Four New Anthraquinones with Histone Deacetylase Inhibitory Activity from Ventilago denticulata Roots

Chromatographic separation of the crude extracts from the roots of Ventilago denticulata led to the isolation of four new anthraquinones, ventilanones L–O (1–4), together with eight known anthraquinones (5–12). Their structures were elucidated by spectroscopic methods (UV, IR, 1H NMR, 13C NMR, and 2D NMR) and mass spectrometry (MS), as well as comparison of their spectroscopic data with those reported in the literature. HDACs inhibitory activity evaluation resulted that compound 2 exhibited moderate antiproliferative activity against HeLa and A549 cell lines but nontoxic to normal cell. Molecular docking indicated the phenolic functionality of 2 plays crucial interactions with class II HDAC4 enzyme.


Introduction
Ventilago is a genus of the plants in the family of Rhamnaceae, which contains around 40 species worldwide [1]. Ventilago denticulata Willd. (Synonym of: Ventilago calyculata Tul.), one of nine species found in Thailand [2], is native to Indian subcontinent to China and Indo-China region. It is a climbing shrub, spreading throughout tropical evergreen forests. The plant is locally recognized in Thai as Rang daeng (Central), Kong kaep (Northern), and Song daeng (Peninsular). Thai traditional medicine uses leaves of V. denticulata for the treatments of diuretics, arthritis, and hyperglycemia, whereas vines are used to treat muscle pain [3,4]. According to previous phytochemical investigations of V. denticulata (V. calyculata), several classes of bioactive compounds have been reported including anthraquinones from vine [4], root bark [5,6], and root [7]; naphthalene derivatives from vine [4]; benzisochromanquinones from vine [4], root [7], and trunk [8]; and flavonoids from the trunk [8]. These natural products demonstrated their biological principles such as antioxidant, cytotoxic, antibacterial, antifungal, and phosphodiesterase inhibitory activities [4,8]. It is obvious that V. denticulata is rich in pharmaceutical active compounds related to the polyketide biosynthetic pathway. As part of our ongoing search for potent anticancer agents from plants, we are interested in anthraquinones, one of the polyketide-derived secondary metabolites from V. denticulata, due to their promising anticancer activity [9][10][11]. Anthraquinones have a planarity of 9,10-dioxoanthracene core structure, which can embed into active site pocket of targeting enzymes, resulting enzyme suppression, arresting cell cycle, and inducing cell apoptosis. To broaden the study of new naturally occurring sion, arresting cell cycle, and inducing cell apoptosis. To broaden the study of new rally occurring anthraquinones and their anticancer potency, we report herein the tion and characterization of anthraquinones from the roots of V. denticulata and ev their histone deacetylase (HDAC) inhibitory activity.

HDAC Inhibitory Activity
Anthraquinones 2, 6, and 9 were selected for screening by the Fluor-de-Lyse™ in vitro fluorescence activity assay kit as measuring total HDAC inhibitory activity in HeLa nuclear extract (Table 3). Among the tested compounds, 2 showed the highest percentage of HDAC inhibition value (61.27%), whereas 6 and 9 demonstrated HDAC inhibition lower than 50%. Table 3. HDAC inhibitory activity of compounds 2, 6, and 9 at 40 µg/mL.

Physicochemical Properties
Drug-likeness is a useful concept in drug design that increases the chances of chemical entities and avoids drug development failure. In this study, the SwissADME web server (http://swissadme.ch, accessed on 30 December 2021) was performed to estimate the physicochemical features of anthraquinones 1-4, to assess their drug-likeness [21]. The results ( Table 4) showed that 1-4 presented no violation of Lipinski's rules. The molecular weights, number of hydrogen-bond acceptors, and number of hydrogen-bond donors were within the accepted values of less than 500, 10, and 5, respectively. Their LogP values were within the range of 2.60 to 3.65. Additionally, 2 showed the lowest topological polar surface area (TPSA), indicating the most favorable drug-likeness.

Molecular Docking Study
To predict the possibility of being HDAC isoform-selective inhibitor, 2 was docked into the catalytic pockets of the representative isoform of class I (HDAC1, HDAC2, and HDAC8) and class II (HDAC4 and HDAC7). The available crystal structures of HDAC1, HDAC2, HDAC4, HDAC7, and HDAC8 were obtained from the Protein Data Bank (https: //www.rcsb.org, accessed on 30 December 2021). The docking results are summarized in Table 5. For HDAC4 and HDAC7 templates, 2 had a low binding energy, which has significance for HDAC class II. The docking studies of 2 with HDAC4 revealed that it has the lowest binding energy of −6.85 kcal/mol. It is also against HDAC4 and HDAC7 with Ki values of 9.49 and 5.29 µM, respectively. domain resembles the substrate and is able to bind the cylindrical pocket of the HDAC active site. Meanwhile, CAP interacts with the surface and closes the cylindrical pocket of the active site. In HDAC4, a Zn 2+ cofactor binds to charge-relay system consisting of two aspartate residues (Asp196 and Asp290) and one histidine residue (His198) [22].
The binding modes and interactions of anthraquinones 1-4 with HDAC4 template were studied in order to obtain more insights into their HDAC inhibitory activity (Figure 3). Compound 1 formed six hydrogen bonds with His159, His198, Asp196, Asp290, Gly331, and Lys20, whereas no interaction with catalytic zinc ion (Figure 3a). Docking mode of 2 shows two hydrogen bonds of the hydroxyl group at C-1 with N-imidazole ring of His198 and Asp290, as the charge-relay system in HDAC4 catalytic site. In addition, this hydroxyl group also approached the zinc ion to establish ionic interaction (Figure 3b). According to the result, the phenolic component of 2 interacts with HDAC4 enzyme through dual binding mode, including CAP and ZBG domains. Although the hydroxyl group at C-1 of 3 interacted with His198 and Asp290, as found in 2, compound 3 showed no hydrophobic interaction with Phe168 and Pro156 residues (Figure 3c). Interestingly, 4 was found to have a hydrogen-bond with His198, together with hydrophobic and ZBG interactions (Figure 3d).

MTT Assay
The MTT assay of 2 was carried out to gain more details regarding the anticancer activity of the potent HDAC inhibitor. To complete the evaluation of this potent HDAC inhibitor, the antiproliferative activity was determined in human cervical cancer (HeLa), human lung cancer (A549), and human breast adenocarcinoma cancer (MCF-7) cells. More-over, 2 was also determined in noncancer cells (Vero cells) and cisplatin was used as drug control (Table 6). Results indicated that 2 possessed potent capacity against HeLa and A549 cell lines for 72 h with IC 50 values of 160.87, and 177.32 µM, respectively. However, 2 showed less active against MCF-7 cell line and appeared less toxic to normal cells.

General Experimental Procedures
Melting points were determined on a SANYO MPU250BM3.5 melting point apparatus and were uncorrected. UV spectra were recorded on an Agilent 8453 UV-visible spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). IR spectra were determined by a Bruker Tensor 27 FT-IR spectrophotometer (Bruker, Ettlingen, Germany). The 1 H and 13 C NMR spectra were obtained from a Bruker AVANCE NEO (400 MHz) spectrometer (Bruker, Rheinstetten, Germany). Chemical shifts were reported on the δ (ppm) scale using chloroform-d 1 , methanol-d 4 , acetone-d 6 , and DMSO-d 6 as the solvent and residual of those solvents as internal standards. HRESITOFMS were recorded on a Finnigan Mat INCOS 50 and Micromass LTC mass spectrometers and a Finnigan MAT-90, a microTOF, Bruker Daltonics, and a Finnigan LC-Q mass spectrometer. Silica gel 60 (Merck, Darmstadt, Germany) 0.063-0.200 mm or less than 0.063 mm and Sephadex LH-20 (Amersham Pharmacia, Biotech AB, Sweden) were used for column chromatography. Preparative thin layer chromatography was carried out on glass supported silica gel plates using silica gel 60 GF254 for thin-layer chromatography (Merck, Darmstadt, Germany). Thin layer chromatography was performed on precoated silica gel 60 PF254, aluminum sheets (Merck, Darmstadt, Germany).

Plant Materials
The roots of V. denticulata were collected from local conservation forest in Lampang province, Thailand, in November 2017. The plant was identified by Surapon Saensouk, Mahasarakham University, Thailand. The voucher specimen (no. SPMSU002) was deposited at Mahasarakham University Herbarium, Thailand.

HDAC Inhibitory Activity Assay
The HDAC inhibitory activity was determined by Fluor-de-Lys HDAC activity assay kit (Biomol, Enzo Life Sciences International, Inc., USA). The assay was carried out according to the manufacturer's instructions. In brief, preparation of the recombinant HeLa nuclear extract was performed by diluted in assay buffer and added to a microliter plate. The Fluor de Lys TM substrate was diluted with assay buffer. HDAC reaction was started by adding the substrate to each well and incubated at 37 • C for 10 min. The reaction was stopped by the addition of a developer and then incubated at room temperature for 10 min. After 10 min of incubation, the samples were monitored by SpectraMax M5 (Molecular Devices, USA). The fluorescence was measured at excitation wavelength 360 nm and emitted light 460 nm. Trichostatin A (TSA) was used as the positive control. All experiments were carried out in triplicate.

In Silico Physicochemical Properties
SwissADME web server [23] was used to assess the physicochemical properties for determination of the good drug candidates. In this study, the physicochemical parameters (molecular weight, topological polar surface area (TPSA), number of rotatable bonds, number of hydrogen-bond acceptors, and number of hydrogen-bond donors), and lipophilicity were checked for the evaluated compounds. Together, the Lipinski's and Veber's rules were used to verify the drug-likeness profile.

Molecular Docking
Molecular docking was performed using AutoDock 4.2 to calculate the binding free energies and to obtain the best orientation of selected compounds with HDAC1, HDAC2, HDAC4, HDAC7, and HDAC8 (PDB entry code: 4BKX [24], 3MAX [25], 2VQW [26], 3C0Z [27], and 1T64 [28], respectively) template. For all of the docking calculations, Lamarkian genetic algorithm search (LGA) was used. Polar hydrogens and Gasteiger charges were assigned by using AutoDockTools (ADT) [29]. All water and non-interacting ions, as well as, ligands were removed. Atomic salvation parameters, based on the Stouten model and fragmental volumes, were added in accordance with the AutoDock force field [30,31]. The grid box site of 60 × 60 × 60 points with grid spacing of 0.375 Å. The cartesian coordination grid box measures 11.058 × 7.784 × 31.524, the center base of the crystal ligand. The AutoGrid 4.2 program was used to generate the grid map files. Maximum energy evaluations of 2.5 × 106 steps were performed with a population size of 200 ligand orientations while the total independent runs were fixed to 200. The final docked structure, RMSD from the bound crystal structure, docked energy, and predicted free energy of binding were used to analyze its interaction with the active site. The best orientations with the lowest docked energies were visualized for their interactions by using LigPlot+ software [32].

MTT Assay
The MTT reduction assay was performed with non-cancer (Vero), human cervical cancer (HeLa), human lung carcinoma (A549) and human breast adenocarcinoma cancer (MCF-7) cell lines according to the method previously described [33][34][35]. Briefly, cells were seeded in a 96-well plate and incubated for 24 h. The cells were treated with the selected compounds and incubated at 37 • C in CO 2 for 24 h, 48 h, and 72 h. After incubation, the culture medium was exchanged with 110 µL of MTT (0.5 mg/mL in PBS medium) and incubated for 2 h. The amount of MTT formazan product was determined after dissolved in DMSO by measuring its absorbance with a microplate reader (Bio-Rad Laboratories, USA) at a test wavelength of 550 nm and a reference wavelength of 655 nm. The cell viability was expressed as a percentage to the viable cells of control culture condition, and IC 50 values of each group were calculated.

Conclusions
Four new anthraquinones, ventilanones L-O (1-4), together with eight known anthraquinones (5)(6)(7)(8)(9)(10)(11)(12) were isolated from the roots of Ventilago denticulata. Among the known compounds, this is the first report of 5 as a naturally occurring anthraquinone. Additionally, 8 was first isolated from the genus Ventilago, whereas 11 was first isolated from the plant V. denticulata. Physicochemical properties analysis revealed that 1-4 displayed no violation of Lipinski's rules and suggested that they have good drug-likeness properties. The molecular docking study demonstrated that 2 is well embedded into HDAC4 enzyme active site, generating hydrogen-bonds to amino acids and ionic interaction to ZBG. The antiproliferative activity evaluation showed that 2 exhibited moderate toxicity against HeLa and A549 cell lines but showed nontoxic to normal cells.