Molecular Docking of Compounds From Chaetomium SP. Against Human Estrogen Receptor Alpha in Searching Anti Breast Cancer

A study on molecular docking-based virtual screening has been conducted to select virtual hit of compounds, reported its existence in fungal endophytes of Chaetomium sp. as cytotoxic agent of breast cancer. The ligands were docked into Human Estrogen Receptor alpha (HERa) as the protein which regulates the breast cancer growth via estradiol-estrogen receptor binding intervention. The results showed that two compounds bearing xanthone and two compounds bearing benzonaphtyridinedione scaffolds were selected as virtual hit ligands for HERa leading to the conclusion that these compounds were good to be developed as anti breast cancer.


INTRODUCTION
In family Chaetomiaceae (Ascomyta), Chaetomium sp. is a genus employing species broadly distributed among hundred marine and terrestrial natural products (Ferlay et al, 2007;Zhang et al, 2012). To date, about 200 compounds were reported as the chemical constituents from this genus (Li et al, 2011). This genus employs either bacterial or fungal endophyte which is currently being a trending topic in searching new source of biological active compounds due to its capability to be cultivated in abundance yields (Yang et al, 2011). This is helpful to overcome the problem in isolating a single compound from raw materials which is normally using multistep extractions as well as fractionations (Hussain et al, 2012).
As characterized in general natural product compounds, these endophyte from Chaetomium sp. also posses diverse biological activities such as antibacterial, antioxidant, anti-inflammatotry, antileishmanial and many others (Ruch, Cheng, and Klaunig, 1989). The fungal endophyte is living symbiotically in between the plant cells (Strobel, 2003). Some plants have been identified its presence of Chaetomium sp. are Vinca rosea, Taxus braxifollia (Zhao et al, 2010), Phyllantus amarus (Kandavel and Sekar, 2015), Ginko biloba (Li et al, 2011), and so forth. Uniquely, the chemicals contained in the endophyte often highly related with the identity compounds in the plant of origins. For example is vincristine, the chemotherapeutics for cancer which is contained in Vinca rosea also found in its fungal endophyte (Kumar et al, 2013). Likewise, taxol was characterized in the fungal endophyte of Taxus braxifollia (Stierle, Strobel, and Stierle, 1993).
Two mentioned plants were used clinically as the anti-cancer, a disease which is too tough to be controlled due its complicated life cycle and molecular mechanism. Breast cancer is one of the high prevalence cancers with about 1.7 million incidents annually (Lane, 2006). Therefore, the discovery and development of new anti-cancer is continuously needed especially to those resistant cancer genes.
Computational studies in a virtual identification were broadly used in the screening of natural product database as bioactive compounds. This reduces the time, budget and safety in wet lab experiment since the sample to be tested is selected based on the mathematical value of drug-receptor interaction associating with its in 36 Maywan Hariono and Rollando vivo pharmacodynamic behaviours (Kitchen et al, 2004). In this present study, we virtually screen 27 compounds being identified in fungal endophyte of Chaetomium sp. to be selected as the hit compounds by docking them into one of the protein targets in the breast cancer cell, i.e., human estrogen receptor alpha (HERD) DNA binding protein domain.

EXPERIMENTAL
The computer used in this study was a PC with Core 2 Duo processor 2.93 GHz and 2 GB of RAM. The operating system of the PC is Windows8. Docking the compounds into human estrogen receptor alpha (HERD; PDB 1G50) was carried out to predict affinity of chemical substances which are reported presenting in Chaetomium sp. from diverse plants. The ligands were sketched and geometrically minimized using ACD Chemsketch (www.acdlabs.com) and Marvin Sketch (www.chemaxon.com), respectively. The ligands and proteins were then prepared using AutoDockTools 1.5.6 (www.autodock.scripps.edu). The proteins were added with polar hydrogen and given by kollman charge whereas the ligands were given by gasteiger charges. The grid was centered on each protein binding site and the docking was then performed using AutoDock Vina embedded in PyRx version 8.0 (www.autodock.scripps.edu). The docking results were obeserved as free energy of binding ('Gbind) and the selected docking pose was visualised using Discovery Studio 3.5 (www.accelyrs.com).

RESULTS AND DISCUSSION
There have been well characterized that estrogen controls the breast cancer by stimulating the expression of certain genes (Horwitz and McGuire, 1978). Estrogen receptor with its helix H12 provides the binding site for estradiol as the messenger to dimerize and expose the SF-2 region. This will attract the Co-activator to bind leading to nucleic acid transcription during the breast cancer cell replication. The side chain containing ASP351 is crucial to antagonism.
The antagonist must contain an amine group of the correct basicity such that it ionizes and forms the interaction with ASP351, and it must be of the correct length and flexibility to place the amine in the correct position for binding (Patrick, 2013). Several crystal structures of human HVWURJHQ UHFHSWRU . OLJDQG-ELQGLQJ GRPDLQ K(5. LBD) complexed with agonist or antagonist molecules have previously been solved. The binding site was characterized where in estradiol bound to, by interacting with GLU353, ARG394, PHE404 and HIS524 via electrostatics, pi-pi and hydrogen bon interaction, respectively (Eiler et al, 2001) The binding of estradiol as the chemical messenger is presented in Figure 1.

WP2
In searching new active ligands, a number of 27 compounds (see Table 1) from Chaetomium sp., was utilized as the database to be virtually screened using 1G50 as the protein target (Marwah et al, 2007;Abdel-Lateff, 2008;Qin et al, 2009;Li et al, 2011;Wang et al, 2012;Wang et al, 2015;Li et al, 2015;). As results, Table 2 presented the 'Gbind, the amino acids which contribute to its binding via Hbonding along with its H-bond distances. The order of compounds was ranked from the lowest 'Gbind to the highest one.
The 'Gbind was observed at range -9.2 to -4.9 kcal/mol for virtual active compounds while inactive compounds were indicated at range -4.2 to 9.4 kcal/mol. There are six compounds found to be low in 'Gbind but they are not bound to the active site as no hydrogen bond being observed in their poses. This might be due to the van der Waals contribution which is normally not as essential as Maywan Hariono and Rollando hydrogen bond. Van der Waals role in the ligandprotein binding is normally to support or stabilize the pose via dipole induced dipole such as pi-pi, pication and or pi-sigma interactions. Van der waals interaction has a weak affinity, therefore, this is normally less priority in the ligand-protein interaction. Although virtual screening has been developed as a fast and accurate scoring function, however, no single scoring can satisfy the peculiarities of each target system (Li et al, 2009).
In further virtual hit selection, the priority goes to the ligands which have lower 'Gbinds and similar binding modes with the reference (Yang et al, 2013). A diverse hydrogen bond interaction was demonstrated by all ligands fit into the active site with the corresponding amino acid residues such as GLU323, PRO324, PRO325, LEU345, LEU346, GLU353, TRP393, ARG394 and LYS449. However, among those mentioned amino acid residues, interactions with GLU353 and ARG394 were the most prevalent and it was suggested to be important as these interaction were also possessed by the reference ligand. Therefore, compounds having interaction with these types of residue were proposed to be the virtual hits ready for further optimization. Figure 2 illustrated the superimposition of 27 ligands in the active site of 1G50, whereas Figure 3 illustrated the ligandsprotein interaction of some representative virtual hit compounds.  Compounds bearing xanthone (Wang3 and Wang4) and benzonaphtyridinedione (Latef4 and Latef5) could be good scaffolds for further lead optimization of cytotoxic agent against breast cancer by targeting HERD DNA binding protein domain. Xanthone has been widely published as anticancer due to triple heteroaromatic ring which acts a DNA intercalating agent. The planar system Maywan Hariono and Rollando of heteroaromatic ring takes over the space in between two layers of nucleic acid pairs and therefore disrupts the shape of the helix. This action occurred at either minor or major groove which prevents the DNA replication as well as its transcription.
On the other hand, benzonaphtyridinedione has been reported as tyrosine kinase inhibitors which play roles in signal transduction which regulates the intercellular communication, thus it is a good approach for the therapeutic intervention against pathological processes such as proliferation, inflammation and cancer.

CONCLUSIONS
Fungal endophyte has been recognized as the new source of bioactive compounds from natural products. One of genus widely cultivate this fungi is Chaetomium sp. spread over plant has been well known in producing high value of compounds for many pharmacological activities. The advantage of this material is its capability to be cultivated in a high yield via biotechnology process compares to the traditional fabrication that needs more plantation. In order to increase the knowledge in developing new anticancer drug from natural product, a molecular docking based virtual screening has been approached to select some hit compounds to be active as breast cancer cytotoxic agent by HERD DNA binding protein domain intervention. Compounds bearing xanthone and benzonaphtyridinedione were selected as the most active ligand against the protein target. In conclusion, Chaetomium sp. is strongly suggested to be developed as the source for bioactive compounds concerning on anticancer agent. However, further retrospective validation is required to have a validated protocol in virtual screening leading to the features of ligand to be suitable as HERD antagonist.