (mammalian MDH), and 0.6 mM NADH analog in a final volume of 0.5 mL. An appropriate amount of ODC was then added to the assay mixture to initiate the reaction, and the decrease in absorbance at 405 nm was continuously traced with a Lambda 25 UV/VIS spectrophotometer (Perkin Elmer, Waltham, MA, USA). In the coupled reaction, 1 mol of CO2 was produced, and 1 mol of the NADH analog was oxidized under the assay conditions. One enzyme unit was defined as the amount of enzyme that catalyzes the production of 1 µmol of NAD per minute. 1 An absorption coefficient of 2410 M− was used for the NADH analog in the calculations. To determine the IC50 values of compounds that inhibited ODC, a dose-response curve was constructed with the following equation using SigmaPlot 10.0 software (Jandel, San Rafael, CA, USA). To determine the Ki values of baicalein and 7,8-DHF, inhibition studies of ODC were conducted with different concentrations of baicalein (0, 0.5, 2, and 4 µM) or 7,8-DHF (0, 1, 2.4, and 8 µM) against a series of concentrations of ornithine. The total dataset was globally fitted using the following equation, which describes noncompetitive inhibition:

Vmax [S] v = × [I] (Km + [S]) 1 + × Ki where v is the observed initial velocity, Vmax is the maximum rate of the reaction, Km is the Michaelis + constant for NAD or malate, and Ki is the inhibition constant for baicalein or 7,8-DHF.

2.9. Measurement of Cellular ODC Activity The enzyme activity of ODC in cells was determined by measuring the increased level of putrescine after adding the substrate L-ornithine based on a luminescence-based coupled reaction assay [4,40]. The lysate protein (0.5–1 mg) was incubated with 1 mM L-ornithine and 0.5 mM pyridoxal-50-phosphate (PLP) or only with 0.5 mM PLP, which was used as the blank representing the endogenous level of putrescine. The samples were incubated at 37 ◦C for 1 h, and the product putrescine was then detected by a PARADIGM Detection Platform microplate reader (Beckman Coulter, Atlanta, GA, USA) in luminescence detection mode. The luminescence of putrescine ranging from 0–40 pmol was determined as the standard, and the increased putrescine levels per hour represent the enzyme activity of ODC (pmol putrescine/mg lysate/h) in the cell. Nutrients 2020, 12, 3867 5 of 18

2.10. In Silico Molecular Docking The docking software PyRx version 0.97, together with AutoDock Vina, was used for all docking calculations [41]. The ligand structures, including baicalein (ZINC3871633), 7,8-DHF (ZINC57657) and myricetin (ZINC3874317), were obtained from the ZINC database (https://zinc.docking.org/). The receptor structure was obtained from the crystal structure of human ornithine decarboxylase from the RCSB Protein Databank (http://www.rcsb.org/) (PDB: 1D7K). Subsequent protein-ligand docking simulations were performed using PyRx. PyRx/AutoDock Vina is based on the Lamarckian genetic algorithm and empirical free energy scoring function. The receptor structure and ligands were prepared, docking was performed into a grid box space with the X, Y and Z axes, and the dimensions were adjusted to 49.43 Å 38.97 Å 40.61 Å. The exhaustiveness parameter was set to 20, × × and the number of modes was set to 9 in the PyRx platform. AutoDock Vina automatically samples different conformations of the ligands to best fit the predicted binding site. The docking results were analyzed based on the binding affinity (kcal/mol) of the protein-ligand complex. The more negative the binding affinity is, the better the orientation of the ligand in the binding site is. Docked complexes were visualized and analyzed using the PyMOL Molecular Graphics System (Ver. 2.2 Schrödinger, Portland, OR, USA), and the interactions between protein and ligand were analyzed using the LigPlot program [42].

2.11. Statistical Analysis All data are expressed as the means SE. Statistical analysis was performed using the ± 95% conﬁdence interval and appropriate post testing from Student’s t-test or one-way ANOVA at signiﬁcance levels of p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). One-way ANOVA followed by Turkey’s multiple comparisons test or t-tests was performed using GraphPad Prism version 8.0.0 for Windows, GraphPad Software, San Diego, CA, USA, www.graphpad.com.

3. Results and Discussion

3.1. Flavone and Flavonol Derivatives Strongly Suppress Human ODC Enzymatic Activity We examined the inhibitory effects of a variety of flavone and flavonol derivatives on ODC enzymatic activity (Figure1). Tables1 and2 show the chemical structures of these derivatives along with their characteristics and IC50 values. Flavone and flavonol, without substitutions on the A or B rings, displayed little ODC suppression activity (Figures1A and2A, respectively). Baicalein, a flavone with three consecutive hydroxyl groups at the 5th, 6th, and 7th positions of the A ring, exhibited significant ODC suppression activity with an IC50 of 0.88 µM (Figure1B and Table1). In addition, myricetin, a flavonol with three consecutive hydroxyl groups at the 3rd, 4th, and 5th positions of the B ring, exhibited substantial ODC suppression activity with an IC50 of 7.3 µM (Figure2B and Table2). The IC50 of DFMO determined in our system was approximately 100 µM (Figure1F). As an enzyme inhibitor of ODC, baicalein is nearly 100-fold more potent than DFMO, and myricetin is nearly 10-fold more potent than DFMO. Nutrients 2020, 12, 3867 6 of 18 Nutrients 2020, 12, x FOR PEER REVIEW 6 of 19

NutrientsNutrients 2020 2020,, , 12 12,, , x xx FOR FORFOR PEER PEERPEER REVIEW REVIEWREVIEW 66 of of 19 19

FigureFigure 1.FigureFigure 1.Dose-dependent Dose-dependent 1. 1. Dose-dependent Dose-dependent inhibition inhibition inhibition inhibition of of flavoneof offlavone flavone flavone derivatives derivati derivati derivativesves towardtoward toward the the the the ornithine ornithine ornithine ornithine decarboxylase decarboxylase decarboxylase decarboxylase (ODC) (ODC) (ODC) (ODC) enzyme. (A) Flavone; (B) baicalein; (C) 7,8-Dihydroxyflavone (7,8-DHF); (D) 6,7-Dihydroxyflavone enzyme.enzymeenzymeenzyme. ((AA)) Flavone;.Flavone;. ((AA)) Flavone;Flavone; ((BB)) baicalein;(baicalein;(BB)) baicalein;baicalein; ((CC )(()CC 7,8-Dihydroxyflavone)7,8-Dihydroxyflavone) 7,8-Dihydroxyflavone7,8-Dihydroxyflavone (7,8-DHF);(7,8-DHF); (7,8-DHF); ((DD ( )D)( D6,7-Dihydroxyflavone6,7-Dihydroxyflavone)) 6,7-Dihydroxyflavone 6,7-Dihydroxyflavone (6,7-DHF);(6,7-DHF); ((EE)) chrysin;chrysin; ((FF)) difluoromethylornithinedifluoromethylornithine (DFMO),(DFMO), anan irreversibleirreversibleirreversible iiinhibitornhibitor ofof ODC.ODC. (6,7-DHF);(6,7-DHF);(6,7-DHF); (E )(E chrysin;) chrysin;(E) chrysin; (F) difluoromethylornithine(F) (difluoromethylornithineF) difluoromethylornithine (DFMO), (DFMO), an irreversible an an irreversible irreversible inhibitor inhibitor i ofnhibitor ODC. of ODC. Enzymeof ODC. EnzymeEnzyme activityactivity ofof ODCODC werewere measuredmeasured withwith aa seriserieses ofof concentrationsconcentrations ofof thethe respectiverespective flavoneflavone activityEnzyme of ODCactivity were of measured ODC were with measured a series of with concentrations a series of of concentrations the respective flavone of the derivativesrespective (flavonen = 3). derivativesderivatives ((nn === 3).3).3). derivatives (n = 3). Table 1. Flavonoids with A ring substitution(s) and with the the half maximal inhibitory concentration TableTable 1.1. FlavonoidsFlavonoids withwith AA ringring substitution(s)substitution(s) anandd withwith thethe thethe halfhalf maximalmaximal inhibitoryinhibitory (IC50) to ornithine decarboxylase (ODC). Table 1. Flavonoids concentrationwithconcentration A ring substitution(s) (IC(IC5050)) toto ornithineornithine an decarboxylasedecarboxylased with the the (ODC).(ODC). half maximal inhibitory Compoundconcentration IC (IC50) to Chemicalornithine Structure decarboxylase (ODC).Characteristics CompoundCompound 50ICIC5050 ChemicalChemicalChemical Structure StructureStructure Characteristics Characteristics Characteristics

Compound IC50 Chemical Structure Characteristics

FlavoneFlavoneFlavone NoNo inhibitionNo inhibition inhibition NoNoNo A A Aringring ring substitution substitution substitution

Flavone No inhibition No A ring substitution

Baicalein BaicaleinBaicalein 0.880.880.88µM µM µM AAA ring ring ring substitution: substitution: substitution: 5,6,7-tri-OH 5,6,7-tri-OH 5,6,7-tri-OH (5,6,7-trihydroxyﬂavone)(5,6,7-trihydroxyflavone)(5,6,7-trihydroxyflavone)

Baicalein 0.88 µM A ring substitution: 5,6,7-tri-OH (5,6,7-trihydroxyflavone)

7,8-Dihydroxyﬂavone7,8-Dihydroxyflavone7,8-Dihydroxyflavone 2.542.542.54µM µM µM AAA ring ring ring substitution: substitution: substitution: 7,8-di-OH 7,8-di-OH 7,8-di-OH (7,8-DHF)(7,8-DHF)(7,8-DHF)

7,8-Dihydroxyflavone 2.54 µM A ring substitution: 7,8-di-OH (7,8-DHF)

6,7-Dihydroxyflavone6,7-Dihydroxyflavone 6,7-Dihydroxyﬂavone 289289 µM µM AA ring ring substitution: substitution: 6,7-di-OH 6,7-di-OH (6,7-DHF) 289289289µM µMµM AAA ringring ring substitution:substitution: substitution: 6,7-di-OH6,7-di-OH 6,7-di-OH (6,7-DHF)(6,7-DHF)(6,7-DHF)

6,7-Dihydroxyflavone 289 µM A ring substitution: 6,7-di-OH (6,7-DHF)

Nutrients 2020, 12, 3867 7 of 18

Table 1. Cont. NutrientsNutrients 2020 2020, ,12 12, ,x x FOR FOR PEER PEER REVIEW REVIEW 77 of of 19 19 Nutrients 2020,, 12,, xx FORFOR PEERPEER REVIEWREVIEW 7 of 19 Compound IC50 Chemical Structure Characteristics Nutrients 2020, 12, x FOR PEER REVIEW 7 of 19 NutrientsNutrients 20202020,, 1212,, xx FORFOR PEERPEER REVIEWREVIEW 77 ofof 1919

ChrysinChrysin NoNo inhibition inhibition AA ring ring substitution: substitution: 5,7-di-OH 5,7-di-OH ChrysinChrysin NoNo inhibition inhibition AA ring ring substitution: substitution: 5,7-di-OH 5,7-di-OH Chrysin No inhibition A ring substitution: 5,7-di-OH ChrysinChrysin NoNo inhibitioninhibition AA ringring substitution:substitution: 5,7-di-OH5,7-di-OH

7,8-Dimethoxyflavone7,8-Dimethoxyflavone NoNo inhibition inhibition AA ring ring substitution: substitution: 7,8-di-OCH 7,8-di-OCH3 3 7,8-Dimethoxyﬂavone7,8-Dimethoxyflavone NoNo inhibition inhibition AA ring ring substitution: substitution: 7,8-di-OCH 7,8-di-OCH33 3

7,8-Dimethoxyflavone7,8-Dimethoxyflavone NoNo inhibition inhibition AA ring ring substitution: substitution: 7,8-di-OCH 7,8-di-OCH33 7,8-Dimethoxyflavone No inhibition A ring substitution: 7,8-di-OCH3

PinocembrinPinocembrin NoNo inhibition inhibition AA ring ring substitution: substitution: 5,7-di-OH 5,7-di-OH PinocembrinPinocembrin NoNo inhibition inhibition AA ring ring substitution: substitution: 5,7-di-OH 5,7-di-OH Pinocembrin No inhibition A ring substitution: 5,7-di-OH PinocembrinPinocembrin NoNo inhibitioninhibition AA ringring substitution:substitution: 5,7-di-OH5,7-di-OH

6-Hydroxyflavone6-Hydroxyflavone NoNo inhibition inhibition AA ring ring substitution: substitution: 6-OH 6-OH 6-Hydroxyﬂavone6-Hydroxyflavone NoNo inhibition inhibition AA ring ring substitution: substitution: 6-OH 6-OH 6-Hydroxyflavone No inhibition A ring substitution: 6-OH 6-Hydroxyflavone6-Hydroxyflavone NoNo inhibitioninhibition AA ringring substitution:substitution: 6-OH6-OH

7-Hydroxyflavone7-Hydroxyflavone NoNo inhibition inhibition AA ring ring substitution: substitution: 7-OH 7-OH 7-Hydroxyﬂavone7-Hydroxyflavone NoNo inhibition inhibition AA ring ring substitution: substitution: 7-OH 7-OH 7-Hydroxyflavone No inhibition A ring substitution: 7-OH 7-Hydroxyflavone7-Hydroxyflavone NoNo inhibitioninhibition AA ringring substitution:substitution: 7-OH7-OH

TableTable 2. 2. Flavonoids Flavonoids with with substitutions substitutions on on bo bothth the the A A and and B B rings rings and and with with the the IC IC5050 to to ODC. ODC. Table 2. Flavonoids with substitutions on bothth thethe AA andand BB ringsrings andand withwith thethe ICIC5050 toto ODC.ODC. Table 2. Flavonoids with substitutions on both the A and B rings and with the IC50 to ODC. Table 2.Compound Flavonoids withIC substitutions50 on boChemicalth the A Structureand B rings and with the IC50 Characteristics to ODC. Table 2.Compound Flavonoids withIC substitutions50 on boChemicalth the A andStructure B rings and with the IC5050 Characteristics to ODC. TableCompound 2. Flavonoids withIC 50substitutions50 on boChemicalChemicalth the A StructureStructure and B rings and with the IC Characteristics Characteristics50 to ODC. Compound IC Chemical Structure Characteristics CompoundCompound ICIC5050 50 ChemicalChemical Structure Structure Characteristics Characteristics Compound IC50 Chemical Structure Characteristics FlavonolFlavonol FlavonolFlavonol NoNo inhibition inhibition Flavonol Flavonol No inhibition NoNo A A or or B B ring ring substitutions substitutions NoFlavonol A or B ring substitutions Flavonol No inhibition FlavonolFlavonolFlavonol FlavonolFlavonolFlavonol NoNoNo inhibitioninhibition inhibition No A or B ring substitutions NoNo AANo oror BB A ringring or B substitutionssubstitutions ring substitutions

FlavonolFlavonol Flavonol MyricetinMyricetin 7.37.3 µM µM AA ring ring substitution: substitution: 5,7-di-OH 5,7-di-OH Myricetin 7.3 µM AFlavonol ring substitution: 5,7-di-OH BB ring FlavonolFlavonolring substitution: substitution:Flavonol 3,4,5-tri-OH 3,4,5-tri-OH Myricetin 7.3 µM A ring substitution:B ring substitution: 5,7-di-OH 3,4,5-tri-OH MyricetinMyricetinMyricetin 7.37.37.3 µMµMµM AA ringringA substitution: ringsubstitution: substitution: 5,7-di-OH5,7-di-OH 5,7-di-OH B ring substitution: 3,4,5-tri-OH BB ringringB substitution: ringsubstitution: substitution: 3,4,5-tri-OH3,4,5-tri-OH 3,4,5-tri-OH

FlavonolFlavonol Flavonol QuercetinQuercetin 8282 µM µM AA ring ring substitution: substitution: 5,7-di-OH 5,7-di-OH Quercetin 82 µM AFlavonol ring substitution: 5,7-di-OH BB FlavonolFlavonolring ring substitution: substitution:Flavonol 4,5-di-OH 4,5-di-OH Quercetin 82 µM A ring substitution:B ring substitution: 5,7-di-OH 4,5-di-OH QuercetinQuercetinQuercetin 828282 µMµMµM AA ringringA substitution: ringsubstitution: substitution: 5,7-di-OH5,7-di-OH 5,7-di-OH B ring substitution: 4,5-di-OH BB ringring substitution:substitution: 4,5-di-OH4,5-di-OH B ring substitution: 4,5-di-OH

FlavoneFlavone Flavone LuteolinLuteolin 9090 µM µM AA ring ring substitution: substitution: 5,7-di-OH 5,7-di-OH Luteolin 90 µM AFlavone ring substitution: 5,7-di-OH BB ringFlavoneFlavone ring substitution: substitution: 3,4-di-OH 3,4-di-OH Luteolin 90 µM A ring substitution:B ring substitution:Flavone 5,7-di-OH 3,4-di-OH LuteolinLuteolin 9090 µMµMµ AA ringring substitution:substitution: 5,7-di-OH5,7-di-OH Luteolin 90 M B ringA substitution: ring substitution: 3,4-di-OH 5,7-di-OH BB ringring substitution:substitution: 3,4-di-OH3,4-di-OH B ring substitution: 3,4-di-OH

Nutrients 2020, 12, 3867 8 of 18

Nutrients 2020, 12, x FOR PEER REVIEW Table 2. Cont. 8 of 19 NutrientsNutrientsNutrients 2020 2020 2020, 12, 12, ,x, 12 xFOR FOR, x FOR PEER PEER PEER REVIEW REVIEW REVIEW 8 8of of 819 19of 19 NutrientsNutrientsNutrients 20202020 2020,,, 1212, ,,12, xxx , FORFOR FORx FOR PEERPEERPEER PEER REVIEWREVIEWREVIEW REVIEW 88 ofof8 19of19 19 Compound IC50 Chemical Structure Characteristics

Flavonol FlavonolFlavonolFlavonolFlavonol Morin 82 µM A ring substitution:FlavonolFlavonolFlavonol 5,7-di-OH MorinMorinMorinMorin 828282 µM µMµ82M µM AA ring ringA ring substitution: ring substitution: substitution: substitution: 5,7-di-OH 5,7-di-OH 5,7-di-OH 5,7-di-OH MorinMorinMorin 8282 82 µMµM µM AA B ringAring ring ring substitution:substitution: substitution: substitution: 5,7-di-OH5,7-di-OH 2,4-di-OH 5,7-di-OH BB ring ringBB ring substitution: ring substitution: substitution: substitution: 2,4-di-OH 2,4-di-OH 2,4-di-OH 2,4-di-OH BB ringringB ring substitution:substitution: substitution: 2,4-di-OH2,4-di-OH 2,4-di-OH

Flavonol FlavonolFlavonolFlavonolFlavonol Fisetin 96 µM A ring Flavonolsubstitution:FlavonolFlavonol 7-OH FisetinFisetinFisetinFisetin 969696 µM µMµ96M µM AA ring ringAA ring substitution: ringsubstitution: substitution: substitution: 7-OH 7-OH 7-OH 7-OH FisetinFisetinFisetin 9696 96 µMµM µM BAA ring ringAring ring substitution: substitution:substitution: substitution: 3,4-di-OH 7-OH7-OH 7-OH BB ring ringBB ring substitution: ring substitution: substitution: substitution: 3,4-di-OH 3,4-di-OH 3,4-di-OH 3,4-di-OH BB ringringB ring substitution:substitution: substitution: 3,4-di-OH3,4-di-OH 3,4-di-OH

FlavonolFlavonol derivative derivative FlavonolFlavonolFlavonolFlavonol derivativederivative derivative derivative A ringFlavonolFlavonol substitution: derivative derivative 5,7-di-OH AA ring ringA ring substitution: substitution: substitution: 5,7-di-OH 5,7-di-OH 5,7-di-OH RutinRutin >120>120 µM µM AA ringAringA ring ring substitution:substitution: substitution: substitution: 5,7-di-OH5,7-di-OH 5,7-di-OH 5,7-di-OH RutinRutinRutinRutin >>120>120120>120 µMµMµM µM B ringB ring substitution: substitution: 3,4-di-OH 3,4-di-OH Rutin >120 µM BB ringringBB ring ring substitution:substitution: substitution: substitution: 3,4-di-OH3,4-di-OH 3,4-di-OH 3,4-di-OH B ManyringB ring substitution: additionalsubstitution: OH3,4-di-OH 3,4-di-OH groups ManyManyMany additional additional additional OH OH OHgroups groups groups ManyManyManyMany additionaladditional additional additional OHOH OH groupsgroups OH groups groups

FlavonolFlavonolFlavonolFlavonol FlavonolFlavonolFlavonolFlavonol KaempferolKaempferolKaempferol 200200 µM200 µM µM AA ringA ringA ring ring substitution: substitution: substitution: substitution: 5,7-di-OH 5,7-di-OH 5,7-di-OH 5,7-di-OH KaempferolKaempferolKaempferolKaempferol 200200200 µMµMµM µM AA ringAringA ring ring substitution:substitution: substitution: substitution: 5,7-di-OH5,7-di-OH 5,7-di-OH 5,7-di-OH BB ringB ring Bring ring substitution: substitution: substitution: substitution: 4-OH 4-OH 4-OH 4-OH BB ringringBB ring ring substitution:substitution: substitution: substitution: 4-OH4-OH 4-OH 4-OH

FlavoneFlavoneFlavoneFlavone FlavoneFlavoneFlavoneFlavone ApigeninApigeninApigenin NoNo inhibitionNo inhibition inhibition AA ringA ringA ring ring substitution: substitution: substitution: substitution: 5,7-di-OH 5,7-di-OH 5,7-di-OH 5,7-di-OH ApigeninApigeninApigeninApigenin NoNoNoNo inhibitioninhibition inhibition AA ringAringA ring ring substitution:substitution: substitution: substitution: 5,7-di-OH5,7-di-OH 5,7-di-OH 5,7-di-OH BB ringB ring Bring ring substitution: substitution: substitution: substitution: 4-OH 4-OH 4-OH 4-OH BB ringringBB ring ring substitution:substitution: substitution: substitution: 4-OH4-OH 4-OH 4-OH

Isoflavone IsoflavoneIsoflavoneIsoflavoneIsoflavoneIsoflavone Biochanin No inhibition A ring substitution:IsoflavoneIsoﬂavone 5,7-di-OH BiochaninBiochaninBiochanin NoNo No inhibitioninhibition inhibition AA A ringringA ring ring substitution:substitution: substitution: substitution: 5,7-di-OH5,7-di-OH 5,7-di-OH 5,7-di-OH BiochaninBiochanin NoNo inhibition inhibition AA ring ring substitution: substitution: 5,7-di-OH 5,7-di-OH3 B ring substitution: 4-OCH 3 BB B ringring Bring ring substitution:substitution: substitution: substitution: 4-OCH4-OCH 4-OCH 4-OCH33 3 3 B ringBB ring ring substitution: substitution: substitution: 4-OCH 4-OCH 4-OCH3 3 3

FlavoneFlavoneFlavone FlavoneFlavoneFlavoneFlavoneFlavone DaidzeinDaidzeinDaidzein NoNo inhibitionNo inhibition inhibition AA ring ringA ring substitution: substitution: substitution: 6-OH 6-OH 6-OH DaidzeinDaidzeinDaidzeinDaidzein NoNoNoNo inhibitioninhibition inhibition AA A ringAring Aring ring ring substitution:substitution: substitution: substitution: substitution: 6-OH6-OH 6-OH 6-OH 6-OH BB ring ringB ring substitution: substitution: substitution: 4-OH 4-OH 4-OH BB B ringringB ringB ring ring substitution:substitution: substitution: substitution: substitution: 4-OH4-OH 4-OH 4-OH 4-OH

FlavoneFlavoneFlavone FlavoneFlavoneFlavoneFlavoneFlavone NaringeninNaringeninNaringenin No No inhibitionNo inhibition inhibition AA ring ringA ring substitution: substitution: substitution: 5,7-di-OH 5,7-di-OH 5,7-di-OH NaringeninNaringeninNaringeninNaringenin NoNoNoNo inhibitioninhibition inhibition AA A ringAring Aring ring ring substitution:substitution: substitution: substitution: substitution: 5,7-di-OH5,7-di-OH 5,7-di-OH 5,7-di-OH 5,7-di-OH BB ring ringB ring substitution: substitution: substitution: 4-OH 4-OH 4-OH BB B ringringB ringB ring ring substitution:substitution: substitution: substitution: substitution: 4-OH4-OH 4-OH 4-OH 4-OH

Nutrients 2020, 12, 3867 9 of 18 Nutrients 2020, 12, x FOR PEER REVIEW 9 of 19

FigureFigure 2.2. Dose-dependentDose-dependent inhibitioninhibition ofof flavonolflavonol derivativesderivatives towardtoward thethe ODCODC enzyme.enzyme. ((AA)) Flavonol;Flavonol; ((BB)) myricetin;myricetin; (C ()C quercetin;) quercetin; (D )( luteolin;D) luteolin; (E) morin; (E) morin; (F) fisetin. (F) fisetin. Enzyme Enzyme activity ofactivity ODC wereof ODC measured were withmeasured a series with of concentrationsa series of concentrations of the respective of the flavonolrespective derivatives flavonol derivatives (n = 3). (n = 3).

3.2. Substitutions to the A or B Rings of the Flavone and Flavonol Derivatives Are Determining Factors for 3.2. Substitutions to the A or B Rings of the Flavone and Flavonol Derivatives Are Determining Factors for Human ODC Enzyme Suppression Activity Human ODC Enzyme Suppression Activity 7,8-DHF, which has two consecutive hydroxyl groups at the 7th and 8th positions of the A ring, 7,8-DHF, which has two consecutive hydroxyl groups at the 7th and 8th positions of the A ring, also exhibited unusual ODC suppression activity, with an IC50 of 2.54 µM (Figure1C and Table1). also exhibited unusual ODC suppression activity, with an IC50 of 2.54 µM (Figure 1C and Table 1). However, 6,7-DHF, which has two consecutive hydroxyl groups at the 6th and 7th positions of the A However, 6,7-DHF, which has two consecutive hydroxyl groups at the 6th and 7th positions of the A ring, displayed much weaker ODC-suppressing activity than 7,8-DHF; the IC50 of 6,7-DHF was 289 µM, ring, displayed much weaker ODC-suppressing activity than 7,8-DHF; the IC50 of 6,7-DHF was 289 approximately 100-fold greater than that of 7,8-DHF (Figure1D and Table1), indicating the significance µM, approximately 100-fold greater than that of 7,8-DHF (Figure 1D and Table 1), indicating the of the positioning of the hydroxy groups. Although 7,8-dimethoxyflavone has two consecutive significance of the positioning of the hydroxy groups. Although 7,8-dimethoxyflavone has two substituents (methyl groups) at the 7th and 8th positions of the A ring, this compound displayed little consecutive substituents (methyl groups) at the 7th and 8th positions of the A ring, this compound ODC suppression activity, indicating the crucial role of the hydroxyl groups (Figure S1A and Table1). displayed little ODC suppression activity, indicating the crucial role of the hydroxyl groups (Figure Chrysin and pinocembrin are B ring stereoisomers that have two hydroxyl groups at the 5th S1A and Table 1). and 7th positions of the A ring; however, they displayed little ODC suppression activity (Figure1E Chrysin and pinocembrin are B ring stereoisomers that have two hydroxyl groups at the 5th and and Figure S1B, respectively; Table1). Therefore, it was not surprising that 6-hydroxyflavone and 7th positions of the A ring; however, they displayed little ODC suppression activity (Figures 1E and 7-hydroxyflavone, which each have a single hydroxyl group at the 6th or 7th position of the A ring, S1B, respectively; Table 1). Therefore, it was not surprising that 6-hydroxyflavone and 7- respectively, did not show any ODC suppression activity (Figure S1C,D; Table1). hydroxyflavone, which each have a single hydroxyl group at the 6th or 7th position of the A ring, We also examined the ODC-inhibiting ability of other flavonols, including quercetin, luteolin, respectively, did not show any ODC suppression activity (Figure S1C,D; Table 1). morin, rutin, kaempferol, apigenin, and biochanin (Figure2C–E and Figure S2A–D, respectively). We also examined the ODC-inhibiting ability of other flavonols, including quercetin, luteolin, These flavonols have two hydroxyl groups at the 5th and 7th positions of the A ring, which have morin, rutin, kaempferol, apigenin, and biochanin (Figures 2C–E and S2A–D, respectively). These common features with myricetin but differ in the positioning and number of hydroxyl groups on the flavonols have two hydroxyl groups at the 5th and 7th positions of the A ring, which have common B ring (Table2). Quercetin, luteolin and morin, which have two hydroxyl groups at the 4th and 5th features with myricetin but differ in the positioning and number of hydroxyl groups on the B ring positions, the 3rd and 4th positions or 2nd and 4th positions of the B ring, respectively, exhibited similar (Table 2). Quercetin, luteolin and morin, which have two hydroxyl groups at the 4th and 5th ODC suppression activity with IC values of approximately 80 to 90 µM, which were 10-fold greater positions, the 3rd and 4th positions50 or 2nd and 4th positions of the B ring, respectively, exhibited than that of myricetin (Figure2C–E; Table2). Rutin, which has a similar structure to luteolin on the A similar ODC suppression activity with IC50 values of approximately 80 to 90 µM, which were 10-fold and B rings but has many hydroxyl groups on the C ring, did not display apparent ODC-suppressing greater than that of myricetin (Figure 2C–E; Table 2). Rutin, which has a similar structure to luteolin activity (Figure S2A and Table2), suggesting that the ODC-inhibiting activity of flavone or flavonol on the A and B rings but has many hydroxyl groups on the C ring, did not display apparent ODC- derivatives is not due to the total number of hydroxyl groups present that may oxidize and modify suppressing activity (Figure S2A and Table 2), suggesting that the ODC-inhibiting activity of flavone or flavonol derivatives is not due to the total number of hydroxyl groups present that may oxidize and modify the ODC protein. Fisetin is similar in structure to luteolin but lacks a single hydroxyl

Nutrients 2020, 12, 3867 10 of 18

Nutrients 2020, 12, x FOR PEER REVIEW 10 of 19 the ODC protein. Fisetin is similar in structure to luteolin but lacks a single hydroxyl group on the group on the 5th position of the A ring, and displayed an IC50 of 96 µM (Figure 2F and Table 2). 5th position of the A ring, and displayed an IC50 of 96 µM (Figure2F and Table2). Furthermore, kaempferol,Furthermore, apigenin, kaempferol, biochanin, apigenin, daidzein, biochanin, and daidzein, naringenin, and each naringenin, of which each have of a which single have substitution a single onsubstitution the B ring, on did the not B ring, show did any not ODC show suppression any ODC activitysuppression (Figure activity S2B–F; (Figure Table 2S2B–F;). Table 2). Additionally, wewe tested tested the the ODC-inhibiting ODC-inhibiting ability ability of PLP of analogs PLP analogs (4-deoxypyridoxine, (4-deoxypyridoxine, 4-pyridoxic 4- acid,pyridoxic and pyridoxamine) acid, and pyridoxamine) and other structurally and other related structurally compounds related (ethyl compounds vanillin, 1,2-naphthoquinone,(ethyl vanillin, 1,2- canaphthoquinone,ﬀeine, sesamol, theobromine,caffeine, sesamol, and theophylline).theobromine, However,and theophylline). none of these However, compounds none wereof these able tocompounds suppress ODCwere activityable to suppress (Table S1), ODC indicating activity that (Table a chemical S1), indicating drug with that a PLP-likea chemical structure drug with is not a suPLP-likeﬃcient structure to be an ODCis not inhibitor. sufficient to be an ODC inhibitor.

3.3. Baicalein and 7,8-DHF DemonstrateDemonstrate NoncompetitiveNoncompetitive Inhibition,Inhibition, and and These These Drugs Drugs May May Bind Bind to to the the Region nearRegion the near Active the SiteActive Pocket Site atPocket the Dimer at the InterfaceDimer Interface of ODC of ODC The inhibition patterns of baicalein and 7,8-DHF with respect to the substrate L-ornithine were examined, and thethe inhibitioninhibition constantsconstants valuesvalues against against ODC ODC (K (i(ornithine)Ki(ornithine))) were determined.determined. KineticKinetic analysis indicatedindicated that that baicalein baicalein and and 7,8-DHF 7,8-DHF displayed displayed a noncompetitive a noncompetitive inhibition inhibition pattern pattern with respect with torespect L-ornithine to L-ornithine against ODCagainst (Figure ODC3 A,B).(Figure The 3A,B).Ki(ornithine) The Ki(ornithine)values ofvalues baicalein of baicalein and 7,8-DHF and 7,8-DHF against ODCagainst were ODC 0.64 were0.18 0.64µM ± and0.18 1.4 µM 0.1andµ M,1.4 respectively, ± 0.1 µM, respectively, which were similarwhich towere their similar corresponding to their ± ± ICcorresponding50 values (0.88 ICµ50M values and 2.54 (0.88µ M,µM respectively; and 2.54 µM, Table respectively;1). Table 1).

Figure 3. Non-competitiveNon-competitive inhibition inhibition of ofhuman human ODC ODC by baicalein by baicalein or 7,8-DHF. or 7,8-DHF. Double Double reciprocal reciprocal plots plotsfor the for inhibition the inhibition of ODC of ODC with with (A) (Abaicalein) baicalein and and (B) ( B7,8-DHF) 7,8-DHF with with respect respect to to the the ODC ODC substrate ornithine. HumanHuman ODCODC activity activity was was measured measured using usin diﬀgerent different concentrations concentrations of ornithine of ornithine with various with concentrationsvarious concentrations of baicalein of baicalein (0, 0.5, 2, (0, and 0.5, 4 2,µM) and or 47,8-DHF µM) or 7,8-DHF (0, 1, 2.4, (0, and 1, 82.4,µM). and 8 µM).

We performedperformed in in silico silico molecular molecular docking docking of baicalein,of baicalein, 7,8-DHF 7,8-DHF and myricetinand myricetin to the to whole the whole dimer ofdimer human of human ODC (PDB ODC code: (PDB 1D7K) code: 1D7K) (Figure (Figures4, Figures 4, S3S3 and S4, respectively). Structural andand functionalfunctional studies revealedrevealed that that residues residues Asp134, Asp134, Thr157, Thr157, Lys169, Lys169, Val198, Val198, Ser200, Ser200, Gly201, Gly201, Arg277, Arg277, Lys294, Lys294, Tyr331, Asp332,Tyr331, Asp332, Gly362, andGly362, Tyr389 and are Tyr389 recognizable are recognizable around the around active the site active in the site dimer in the interface dimer andinterface play signiﬁcantand play significant roles in either roles dimerin either formation dimer formation or enzymatic or enzymatic activity [39 activity]. [39].

Nutrients 2020, 12, 3867 11 of 18 Nutrients 2020, 12, x FOR PEER REVIEW 11 of 19

FigureFigure 4. 4. MolecularMolecular docking docking simulations simulations of of the the ODC ODC dimer dimer with with baicalein. baicalein. ( (AA)) The The best best docking docking result result ofof the ODCODC dimerdimer with with baicalein. baicalein. The The yellow yellow circle circle indicates indicates the bindingthe binding pocket pocket of baicalein. of baicalein. (B) Ligand (B) Ligandinteraction interaction diagram diagram of ODC of with ODC baicalein. with baicalein. (C) Ligand (C interactions) Ligand interactions between baicalein between andbaicalein ODC wereand ODCdrawn were by thedrawn LigPlot by the program LigPlot [41 program]. The green [41]. dotted The green line represents dotted line a hydrogenrepresents bond. a hydrogen Hydrophobic bond. Hydrophobicinteraction contributions interaction contribu from nonligandtions from residues nonligand are indicated residues byare open indicated spokes. by open spokes.

TheThe docking docking results results showed showed that that baicalein baicalein was was buri burieded near near the the active active site site pocket pocket in in the the dimer dimer interfaceinterface of of the the enzyme enzyme (yellow (yellow circles, circles, Figure Figure 44A).A). Baicalein was surrounded by amino acid residues fromfrom the AA andand BB chains, chains, which which were were hydrogen hydrogen bonded bonded to Ser200,to Ser200, Arg277, Arg277, and and Asp332 Asp332 from from the A the chain A chainand Gly362 and Gly362 from thefrom B chainthe B (Figurechain (Figure4B,C, and 4B,C, Table and3). Table A negative 3). A bindingnegative energy binding of energy8.3 kcal of /−mol8.3 − kcal/molwas calculated was calculated for the binding for the of baicaleinbinding of to ODCbaicalein (Table to3 ),ODC which (Table coincided 3), which with thecoincided inhibition with study the inhibitionsince baicalein study was since determined baicalein towas be determined a non-competitive to be a ODC non-competitive inhibitor with ODC a K i(ornithine)inhibitorvalue with ofa K0.64i(ornithine)µM; value this result of 0.64 showed µM; this that result baicalein showed is a high-athat baicaleinﬃnity ODC is a inhibitorhigh-affinity capable ODC of inhibitor strong binding capable to ofthe strong enzyme. binding The simulationto the enzyme. study The showed simulation that the study hydroxyl showed moiety that atthe C-6 hydroxyl on the A moiety ring of at baicalein C-6 on theconcurrently A ring of baicalein acted as aconcurrently hydrogen bond acted donor as a hydrogen to Asp332 bond and donor an acceptor to Asp332 to Arg277; and an additionally, acceptor to Arg277;the oxygen additionally, on C-5 of the the oxygen A ring acceptedon C-5 of athe hydrogen A ring accepted bond from a hydrogen Arg277. Sincebond Arg277from Arg277. and Asp332 Since Arg277are critical and residues Asp332 forare ODCcritical activity residues and for dimerization, ODC activity hydrogen and dimerization, bonding of hydrogen baicalein withbonding Arg277 of baicaleinand Asp332 with may Arg277 disturb and theAsp332 geometry may disturb of the activethe geom siteetry in theof the dimer active interface, site in the thereby dimer inhibitinginterface, therebyODC activity. inhibiting ODC activity.

Nutrients 2020, 12, x FOR PEER REVIEW 12 of 19

Table 3. Molecular interactions between the ODC dimer and baicalein, 7,8-DHF, and myricetin.

Nutrients 2020, 12, 3867 1 Binding 2 Number of 12 of 18 2 Hydrogen Bonding 2 Interacting Protein Ligand Affinity Hydrogen Residues Hydrophobic Residues (kcal/mol) Bonds Table 3. Molecular interactions between the ODC dimer and baicalein, 7,8-DHF, andArg154(A), myricetin. Val168(A) Baicalein Ser200(A), Arg277(A) Phe170(A), His197(A) 1 Binding− A8.3ﬃnity 2 Number4–6 of 2 Hydrogen Bonding 2 Interacting Protein(ZINC3871633) Ligand Asp332(A), Gly362(B) Gly199(A), Tyr389(A) (kcal/mol) Hydrogen Bonds Residues HydrophobicAsp361(B), ResiduesGly362(B) Arg154(A),Val198(A), Val168(A) Gly199(A) Baicalein Ser200(A), Arg277(A) Phe170(A), His197(A) ODC 7,8-DHF 8.3 4–6 Ser200(A), Gly201(A) Tyr331(A), Asp332(A) (ZINC3871633) − −8.1 3–4 Asp332(A), Gly362(B) Gly199(A), Tyr389(A) (1D7K) (ZINC57657) Gly362(B) Asp361(B),Tyr389(A) Gly362(B) Tyr323(B), Asp361(B) Val198(A), Gly199(A) ODC 7,8-DHF Cys360(A),Ser200(A), Gly362(A) Gly201(A) Tyr331(A), Asp332(A) 8.1 3–4 (1D7K) (ZINC57657)Myricetin − Arg154(B),Gly362(B) Thr157(B) Tyr389(A)Asp361(A) −9.2 9–10 (ZINC3874317) Val198(B), Arg277(B) Tyr323(B),Phe170(B), Asp361(B) His197(B) Asp332(B),Cys360(A), Tyr389(B) Gly362(A) Myricetin Arg154(B), Thr157(B) Asp361(A) 1 9.2 9–10 2 Binding(ZINC3874317) affinity of the docked− complex was determined withVal198(B), PyRx/AutoDock Arg277(B) Vina.Phe170(B), The numbers His197(B) of hydrogen bonds, hydrogen bonding residues, and interactingAsp332(B), hydrophobic Tyr389(B) residues of the docked 1complexBinding awereﬃnity analyzed of the docked with complex the PyMOL was determined and LigPlot with programs PyRx/AutoDock and are Vina. visualized2 The numbers in Figures of hydrogen 4, S3 bonds,and S4. hydrogen bonding residues, and interacting hydrophobic residues of the docked complex were analyzed with the PyMOL and LigPlot programs and are visualized in Figure4, Figures S3 and S4.

7,8-DHF7,8-DHF waswas alsoalso boundbound nearnear thethe activeactive sitesite pocketpocket ofof thethe dimerdimer interfaceinterface ofof thethe enzymeenzyme (yellow(yellow circles,circles, FigureFigure S3A).S3A). TheThe molecularmolecular interactionsinteractions werewere attributedattributed toto hydrogenhydrogen bondingbonding betweenbetween thethe hydroxylhydroxyl moietymoiety at at C-7 C-7 and and C-8 onC-8 the on A the ring A of ring 7,8-DHF of 7,8-DHF with Gly201 with and Gly201 Ser200 and of ODC, Ser200 respectively of ODC, (Figurerespectively S3B,C, (Figure and Table S3B,C,3). Aand negative Table 3). binding A negative energy binding of 8.1 energy kcal/mol of − was8.1 kcal/mol observed was for observed 7,8-DHF − bindingfor 7,8-DHF with binding the ODC with dimer the (Table ODC3 ),dimer which (Table acted 3), as awhich high-a actedﬃnity as noncompetitive a high-affinity inhibitornoncompetitive with a Kinhibitori(ornithine) withvalue a K ofi(ornithine) 1.4 µM. value Interestingly, of 1.4 µM.although Interestingly, the chemical although structures the chemical of baicalein structures and of 7,8-DHFbaicalein andand binding7,8-DHF energies and binding of these energies two compoundsof these two toward compounds ODC toward are quite ODC similar are quite and both similar compounds and both displayedcompounds noncompetitive displayed noncompetitive inhibition, the simulationinhibition, studythe simulation showed that study the bindingshowed geometrythat the ofbinding these twogeometry compounds of these in thetwo enzyme compounds were quitein the di enzymeﬀerent (Figurewere quite5). In different 7,8-DHF, (Figure the oxygen 5). In atom 7,8-DHF, at C-4 the of theoxygen C ring atom forms at aC-4 hydrogen of the C bond ring withforms Gly362 a hydrogen (Figure 5bondB), but with the Gly362 oxygen (Figure atom at 5B), C-4 ofbut the the C ringoxygen of baicaleinatom at C-4 forms of the a hydrogen C ring of bondbaicalein with forms Ser200 a hydrogen (Figure5A). bond Additionally, with Ser200 Ser200 (Figure is hydrogen 5A). Additionally, bonded toSer200 7,8-DHF is hydrogen by the hydroxyl bonded to moiety 7,8-DHF at C-8 by the on thehydroxyl A ring moiety (Figure at5B) C-8 and on thisthe sameA ring residue (Figure forms 5B) and a hydrogenthis same bondresidue to forms baicalein a hydrogen through thebond hydroxyl to baical moietyein through at C-5 the on thehydroxyl A ring moiety (Figure at5A). C-5 The on basicthe A structuralring (Figure elements 5A). The that basic drive structural the potential elements inhibition that drive of ODC themay potential be as inhibition follows: the of twoODC consecutive may be as hydroxylfollows: the moieties two consecutive at C-5 and hydroxyl C-6 on the moieties A ring andat C-5 the and oxygen C-6 atomon the within A ring the and C ringthe oxygen of baicalein, atom whichwithin are the similar C ring to of the baicalein, two consecutive which are hydroxyl similar to moieties the two at consecutive C-7 and C-8 hydroxyl on the A ringmoieties and the at C-7 oxygen and atomC-8 on at the C-4 A on ring the and C ring the ofoxygen 7,8-DHF. atom at C-4 on the C ring of 7,8-DHF.

FigureFigure 5.5. SchematicSchematic diagramdiagram forfor thethe chemicalchemical structurestructure ofof baicaleinbaicalein andand 7,8-DHF7,8-DHF andand thethe interactinginteracting residuesresidues ofof ODC. ODC. ( A(A)) Hydrogen Hydrogen bonding bonding of of the the hydroxyl hydroxyl moieties moieties at C-5at C-5 and and C-6 C-6 on theon the A ring A ring and and the oxygenthe oxygen atom atom on the on C the ring C of ring baicalein of baicalein with Ser200, with Ser200, Arg277, Arg277, Asp332, Asp332, and Gly362 and in Gly362 the dimer in the interface dimer of ODC. (B) Hydrogen bonding of the hydroxyl moieties at C-7 and C-8 on the A ring and the oxygen

atom at C-4 on the C ring of 7,8-DHF with Ser200, Gly201, and Gly362 in the dimer interface of ODC. Nutrients 2020, 12, 3867 13 of 18

Myricetin also formed hydrogen bonds near the active site pocket in the dimer interface of the enzyme (yellow circles, Figure S4A) through its hydroxyl moieties on the A, B, and C rings that served as hydrogen bond donors or acceptors with the residues essential for ODC activity and dimerization (Figure S4B,C and Table3). Among the three compounds, myricetin was shown to bind to ODC the tightest, with the lowest energy of 9.2 kcal/mol, but did not display the most effective inhibitory ability − toward ODC, suggesting the specificity of the individual compounds toward the inhibition of ODC.

3.4. Baicalein and Myricetin Suppress Cell Growth and Induce Cellular Apoptosis of the Cell When HL-60 cells were treated with baicalein at concentrations of 0–70 µM, cell death was dose-dependent (Figure6A). The IC 50 value for baicalein in HL-60 cells after 24 h was approximately 50 µM (Figure6A), and when these cells were treated with 50 µM baicalein, the cells exhibited apoptosis by displaying lobular apoptotic cell bodies (arrows in Figure6B) and DNA fragmentation (Figure6C). A similar result was observed in myricetin-treated HL-60 cells. The IC50 of myricetin in HL-60 cells after 24 h was approximately 75 µM (Figure6D). Myricetin induced apoptosis of HL-60 cells at 24 h with a clearly visible apoptotic cell bodies (arrows in Figure6E), and the DNA fragmentation of HL-60 cells occurred in a dose-dependent manner after HL-60 cells were treated with various concentrations of myricetin (Figure6F). Additionally, both baicalein and myricetin suppressed the growth of Jurkat cells and induced cellular apoptosis with DNA fragmentation (Figure S5), indicating the cytotoxicity of these compounds toward leukemic cells.

3.5. Baicalein and Myricetin Suppress ODC-Evoked Cellular Anti-Apoptosis To determine whether baicalein- or myricetin-induced apoptosis was related to ODC expression, we introduced ODC cDNA into the pcDNA3 mammalian expression plasmid to produce the ODC-empty vector (ODC-empty) and ODC-overexpressing vector (ODC-WT) in parental HL-60 cells. Therefore, the protein expression and enzyme activity of ODC were expressed at a higher level in ODC-WT cells than in the parental HL-60 and ODC-empty cells (Figure S6). Without baicalein, both ODC-empty and ODC-WT HL-60 cells grew very fast, approximately 2- to 2.1-fold after 24 h (Set 1 and Set 2, respectively, Figure7A). However, when ODC-empty or ODC-WT HL-60 cells were treated with 50 µM baicalein, the cells exhibited growth arrest within 24 h (Set 3 and Set 4, respectively, Figure7A). The baicalein-induced growth arrest of the ODC-empty HL-60 cells coincided with the appearance of apoptotic bodies and DNA fragmentation (upper panels of Figure7B,C). Furthermore, ODC-WT HL-60 cells exhibited a normal cell morphology and slight DNA fragmentation (lower panels of Figure7B,C), indicating that overexpression of ODC could prevent baicalein-induced apoptosis. Similar to baicalein, myricetin had cytotoxic effects toward ODC-empty or ODC-WT HL-60 cells. Myricetin-inhibited cell growth in ODC-empty HL-60 cells was more effective than in ODC-WT HL-60 cells (Set 3 and Set 4, respectively, Figure7D). Myricetin treatment induced apoptotic bodies in ODC-empty cells but not in ODC-WT HL-60 cells (Figure7E). In addition, myricetin-induced DNA fragmentation in parental and ODC-empty cells was more evident than that in ODC-WT HL-60 cells (Figure7F). These data further supported that baicalein and myricetin are able to suppress ODC-induced anti-apoptotic effects in HL-60 cells.

3.6. Baicalein, 7,8-DHF, and Myricetin Can Be Used as Chemopreventive Agents by Targeting the ODC Enzyme ODC is highly expressed in many cancers, promoting cell growth and tumorigenesis [43,44]. Elevated ODC activity in the cell leads to uncontrolled levels of cellular polyamines, and polyamines are critical for DNA folding and replication and cell proliferation [44,45]. Therefore, ODC has been considered a valid target for chemoprevention and cancer treatment. A well-known ODC irreversible inhibitor, DFMO, is an FDA-approved drug for cancer treatment. However, high doses of DFMO in humans will lead to diﬀerent degrees of hearing loss. Our present study revealed several small molecule inhibitors of ODC from natural sources that are nontoxic and more potent than DFMO inhibitors of Nutrients 2020, 12, 3867 14 of 18

ODC activity and therefore could be good candidates as substitutes for DFMO to speciﬁcally inhibit ODCNutrients enzymatic 2020, 12, x activity.FOR PEER REVIEW 14 of 19

FigureFigure 6.6. BaicaleinBaicalein and and myricetin myricetin suppress suppress cell cell growth grow andth and induce induce apoptosis apoptosis of HL-60 of HL-60 cells. cells. (A,D )(A Cell,D) growthCell growth was was suppressed suppressed in a in dose-dependent a dose-dependent manner manner with with baicalein baicalein and and myricetin,myricetin, respectively. ****p p< < 0.01,0.01, ****** pp << 0.0010.001 ((nn == 3). ( B,,E)) Apoptotic Apoptotic bodies (ind (indicatedicated by arrows) inin thethe cellscells werewere presentpresent µ µ afterafter treatmenttreatment with with 50 50 µMM of of baicalein baicalein and and 75 75 MµM myricetin, myricetin, respectively. respectively. (C ,(FC),F DNA) DNA fragmentation fragmentation in µ µ thein the cells cells after after treatment treatment with with 50 50M µM baicalein baicalein and and 0-75 0-75M µM myricetin. myricetin.

Nutrients 2020, 12, x FOR PEER REVIEW 15 of 19 appearance of apoptotic bodies and DNA fragmentation (upper panels of Figure 7B,C). Furthermore, ODC-WT HL-60 cells exhibited a normal cell morphology and slight DNA fragmentation (lower Nutrientspanels 2020of Figure, 12, 3867 7B,C), indicating that overexpression of ODC could prevent baicalein-induced15 of 18 apoptosis.

FigureFigure 7.7. BaicaleinBaicalein andand myricetinmyricetin battledbattled againstagainst ODC-evokedODC-evoked cellularcellular anti-apoptosis.anti-apoptosis. ((AA,,DD)) FoldFold changechange inin growth growth of of HL-60 HL-60 cells cells with with ODC-empty ODC-em vectorpty vector (ODC-empty) (ODC-empty) and ODC-overexpressing and ODC-overexpressing vector (ODC-WT)vector (ODC-WT) in the absence in the absence or presence or presence of 50 µ Mof baicalein50 µM baicalein or 75 µ Mor myricetin,75 µM myricetin, respectively. respectively. * p < 0.01, * p ***< 0.01,p < 0.001 *** p (n< =0.0013). (B(n, E=) Apoptotic3). (B,E) Apoptotic bodies in bodies the ODC-empty in the ODC-empty and ODC-WT and HL-60ODC-WT cells HL-60 (indicated cells by(indicated arrows) by were arrows) present were after present treatment after with treatm 50 entµM with baicalein 50 µM and baicalein 75 µM myricetin,and 75 µM respectively. myricetin, (respectively.C,F) DNA fragmentation (C,F) DNA fragmentation in the ODC-empty in the andODC-empty ODC-WT an HL-60d ODC-WT cells (indicated HL-60 cells by (indicated arrows) was by presentarrows) after was treatmentpresent after with treatment 50 µM baicalein with 50 µM and baicalein 75 µM myricetin, and 75 µM respectively. myricetin, respectively.

4. Conclusions Our present study indicated that the flavonoids baicalein, 7,8-DHF, and myricetin could effectively inhibit ODC activity, suppress cell growth and induce cellular apoptosis. Therefore, we suggest that these three compounds are potent chemopreventive and chemotherapeutic agents that work by Nutrients 2020, 12, 3867 16 of 18 targeting ODC. Besides three flavonoids we report in this paper, herbacetin, a natural flavonoid from flaxseed, has also demonstrated as an allosteric ODC inhibitor [31]. Another natural product allicin also demonstrates potent ODC-inhibitory effect, thus this natural product is also an ODC inhibitor potentially used as chemopreventive or chemotherapeutic agents [46]. Since ODC and polyamines are highly associated with the initiation of tumorigenesis, these nature products may be used as promising lead compounds for the development of pharmaceutical anticancer agents or as daily nutraceutical supplements to prevent cancer initiation and promotion. Further studies for the functional regulation of flavonoids baicalein, 7,8-DHF, and myricetin toward the homeostasis of cellular polyamines in various cancer cells that are overexpressed ODC will be discussed in the near future.

Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6643/12/12/3867/s1, Figure S1. Dose-dependent inhibition of flavone derivatives toward the ODC enzyme. Figure S2. Dose-dependent inhibition of flavonol derivatives toward the ODC enzyme. Figure S3. Molecular docking simulations of the ODC dimer with 7,8-DHF. Figure S4. Molecular docking simulations of the ODC dimer with myricetin. Figure S5. Baicalein and myricetin suppress cell growth and induce apoptosis in Jurkat cells. Figure S6. Protein levels and enzyme activity of ODC in ODC-overexpressing HL-60 cells. Table S1. PLP analogs and other compounds that cannot inhibit ODC activity Author Contributions: Y.-C.L. and J.-Y.H. carried out the kinetics experiments; C.-H.W. and Y.-L.L. performed the cell-based studies; J.-Y.H., Y.-L.L., C.-L.L., G.-Y.L., and H.-C.H. analyzed the data and aided in interpreting the results. C.-L.L. co-supervised the project, and G.-Y.L. and H.-C.H. contributed to the design and implementation of the research, the analysis of the results, and the writing of the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This work was financially supported by the Ministry of Science and Technology, ROC (108-2311-B-005-006 and 108-2320-B-040-020-MY3); partly supported by the “iEGG & Animal Biotechnology Center” from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) of Taiwan; and partly supported by the bilateral project from National Chung Hsing University and Chung Shan Medical University (NCHU-CSMU-10701). We also appreciate the Structural Proteomics and Pharmaceutical Application Service provided by the BP Bioinformatics Core (http://www.tbi.org.tw/) funded by the National Core Facility for Biopharmaceuticals (NCFB), MOST 109-2740-B-400-002. Conflicts of Interest: The authors declare no conflict of interest.

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