Identification of α-Mangostin as a Potential Inhibitor of Microtubule

Jul 25, 2019 - Microtubule affinity regulating kinase 4 (MARK4) is a potential drug target for neuronal disorders and several types of cancers. Filtra...
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Identification of α‑Mangostin as a Potential Inhibitor of Microtubule Affinity Regulating Kinase 4 Parvez Khan,†,§ Aarfa Queen,†,‡,§ Taj Mohammad,† Smita,† Nashrah Sharif Khan,† Zubair Bin Hafeez,∥ Md. Imtaiyaz Hassan,*,† and Sher Ali*,† †

Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, India Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India ∥ Department of Biosciences, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India Downloaded via BUFFALO STATE on July 25, 2019 at 22:29:03 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

S Supporting Information *

ABSTRACT: Microtubule affinity regulating kinase 4 (MARK4) is a potential drug target for neuronal disorders and several types of cancers. Filtration of naturally occurring compound libraries using high-throughput screening and enzyme assay suggest α-mangostin is a potential inhibitor of MARK4. Structure-based docking and 100 ns molecular dynamics simulation revealed that the binding of α-mangostin stabilizes the MARK4 structure. Enzyme inhibition and binding studies showed that α-mangostin inhibited MARK4 in the submicromolar range with IC50 = 1.47 μM and binding constant (Ka) 5.2 × 107 M−1. Cell-based studies suggested that α-mangostin inhibited the cell viability (MCF-7 and HepG2), induced apoptosis, arrested the cell cycle in the G0/G1 phase, and reduced tau-phosphorylation. This study implicates MARK4 as a new target of α-mangostin, adding an additional lead molecule to the anticancer repertoire.

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compounds have been found to reduce the risk of tumors, diabetes, and neurodegeneration.20 Natural compounds possess anticancer, antioxidant, and anti-inflammatory activities. These compounds have been used as leads for drug discovery.21−24 It is difficult to predict how a small molecule will interact with the myriad of targets driving fundamental biological processes. High-throughput methods or structure-based drug design make it feasible to identify molecules having binding affinities.25,26 For drug discovery, millions of natural products that have been stored in libraries such as DrugBank, the NCI database, and the ZINC library are screened by various computational approaches such as high-throughput screening (HTS) or virtual screening to find potential leads against selected targets.25−27 Here, in search of inhibitors of MARK4, α-mangostin was selected with the help of structure-based vHTS of naturally occurring compounds (from different databases and an inhouse library of natural compounds) followed by 100 ns molecular dynamics (MD) simulations. Inferences of an in silico study were extended to in vitro studies that suggested αmangostin inhibits MARK4. More importantly this study provided a direction for the repurposing of α-mangostin and αmangostin-based inhibitors in the treatment of MARK4oriented cancers and related disorders.

ancer is an array of disorders, and often the anticancer strategies are based on pursuing the different flaws including oncogenic or non-oncogenic genetic defects.1,2 Personalized strategies targeting genetic disorders are focused on intratumor heterogeneity, high rate of somatic mutations, and adaptation of cancer cells to a new environment.3 A careful analysis of different types of kinases showed that microtubule affinity regulating kinase 4 (MARK4) is overexpressed in a large number of cancers, suggesting its involvement in cancer progression and metastasis.4−6 Initially MARK4 was recognized with its primary function to phosphorylate tau and other microtubule-associated proteins (MAPs) at Ser residues in KXGS motifs.5 Detailed studies suggested the diverse role of MARK4 such as guiding neuronal migration, cell polarity, microtubule dynamics, apoptosis, cell cycle regulation, cell signaling, and differentiation.7,8MARK4 is found to be overexpressed in metabolic disorders including diet-induced obesity, cardiovascular diseases, type-II diabetes, Alzheimer’s disease, hepatocellular carcinoma, glioma, and metastatic breast carcinomas.9−12 Recent reports suggested the role of MARK4 in breast cancer cell proliferation and migration through hippo signaling.6 It also regulates miR515-5p, implicated in cancer cell migration and metastasis.13 Thus, MARK4 qualifies to be a potential target for cancer drug discovery.14−16 Due to their enormous structural and chemical diversity, natural compounds/products continue to be the best sources of chemical entities employed in the search for drugs and drug leads.14,17,18 Natural products exemplify the richest source of novel molecular scaffolds for synthetic chemists as well.17,19 A number of natural molecules and their derivatives have been tested for their therapeutic properties. Several of these © XXXX American Chemical Society and American Society of Pharmacognosy

Received: April 28, 2019

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DOI: 10.1021/acs.jnatprod.9b00372 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 1. Structure and interacting residues of MARK4. (A) Chemical structure of α-mangostin. (B) 2D representation of MARK4 residues interacting with α-mangostin. (C) Three-dimensional view of MARK4 residues interacting with α-mangostin (α-mangostin is shown in ball-andstick representation; residues participating in polar contacts and other interactions are shown in green element sticks and lines, respectively).

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RESULTS AND DISCUSSION Screening of Natural Molecules. We have screened naturally occurring compounds from the ZINC database and our in-house library of natural compounds. It was found that many natural molecules bind efficiently with MARK4. The inhibitory potential of these compounds was evaluated with MARK4. Selected molecules were screened with recombinant MARK4 at a single concentration of 10 μM (Figure S1). Enzyme activity results suggested that at this concentration ellagic acid and α-mangostin inhibit MARK4. Further, it was found that α-mangostin inhibits MARK4 more potently than ellagic acid (Figure S1). Since α-mangostin displays the best potency against MARK4, it was evaluated through detailed in silico and in vitro studies. Molecular Docking. Molecular docking studies revealed that α-mangostin binds at the binding pocket of MARK4 with an affinity of −9.2 kcal/mol. The compound α-mangostin is present in the deep cavity of MARK4 and has several close interactions with binding pocket residues (Figure 1, Figure S2, Supporting Information). It forms three hydrogen bonds with Lys64, Lys85, and Asp196 and interacts with Ile62, Gly63,

Asn66, Phe67, Ala68, Val70, Ala83, Leu84, Val116, Met132, Tyr134, Ala135, Gly138, Glu182, Leu185, and Ala195 (Figure 1). It was found that the oxygen atom of two hydroxyl groups of α-mangostin interacts with Lys64, Lys85, and Asp196 via hydrogen bonding, and these residues belong to the catalytic domain of MARK4.28 The crystal structure of MARK4 suggested that Asp196 of the DFG motif is mainly responsible for the interaction of the known inhibitor of MARK4, and these interactions stabilize the switching of the activation loop and positions the substrate in the binding cavity.28 The binding of α-mangostin also involves similar types of interactions with the catalytic domain of MARK4 and thus reduces the catalytic activity of MARK4. Moreover, the surface representations also indicate that α-mangostin occupies the internal cavity and binds MARK4 with great affinity (Figure S2, Supporting Information). Average Potential Energy of the Systems. After performing the MD simulation studies, the average potential energy of free MARK4 and MARK4−α-mangostin were calculated to evaluate the stability of free MARK4 and MARK4 complexed with α-mangostin during the simulations. B

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Table 1. Calculated Parameters for Both the Systems during MD Simulations (100 ns) complex MARK4 MARK4−αmangostin

average potential energy (kJ/mol)

radius of gyration (nm)

average RMSD (nm)

average SASA (nm2)

free energy of solvation (kJ/mol/nm2)

volume (nm3)

density (g/L)

−1 147 344.54 −1 147 218.36

1.95 2.03

0.30 0.33

146.53 151.15

200.26 201.82

58.88 59.21

988.73 981.48

Figure 2. Structural changes in MARK4 upon binding of α-mangostin. (A) RMSD plot of native MARK4 and complexed with α-mangostin in black and red color, respectively. (B) Rg plot of native MARK4 and complexed with α-mangostin in black and red color, respectively. (C) RMS fluctuations in MARK4 and upon binding of α-mangostin in black and red color, respectively. (D) SASA plot of native MARK4 and complexed with α-mangostin in black and red color, respectively.

mangostin throughout the simulations in a region spanning from the N-terminal to the C-terminal. The radius of gyration (Rg) is a parameter linked to the tertiary structural volume of a protein and has been applied to obtain insight into the stability of a given protein in a biological system. A protein with a higher radius of gyration is less tightly packed. The average Rg values for free MARK4 and MARK4−α-mangostin were found to be 1.95 and 2.03 nm, respectively. The Rg data suggest that MARK4 became less compact in the presence of α-mangostin (Figure 2C). Solvent-Accessible Surface Area. The solvent-accessible surface area (SASA) of a protein is the surface area of a given protein that interacts with its surrounding solvent. The SASA is directly related to the Rg of a protein. The average SASA values for MARK4 and MARK4−α-mangostin complexes were calculated during 100 ns MD simulations. The average SASA values for MARK4 protein and MARK4 complexed with αmangostin were found to be 146.53 and 151.15 nm2, respectively. A slight increase in SASA values was observed in the presence of α-mangostin, which was possibly due to less tight packing of MARK4. However, no major change was seen in the SASA values upon α-mangostin binding to MARK4 (Figure 2D). During SASA calculations, the free energies of solvation of MARK4 and MARK4−α-mangostin were found to be 200.26 and 201.82 kJ/mol/nm2, respectively.

An average potential energy for free MARK4 and MARK4−αmangostin was found to be −1 147 344.54 and −1 147 218.36 kJ/mol, respectively. Structural Deviations and Compactness. Binding of a small molecule in the binding pocket of a protein can lead to large conformational changes.29 Root mean square deviation (RMSD) is one of the most important fundamental properties to evaluate the structural stability of a protein.25,30 The average RMSD values for MARK4 and MARK4−α-mangostin were found to be 0.30 and 0.33 nm, respectively (Table 1). The RMSD data suggested that the binding of α-mangostin stabilized MARK4 and leads to less structural deviations from its native conformation (Figure 2A). However, some fluctuation can be seen in the RMSD plot upon binding of αmangostin possibly due to its orientation in the active pocket of MARK4. Binding of α-mangostin led to lower RMSD values at several parts and showed equilibration throughout the 100 ns MD simulations (Figure 2A). To further assess local structural flexibility, the average fluctuation of all residues and the root-mean square fluctuations (RMSF) of MARK4 upon ligand binding were plotted during the simulation as a function of residue number (Figure 2B). The resulting RMSF plot revealed several residual fluctuations in MARK4 at several regions of protein structure. These residual fluctuations were minimized upon binding of αC

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Table 2. Percentage of MARK4 Residues Participating in Average Structure Formation percentage of protein secondary structure (SS %) complex

structurea

coil

β-sheet

β-bridge

bend

turn

α-helix

310-helix

MARK4 MARK4−α-mangostin

63 62

23 24

14 13

1 1

13 12

10 11

38 37

1 1

Structure = α-helix + β-sheet + β-bridge + turn.

a

Figure 3. Enzyme inhibition and tau phosphorylation studies of MARK4 with α-mangostin. (A) Hydrolysis of Pi from ATP. The positions of Pi and ATP spots are indicated. Lane 1, negative control (no protein); lane 2, 100 nM MARK4 (positive control); and lanes labeled as 0.5, 1, 2, 3, 5, and 10 show the concentration of α-mangostin. (B) ATPase inhibition (% hydrolysis of Pi) with increasing concentrations of α-mangostin (μM) shown as a function of concentration calculated by comparing with positive control. Bar graph represents the normalized intensity of ATP hydrolysis ± SD; *p < 0.05; **p < 0.01, compared with the control. Statistical analysis was done using Student’s t test for unpaired samples. (C) Representative flow cytometry histogram of SH-SY5Y cells stained with phosphorylated anti-tau antibodies. Each histogram represents the phosphorylation status of tau under varying treatment conditions mentioned in the inset.

Hydrogen Bond Analysis. Hydrogen bonding between a protein and ligand provides a directionality and specificity of interaction that is a fundamental aspect of molecular recognition. To validate the stability of a docked complex, the hydrogen bonds paired within 0.35 nm between MARK4 and α-mangostin were calculated in a solvent environment during the 100 ns MD simulations. The results suggest that the α-mangostin binds to the active pocket of MARK4 with 3−5 hydrogen bonds with higher fluctuations and 2 or 3 hydrogen bonds with the least fluctuations, which also supports our molecular docking results (Figure S3, Supporting Information). Secondary Structural Changes upon Ligand Binding. Secondary structural changes upon ligand binding were calculated to measure the secondary structure of MARK4 as a function of time. The average number of residues

participating in secondary structure formation in the MARK4−α-mangostin complex was found to be slightly reduced due to an increase in the percentage of residues participating in coils and a decrease in residues participating in β-sheets in comparison with free MARK4 (Table 2, Figure S4, Supporting Information). These in silico data suggest that α-mangostin has a strong binding affinity with MARK4 as compared to other molecules. The α-mangostin−MARK4 complex is stabilized by hydrogen bonding as well as other noncovalent interactions such as π−π and van der Waals interactions (Figure 1). Molecular docking and MD simulation studies have shown that α-mangostin binds in the binding cavity of MARK4 to which its substrate binds, as reported by crystal studies of MARK4.28 Secondary structure evaluation and hydrogen bond analysis of the αmangostin−MARK4 complex through simulation studies D

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Figure 4. Binding studies of α-mangostin with MARK4. (A) Fluorescence emission spectra of MARK4 (10 μM) with the increasing concentration of α-mangostin showing quenching of MARK4 internal fluorescence with increasing concentration of α-mangostin. Excitation wavelength is 280 nm, and emission range is 300−400 nm. (B) Modified Stern−Volmer plot obtained from the quenching of MARK4 fluorescence with increasing concentration of α-mangostin and used to calculate binding affinity (Ka).

mangostin. A substantial decrease in the fluorescence intensity of MARK4 with increasing concentrations of α-mangostin was analyzed using a modified Stern−Volmer equation, and the value of the binding constant (Ka) was estimated (Figure 4A,B). The value of Ka for this binding was found to be 5.2 × 107 M−1. These results clearly indicated that α-mangostin binds with MARK4 and suggests that this binding leads to a slight blue shift in the emission spectrum of MARK4. This further suggests that α-mangostin forms a stable complex with MARK4. Thus, fluorescence-based binding studies of αmangostin with recombinant MARK4 also confirm the strong binding between the two. Inhibition of Cancer Cell Proliferation. To evaluate cancer cell proliferation inhibition potential of α-mangostin, human breast cancer cells (MCF-7) and human hepatic cancer cells (HepG2) were exposed to different concentrations of αmangostin. These cell lines have been chosen as these are known to express MARK4.6,13 For normal control, human kidney embryonic cells (HEK293) were selected. Treatment with α-mangostin suppressed the cell proliferation of MCF-7 and HepG2 cells (Figure 5A). The IC50 values of α-mangostin for the MCF-7 and HepG2 cells were 9.95 ± 0.45 and 18.55 ± 1.33 μM, respectively. Interestingly, it was found that αmangostin did not inhibit the growth of normal cells (HEK293) (Figure S5, Supporting Information). Studies on anticancer/apoptotic potential of α-mangostin showed that it inhibits the growth of cancerous cells. Previous studies also reported the anticancer properties of α-mangostin in different types of cancer cells and animal models.33−35 These reports generalized the activity of α-mangostin and suggested that it affects the viability of cancerous cells.34 Recently, it was found that MARK4 overexpression also supports the growth and evasion of cancerous cells.6 These results corroborate previous observations that MARK4 acts as a regulator of cell proliferation and migration in cancer cells,11 and its inhibition suppresses MCF-7 cell proliferation. Induction of Apoptosis in Cancer Cells. Although apoptosis is an essential process that regulates the typical growth of cells, it is compromised in cancerous cells, and thus

support the existence of 2 to 3 hydrogen bonds throughout the 100 ns simulation time scale. This means that α-mangostin− MARK4 is a stable complex and α-mangostin may decrease the accessibility of MARK4 for its substrate and thus behaves as an inhibitor. Enzyme Inhibition Assay. In silico studies suggest that αmangostin binds with MARK4 and the complex is stabilized by numerous interactions. This binding affinity may also be responsible for inhibition of MARK4 as shown by kinase assay screening results. The inhibition potential of α-mangostin with MARK4 was estimated in a concentration-dependent manner using an ATPase assay. Enzyme activity was performed with recombinant MARK4 as described in our previous communications.14,31 Results of the kinase assay showed that αmangostin inhibits MARK4 in a concentration-dependent manner with IC50 value of 1.77 ± 0.12 μM (Figure3A,B). Interestingly, these results showed that α-mangostin inhibits MARK4 more strongly than some of the previously reported inhibitors.14,31 A kinase inhibition assay suggested that α-mangostin inhibited MARK4; so to further validate these results, a cellbased tau phosphorylation assay was carried out as described previously.14,32 Cells were incubated with the IC50 concentration of α-mangostin, and tau phosphorylation status was studied using flow cytometry. The results indicate that treatment of α-mangostin shifts the position of the histogram left (lower value), which suggests that the level of tau phosphorylation decreased as compared to untreated controls (Figure 3C). These results support our MARK4 inhibition observation, as tau also acts as the substrate of MARK4. Inhibition of MARK4 as well as tau phosphorylation by αmangostin is consistent with earlier reports that inhibition of MARK4 decreases the phosphorylation of tau.5,37 Fluorescence-Based Binding Studies. Encouraged by the enzyme assay and in silico studies, the actual binding affinity of α-mangostin with MARK4 was estimated using a fluorescence-based binding assay. For this, the protein sample was excited at 280 nm, and emission spectra were recorded in the range 300−400 nm with increasing concentrations of αE

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Figure 5. Binding of α-mangostin with MARK4 inhibits cell growth, induces apoptosis and G0/G1 cell cycle arrest in MCF-7 and HepG2 cells. (A) Effect of α-mangostin on the viability of MCF-7 and HepG2 cells. Cells were treated with increasing concentrations of α-mangostin (0−50 μM) for 48 h. Cell viabilities are presented as a percentage of the number of viable cells to that of the control. Each data point shown is the mean ± SD from n = 3. *p < 0.05; **p < 0.01 (for anticancer activities, paclitaxel has been taken as the positive control). (B) Apoptosis studies of MCF-7 and F

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Figure 5. continued HepG2 cells treated with IC50 concentrations of α-mangostin for 24 h and processed for apoptosis analysis using an annexin-V/PI apoptosis kit. Histogram showing the anti-FITC-annexin-V and PI stained cells after the treatment with α-mangostin. Name of cell line is indicated inside each histogram. (C) Bar graphs represent the percentage of apoptotic cells stained with annexin-V for triplicate measurements ± SD. (D) Representative histogram showing the cell cycle distribution of MCF-7 and HepG2 cells after α-mangostin treatment as mentioned on each histogram. (E) Graphical presentation of cell cycle distribution in each phase and the respective treatment shown on the x-axis.

Figure 6. Treatment of α-mangostin decreases the production of ROS and inhibits cancer cell migration. (A) Bar graphs represent the relative intensity of DCF fluorescence for triplicate measurements ± SD; *p < 0.05, compared with the control. Statistical analysis was done using one-way ANOVA and t test for unpaired samples. Cells were treated with the IC50 concentration of α-mangostin for 5−6 h and processed for ROS measurements using DCFDA staining using spectroflourimetery. (B) Representative images (20× original magnification) showing inhibition of MCF-7 cell migration as determined by the wound healing assay.

evasion of apoptosis is a striking hallmark.36 MARK4 has a prominent role in the growth and evasion of different cancerous cells. Therefore, the effect of MARK4 inhibition on cell apoptosis was examined. Serum-starved cells were treated with the IC50 concentration of α-mangostin for 24 h, and apoptosis induction was studied through annexin-V staining. Treated cells were stained and analyzed by flow cytometry. The results showed that the treatment with αmangostin induces apoptosis in MCF-7 and HepG2 cells (Figure 5B). It was found that α-mangostin induces apoptosis in 25.90% and 19.70% of MCF-7 and HepG2 cells, respectively, as compared to the untreated control (Figure 5C). These results clearly suggested that inhibition of MARK4 using α-mangostin also inhibits the proliferation of MCF-7 and HepG2 cells and induces apoptosis. α-Mangostin Induces G0/G1-Phase Arrest of the Cell Cycle. MARK4 has an important role in cell cycle progression; thus the effect of α-mangostin treatment on the cell cycle progression of MCF-7 and HepG2 cells was studied. The cells were treated with α-mangostin (IC50 concentration) for 24 h, harvested, and processed for cell cycle analysis using flow cytometry. It was found that α-mangostin treatment increases the percentage of G0/G1 phase and decreases the percentage of S and G2 phase of the cell cycle as compared to control cells (Figure 5D,E). Thus, treatment of α-mangostin induces G0/ G1 arrest (Figure 5D,E). These observations suggesting the inhibitory effect of α-mangostin on MARK4 might be

responsible for G0/G1 phase cell cycle arrest of MCF-7 and HepG2 cells. The present study also showed that α-mangostin acts as a barricade in the cell cycle progression of highly aggressive MCF-7 and HepG2 cells. Uncontrolled progression of the cell cycle is a characteristic feature of cancer cells.36 To maintain the proper cell division, the cell cycle check-points have evolved to ensure accurate cell cycle progression. However, these check-points are compromised in many types of cancer cells.36,37 The G0/G1 check-point is an important target for anticancer therapies. Retraction or arrest of cancer cells from the G0/G1 check-point of the cell cycle inhibits DNA repair and S-phase entry. Interestingly, it was observed that treatment with α-mangostin arrested the MCF-7 and HepG2 cells in the G0/G1 phase of the cell cycle and induced apoptosis (Figure 5D,E). It was also an established fact that MARK4 is a key component in cell cycle regulation and is responsible for G1/S phase transition.8 Thus, the results of G1 arrest by treatment with α-mangostin were found to be consistent with these findings that inhibition of MARK4 arrested the cells in the G1 phase.8 α-Mangostin Inhibits Reactive Oxygen Species Production and Cancer Cell Migration. Each type of cell has its own microenvironment, and ROS are an important component of that microenvironment. Any change in this microenvironment may lead to cell death. In this context, ROS have a potential role in cell survival and apoptosis.14 Thus, we G

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evaluated the effect of α-mangostin treatment on the production of ROS. MCF-7 and HepG2 cells were treated with α-mangostin (IC50 concentration) for 5−6 h, and ROS levels were quantified by using 2′,7′-dichlorofluorescein diacetate (DCFDA) staining. Interestingly, the results showed that treatment with α-mangostin decreases the production of ROS (Figure 6A). These results suggested that α-mangostin behaves as an antioxidant and acts as a contributing factor to cancer cell death. MARK4 is also known to promote oxidative stress in adipocytes;12 thus inhibition of MARK4 may help to relieve oxidative stress.14 The results so obtained suggested that α-mangostin treatment leads to MARK4 inhibition and decreases the levels of ROS and also supports previous observations that MARK4 inhibition relieves oxidative stress.12 Further, the effect of α-mangostin on the migration potential of MCF-7 cells was evaluated, and it was found that the untreated cells migrated more rapidly compared to cells treated with αmangostin (Figure 6B). The results of the wound healing assay suggest that α-mangostin inhibited the migration of MCF-7 cells (Figure 6B). Concluding Remarks. MARK4 inhibition appears to be an interesting approach considering many cancer regulating pathways are associated with its expression and activity. Further, severe side effects associated with chemically synthesized molecules or chemotherapies encouraged the evaluation of dietary molecules and phytonutrients as anticancer molecules. New mechanisms of resistance to existing kinase-targeting drugs have encouraged and escalated the identification of novel molecules and alternative drug targets. Results from the present study provide new insight into the mechanism of action for α-mangostin and implicate MARK4 as a potential target for cancer treatment. Exploiting different experimental approaches, α-mangostin, a dietary xanthone, was established as a potent inhibitor of MARK4 with a moderate ability to inhibit cancer cell proliferation. The results provide an important scaffold for the design and development of promising MARK4 inhibitors. The perceived potency of α-mangostin indicates the remarkable significance for future drug development and cancer therapeutics in terms of MARK4 inhibitors.

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obtained from Gibco-Life Technologies, Thermo Fisher Scientific (USA). Preparation of Library and vHTS. Atomic coordinates of MARK4 were retrieved from the Protein Data Bank (PDB ID: 5ES1).28 Subsequently, target/library preparation and vHTS were carried out as per our previously published protocol.25 Docking and Molecular Dynamics Simulation Studies. Molecular docking and MD simulation studies were carried out on a DELL workstation with an Intel Xeon CPU E5-2609 v3 @ 1.90 GHz processor, 32 GB RAM, and one TB hard disk running on a Ubuntu 14.04.5 LTS operating system. Atomic coordinates of the three-dimensional structure of MARK4 were taken from the Protein Data Bank (PDB ID: 5ES1)38 and preprocessed in SPDBV39 and MGL tools40 using AutoDockVina41 on the PyRx42 platform to perform molecular docking. Computational tools such as PyMOL and DS visualizer43,44 were used for visualization, evaluation, and analysis purposes. MD simulations for 100 ns were performed on MARK4 without and with α-mangostin at 300 K at the molecular mechanics level using the GROMOS96 43a1 force field in GROMACS. Both the systems were minimized using 1500 steps of steepest descent for energy minimization. The resulting trajectories were analyzed using gmx energy, gmx rms, gmx rmsf, gmx gyrate, and gmx sasa utilities of GROMACS.45 The GROMACS 5.1.2 program was used for MD simulations, and all graphs were prepared using Qt Grace. Expression and Purification of MARK4. Human MARK4 was cloned, expressed, and purified by following our previously reported protocols.46,47 MARK4 was purified using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and confirmed with the help of Western blot using peptide-specific primary antibodies.48 ATPase and Tau-Phosphorylation Inhibition Assay. To analyze the inhibition impact of α-mangostin on MARK4, an ATPase assay was carried out in the presence of increasing concentrations of α-mangostin by following our previous protocol.49,50 In brief, the released 32Pi from MARK4-mediated hydrolysis of [γ-32P] ATP was measured. MARK4 and ice-cold ATP (1 mM) and [γ-32P] ATP (specific activity 222 TBq mmol−1) with or without α-mangostin were incubated for 2 h at 37 °C, and reaction products were separated by using thin layer chromatography. The observed spots of reaction products were captured and used to quantify MARK4 inhibition in terms of percentage hydrolysis of ATP with the help of Image J tools (https://imagej.nih.gov/ij/index.html). Tau-phosphorylation inhibition studies were also performed by following a previously published protocol.14 In a six-well cell culture plate the SH-SY5Y cells were grown and incubated with α-mangostin (IC50 concentration). Nearly 1 × 106 cells were collected after 24 h, fixed, washed, and suspended in antibody incubation buffer with anti-tau primary antibodies at 25 °C for 2−3 h. Following the incubation, cells were analyzed by flow cytometry. Fluorescence Measurements. To study the binding affinity of α-mangostin with recombinant MARK4, a fluorescence-based experiment was carried out as per our previous protocol.51 Each titration of protein with α-mangostin was carried out in three independent triplicates, and the average was taken for analysis of binding parameters. In order to know the binding constant (Ka) and number of binding sites (n) on the protein molecule, the quenching of fluorescence intensity of MARK4 with an increase in the concentration of α-mangostin was used as the basic criterion as per the modified Stern−Volmer equation:52

EXPERIMENTAL SECTION

Computational. In silico studies were carried out on an Intel Workstation with a 4× 2.13 GHz processor, 64 GB RAM, and two TB hard disks running on a Ubuntu 14.04.5 LTS operating system. Different bioinformatics tools named AutoDockVina (Trott and Olson 2010), PyRx 0.8 and Discovery Studio (Biovia2015), VMD (visual molecular dynamics) (Humphrey, Dalke, and Schulten 1996), Pymol (Schrödinger 2016), and Discovery Studio Visualizer were used for vHTS, molecular docking, and visualization purposes. Retrieval, evaluation, and analysis of data were performed using online servers/resources such as NCBI, PDB, the ZINC database, and PubChem. Chemicals and Reagents. α-Mangostin was procured from Sigma-Aldrich/Merck Darmstadt, Germany. The bacterial culture medium, Difco LB broth Miller (Luria−Bertani), was purchased from Becton, Dickinson and Company, Sparks, MD, USA. The Ni-NTA resin column and gel filtration column (Superdex-75) were procured from GE Healthcare (GE Healthcare Life Sciences, Uppsala, Sweden). Human hepatoma cells (HepG2), a breast cancer cell line (MCF-7), and an embryonic kidney (HEK-293) cell line were obtained from National Centre for Cell Sciences, Pune, India. MTT (3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), fetal bovine serum, TrypLE express cell detachment enzyme, propidium iodide, antibiotic cocktail, and Dulbecco’s modified Eagle’s medium were

log(Fo − F )/F = log K a + n log[L]

(1)

where Fo = fluorescence intensity of native protein, F = fluorescence intensity of protein in the presence of ligand, Ka = binding constant, n = number of binding sites, [L] = concentration of ligand. The values for the binding constant (Ka) and number of binding sites (n) were derived from the intercept and slope, respectively. Cell Culture and Viability Assay. HEK-293, MCF-7, and HepG2 human cell lines were maintained in DMEM, while SH-SY5Y were maintained in a 1:1 mixture of Eagle’s minimum essential medium and F12 medium supplemented with 10% heat-inactivated H

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fetal bovine serum (Gibco) and 1% penicillin−streptomycin solution (Gibco), in a 5% CO2 humidified incubator at 37 °C. The cells were plated in a 96-well plate at a density of 9000−10 000 cells/well and incubated for 24 h for growth in a CO2 incubator. Each cell type was treated with increasing concentrations of α-mangostin (0−50 μM) for 48 h at 37 °C in a CO2 incubator, and a standard MTT assay was carried out.53,54 Paclitaxel was used as positive control. Cell Apoptosis Assay. The apoptotic potential of α-mangostin was analyzed using annexin-V staining as described previously.53,54 In brief, cells were treated with the IC50 concentration of α-mangostin for 24 h at 37 °C, while control cells were treated with media only. After 24 h of incubation, ∼(2.2−2.5) × 106 cells were collected by trypsinization and centrifugation. Collected cells were washed three times with 5−6 mL of phosphate-buffered saline (PBS) and stained with FITC-labeled annexin-V using a FITC-annexin-V kit as per the guidelines of the manufacturer (BD-Biosciences, USA). Cells were analyzed on a BD LSR II flow cytometry analyzer with FlowJo. Cell Cycle Analysis by Propidium Iodide Staining. Propidium iodide (PI) staining was used to investigate the cell cycle distribution by following a previously published protocol.51 Briefly, HepG2 and MCF-7 cells were incubated with IC50 concentrations of α-mangostin for 24 h at 37 °C; the control cells were treated with media only. After 24 h of incubation with α-mangostin, the cells were collected by trypsinization and washed twice using 5−6 mL of PBS at 37 °C by centrifugation. The homogeneous cell suspension of treated and control cells was fixed using chilled 70% ethanol by gentle mixing and was further incubated overnight at 4 °C. The fixed cells were washed twice with 5 mL of PBS, suspended in 50 μL of PBS having 200 μg/ mL RNase A, and incubated at 37 °C for 45 min. Cells were incubated for 5 min with PI solution (50 μL from a 2 μg/mL solution mixed with 450 μL of citrate buffer). Approximately 10 000 events were collected for each sample by the BD LSR II flow cytometry analyzer, and data were analyzed using FACS DIVA software. Measurement of Reactive Oxygen Species. Reactive oxygen species (ROS) level was measured using DCFDA staining as described earlier.14 Briefly, the MCF-7 and HepG2 cells (nearly 70−80% confluent) were treated with an IC50 concentration of αmangostin, and the positive control was treated with H2O2. After 5−6 h of incubation with α-mangostin, cells were washed gently with 500 μL of prewarmed (at 37 °C) Krebs Ringer buffer (20 mM HEPES, 10 mM dextrose, 2 mM MgSO4, 5.5 mM KCl, 127 mM NaCl, and 1 mM CaCl2). Next, 10 μM DCFDA (Invitrogen Grand Island, NY, USA) was added to each well, and the plate was incubated for 30 min in the dark at 37 °C in a humidified CO2 incubator. Following the 30 min incubation period, cells were trypsinized and collected. The levels of ROS were estimated by measuring the fluorescence of cells with a Jasco spectrofluorimeter (FP-6200) using a 5 mm quartz cuvette with excitation and emission filters set at 485/500−550 nm, respectively. Wound Healing Assay. The cell migration inhibition potential of α-mangostin on MCF-7 cells was studied using the wound healing assay as described previously.51 In brief, MCF-7 cells were plated in a six-well plate, and a scratch was introduced at ∼60% confluency by scraping the cell monolayer with a 200 μL sterile micropipet tip. Detached cells were removed by washing with incomplete medium, complemented with fresh complete medium with α-mangostin (IC50concentration), and incubated for 48 h. Cells were then fixed in 4% paraformaldehyde and photographed on a phase contrast inverted microscope. Statistical Analysis. Data are presented as means ± SD from at least three independent experiments. Statistical analysis of data was performed using Student’s t test for unpaired samples and ANOVA (one- and two-way). Differences were considered significant at p < 0.05.

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ATPase enzyme assay of MARK4 with in silico screened natural molecules (S1); binding of α-mangostin to MARK4 (S2); stability of the number of hydrogen bonds between MARK4 and α-mangostin with respect to time (S3); secondary structure content of native MARK4 and MARK4 complexed with α-mangostin (S4); cytotoxicity studies on normal human cell line (S5) (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (S. Ali.). Tel: +91-11-26981717/0108 (extn). *E-mail: [email protected] (M. I. Hassan). Tel: +919990323217. ORCID

Aarfa Queen: 0000-0001-9750-5301 Nashrah Sharif Khan: 0000-0002-8566-0248 Md. Imtaiyaz Hassan: 0000-0002-3663-4940 Author Contributions §

P.K. and A.Q. contributed equally to this publication.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Department of Biotechnology, Government of India (Grant No. BT/PR12828/AAQ/1/622/ 2015) (S.A.), Science and Engineering Research Board, Government of India (Grant No. SR/S2/JCB-49/2011) to S.A., and (Grant No. EMR/2015/002372) to M.I.H. A.Q. thanks the Indian Council of Medical Research (Government of India) for the award of a senior research fellowship (No. 45/ 63/2018-PHA/BMS/OL). We sincerely acknowledge Harvard University plasmid providing facility for providing the MARK4 gene. S.A. acknowledges the award of the J.C. Bose National Fellowship by SERB, New Delhi, India.

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DOI: 10.1021/acs.jnatprod.9b00372 J. Nat. Prod. XXXX, XXX, XXX−XXX