The potential of the metabolites active from Moringa leaves ( Moringa oleifera , Lam) on sensitivity of doxorubicin towards breast cancer: in silico studies

. Breast cancer is one type of cancer with the highest incidence suffered by women. Doxorubicin is a chemotherapy that is often used as the main chemotherapy and combination chemotherapy, but the use of doxorubicin is often complained of side effects that cause auto resistance. Combination with chemopreventives from natural ingredients has become an option to increase therapeutic response and to minimize side effects and resistance to chemotherapy use. This study aims to screen several active compounds of the phenolic-flavonoid group contained in Moringa leaves ( Moringa oleifera , Lam) against NFκβ receptors in silico using a molecular docking technique. The material in the form of “Canonical smiles” data is quercetin, quercetin-3 glycoside (Q3G), rutin, kaempferol, myricetin, isorhamnetin, deoxyelephantopin and doxorubicin which were downloaded from www.pubchem.org and converted to 3D structures using MOE software. While the 3D structure of the receptor (1VKX) was downloaded from www.rscb.org. The results of the docking of the active compounds contained in Moringa leaves ( Moringa oleifera , Lam) showed a fairly strong affinity by releasing energy when forming a ligand-receptor complex. Quercetin 3-glycoside has the best potential as an NF inhibitor with an affinity of -14.23 kcal/mol. Quercetin 3-glycoside also has a good pharmacokinetic profile with low toxicity. While the phenolic-flavonoid compounds contained in other Moringa leaves are only able to reduce the affinity of doxorubicin for the NF receptor by changing the "site binding" conformation of the receptor. In conclusion, quercetin 3-glycoside deserves to be a drug candidate or a companion to the chemotherapy of doxorubicin.


INTRODUCTION
Cancer is a disease characterized by the abnormal and uncontrolled growth of body tissue cells. Cells are formed due to gene mutations so that they experience changes in shape, size, and function from the original cell [1]. Breast cancer is one type of cancer with the highest incidence in women in various parts of the world. Based on the incidence of breast cancer, it shows the second largest percentage (11.9%) after lung cancer (13%) of all types of cancer, and specifically in women, it is the most common case of 43.3% with a mortality rate of 12.9% [2].
Not unlike humans, in several types of animals, the incidence of cancer has also been reported. Nearly 50% of cancers occurring in dogs and 85% in cats are maglina. Mammary cancer cases in female dogs have the second highest incidence after skin cancer cases. It is estimated that 165-198 out of 10,000 female dogs have mammary cancer, of which 50% are benign and 50% are malignant, while in cats cases of mammary gland cancer are rare, but several cases of mammary gland cancer in cats it is recorded that 85% are malignant and the rest are benign [3].
The exact cause of breast cancer is not yet known, but the accumulation of genetic disorders, hormonal disturbances and *Corresponding Author: frengki_fkh@unsyiah.ac.id environmental factors are suspected as triggering factors for accelerating the occurrence of breast cancer and worsening the prognosis of this disease [4]. As a result, it will trigger mutations in genes that are considered markers of this disease, such as p53, BRCA1 and BRCA2, etc. Mutations in the suppressor gene (p53) are found in up to 35% of breast cancer cases [5], and mutations in the DNA repair gene (BRCA1, and BRCA2) are identified as the main factor causing familial breast cancer cases with the highest rates (80-90%) [6], hormonal factors are related to their role in activating cell growth signals through the PI3K/Akt and MAPK pathways. Almost 70% of cases of breast cancer, were found increased levels of estrogen [7]. Chronic infection of the breast is also considered to trigger the formation of breast cancer. Uncontrolled regulation of inflammatory factors can lead to increased expression of growth & survival factors in these cells so that they can initiate the formation of cancer cells [4].
Doxorubicin is one of the most effective anthracycline chemotherapeutics and is often used as the main chemotherapy or as part of combination chemotherapy [8]. The mechanism of action of doxorubicin as a cytotoxic agent includes; inhibition of topoisomerase II enzyme action and inhibition of DNA/RNA synthesis through DNA intercalation [9]. However, its use also has quite dangerous side effects such as cardiomyopathy and congestive heart failure [10]. Doxorubicin also has toxicity in the form of myelodysplasia and leukemia if used in the long term [11], it is also reported to trigger resistance events [12].
Resistance to the use of doxorubicin is closely related to overexpression and increased activity of Pgp in cancer cells which causes the accumulation of intracellular chemotherapy to be low and not strong enough to trigger apoptotic events [13]. NFκβ as a proinflammatory transcription factor and immune response also directly triggers chemotherapy resistance through Pgp activation and overexpression [14]. Doxorubicin was reported as one of the chemotherapeutics that can trigger the activation of the NFkB transcription factor [15]. Thus, apart from being a chemotherapeutic agent, it turns out that doxorubicin also triggers resistance against itself, known as auto resistance through NFκβ stimulation. Therefore, the use of doxorubicin should be given in combination with compounds capable of suppressing the function of this NFκβ.
Moringa oleifera, Lam is a nutritious plant, this plant grows a lot and is easily cultivated in Indonesia. This plant has different names in each region in Indonesia, for example (Sumatra) murong, kacanggai, kelor, marungga, marunggai, mungai, kelor (Javanese) kelor, marongghi [16]. Moringa leaves are also reported to contain several types of phenolicflavonoids including quercetin-3 glycosides, rutin, kaempferol glycosides, and chlorogenic acid. Flavonoids in vitro have been widely reported to act as antioxidants by capturing radicals, where free radicals are suspected as the cause of various chronic diseases [17]. Quercetin is reported to be able to prevent the growth of cancer cells with one of its mechanisms as scavenger of free radicals [18]. Rutin as a type of flavonoid that has a glycosidic bond is also reported as an antioxidant, antiinflammatory and anti-platelet aggregation, and blood clotting [19]. Likewise, with kaempferol and its glycosides which have multiple roles as antioxidants, anti-inflammatories, anticancer [20].
Therefore, it is necessary to explore the active compounds of this plant more deeply as an anticancer. One of the efforts is by utilizing digital techniques or known as "in silico" tests using the "reDocking" technique in predicting the effect of active metabolites compounds in Moringa leaves in increasing chemotherapy sensitivity by suppressing the interaction of doxorubicin to NFκβ receptors. The in silico test has its own advantages, without the use of test animals, without consuming a lot of time and money and the analysis results are obtained more quickly. In silico is usually used to confirm in vitro, in vivo assays and to develop further research [21]. The in silico method can help pre-clinical tests (in vitro and in vivo) to be more directed and efficient so that trial and error can be avoided. Selecting thousands of active compounds for certain receptor targets can be done in a short time and at very minimal cost by simply screening thousands of active compounds stored in databases using molecular docking techniques. Likewise, to find out the derivatives of active compounds that are more potential to be developed as drug candidates, you only need to use pharmacopore and QSAR techniques, such as curcumin analogue derivatives that have been reported by Singh et al [22].

Materials
The test material is a 3D structure of the NFκβ receptor downloaded from the 3D structure provider site www.rscb.org (pdb id.1VKX).
The potential of the metabolites active from Moringa leaves (Moringa oleifera, Lam) on sensitivity … (Frengki Frengki, KMS M Amin Alqadri, Siti Aisyah, Heni Vanda) ___________________________________________________________________________________________________ Meanwhile, the 3D structure of the active compound contained in Moringa oleifera leaves was downloaded in the form of "Canonical SMILES" data from PubChem and converted to a 3D structure using "MOE builder software" 10. 2008. The device used is a 1.80 GHz Intel Core(TM) hardware processor with 4GB memory capacity and Windows 10 operating system and is equipped with internet access. While the software used is ChemOffice 10 (Cambridge) and Molecular Operating Environment (MOE 2008 version. 10) (developed by Chemical Computing Group, Inc. (Montreal, Canada).

Docking file preparation
This research was conducted in silico using a molecular docking protocol modified by Frengki, et al., [23]. Docking file preparation was carried out by optimizing the geometry and minimizing the energy of the three-dimensional structure of the ligand and receptor using MOE software running on a single dual-core computer. The geometry optimization and minimization processes were carried out using the MOE software. The first step is the addition of hydrogen atoms and protonation using the 3D protonate function. After that, the partial charge setting is carried out using a partial charge, with the parameter method used being the current force field. Furthermore, energy minimization is carried out with the MMF94x force field, the solvation used is the gas phase, the Root Mean Square Deviation (RMSD) gradient is 0.001 kcal/mol using the default and the output file is in moe format. Ligand optimization was performed on the MOE database viewer (dv) with the mdb format. The optimization process begins with washing the ligands. Then, the partial charge of the ligand was adjusted using a partial charge, with the parameter method used being MMFF94x. The solvation used during the ligand optimization process is in the gas phase. After that, the energy minimization process was carried out with an RMS gradient of 0.001 kcal/mol.

Docking of Receptor-ligand
This stage begins with the preparation of the docking file and selecting the amino acid residues that are the target for docking. Followed by opening the ligand candidate file in mdb format. After the preparation is complete, docking is carried out with the simulation-dock program. The placement method used is the triangle matcher with a several rounds of 1000. The scoring function used is London dG displaying the best data of 30. Then from the 30 best data displays, a refinement is carried out using a refinement force field with a population repeat size configuration of 1000 according to the default MOE. The display of the overall results of the selected docking process is 1 best data. The rest of the parameters correspond to the defaults of the MOE and the output files are in .mdb format.

ReDocking receptor-ligand
The process is the same as the "receptor-ligand docking" above, the difference is that the target receptor has previously formed a complex with the active compound Areca catechu.

Evaluate docking results
The binding free energy of the docking results is seen in the output in mdb format. The selected ligand-enzyme complex is a complex that has a bond-free energy value. Residual contact and hydrogen bonding that occurred in the bestdocked ligand-enzyme complexes were identified and analyzed in three-dimensional media using MOE-ligPlot, then visualized in the ligand interaction program.

Pharmacokinetic Predictions and Toxicity of All Ligands
The pharmacokinetic and toxicity profiles of all tested and control ligands were traced via the http://www.swissadme.ch/index.php and https://tox-new.charite/servers. Based on these data it can be analyzed the level of adsorption,

Ligand and Receptor Download
The in silico study or digital test in this study is intended as an initial screening to select the active compounds contained in Moringa oleifera leaves. We selected 6 flavonoid compounds (Kaempferol, Rutin, Quercetin, Quercetin glycoside; Myricetin, and Isorhamnetin) contained in Moringa oleifera leaves as reported [24,25,26]. The native ligand and control ligand (Substrate) were deoxyelephantopin and doxorubicin, respectively. The ligands were downloaded based on PubChem's "SMILES" canonical data. Meanwhile, the receptor (Pdb id.1VKX) was selected based on a report written by Frengki, et al., [23]. Furthermore, the "smiles" format above is converted to the "pdb" format as the default molecular docking format. Ligand modeling was carried out through a series of conversions of the "SMILES" ligand structure to the "cdx" (2D) format using the ChemDraw program and then to the "pdb" (3D) format using the ChemD program ( Table 2).

Results of docking of each ligand to the NFκβ receptor
The docking process on the NFκβ receptor begins with the validation of the docking of the deoxyelephantopin ligand against the NFκβ receptor (pdb id. 1VKX). Validation was carried out using the deoxyelephantopin redocking technique at the same binding site for the 1VKX deoxyelephantopin complex. The validation results show an RMSD value of 1.277Å. Details are shown in Figure 1.  Table  3.
Doxorubicin substrate interactions form 5 hydrogen bonds with 4 amino acid NFκβ receptors (Cys 120, Lys 37, Lys 122(2), and Gln 132). It is thought that this hydrogen bond plays an important role in producing the affinity between doxorubicin for the NFκβ receptor. The same conditions were also shown by the test ligands kaempferol, rutin, quercetin, quercetin 3-glucoside, myricetin, and isorhamnetin, as well as the control deoxyelephantopin. Each interacts with a polar amino acid to form a hydrogen bond. The quercetin 3-glucoside test ligand showed the strongest affinity of -14.23 kcal/mol by forming 5 hydrogen bonds with 4 polar amino acids (Glu 89, Lys 37(2), Lys 122(2), and Gln 128). Furthermore, myricetin with an affinity of 13.57 kcal/mol forms hydrogen bonds with 2 polar amino acids (Cys 120, Gln 132(2)), and the routine test ligand with an affinity of 13.18 kcal/mol forms hydrogen bonds with 4 polar amino acids (Glu 89, Asp 125, Lys 37, Lys 122). The three tested ligands were able to shift the position of the doxorubicin substrate towards the NFκβ receptor.
In contrast, the test ligands kaempferol, quercetin, and isorhamnetin were unable to shift the position of the doxorubicin substrate when interacting with the NFκβ receptor. However, the three tested ligands were able to decrease the affinity of the doxorubicin substrate for the NFκβ receptor as well as the control deoxyelephantopin. Details are shown in Table  4.
The results of the interaction model of doxorubicin substrate and quercetin 3-glucoside as best ligand on NFκβ receptors are visualized in Figure 2. The results of the quercetin interaction as a model in reducing the affinity of doxorubicin at the NFκβ receptor are visualized in Figure 3.     Besides having a strong affinity for interacting with the NFκβ receptor, the routine test ligand and quercetin 3-glucoside also showed the formation of hydrogen bonds between the 16th carbonyl group of the sesquiterpene lactone and the N atom of the Lys 122 residue of the NFκβ receptor. This bond is so important that it is a prerequisite for inhibitory activity against NFkB where this bond can block the translocation of P65 to the cell nucleus and the binding consensus DNA element of TNFα [27].
The rutin ligand and quercetin 3-glycoside also showed interactions with the Lys 122 amino acid residue, so the two test ligands fulfilled 2 things that confirmed them as NFκβ receptor inhibitors. Whereas myricetin shows interaction with Cys 120, and Gln 132(2) amino acid residues so myricetin only has 1 evidence which is the reason for its designation as an NFκβ receptor inhibitor. Similar conditions also apply to other ligands that do not have direct interaction with Lys 122 so the designation as an NFκβ inhibitor is only based on evidence of decreased affinity. The pharmacokinetic profile and toxicity of the ligand contained in Moringa oleifera leaves (Moringa oleifera, Lam) are shown in Table 5.
Based on the pharmacokinetic and toxicity profiles in Table 5, quercetin 3-glycosides have a low level of absorption and distribution, however, quercetin 3-glycosides do not affect the function of metabolic enzymes at all and have low toxicity and are categorized as safe for consumption (level V) as rutin. What distinguishes it from rutin is that quercetin 3glycoside is also not a Pgp substrate, so it has no resistance potential when used as a chemotherapy agent. With the presence of the Moringa leaf compounds above, the interaction of doxorubicin can be blocked or reduced against NFκβ receptors, thereby increasing the proliferation-differentiation of cancer cells, and