In Silico Discovery of Potential Uridine-Cytidine Kinase 2 Inhibitors from the Rhizome of Alpinia Mutica

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Article In Silico Discovery of Potential Uridine-Cytidine Kinase 2 Inhibitors from the Rhizome of Alpinia mutica

Ibrahim Malami 1,*, Ahmad Bustamam Abdul 1,*, Rasedee Abdullah 2, Nur Kartinee Bt Kassim 3, Peter Waziri 1 and Imaobong Christopher Etti 4 1 MAKNA-Cancer Research Laboratory, Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia; [email protected] 2 Department of Veterinary Pathology and Microbiology, Faculty of Veterinary, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia; [email protected] 3 Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia; [email protected] 4 Department of Pharmacology and Toxicology, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia; [email protected] * Correspondence: [email protected] (I.M.); [email protected] (A.B.A.); Tel.: +60-103-300-534 (I.M.); +60-126-894-693 (A.B.A.)

Academic Editor: Derek J. McPhee Received: 10 February 2016; Accepted: 22 March 2016; Published: 8 April 2016 Abstract: Uridine-cytidine kinase 2 is implicated in uncontrolled proliferation of abnormal cells and it is a hallmark of cancer, therefore, there is need for effective inhibitors of this key enzyme. In this study, we employed the used of in silico studies to find effective UCK2 inhibitors of natural origin using bioinformatics tools. An in vitro kinase assay was established by measuring the amount of ADP production in the presence of ATP and 5-fluorouridine as a substrate. Molecular docking studies revealed an interesting ligand interaction with the UCK2 protein for both flavokawain B and alpinetin. Both compounds were found to reduce ADP production, possibly by inhibiting UCK2 activity in vitro. In conclusion, we have identified flavokawain B and alpinetin as potential natural UCK2 inhibitors as determined by their interactions with UCK2 protein using in silico molecular docking studies. This can provide information to identify lead candidates for further drug design and development.

Keywords: UCK2; in silico; ﬂavokawain B; alpinetin; Alpinia mutica; nucleoside analogues; amino acid active site residues

1. Introduction Nucleotides are the foundation of all physiological functions required for cell growth and replication and any genetic changes will therefore lead to disturbances in nucleotide pools [1,2]. In cancer cells, these genetic changes can show characteristic DNA/RNA modifications and activities of modifying enzymes, that results in fluctuations in nucleotide levels [3]. In mammalian cell, RNA is in constant turned over in cells, both during the production of mature RNAs from longer precursors and to regulate the amounts of mRNA expressed in cells. During the metabolic breakdown of polymeric RNA and DNA, the resulting NMPs are released which can then be recycled by the action of the uridine-cytidine kinase 2 (UCK2) via alternative salvage pathway [4]. Drugs such 5-fluorouracil and hydroxyurea inhibit de novo nucleotide biosynthesis. However, inhibition of de novo pathway alone is insufficient to produce effective treatment, as such, unutilized nucleotides are salvaged via pyrimidine synthesis. UCK2 is an enzyme that catalyzes the phosphorylation of uridine and cytidine nucleotides to their corresponding uridine monophosphate (UMP) and cytidine monophosphate (CMP) [5].

Molecules 2016, 21, 417; doi:10.3390/molecules21040417 www.mdpi.com/journal/molecules Molecules 2016, 21, 417 2 of 10

MoleculesUCK22016 ,is21 ,an 417 enzyme that catalyzes the phosphorylation of uridine and cytidine nucleotides2 of 10to their corresponding uridine monophosphate (UMP) and cytidine monophosphate (CMP) [5]. Phosphorylation of UMP and CMP are an essential requirement to form 5′-triphosphate nucleotides Phosphorylation of UMP and CMP are an essential requirement to form 51-triphosphate nucleotides required for gene synthesis. UCK2 have been reported to be expressed only in the placenta and its required for gene synthesis. UCK2 have been reported to be expressed only in the placenta and its over expression have been implicated in several rapidly proliferating cancer cells [6,7]. Therefore, the over expression have been implicated in several rapidly proliferating cancer cells [6,7]. Therefore, the selective expression of UCK2 in cancer cells makes it a potential target for cancer chemotherapy. selective expression of UCK2 in cancer cells makes it a potential target for cancer chemotherapy. Nucleoside analogues are being used to treat different cancer cells via their phosphorylation Nucleoside analogues are being used to treat different cancer cells via their phosphorylation catalyzed UCK2. Such nucleoside analogues under investigation in clinical trials either as a single catalyzed UCK2. Such nucleoside analogues under investigation in clinical trials either as a single drug entity or in combination with other cytotoxic agents includes 1-(3-C-ethynyl-beta-D-ribo- drug entity or in combination with other cytotoxic agents includes 1-(3-C-ethynyl-beta-D-ribo- pentofuranosyl)cytosine (ECyd, TAS 106) [8,9] and fluorocyclopentenylcytosine (RX-3117) [10]. pentofuranosyl)cytosine (ECyd, TAS 106) [8,9] and fluorocyclopentenylcytosine (RX-3117) [10]. These nucleoside analogues depend on UCK2 for phosphorylation to their triphosphate form to exert These nucleoside analogues depend on UCK2 for phosphorylation to their triphosphate form to exert their pharmacological activity and once phosphorylated, the nucleoside analogues interfere with the their pharmacological activity and once phosphorylated, the nucleoside analogues interfere with the synthesis of RNA or DNA which are vital cellular processes required for growth and development [6]. synthesis of RNA or DNA which are vital cellular processes required for growth and development [6]. Hence, TAS 106 use is accompanied by several serious toxicity issues such as neurotoxicity, neutropenia, Hence, TAS 106 use is accompanied by several serious toxicity issues such as neurotoxicity, neutropenia, febrile neutropenia, pneumonia, leukopenia, thrombocytopenia, tremor, pain, hyperesthesia, asthenia, febrile neutropenia, pneumonia, leukopenia, thrombocytopenia, tremor, pain, hyperesthesia, asthenia, anorexia, nausea, vomiting, myelosuppression, as well as dermatological effects [8,9,11–13]. anorexia, nausea, vomiting, myelosuppression, as well as dermatological effects [8,9,11–13]. Low molecular weight natural products are capable of inhibiting enzyme catalytic activity due to Low molecular weight natural products are capable of inhibiting enzyme catalytic activity due to the remarkable complexity of their chemical structures. Some natural anticancer agents used nowadays the remarkable complexity of their chemical structures. Some natural anticancer agents used nowadays exert their pharmacological action by inhibiting a human enzyme, particularly ones involved in exert their pharmacological action by inhibiting a human enzyme, particularly ones involved in metabolic pathways [14]. Scientists have for long focused only on the design and synthesis of drugs metabolic pathways [14]. Scientists have for long focused only on the design and synthesis of drugs that that are UCK2 dependent, hence, forgetting that bioactive natural products which are capable of are UCK2 dependent, hence, forgetting that bioactive natural products which are capable of reducing reducing or completely inhibiting enzyme catalytic activity can be used as a chemotherapeutic or completely inhibiting enzyme catalytic activity can be used as a chemotherapeutic targeting UCK2 targeting UCK2 activity. Due to the serious side effects evidenced by these ribonucleoside analogues, activity. Due to the serious side effects evidenced by these ribonucleoside analogues, effective drugs of effective drugs of natural origin with less potential side effect to patients would be highly desirable. natural origin with less potential side effect to patients would be highly desirable. To date, there has To date, there has not been any research on any single natural bioactive compound targeting the not been any research on any single natural bioactive compound targeting the UCK2 enzyme and no UCK2 enzyme and no inhibitors of this enzyme have been so far reported. In our search for natural inhibitors of this enzyme have been so far reported. In our search for natural bioactive compounds bioactive compounds targeting UCK2, we report here for the first time using in silico visual screening targeting UCK2, we report here for the first time using in silico visual screening that flavokawain B and that flavokawain B and alpinetin inhibit UCK2. alpinetin inhibit UCK2.

2. Results Results and and Discussion Discussion

2.1. Cell Viability Studies To determinedetermine thethe percentagepercentage cell cell viability viability of of HT HT 29 29 cells, cells, the the MTT MTT colorimetric colorimetric assay assay was was used used in thisin this investigation investigation to measureto measure the amountthe amount of viable of viable cells cells after after 72 h of72 incubation. h of incubation. Flavokawain Flavokawain B (FKB) B and(FKB) alpinetin and alpinetin (APN) (APN) inhibit inhibit 50% 50% cell proliferationcell proliferation at an at ICan50 ICof50 of 29.84 29.84µM µM (8.47 (8.47µ g/mL)µg/mL)and and 48.5848.58 µµMM (13.12(13.12 µµg/mL),g/mL), respectively respectively (S1 (S1 files) ﬁles) (Figure 11).). 5-Fluorouracil5-Fluorouracil (5FU)(5FU) waswas usedused asas positivepositive controlcontrol inin this study.study. TheThe inhibition inhibition of of proliferating proliferating cells cells by by FKB FKB at aat very a very low low concentration concentration has previouslyhas previously been reportedbeen reported in colon in colon cancer cancer [15,16 [15,16].].

0.8 400 200 0.6 100 50 μM 0.4 25 12.5 0.2 6.25 0 percentage cell (%) viability 0.0

N P 5FU FKB A

Figure 1.1. PercentagePercentagecell cell viability viability of of HT HT 29 29 cells cells treated treated with with 5FU, 5FU, FKB, FKB, and and APN. APN. MTT MTT assay assay was usedwas used to determine the IC50 of the drugs at different concentrations in µM for 72 h. to determine the IC50 of the drugs at different concentrations in µM for 72 h.

Molecules 2016, 21, 417 3 of 10

2.2. Molecular Docking Studies

2.2.1. Redocking Analysis Autodock is an effective tool used to obtain unbiased docking of flexible inhibitors in enzyme active sites [17]. Autodock 4 uses a semiempirical free energy force field to predict the binding free energies of small molecules to macromolecular targets [18]. In order to validate our data sets using Autodock 4, a control docking was performed on UCK2 protein in complex with the inhibitor CTP using a root-mean-square deviation (RMSD) tolerance of 2.0 Å. The results of the redocking study are shown in Table1. Autodock 4 successfully redocks the complex with the lowest energy conformation showing a RMSD of 0.829 Å from the reference structure. The lowest energy docked conformation has an estimated free binding energy of ´14.27 kcal/mol, intermolecular energy of ´16.66 kcal/mol, and inhibition constant (Ki) of 34.63 pM. The obtained RMSD of less than 2.0 Å confirmed the validity of our docking data sets (S2 files).

Table 1. The lowest energy docked conformation from each ligand.

Free Binding Inhibition Intermolecular vdW + Hbond + Electrostatic Ligand Energy * Constant, Ki Energy * Desolv Energy * Energy * CTP ´14.27 34.63 pM ´16.66 ´12.59 ´4.07 FKB ´8.47 618.12 nM ´10.26 ´9.96 ´0.30 APN ´8.86 321.38 nM ´9.75 ´9.04 ´0.71 * kcal/mol; CTP: cytosine-51-triphosphate, FKB: ﬂavokawain B, APN: alpinetin.

2.2.2. Docking Analysis of FKB and APN on UCK2 Protein FKB was estimated to have a lowest binding energy of ´8.47 and intermolecular energy of ´10.26 kcal/mol, and inhibition constant (Ki) of 618.12 nM (Table1). On the other hand, alpinetin had ´8.86 binding and ´9.75 kcal/mol, intermolecular energy of, and a Ki of 321.38 nM. The intermolecular hydrogen interactions between the amino acid residues in the catalytic active site of UCK2 and its inhibitors are tabulated in Table2.

Table 2. Intermolecular hydrogen interaction between UCK2 protein and its inhibitors.

UCK2 HB with CTP HB with FKB HB with APN Active Residues (BD ď 3.5 Å, DHA ě 90˝) (BD ď 3.5 Å, DHA ě 90˝) (BD ď 3.5 Å, DHA ě 90˝) Thr29 Hβ with 31OOγ1 with 2 H Hα with 3 O Ala30 Hα with Oγ3, HN with Oα2 HN with 3 O Gly32 HN with Oγ2 Hζ2 with Oγ2, HN with Oγ2, Hζ1 Lys33 Hζ2 with 4 O Hζ1 with 5 O with Oα1, Hζ1 with Oβ3 Ser31 HN with Oγ2 HN with Oγ1, Hα with Oβ1, Hβ2 Ser34 Oγ with 41 H with Oγ1 Asp62 HN with 7 O Tyr65 Hη with 91 OHη with 10 O Phe83 Asp84 Oγ1 with 21O, Oγ2 with 31O Tyr112 Oη with 4N His117 Nδ1 with 4N Glu135 Oε1 with 101 H, 41 HOε1 with 5 H O with 101 H O with 7 H Oη12 with 31O, Oη22 with 21O, Arg166 Oη22 with 31O Arg169 Hη22 with Oγ2 Hη22 with 2 O Hη12 with 3O Arg174 Oη11 with 41O Arg179 HB: hydrogen bond, BD: bond distance, DHA: D, hydrogen donor; H, hydrogen; A, hydrogen acceptor. Molecules 2016, 21, 417 4 of 10

MoleculesFigures 20162 and, 21, 3417 show a total of eight hydrogen bond interactions formed between the4 catalytic of 10 activeMolecules site of 2016 UCK2, 21, 417 protein and flavokawain B as well as alpinetin, respectively. The hydrogen4 of 10 and oxygen ofFigures the 4-methoxy 2 and 3 show group a total of flavokawainof eight hydrogen B form bond hydrogen interactions bonds formed with between the O γtheatom catalytic of Ser-34 active site of UCK2 protein and flavokawain B as well as alpinetin, respectively. The hydrogen and and Hζ2Figures atom of 2 Lys-33,and 3 show while a total the of 2-hydroxyl eight hydrogen group bond forms interactions a hydrogen formed bond between with the the catalytic Hη22 atom oxygen of the 4-methoxy group of flavokawain B form hydrogen bonds with the Oγ atom of Ser-34 of Arg-169.active site These of UCK2 amino protein acid and residues flavokawain form B the as bindingwell as alpinetin, site for respectively. the γ- and βThe-phosphate hydrogen moietiesand and Hζ2 atom of Lys-33, while the 2-hydroxyl group forms a hydrogen bond with the Hη22 atom of oxygen of the 4-methoxy group of flavokawain B form hydrogen bonds with the Oγ atom of Ser-34 of ATP.Arg-169. ATP These has been amino shown acid residues to adopt form a the pentacoordinate binding site for the transition γ- and β-phosphate state and moieties produce of an ATP. anionic and Hζ2 atom of Lys-33, while the 2-hydroxyl1 group forms a hydrogen bond with the Hη22 atom of chargeATP upon has been nucleophilic shown to adopt attack a pentacoordinate by the 5 -oxygen tran atomsition atstate the andγ-phosphate produce an anionic moiety charge of ATP upon and the Arg-169. These amino acid residues form the binding site for the γ- and β-phosphate moieties of ATP. sidenucleophilic chains of Lys-33, attack by Arg-169 the 5′-oxygen and Arg-174 atom at the are γ the-phosphate potential moiety candidates of ATP and to stabilizethe side chains this anionicof ATP has been shown to adopt a pentacoordinate transition state and produce an anionic charge upon Lys-33, Arg-169 and Arg-174γ are theβ potential candidates to stabilize this anionic charge [19]. Apart chargenucleophilic [19]. Apart attack from by thethe 5′-oxygen- and -phosphateatom at the γ moieties-phosphate of moiety ATP, flavokawainof ATP and the B side also chains forms of three from the γ- and β-phosphate moieties of ATP, flavokawain B also forms three important hydrogen 2+ importantLys-33, hydrogen Arg-169 and bonds Arg-174 with are Glu-135 the potential involved candidat ines coordination to stabilize this with anionic a magnesium charge [19]. ion Apart (Mg ). bonds2+ with Glu-135 involved in coordination with a magnesium ion (Mg2+). A Mg2+ is found to play A Mgfromis the found γ- and to play β-phosphate a crucial moieties role in theof ATP, stabilization flavokawai ofn the B also transition forms three state important by its coordination hydrogen with a crucial role in the stabilization of the transition state by its coordination with the γ- and β-phosphate the γ-bonds and βwith-phosphate Glu-135 involved oxygens in of coordination ATP [7,19]. Otherwith a magnesium hydrogen bonds ion (Mg formed2+). A Mg between2+ is found flavokawain to play B oxygens of ATP [7,19]. Other hydrogen bonds formed between flavokawain B and the amino acid and thea crucial amino role acid in the side stabilization chains of UCK2of the transition protein involvestate by its the coordination 2-hydroxyl with group the andγ- and 91 βketone-phosphate group of side chains of UCK2 protein involve the 2-hydroxyl group and 9′ ketone group of flavokawain B with flavokawainoxygens Bof withATP O[7,19].γ1 and Other the hydrogen Hη atom bonds of Thr-29 formed and between Tyr-65, flavokawain respectively. B and the amino acid Oγ1 and the Hη atom of Thr-29 and Tyr-65, respectively. side chains of UCK2 protein involve the 2-hydroxyl group and 9′ ketone group of flavokawain B with Oγ1 and the Hη atom of Thr-29 and Tyr-65, respectively.

(a) (b)(c)

Figure 2.(a )In silico redocking of UCK2( bprotein)( in complex with inhibitor CTP. (ca)) Complete x-ray Figure 2. In silico redocking of UCK2 protein in complex with inhibitor CTP. (a) Complete x-ray structure of UCK2 receptor protein shown as a cartoon; (b) Amino acid residues in the active site of structureFigure of 2. UCK2 In silico receptor redocking protein of UCK2 shown protein as a cartoon;in complex (b )with Amino inhibitor acid residuesCTP. (a) Complete in the active x-ray site of UCK2; (c) Interactions of CTP with UCK2 as identified by in silico docking analysis. CTP shown in structure of UCK2 receptor protein shown as a cartoon; (b) Amino acid residues in the active site of UCK2;ball-and-stick (c) Interactions model ofwith CTP purple with indicating UCK2 as carbon identiﬁed atoms, by whitein silico for hydrogendocking, blue analysis. for nitrogen, CTP shown red in UCK2; (c) Interactions of CTP with UCK2 as identified by in silico docking analysis. CTP shown in ball-and-stickfor oxygen, model and phosphorus with purple in orange. indicating carbon atoms, white for hydrogen, blue for nitrogen, red ball-and-stick model with purple indicating carbon atoms, white for hydrogen, blue for nitrogen, red for oxygen, and phosphorus in orange. for oxygen, and phosphorus in orange.

(a)

(b)

Figure( b3.) Cont. Figure 3. Cont. Figure 3. Cont.

Molecules 2016, 21, 417 5 of 10 Molecules 2016, 21, 417 5 of 10

Molecules 2016, 21, 417 5 of 10

(c)

Figure 3. (a) Interactions of ligands with UCK2 protein as identified by in silico docking analysis; (b) Figure 3. (a) Interactions of ligands with UCK2 protein as identified by in silico docking analysis; Ligands interacting with the amino acids residues in the active sites of UCK2 protein; (c) Surface (b) Ligands interacting with the amino acids residues in the active sites of UCK2 protein; (c) Surface representation of the hydrophobic contacts of bound ligand and the ligand binding pocket of UCK2 representationprotein shown of the as hydrophobictranslucent blue contacts surface. of Ligand bounds shown ligand as and a ball-and-stick the ligand binding model pocketwith purple of UCK2 protein shown as translucent blue surface. Ligands shown as a ball-and-stick model with purple indicating carbon atoms, white for hydrogen, an(dc )red for oxygen Hydrogen bonds shown in green indicatingdotted carbonlines, electrostatic atoms, white in yello forw, hydrogen, and hydrophobic and red in forpurple. oxygen Hydrogen bonds shown in green dotted lines,Figure electrostatic3. (a) Interactions in yellow, of ligands and with hydrophobic UCK2 protein in as purple. identified by in silico docking analysis; (b) OnLigands the other interacting hand, like with flavokawai the aminon acidsB, alpinetin residues also in thebinds active to thesites γ -of and UCK2 β-phosphate protein; (c )moieties Surface of ATP representation of the hydrophobic contacts of bound ligand and the ligand binding pocket of UCK2 (S2 files). Three hydrogen bonds are formed with the amino acid side chains of HN, Hζ1 and Hη12 atom of Ala-30, On theprotein other shown hand, as like translucent flavokawain blue surface. B, alpinetin Ligands shown also as binds a ball-and-stick to the γ- model and β with-phosphate purple moieties Lys-33 and Arg-169, respectively. The Oε1 atom and carboxyl group of Glu-135 also form two hydrogen bonds indicating carbon atoms, white for hydrogen, and red for oxygen Hydrogen bonds shown in green of ATPwith (S2 the files). 5-hydroxyl Three group hydrogen and methoxy bonds group are of formed alpinetin. with In addition, the amino three acidhydrog sideen bonds chains are of also HN, formed Hζ1 and dotted lines, electrostatic in yellow, and hydrophobic in purple. Hη12with atom the ofside Ala-30, chain of Lys-33 Thr-29, andTyr-65 Arg-169, and Asp- respectively.62. Asp-62 has Thebeen Oknownε1 atom to activate andcarboxyl the 5′-hydroxyl group group of Glu-135 of also formribonucleosides twoOn hydrogenthe other because hand, bonds it’s like the flavokawai with only the aminon 5-hydroxyl B, alpinetinacid residu also groupe bindsthat and canto the methoxyfunction γ- and asβ group-phosphate a catalytic of alpinetin.moieties base around of ATP In addition, the three5′ hydrogen-hydroxyl(S2 files). Threegroup bonds hydrogen [7,19]. are Therefore, bonds also are formed thformede hydrogen with with the bond the amino sidebetween acid chainside the chains hydroxyl of Thr-29,of HN, group Hζ1 Tyr-65 and of alpinetinHη12 and atom and Asp-62. of Ala-30,HN atom Asp-62 has beenof Lys-33Asp-62 known and may Arg-169,to strongly activate respectively. inhibit the the5The1-hydroxyl 5O′-hydroxylε1 atom and group group carboxyl ofinteraction group ribonucleosides of Glu-135of ribonucleosides, also becauseform two evenhydrogen it’s if the they bonds only were amino phosphorylated. In comparison to the control feedback inhibitor CTP, the binding mode of UCK2 inhibitors acid residuewith the that 5-hydroxyl can function group and as methoxy a catalytic group baseof alpineti aroundn. In addition, the 51-hydroxyl three hydrog groupen bonds [ 7are,19 also]. Therefore,formed the positionedwith the inside such chain a way of Thr-29, that the Tyr-65 inhibitors and Asp- buried62. Asp-62 themselves has been deep known in the to ATP activate binding the 5 pocket′-hydroxyl surrounded group of by hydrogenaminoribonucleosides bondacid side between chains because of the the it’s hydroxyl ATP the bindingonly amino group site (Figureacid of alpinetinresidu 4). e that and can HNfunction atom as ofa catalytic Asp-62 base may around strongly the inhibit 1 the 5 -hydroxyl5′-hydroxyl group group [7,19]. interaction Therefore, of th ribonucleosides,e hydrogen bond between even the if they hydroxyl were group phosphorylated. of alpinetin and HN In atom comparison to the controlof Asp-62 feedback may strongly inhibitor inhibit CTP, the 5 the′-hydroxyl binding group mode interaction of UCK2 of ribonucleosides, inhibitors positioned even if they in were such a way that thephosphorylated. inhibitors buried In comparison themselves to the deepcontrol in feedback the ATP inhibitor binding CTP,pocket the binding surrounded mode of UCK2 by aminoinhibitors acid side positioned in such a way that the inhibitors buried themselves deep in the ATP binding pocket surrounded by chains of the ATP binding site (Figure4). amino acid side chains of the ATP binding site (Figure 4).

(a) (b)

Figure 4. Comparison of the binding mode of UCK2 inhibitors in the ligand binding pocket of UCK2 surface. (a) FKB shown in purple color and CTP in maroon color; (b) APN shown in purple color and CTP in maroon color. (a) (b)

It hasFigure been 4. shownComparison that ligand of the binding modeat both of the UCK2 phosphate inhibitors donor in the and ligand acceptor binding binding pocket sites of UCK2competitively Figure 4. Comparison of the binding mode of UCK2 inhibitors in the ligand binding pocket of UCK2 inhibit UCKsurface. [19]. (a Therefore,) FKB shown the in inhi purplebitors color are and bound CTP to in the maroon site where color; γ(b-) and APN β -phosphateshown in purple moieties color of and ATP bind, surface.thus, theCTP (inhibitiona) in FKB maroon shown may color. be in competitive. purple color Flavokawain and CTP in B maroonand alpinetin color; are (b )therefore APN shown estimated in purple to inhibit color UCK2 and CTPprotein in by maroon binding color. to the catalytic active site of ATP, thus inhibiting ATP from binding to its active site in the UCK2 protein.It has been shown that ligand binding at both the phosphate donor and acceptor binding sites competitively inhibit UCK [19]. Therefore, the inhibitors are bound to the site where γ- and β-phosphate moieties of ATP bind, It has been shown that ligand binding at both the phosphate donor and acceptor binding 2.3.thus, In Vitro the inhibition Kinase Activity may be competitive. Flavokawain B and alpinetin are therefore estimated to inhibit UCK2 sites competitivelyprotein by binding inhibit to the catalytic UCK [ 19active]. Therefore,site of ATP, th theus inhibiting inhibitors ATP arefrom boundbinding to to its the active site site where in the γ- and β-phosphateUCK2The protein. fluorimetric moieties of kinase ATP assay bind, is thus, a non-radioactiv the inhibitione determination may be competitive. of kinase activity Flavokawain based on the B and alpinetinamount are of therefore ADP produced estimated by an to enzyme inhibit reaction UCK2 protein in the presence by binding of ATP, to the where catalytic the amount active of site ADP of ATP, formed2.3. In is Vitro directly Kinase proportional Activity to the enzyme activity. To validate our molecular docking result, we thus inhibiting ATP from binding to its active site in the UCK2 protein. carried Theout fluorimetrica preliminary kinase kinase assay activity is a non-radioactiv by measuringe determination the amount of of kinase ADP activityproduced based by onutilizing the 2.3. In5-fluorouridine Vitroamount Kinase of ADPActivity as produced a substrate by inan the enzyme presence reaction of either in the FKBpresence or APN. of ATP, Interestingly, where the amount both compounds of ADP reducedformed enzyme is directly activity proportional in a dose-dependent to the enzyme activity.manner To(S3 validate files, Figure our molecular 5). A significant docking reductionresult, we in The carried ﬂuorimetric out a preliminary kinase assay kinase is activity a non-radioactive by measuring determinationthe amount of ADP of kinase produced activity by utilizing based on the amount5-fluorouridine of ADP produced as a substrate by an enzymein the presence reaction of either in the FKB presence or APN. ofInterestingly, ATP, where both the compounds amount of ADP formedreduced is directly enzyme proportional activity in a to dose-dependent the enzyme activity.manner (S3 To files, validate Figure our 5). A molecular significant dockingreduction result,in we carried out a preliminary kinase activity by measuring the amount of ADP produced by utilizing Molecules 2016, 21, 417 6 of 10

5-fluorouridine as a substrate in the presence of either FKB or APN. Interestingly, both compounds reducedMolecules enzyme 2016, activity 21, 417 in a dose-dependent manner (S3 files, Figure5). A significant reduction6 of 10 in enzyme activity at a concentration of 25 µM (7.1 µg/mL) and 50 µM (14.2 µg/mL) was observed in a dose-dependentenzyme activity manner at a concentration when incubated of 25 µM with (7.1 µg/mL) FKB. A and significant 50 µM (14.2 difference µg/mL) was was observed also observed in a in dose-dependent manner when incubated with FKB. A significant difference was also observed in a a dose-dependent manner compared to DMSO treated control. This shows that the solvent (DMSO) dose-dependent manner compared to DMSO treated control. This shows that the solvent (DMSO) used asused vehicle as vehicle does does not contributenot contribute significantly significantly to the the enzyme enzyme activity activity since since enzyme enzyme assays assayscan can toleratetolerate high DMSO high DMSO concentrations concentrations up up to to 5%–10% 5%–10% [[20].20].

4000 4000 ** ns * ns ns ns 3000 3000 ns ns ns ns * 2000 * 2000 ns

** ** ** 1000 1000 UCK2 activity (µM ADP) (µM activity UCK2 UCK2 activity (µM ADP) (µM activity UCK2

0 0 l M M M er O M M µ µ µ MSO .5µ 0 uff .5 0µ Buffer 25 5 B MS 25µM 5 Control D 12 Contro D 12 DP DP A A (a) (b)

Figure 5. UCK2 enzyme activity was measured based on the amount of ADP produced from the Figure 5. UCK2 enzyme activity was measured based on the amount of ADP produced from the enzyme reaction (a) Cell lysate incubated in the presence of FKB (b) Cell lysate incubated in the

enzymepresence reaction of (APN.a) Cell Florescence lysate incubated intensity (λex in = the450nm/ presenceλem = 590) of was FKB monitored (b) Cell using lysate a fluorescence incubated in the presenceplate of APN.reader. Florescence Results were intensity expressed ( λasex mean= 450 ± nm/SD ofλ emthree= 590)independent was monitored experiments, using * pa <fluorescence 0.05, plate reader.** p < 0.01, Results ns: non-significant were expressed when compared as mean to˘ theSD control. of three independent experiments, * p < 0.05, ** p < 0.01, ns: non-significant when compared to the control. On the other hand, a significant reduction in enzyme activities was only observed when incubated with 50 µM (13.5 µg/mL) of APN, but this shows no significant difference compared to DMSO treated Oncontrol. the other Thus, hand, the potent a significant enzyme reductioninhibition of in APN enzyme is about activities 2-fold lower was compared only observed to that whenof FKBincubated at with 5025µ MµM. (13.5 Researchµg/mL) has shown of APN, that butUCK2 this also shows phosphorylates no significant uridine differencenucleoside analogs compared such toas DMSO treated5-fluorouridine. control. Thus, The the percentage potent enzyme efficiency inhibition of phosphorylation of APN of is 5-fluorouridine about 2-fold have lower been compared shown to to that of FKBbe at more 25 µ M.than Research 100% efficiency has shownwhen correlated that UCK2 to the also efficiency phosphorylates of uridine phosphorylation uridine nucleoside catalyzed analogs by UCK2 [6]. The results of the current investigation shown that 5-fluoroudine may have been such as 5-fluorouridine. The percentage efficiency of phosphorylation of 5-fluorouridine have been phosphorylated by the presence of UCK2 in the untreated cell lysate and the enzyme may have been shown toinhibited be more in HT than 29 100%cells treated efficiency with either when FKB correlated or APN. to the efficiency of uridine phosphorylation catalyzed by UCK2 [6]. The results of the current investigation shown that 5-fluoroudine may have been phosphorylated3. Materials and byMethods the presence of UCK2 in the untreated cell lysate and the enzyme may have been inhibited in HT 29 cells treated with either FKB or APN. 3.1. General Information 3. MaterialsThe and following Methods instruments were issued: Electrothermal IA 9100 series melting point apparatus (Staffordshire, UK), glass vacuum column, aluminium TLC plates (silica gel 60 F254, Merck Millipore, 3.1. GeneralDarmstadt, Information Germany), Bruker Avance 500 MHz Nuclear Magnetic Resonance (NMR) spectrometer (Billerica, MA, USA). NMR spectra were recorded in CDCl3 (δ 7.26 as internal reference) and reported Thein followingppm (δ), and instruments coupling constants were in issued: Hertz, NMR Electrothermal peak patterns IA are 9100 described series as melting broad (br), point singlets apparatus (Staffordshire,(s), doublets UK), (d), glass double-doublets vacuum column, (dd) triplets aluminium (t) and multiplets TLC plates (m). (silica GC-MS gel (Agilent 60 F254 Technologies,, Merck Millipore, Darmstadt,Santa Germany),Clara, CA, USA), Bruker fluorescence Avance mucriplate 500 MHz read Nuclearer (BioTek, Magnetic Winnoski, Resonance VT, USA), ELISA (NMR) microplate spectrometer reader (Beckman, Brea, CA, USA). All other chemicals and reagents were of analytical grade and (Billerica, MA, USA). NMR spectra were recorded in CDCl3 (δ 7.26 as internal reference) and reported obtained from Sigma-Aldrich (St. Louis, MO, USA). in ppm (δ), and coupling constants in Hertz, NMR peak patterns are described as broad (br), singlets (s), doublets3.2. Plant (d), Material double-doublets (dd) triplets (t) and multiplets (m). GC-MS (Agilent Technologies, Santa Clara, CA, USA), fluorescence mucriplate reader (BioTek, Winnoski, VT, USA), ELISA microplate About 6 kg of the rhizomes of Alpinia mutica was collected from the Biodiversity Unit of the reader (Beckman,Institute of Bioscience, Brea, CA, Universiti USA). All Putra other Malaysia chemicals Serdang. and The reagents rhizomes were were ofwashed analytical to remove grade and obtained from Sigma-Aldrich (St. Louis, MO, USA).

3.2. Plant Material About 6 kg of the rhizomes of Alpinia mutica was collected from the Biodiversity Unit of the Institute of Bioscience, Universiti Putra Malaysia Serdang. The rhizomes were washed to remove Molecules 2016, 21, 417 7 of 10 extraneous matter, sliced into small pieces, and air-dried at room temperature. The dried rhizome was Molecules 2016, 21, 417 7 of 10 then ground into a fine powder using a grinding machine. extraneous matter, sliced into small pieces, and air-dried at room temperature. The dried rhizome 3.3. Extractionwas then ground and Isolation into a fine of FKB powder and APNusing a grinding machine. Extraction and isolation of FKB and ALP were performed based on the reported procedure [21] 3.3. Extraction and Isolation of FKB and APN with some modifications. Ground plant material weighing 628.35 g was soaked and allowed to macerateExtraction at room temperatureand isolation of for FKB 72 and h sequentially ALP were pe withrformed solvents based on of the increasing reported polarity,procedure first [21] with hexanewith (3.2 some L) and modifications. then chloroform Ground (3.2 plant L) formaterial three consecutiveweighing 628.35 times. g was Each soaked of the and solutions allowed obtained to macerate at room temperature for 72 h sequentially with solvents of increasing polarity, first with was filtered and concentrated in a rotatory evaporator at a reduced pressure to obtain a crude extract. hexane (3.2 L) and then chloroform (3.2 L) for three consecutive times. Each of the solutions obtained The final concentrate was further allowed to dry at room temperature to complete dryness. Each of was filtered and concentrated in a rotatory evaporator at a reduced pressure to obtain a crude extract. the crudeThe final fractions, concentratei.e., hexane was further (18.22 allowed g, 2.90%) to dry and at chloroformroom temperature (24.41 to g, complete 3.88%) was dryness. was subjectedEach of to columnthe chromatographycrude fractions, i.e., forhexane isolation (18.22 andg, 2.90%) purification and chloroform to obtain (24.41 pure g, 3.88%) compounds was was using subjected silica to gel as stationarycolumn phase chromatography and mixtures for ofisolation petroleum and purification ether, ethyl to acetate obtain andpure methanolcompounds in using various silica proportions gel as as mobilestationary phases. phase All and the mixtures fractions of petroleum were monitored ether, ethyl by acetate TLC, spottedand methanol under in UVvarious and proportions the major spot ˝ identifiedas mobile by sprayingphases. All with the fractions 10% H2SO were4 and monitore heatedd by at TLC, 100 spottedC. The under purified UV naturaland the productsmajor spot were thenidentified analyzed by by spraying spectroscopic with 10% analysis, H2SO4 and in heated particular, at 100 °C.1H- The and purified13C-NMR natural spectroscopy products were as then well as 1 13 directanalyzed infusion by mass spectroscopic spectrometry analysis, (DIMS). in particular, Further H- washing and C-NMR of the hexanespectroscopy and chloroformas well as direct fractions infusion mass spectrometry (DIMS). Further washing of the hexane and chloroform fractions with with petroleum ether afforded FKB (melting point 82–84 ˝C; 200 and 226.4 mg, 1.1% and 0.93%, petroleum ether afforded FKB (melting point 82–84 °C; 200 and 226.4 mg, 1.1% and 0.93%, respectively)respectively) while while APN APN was was obtained obtained from from thethe chloroformchloroform fraction fraction after after washing washing with with petroleum petroleum ˝ etherether (melting (melting point point 212–214 212–214C; °C; 32.4 32.4 mg, mg, 0.13%). 0.13%). Both FKB FKB and and APN APN (Figure (Figure 6) 6were) were identified identified and and matchedmatched previously previously established established NMR NMR and and mass mass spectralspectral data data (S4 (S4 files files and and S5 S5files) files) [22–26]. [22–26 ].

Figure 6. Skeletal structures of flavokawain B and alpinetin. Figure 6. Skeletal structures of flavokawain B and alpinetin. 3.4. Cell Culture 3.4. Cell Culture The HT-29 cell line (ATCC) used in this study was obtained from the MAKNA Cancer TheResearch HT-29 Laboratory cell line of (ATCC)the Institute used of Bioscience. in this study The cell was line obtained was sub-cultured from the in DMEM MAKNA media Cancer Research(Sigma-Aldrich) Laboratory supplemented of the Institute with 10% of fetal Bioscience. bovine serum The (FBS) cell (PAA, line wasFreibug, sub-cultured Germany) and in 1% DMEM media100 (Sigma-Aldrich) IU penicillin and supplemented 100 µg/mL streptomycin with 10% (Sigma). fetal bovine The starting serum culture (FBS) (PAA,was at 1 Freibug, × 104 cells/mL Germany) and 1%and 100maintained IU penicillin at a temperature and 100 ofµ 37g/mL °C in streptomycin a humidified incubator (Sigma). containing The starting 5% CO culture2. Cultures was at 1 ˆ 10were4 cells/mL continuouslyand maintainedmaintained by at routine a temperature trypsinization of 37 (0.05%˝C in Trypsin-EDTA) a humidified of incubator cells at 70%–80% containing confluence. 5% CO2. Cultures were continuously maintained by routine trypsinization (0.05% Trypsin-EDTA) of cells at3.5. 70%–80% Drugs confluence.

3.5. Drugs5-Fluorouracil and bioactive compounds isolated from rhizome of Alpinia mutica was used in this investigation. A stock solution of 5-fluorouracil/bioactive compound was prepared in a 5-Fluorouracilconcentration of and 400 bioactiveµM in 50 µL compounds dimethyl sulfoxide isolated (DMSO) from rhizome and the of finalAlpinia concentration mutica was of usedDMSO in this investigation.will be 0.1% A stock(v/v). solution of 5-ﬂuorouracil/bioactive compound was prepared in a concentration of 400 µM in 50 µL dimethyl sulfoxide (DMSO) and the ﬁnal concentration of DMSO will be 0.1% (v/v).

3.6. Cell Viability Assay The effect of bioactive compounds on colorectal cancer was determined by a MTT assay. Brieﬂy, HT 29 cells were seeded in 96-well microplates at a density of 0.5 ˆ 104 cells/mL. Cells were treated Molecules 2016, 21, 417 8 of 10 after 24 h incubation at different concentrations with FKB, APN or 5FU (400, 200, 100, 50, 25, 12.5, and 6.25 µM) in serial dilution for 72 h. After 72 h incubation, 20 µL of MTT stock solution (5 mg/mL) was added to each well and 100 µL of DMSO was added to each well after 4 h incubation in the dark. The amount of purple formazan formed was measured colorimetrically at 570 nm. Cell viability was expressed as the percentage of amount of viable cells to that of the amount of the total cell population and the potency of testing drugs to inhibit cell growth by 50% was expressed as IC50. The cell viability assay was carried out in three independent experiments.

3.7. Molecular Docking Simulation Studies Molecular docking studies were performed using Autodock version 4.2 [18], analyzed and visualized using Discovery Studio visualizer version 4.5. The X-ray protein crystal structure (2.6 Å resolution) of human uridine-cytidine kinase 2 in complexed with a feedback inhibitor, CTP (1UDW.pdb ID) [19] was obtained from the Protein Data Bank (www.pdb.org). The three dimensional structures of FKB and APN was obtained from National Centre for Biotechnology information PubChem database [27] in an SDF file format and converted into the PDB file format using DS visualizer 4.5. Gasteiger charges were added to the ligand and all non-polar hydrogen atoms were deleted and their charges merged with the carbon atoms. The root of the molecule was detected, rotatable bonds were defined, and the number of torsions was set to 6. Prior to molecular docking, all solvent molecules, heteroatoms and the co-crystallized ligand (CTP) were removed from the structure [28,29] and all missing hydrogen atoms in the protein were added. The grid parameter file was prepared by setting the grid maps of 40 ˆ 40 ˆ 40 Å grid points in xyz, 0.375 Å spacing, and the grid box was positioned directly at the center on CTP-binding site of the crystal structure 1UDW (grid center 11.359 Å, 38.57 Å, and 33.111 Å in xyz-coordinates). Lamarckian genetic algorithm was used to carry out conformational searching in molecular docking simulation studies [18]. A molecular docking experiment was performed using 2,500,000 energy evaluations for 100 numbers of GA Runs per ligand with a population size set at 150.

3.8. Preparation of Cell Lysate HT 29 cells were collected by trypsinization at 70%–80% conﬂuence and centrifuged at 1000 rpm for 5 min at 4 ˝C. Supernatant was discarded, re-suspended in ice cold PBS and centrifuge at 1000 rpm for 5 min. Supernatant was discarded and the cell pellet was re-suspended in an appropriate volume of ProteoJET mammalian cell lysis reagent (Fermentas, Burlington, ON, Canada) and vortex for 5–10 s. The cells were allowed to incubate at room temperature on a shaker at approximately 900 rpm for 10 min and centrifuge at 18,000ˆ g for 15 min. The supernatant was carefully collected, aliquots in PCR tubes and stored at ´80 ˝C until use. Total protein concentration in the cell lysate using Bradford reagent (Bio-Rad Laboratories, Hercules, CA, USA).

3.9. In Vitro Kinase Activity Assay The inhibitory activity of bioactive compounds on UCK2 were assayed using a Universal fluorimetric kinase assay kit (Sigma). A total 80 µL volume of the kinase reaction mixture was set up containing 20 µL of ADP Buffer, 25 µL of cell lysate/H2O, 20 µL of 12.5, 25, and 50 µM test drug/DMSO/H2O, 10 µL of 0.5 mM 5-fluorouridine/H2O, and 5 µL of 1 mM ATP/H2O. The reaction mixture was incubated in a water bath at 37 ˝C for 30 min. 20 µL of the kinase reaction was added into 96 black well microplate. For each well containing 20 µL kinase reaction, 20 µL of ADP sensor buffer was added, followed by 10 µL of the ADP sensor solution and the assay mixture was allowed to incubate for 15 min in the dark at room temperature. Florescent intensity (λex = 450 nm/λem = 590) was monitored using a fluorescence plate reader. This assay was carried out in three independent experiments. Molecules 2016, 21, 417 9 of 10

3.10. Statistical Analysis The results of the experiments are reported as mean ˘ SD for at least three replicate analyses for each sample. Statistical analysis was performed using GraphPad Prism 5.0. Analyses of variance are performed using the ANOVA procedure followed by Dunnett’s and Bonferroni’s test for multiple comparison.

4. Conclusions In conclusion, we have shown for the ﬁrst time that ﬂavokawain B and alpinetin act as potential UCK2 inhibitors in silico. Flavokawain B and alpinetin have shown interesting inhibition of this key enzyme involved in gene synthesis. This inhibition may provide information supporting their use as lead candidates for further drug development. Moreover, further studies are currently under way to determine the molecular mechanism of action by which FKB and ALP inhibit UCK2 from further phosphorylation of nucleosides during gene replication.

Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/ 21/4/417/s1. Acknowledgments: This study was co-supported by TETFund and Universiti Putra Malaysia Geran Pura IPS (Vote No.: 9457100). The authors gratefully acknowledge the costs of publication covered by the Research Management Centre at the Universiti Putra Malaysia. Author Contributions: Ibrahim Malami formulated the idea, carried out the experiments, analyzed data and prepared the manuscript; Ahmad Bustamam Abdul designed the experiments and contributed to the manuscript preparation; Rashedee Abdullah and Nur Kartinee Bt Kassim contributed materials and reagents; Peter Waziri, and Imaobong Christopher Etti participated in the research. Conﬂicts of Interest: All authors declared no conﬂict of interest.

References

1. Wang, F.; Liu, X.; Liu, C.; Liu, Z.; Sun, L. Effects of antibiotic antitumor drugs on nucleotide levels in cultured tumor cells: An exploratory method to distinguish the mechanisms of antitumor drug action based on targeted metabolomics. Acta Pharm. Sin. B 2015, 5, 223–230. [CrossRef][PubMed] 2. Berg, J.M.; Tymoczko, J.L.; Stryer, L. Biochemistry, 5th ed.; W H Freeman: New York, NY, USA, 2002. 3. Zhang, C.; Liu, Z.; Liu, X.; Wei, L.; Liu, Y.; Yu, J.; Sun, L. Targeted metabolic analysis of nucleotides and identiﬁcation of biomarkers associated with cancer in cultured cell models. Acta Pharm. Sin. B 2013, 3, 254–262. [CrossRef] 4. Lane, A.N.; Fan, T.W.-M. Regulation of mammalian nucleotide metabolism and biosynthesis. Nucleic Acids Res. 2015, 43, 2466–2485. [CrossRef][PubMed] 5. Connolly, G.P.; Duley, J.A. Uridine and its nucleotides: Biological actions, therapeutic potentials. Trends Pharmacol. Sci. 1999, 20, 218–225. [CrossRef] 6. Van Rompay, A.R.; Norda, A.; Lindén, K.; Johansson, M.; Karlsson, A. Phosphorylation of uridine and cytidine nucleoside analogs by two human uridine-cytidine kinases. Mol. Pharmacol. 2001, 59, 1181–1186. [PubMed] 7. Appleby, T.C.; Larson, G.; Cheney, I.W.; Walker, H.; Wu, J.Z.; Zhong, W.; Hong, Z.; Yao, N. Structure of human uridine-cytidine kinase 2 determined by SIRAS using a rotating-anode X-ray generator and a single samarium derivative. Acta Crystallogr. D Biol. Crystallogr. 2005, 61, 278–284. [CrossRef][PubMed] 8. Friday, B.; Lassere, Y.; Meyers, C.A.; Mita, A.; Abbruzzese, J.L.; Thomas, M.B. A phase I study to determine the safety and pharmacokinetics of intravenous administration of TAS-106 once per week for three consecutive weeks every 28 days in patients with solid tumors. Anticancer Res. 2012, 32, 1689–1696. [PubMed] 9. Naing, A.; Fu, S.; Zinner, R.G.; Wheler, J.J.; Hong, D.S.; Arakawa, K.; Falchook, G.S.; Kurzrock, R. Phase I dose-escalating study of TAS-106 in combination with carboplatin in patients with solid tumors. Investig. New Drugs 2013, 32, 154–159. [CrossRef][PubMed] 10. Peters, G.J.; Julsing, J.R.; Smid, K.; De Klerk, D.; Sarkisjan, D.; Yang, M.Y.; Lee, Y.B.; Kim, D.J. Fluorocyclopentenylcytosine (RX-3117) is activated by uridine-cytidine kinase 2, a potential biomarker. In Proceedings of the 106th Annual Meeting of the American Association for Cancer Research, Philadelphia, PA, USA, 18–22 April 2015. Molecules 2016, 21, 417 10 of 10

11. Hammond-Thelin, L.A.; Thomas, M.B.; Iwasaki, M.; Abbruzzese, J.L.; Lassere, Y.; Meyers, C.A; Hoff, P.; de Bono, J.; Norris, J.; Matsushita, H.; et al. Phase I and pharmacokinetic study of 3’-C-ethynylcytidine (TAS-106), an inhibitor of RNA polymerase I, II and III, in patients with advanced solid malignancies. Investig. New Drugs 2012, 30, 316–326. [CrossRef][PubMed] 12. Tsao, A.; Hui, E.P.; Juergens, R.; Marur, S.; Huat, T.E.; Cher, G.B.; Hong, R.-L.; Hong, W.K.; Chan, A.T.-C. Phase II study of TAS-106 in patients with platinum-failure recurrent or metastatic head and neck cancer and nasopharyngeal cancer. Cancer Med. 2013, 2, 351–359. [CrossRef][PubMed] 13. Mayer, R.J.; Van Cutsem, E.; Falcone, A.; Yoshino, T.; Garcia-Carbonero, R.; Mizunuma, N.; Yamazaki, K.; Shimada, Y.; Tabernero, J.; Komatsu, Y.; et al. Randomized trial of TAS-102 for refractory metastatic colorectal cancer. N. Engl. J. Med. 2015, 372, 1909–1919. [CrossRef][PubMed] 14. Pandit, N.K. Introduction to the Pharmaceutical Sciences, 1st ed.; Lippincott Williams &Wilkins: Philadelphia, PA, USA, 2007. 15. Kuo, Y.F.; Su, Y.Z.; Tseng, Y.H.; Wang, S.Y.; Wang, H.M.; Chueh, P.J. Flavokawain B, a novel chalcone from Alpinia pricei Hayata with potent apoptotic activity: Involvement of ROS and GADD153 upstream of mitochondria-dependent apoptosis in HCT116 cells. Free Radic. Biol. Med. 2010, 49, 214–226. [CrossRef] [PubMed] 16. Malek, S.N.A.; Phang, C.W.; Ibrahim, H.; Wahab, N.A.; Sim, K.S. Phytochemical and cytotoxic investigations of Alpinia mutica rhizomes. Molecules 2011, 16, 583–589. [CrossRef][PubMed] 17. Morris, G.M.; Goodsell, D.S.; Halliday, R.S.; Huey, R.; Hart, W.E.; Belew, R.K.; Olson, A.J. Automated docking using a lamarckian genetic algorithm and an empirical binding free energy function. J. Comput. Chem. 1998, 19, 1639–1662. [CrossRef] 18. Morris, G.M.; Huey, R.; Lindstrom, W.; Sanner, M.P.; Belew, R.K.; Goodsell, D.S.; Olson, A.J. AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J. Comput. Chem. 2009, 30, 2785–2791. [CrossRef][PubMed] 19. Suzuki, N.N.; Koizumi, K.; Fukushima, M.; Matsuda, A.; Inagaki, F. Structural basis for the specificity, catalysis, and regulation of human uridine-cytidine kinase. Structure 2004, 12, 751–764. [CrossRef][PubMed] 20. Glickman, J.F. Assay Development for Protein Kinase Enzymes. In Assay Guidance Manual; The Rockefeller University: New York, NY, USA, 2004, (available in NCBI: NBK53196). 21. Mustahil, N.A.; Sukari, M.A.; Abdul, A.B.; Ali, N.A.; Ee, G.; Lian, C. Evaluation of biological activities of Alpinia mutica Roxb. and its chemical constituents. Pak. J. Pharm. Sci. 2013, 26, 391–395. [PubMed] 22. Sirat, H.M.; Masri, D.; Rahm, A. Constituents of the Rhizomes of Alpinia rafflesiana. Pertanika J. Sci. Technol. 1996, 4, 17–22. 23. Ranjith, W.; Dharmaratne, H.; Dhammika Nanayakkara, N.P.; Khan, I.A. Kavalactones from Piper methysticum, and their 13C NMR spectroscopic analyses. Phytochemistry 2002, 59, 429–433. [CrossRef] 24. Yap, A.; Ching, L.; Wah, T.S.; Sukari, M.A.; Ee, G.; Lian, C. Characterization of flavonoid derivatives from Boesenbergia rotunda (L.). Malays. J. Anal. Sci. 2007, 11, 154–159. 25. Zhou, P.; Gross, S.; Liu, J.-H.; Yu, B.-Y.; Feng, L.-L.; Nolta, J.; Sharma, V.; Piwnica-Worms, D.; Qiu, S.X. Flavokawain B, the hepatotoxic constituent from kava root, induces GSH-sensitive oxidative stress through modulation of IKK/NF-kappaB and MAPK signaling pathways. FASEB J. 2010, 24, 4722–4732. 26. Xiao, X.; Si, X.; Tong, X.; Li, G. Preparation of flavonoids and diarylheptanoid from alpinia katsumadai hayata by microwave-assisted extraction and high-speed counter-current chromatography. Sep. Purif. Technol. 2011, 81, 265–269. 27. PubChem. Available online: NCBI: http://www.ncbi.nlm.nih.gov/pccompound (accessed on 29 June 2015). 28. Powers, C.N.; Setzer, W.N. A molecular docking study of phytochemical estrogen mimics from dietary herbal supplements. In Silico Pharmacol. 2015, 3, 4. [CrossRef] 29. Saha, S.; Islam, M.; Shilpi, J.A.; Hasan, S. Inhibition of VEGF: A novel mechanism to control angiogenesis by Withania somnifera’s key metabolite Withaferin A. In Silico Pharmacol. 2013, 1, 11. [CrossRef]

Sample Availability: Samples of ﬂavokawain B and alpinetin are available from the authors.

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