Molecular docking of Luteolin compounds from Physalis

angulata as anticancer
Abd Rukman, R. Wardhani, and Sulfahri

Department of Biology, Faculty of Mathematics and Natural Sciences, Hasanuddin

University, Makassar, Indonesia

E-Mail: sulfahri@unhas.ac.id

Abstract. Cancer was known as the second-highest number of deaths after heart disease in the
world. The International Agency for Cancer Research 2012 (IARC) found that cancer
accounted for 7.6 million deaths. Cancer chemoprevention is the latest approach that is
developing rapidly by using natural ingredients or synthetic ingredients to prevent, inhibit, or
reverse tumorigenesis and suppress the development of invasive cancer. This study aims to
determine the bioactivity of potential compounds from several samples of Indonesian plant
namely Physalis angulata to prevent cancer based on reverse docking studies by using PyMOL
v1.7.4.5 Software (Schrödinger), PyRx 0.8 software and Drug-Likeness Test. The compounds
of Physalis angulata has the potential as a cancer inhibitor with BCL-2, with the binding
affinity value of -7.6.

1. Introduction
The World Health Organization (WHO) said that cancer is a disease, a major disease that can affect all
parts of the body and is usually known as malignant tumors and neoplasms. According to the National
Cancer Institute (2009) cancer is a disease of cell growth that is not normal and there is no control in
the body resulting in infections around it.
Cancer is a disease with the second-highest number of deaths after heart disease in the world. The
2012 International Agency for Research on Cancer (IARC ) explained that cancer has caused 7.6
million lives in human mortality in data provided by (American Cancer Society (ACS), 2011). The
World Health Organization (2013) estimates that the number of deaths due to predicts that cancer will
experience a very rapid increase in number worldwide around 13.1 million deaths by 2030. 70% of the
death cases were in low and middle-income countries such as Indonesia. [1]
Cancer as a health barrier that is very influential in people's lives and the main treatment for cancer
still consists of surgery, chemotherapy, radiation therapy, and immunotherapy. However, dangerous
side effects from cancer drugs and radiation are still a threat to cancer sufferers, while not all cancer
patients can be cured by surgery. Cancer chemoprevention is a rapidly increasing contemporary
innovation using natural materials, or synthetic substances to prevent, inhibit or reverse tumorigenesis
and suppress the development of invasive cancer. [2]
Alternative cancer treatments that can be done to reduce the side effects of the above treatments
and which safer to use are through the use of medicinal plants. Physalisangulata, is one of the plants
that has been widely studied, has a cytotoxic effect, and can inhibit the growth of cancer cells.
Physalisangulatacontains saponins, flavonoids, polyphenols, and physalin (3) which play a role in the
inhibition of cancer cells. Saponin compounds have been known to inhibit the formation of Bcl-2
which is expressed too high, induce caspase-3 proteins that are expressed too low, increase expression
of p53, and can also trigger G1 cell cycle arrest.[3]
 Physalisangulata is an annual plant that is found in many tropical and subtropical regions. In Java,
this plant grows wild in gardens, dry fields, roadsides, and in forests [4]. Based on existing research,
Physalis angulatacan function as an immune system stimulator has a cytotoxic effect on several types
of cancer cells and has antiviral activity [5]. From the literature review results, it was found that
Flavarnoid (Luteolin) was known to be able to overcome cancer [6]. Therefore, in this study, we using
Physalis angulata to know that these plants are compounds capable of preventing and treating cancer
using in silico docking method and this method is one of the methods in the science of bioinformatics.

2. Materials and Method

2.1. Ligand Preparation
The chemical structure of luteolin in Physalisangulata was obtained from literature studies of 3D
structure, ligand smile, and CID numbers were obtained from PubChem. Luteolin has a conical smile
C1 = CC (= C (C = C1C2 = CC (= O) C3 = C (C = C (C = C3O2) O) O) O) O) O, obtained from the
PubChem database (https: // PubChem.ncbi .nlm.nih.gov /) with number CID :: CID 5280445 and 5-
Fluorouracil (5 - Fu) as a control have a smile C1 = C (C (= O) NC (= O) N1) F with a CID number:
CID 3385 Ligands are then processed with Avogadro and saved in PDB format.

2.2. Target Selection

At this stage, three types of methods are used to predict protein targets, namely by using The
Pharmmapper ((http://lilab.ecust.edu.cn), superpred (http://prediction.charite.de), and swiss targets
(www swisstargetprediction.ch). After that, checking is done using UniProt (https://www.uniprot.org)
and for taking protein structures obtained in protein data banks (https://www.rcsb.org/) or literacy
studies. to find out the target protein, then using the Pymol v1.7.4.5 application to convert non-protein
molecules, this study uses a target protein based on literacy studies, namely BCL 2.

2.3. Molecular Docking

Molecular docking is carried out using PyRx 0.8 by reacting the protein BCL 2 , Physalis angulata,
and Luteolin ( Physalis angulata ) and controlled by reacting the protein BCL2, and 5- Fluorouracil (5
- Fu).

2.4. Molecular Visualization and Small Molecule Interaction

At this stage, combining the ligand, the target protein and, the control protein with PyMOL v1.7.4.5.
to determine the attachment between the third test.

2.5. Drug-Likeness Test

The similarity test drugs using the physicochemical properties of the ligands and identified by
physicochemical carrying drugs and drug use in common law Lipnski.

3. Result and Discussion

Luteolin is a flavone compound in a medicinal plant as well as some vegetables and spices, it
functions as a natural anti-oxidant with less pro-oxidant potential but the safety for the body is much
better. Luteolin can inhibit osteosarcoma cell proliferation and effectively induce apoptosis in a dose-
dependent manner by decreasing the regulation of the protein levels of BCL-2, Caspase-3, and
Survivin and regulating the expression of BAX protein levels. These findings indicate that luteolin can
be used as a new herbal medicine[7].
The Bcl-2 family of proteins functions to control cell death and includes members of anti-apoptosis
and pro-apoptosis [8, 9]. For each of these proteins has at least one of the four motifs called the Bcl-2
homology (BH) domain. Anti-apoptotic proteins namely Bcl-2, Bcl-xL, and Mcl-1 have the BH1-4
domain. On the other hand, Proclaposis Bcl-2 family members are divided into two subclasses, namely
from BH3 proteins such as Noxa and Puma and multi-domain proteins such as Bak and Bax [10]. The
BH3 domain is a reference to cell death [11]. many types of anticancer such as doxorubicin which kill
tumors by affecting the activity of BCL family members through the apoptotic pathway [12,13,14].
Also, only the expression of BH3 protein induced by certain anticancer agents such as paclitaxel
results in Bak / Bax activation and induction of apoptosis [15]. an increase in the form of anti-
apoptotic proteins such as BCL-2 can overcome apoptosis induction by stopping Bak / Bax[16].
Increased regulation of Bcl-2, and other anti-apoptotic proteins, is the potential way to obtain
resistance due to therapeutic agents [17]. Damage to the BCL-2 gene has been identified as the cause
of many cancers, including melanoma, breast cancer, and prostate cancer. The Caspase 3 protein, a
member of the cysteine-aspartic acid protease (caspase) family, is an important mediator of
programmed cell death. Survivin, a member of the apoptosis inhibiting family (IAP), can inhibit
caspase activation to regulate negative apoptosis or programmed cell death. Luteolin (3 ', 4', 5,7-
tetrahydroxyflavone) is are flavanoid compounds obtained naturally or natural compounds obtained
from plants extracted from various traditional Chinese medicines, such as Lonicera japonica,
Schizonepetatenuifolia, and AjugadecumbensThunb. Important structural features of luteolin,
including 2-3 carbon double bonds and hydroxyl groups at the carbon positions 5, 7, 3 ', and 4', are
related to the biological and biochemical activity of luteolin [18, 19]. The anticancer properties of
luteolin are related to the biological effects of luteolin, such as angiogenesis, anti-inflammatory
activity, inhibition of cell proliferation, induction of apoptosis, and metastasis [20]. Previous reports
have shown that luteolin has various biological activities, such as immunoregulation [21], anti-
oxidants, anti-inflammatory [22], cardiovascular protection, and enzyme inhibition [24]. In recent
years, luteolin has been shown to inhibit leukemia cell proliferation and prostate cancer cell invasion
[24,25].
5-Fluorouracil (5-FU) as a control in this study is a type of chemotherapy drug that is often used
to block colorectal cancer. however, advanced colorectal cancer often experiences chemotherapy
incompatibility and poor results. The phosphorylated paxillin (PXN) protein adapter at Y31 / Y118
(pPXN-Y31 / Y118) by Src contributes to cell movement and Ser (S) 272 of PXN in the LD4 domain
important for interactions between PXN and Bcl-2[27].

3.1. Ligand Preparation

Ligands are active compounds to be tested in the target based on the literature, there are two ligands
used in this study is luteolin and 5-fu used as a control for research. Once obtained from the PubChem
website, the ligand format must be changed from SDF to PDB format to facilitate the molecular
docking process. Figure 1 shows the 3D structure of each ligand.

A B
Figure 1. The compound from Physalis angulata
.A. Luteolin, B. 5- Fluorouracil (5 - Fu) (Control)

3.2. Target selection

This study uses a target protein, B-cell lymphoma 2 with the BCL-2 PDB code. This protein can be
dimerized and has a function as an apoptotic regulator. Also BCL-XL, BCL-2, and BAX are a protein
that functions to change the ion-conductive pore in an artificial membrane. The BCL-2 family consists
of members of pro-apoptosis and anti-apoptosis, which act as a place to examine the ends of caspases
and mitochondrial dysfunction. BID and BAD have a minimum BH3 mortality domain, and BAD
phosphorylation links signals between proximal survival signals with BCL-2 and BCL-XL families
that display reciprocal expression patterns during lymphocyte development. The gain and loss-of-
function models play a role in uncovering the specific stages for BCL-2 and BCL-XL. BCL-2 can
reduce changes at several lymphocyte developmental points. Other functions The BCL-2 family
provides information about the coordination of autonomous cells between the proliferation pathway
and opposing dead cells. [28] After the target is taken from the Protein Data Bank, the molecules are
cleaned of water molecules and other residues using PyMol v1.7.4.5. The results are shown in Figure
2.

Figure 2. B-cell lymphoma 2 (BCL-2) after

being processed with PyMol v1.7.4.5

3.3. Molecular Docking Result

The silico method is a method obtained from databases and software in conducting bioinformatics
research. One technique for this method is molecular docking with computational methods to predict a
potential compound before testing. The benefit of this method is to prevent errors in vivo results by
estimating potential combined activities. The binding affinity results from docking molecules of
Luteolin compounds in Physalis angulata are shown in Table 1. Affinity bonds calculate the ability of
a compound to coexist in a receptor. If the number is smaller, then the affinity relationship with the
receptors and ligands is greater and vice versa, if the number is greater then the relationship is less
[29], from tests, it is carried out that luteolin has the lowest and closest bond to 5- Fluorouracil (5-Fu)
as a control. This means that Luteolin in Physalis angulata has a high potential to be a medicinal
compound to be used as an anti-cancer, with interactions between Luteolin and BCl-2. Whereas in
Figure 1 we can see that Luteolin and 5- Fluorouracil (5-Fu) have the same binding site.

Table 1. The binding affinity of Physalis angulata compound

No Compound Binding Affinity
1 5- Fluoroucil (control) -5,3
2 Luteolin -7,6

3.4. Ligand-Macromolecule Interaction Visualization

The results of visualization of ligand-macromolecular interactions are shown in Figure 3. Based on the
binding of affinity value, luteolin has a low binding affinity value. From the visualization, it appears
that luteolin and 5-Fu (control) compounds bind to one another in macromolecules which mean that
both compounds have the same ability to be used as anti-cancer drugs.
 Figure 3. The binding site of BCL-2 (Blue), Luteolin (Red) and 5-
Fluorouracil (Yellow) PyMol v1.7.4.5

3.5. Drug-Likeness Test

At the stage of the molecule docking process, the next step is to test the similarity of the drug. Drug
similarity is a way to provide information on the physicochemical properties of a compound that can
change the molecular properties in vivo. The way to find out drug similarity tests uses the
physiochemical properties of molecular structure and compatibility with registered drugs. Namely
using the Lipinski rule, where the molecular weight is ≤500 kDa, LogP is ≤5, the hydrogen bond
donor is ≤5 and the hydrogen bond acceptor is ≤10. This criterion is similar to good oral
bioavailability. The log P-Value if the ratio of solubility in fat/water has a value of around -0.4 - 5.
Molecular weights of more than 500 Da cannot mix through cell membranes. A large log P number
shows the hydrophobicity of the molecule. Molecules that are too hydrophobic will experience a high
level of toxicity due to a longer containment process in the fat layer and more channeling into the body
so that the selectivity of the binding to the target enzyme decreases. A P value indicating a negative
log indicates a blockage in the lipid bilayer membrane [30,31]. If between many givers and hydrogen
bonding paths where the higher hydrogen capacity bonds are, the higher the energy needed in the
absorption process occurs. The Lipinski rule explains that the mixing of certain soluble compounds to
be able to pass through cell membranes that diffuse passively [32,33]. Drug similarity test results on
Luteolin compounds are shown in Table 2. Based on Lipinski's rules, it meets the compound
requirements. Where the weight of the compound at luteolin is 286.24 g / mol, the hydrogen's
pronouncement is 4, the hydrogen bond acceptor is 6 with a LogP value of 1.37.

Tabel 2. Drug-likeness result of Physalis angulata compound Luteolin and 5- Fluorouracil (5 –F)
(Control)
Hydrogen Hydrogen
Compound Moleculer Bioavailability
Bond Bond LogP
Weight score
Donor Aceptor
5- Fluorouracil
(5 –Fu) 130,08 g/mol 2 3 0,13 0,55
(Control)
Luteolin 286,24 g/mol 4 6 1,37
0,55

4. Conclussion
The results of research carried out by molecular docking, that the compound luteolin in Physalis
angulata has the lowest binding affinity value of the binding of -7, 6, so it has the potential to be used
as an anti-cancer and drug test results show that the compound luteolin meets the criteria as a drug
anti-cancer based on Lipinski’s rules with a bioavailability score of 0.55. Where the weight of the
compound in luteolin is 286.24 g / mol, the hydrogen statement is 4, the hydrogen bond acceptor is 6
with a LogP value of 1.37.
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