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The 5th International Conference on Agriculture, Environment, and Food Security IOP Publishing
IOP Conf. Series: Earth and Environmental Science 977 (2022) 012017 doi:10.1088/1755-1315/977/1/012017

Molecular docking analysis of Allium chinense compounds as

Secreted Aspartyl Proteinase-5 (SAP5) inhibitor

A Hartanto1, F G Naibaho2, D Panjaitan2, A Lutfia1 and E Munir1,*

1
Department of Biology, Faculty of Mathematics and Natural Sciences,
Universitas Sumatera Utara, Medan, Indonesia
2
Department of Biology, Faculty of Mathematics and Natural Sciences,
Universitas Palangka Raya, Palangka Raya, Indonesia
*
E-mail: erman@usu.ac.id

Abstract. Secreted Aspartyl Proteinase-5 (SAP5) or candidapepsin-5 is known as the current

and major virulence factor in the biofilm formation of Candida albicans. The protein is secreted
into the environment to disrupt the host immune cells and degrade keratin then penetrating the
host defense to express its pathogenicity. SAPs has been targeted for many studies including in
vitro test and in silico analysis of potential inhibitory agents. In the current study, we tested six
selected compounds in the aqueous extract of Allium chinense G. Don. namely 1-tetradecanol,
anozol, hyacinthin, isosorbide, mannitan and oleic acid for in silico analysis along with pepstatin
A as the most potent inhibitor or control. The results obtained that oleic acid displayed the most
stable bonding with the SAP5 based on molecular docking, visualization and data analysis
although slightly lower than anozol in terms of binding affinity. Oleic acid also produced the
most similar number of binding residues with pepstatin A based on 2D feature with also similar
region in the pocket of SAP5 based on 3D visualization. Hence, the compound may be potentially
developed as leading compound in treating C. albicans infections.

1. Introduction
Candida spp are common mycobiota which colonize various mucosal surfaces, including human skin,
vagina, gut, and the oral cavity [1]. Candidiasis has been a global phenomenon with increasing case due
to the invasive fungal diseases or candidemia caused by the fungal pathogens [2]. The incidence of
candidemia has grown in numbers leading to high mortatily rates ≥70% in certain patient populations.
Notable species such as C. albicans, C. krusei, C. glabrata, C. parapsilosis, and C. tropicalis have been
related in many cases of Candida-related infections [3].
Candida albicans is known as the most common causal agent of candidemia in the hospital
environment [4]. The establishment of fungal infection by opportunistic Candida spp especially C.
albicans in the immunocompromised host rely on a complex transition between yeast form and mold-
like growth or hyphal forms as an initial way of expressing pathogenicity and virulences [5]. Upon
attachment, Candida biofilms will actively penetrate into host cells and secrete specific fungal
hydrolytic enzymes such as secreted aspartyl proteinases (SAPs), phospholipases, lysophospholipase,
and other proteases [6,7]. Recent findings on the pivotal role among these enzymes revealed that SAP5
or Candidapepsin-5 (EC: 3.4.23.24) is highly upregulated in C. albicans biofilm while the deleterious
mutant was unable to develop a proper biofilm in vitro and in vivo yet less virulent than usual [8,9].

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The 5th International Conference on Agriculture, Environment, and Food Security IOP Publishing
IOP Conf. Series: Earth and Environmental Science 977 (2022) 012017 doi:10.1088/1755-1315/977/1/012017

Anti-Candida activity is well documented from the extracts of Allium (Amaryllidaceae) species and
has been reported to effectively downregulate the expression of SAPs in C. albicans based on in vitro
test and in silico analysis [10,11]. The present study investigates the possibility of finding potent SAP5
inhibitors in the aqueous extract of local shallot (Allium chinense G. Don.) cultivated in North Sumatra
which displayed a high antifungal activity against C. albicans in our previous study [12].

2. Method

2.1. Instrumentation
A laptop with specifications of processor (CPU) AMD Ryzen 4500U 4GHz, graphics processing unit
(GPU) AMD RADEON Vega 6 and 8 GB RAM with Windows 10 Home Single Language 64-bit was
used.

2.2. Protein database search

Protein target of C. albicans namely secreted aspartyl proteinase-5 (SAP5) was retrieved from Protein
Data Bank (PDB; https://www.rscb.org) with an accession number, 2QZX [13]. The protein file (.pdb)
was prepared by removing the water and existing ligands of the 3D structure using BIOVIA Discovery
Studio Visualizer (http://www.discover.3ds.com).

2.3. Ligand database search

Six compound databases were selected based on their molecular weight and used to predict the
antifungal activity of Allium chinense [12]. The 3D structure of selected molecules namely 1-
tetradecanol, anozol, hyacinthin, isoborbide, mannitan, oleic acid and pepstatin A (positive control) were
retrieved and saved from PubChem (https://www.pubchem.ncbi.nlm.nih.gov) as a single file (.sdf) as
presented in Table 1. The ligands were minimized in energy and prepared for molecular docking (.pdb)
using Open Babel on PyRx.

Table 1a. Selected compounds in the aqueous extract of A. chinense bulbs for in silico analysis
PubChem Chemical Chemical
No. Compound
ID Formula Structure
1. 1-Tetradecanol 8209 C14H30O

2. Anozol 6781 C12H14O4

3. Hyacinthin 998 C8H8O

4. Isosorbide 12597 C6H10O4

2
The 5th International Conference on Agriculture, Environment, and Food Security IOP Publishing
IOP Conf. Series: Earth and Environmental Science 977 (2022) 012017 doi:10.1088/1755-1315/977/1/012017

Table 1b. Selected compounds in the aqueous extract of A. chinense bulbs for in silico analysis
PubChem Chemical Chemical
No. Compound
ID Formula Structure

5. Mannitan 10909888 C6H12O5

6. Oleic acid 445639 C18H34O2

7. Pepstatin A 5478883 C34H63N5O9

2.4. Molecular docking

Molecular docking was executed using AutoDock Vina in PyRx to identify the interaction mode
between SAP5 and ligands. The dimensions of the search scape were 17 Å × 22 Å × 16 Å covering two
active sites i.e. Asp32 and Asp218 [14]. The center of coordinate was X: 7.5421, Y: 33.9386, Z: 26.0348.
Each molecular docking output was saved as a single file (.pdb).

2.5. Interaction of protein-ligand

The interaction between the 3D structure of C. albicans SAP5 with each ligand was visualized and
combined as a single file (.pdb) following the determination of protein-ligand complex using BIOVIA
Discovery Studio Visualizer.

3. Results and Discussion

The SAPs have been recognized as the most important virulence traits in biofilm-forming strain of C.
albicans, which are encoded by a family of 10 sap genes producing ten proteins (SAP1-SAP10) [15-
17]. Therefore, the secreted protein may be a promising target for potential inhibitor to inactive the
protein and reducing the virulence of C. albicans. In this study, six compounds detected in previous GC-
MS analysis of the aqueous extract of A. chinense bulbs were visualized for its ligand-receptor
interactions compared with the native ligand, Pepstatin A. The results of molecular docking using
AutoDock Vina is presented in Table 2.

3
The 5th International Conference on Agriculture, Environment, and Food Security IOP Publishing
IOP Conf. Series: Earth and Environmental Science 977 (2022) 012017 doi:10.1088/1755-1315/977/1/012017

Table 2. Molecular docking result of bioactive compounds in the aqueous extract of A. chinense
Binding No. of H bond Van der Waal’s
No. Compound Interacting residues
affinities H bonds interacting residues
THR 13, ASP 32, GLY
1. 1-Tetradecanol -4.9 2 ILE 12, THR 222 85, ASP 86, GLY 220,
THR 221, ASP 308
ASP 32, GLY 34, LYS
83, GLY 85, ILE 123,
2. Anozol -5.8 1 ASP 86
ASP 218, GLY 220, THR
221, THR 222
ASP 32, ASP 86, SER 88,
3. Hyacinthin -5.3 1 ARG 120 ALA 119, GLY 220
ALA 11, ILE 12, ALA
162, THR 222, ILE 223,
4. Isosorbide -5.1 2 ARG 297, ARG 312
GLU 278, PHE 281, ASN
309
ILE 12, ILE 30, TYR 84,
THR 13, ASP 86, SER
5. Mannitan -5.2 5 ALA 119, ARG 120, ILE
88, GLY 220, THR 222
123, THR 221
ILE 12, THR 13, ASP 32,
TRP 51, GLY 85, SAP
SER 88, ALA 119, ARG
6. Oleic acid -5.4 3 86, SER 88, SER 118,
120
GLY 220, THR 221, THR
222
GLY 85, ASP 86, GLY
220, THR 221, THR 222,
ASP 32, LYS 50, SER 88,
7. Pepstatin A -7.1 9 TYR 225, ARG 299, SER
ILE 223, GLU 300
301,
ILE 205

The binding affinities (kcal/mol) indicate binding stability through an inter- and intramolecular
interaction among pairs of atoms during ligand-receptor complex state. The more negative its value will
result in a more stable binding. The stable interaction through numerous H-bonds will ensure the
possibility of conformational alteration of SAP5 protein leading to the disruption of structural rigidity
and loss of biological function (virulence). Based on the results, the highest binding affinity was anozol
(-5.8 kcal/mol), followed by oleic acid, hyacinthin, mannitan, isosorbide and 1-tetradecanol (-4.9
kcal/mol). Here, pepstatin A is still proven as the most potent inhibitor with -7.1 kcal/mol of binding
affinity. Based on the interacting H bonds, mannitan showed a higher potential among others with 5 H-
bonds, followed by oleic acid (3 bonds) and other compounds with only 1 to 2 bonds. To summarize the
interaction, each bond was checked for any similarity with pepstatin A as presented in Table 3.

Table 3a. Similarities of molecular interaction based on H-bond interacting residues

Binding
No. Compound Similar binding site residues
similarities (%)
ASP 32, LYS 50, GLY 85, ASP 86, SER 88, GLY 220,
Pepstatin A
1. THR 221, THR 222, ILE 223, TYR 225, ARG 299, GLU 100%
(Control)
300, SER 301, ILE 205,
2. 1-Tetradecanol ASP 32, GLY 85, ASP 86, GLY 220, THR 221 35%
ASP 32, ASP 86, GLY 85, GLY 220, THR 221,
3. Anozol 42%
THR 222

4
The 5th International Conference on Agriculture, Environment, and Food Security IOP Publishing
IOP Conf. Series: Earth and Environmental Science 977 (2022) 012017 doi:10.1088/1755-1315/977/1/012017

Table 3b. Similarities of molecular interaction based on H-bond interacting residues

Binding
No. Compound Similar binding site residues
similarities (%)
4. Hyacinthin ASP 32, ASP 86, SER 88, GLY 220 28%
5. Isosorbide THR 222, ILE 223 14%
6. Mannitan ASP 86, SER 88, GLY 220, THR 221, THR 222 35%
ASP 32, SER 88, GLY 85, ASP 86, SER 88, GLY 220,
7. Oleic acid THR 221, THR 222 57%

Based on the binding similarities, oleic acid was revealed as the most potential as it interacted
similarly to 8 out of 14 residues in pepstatin A namely ASP 32, SER 88, GLY 85, ASP 86, SER 88,
GLY 220, THR 221, THR 222 followed by anozol (42%). The 2D illustration of interacting residues
inoleic acid and pepstatin A is presented in Figure 1. Both mannitan and 1-tetradecanol displayed 35%
ofbinding similarities to the residues which were slightly lower than oleic acid and anozol. Other
remaining compounds were less potent. The searching of binding similarities may contribute to the
synthesis of analogous compound similar to pepstatin A or modification of existing compounds to
obtaina more effective ligand. We assumed that the most similar number of binding sites between
oleic acid and pepstatin A may be originated from its high molecular mass since the oleic acid was
known as the highest molecular weight compound analyzed in our study (282.47 g/mol). The 3D
visualization of ligand-protein interaction of the most potent compound is presented in Figure 2.
Based on Figure 2, it was revealed that the binding site region was similar between pepstatin A as
selective inhibitor and oleic acid which also strenghten the possibility of using oleic acid as a
competitive inhibitor to SAP5.

Figure 1. SAP5 in complex with competitive inhibitor, oleic acid (A)and

selective inhibitor, pepstatin A (B). Dashed lines represent the known
hydrogen bonds with the residues.

5
The 5th International Conference on Agriculture, Environment, and Food Security IOP Publishing
IOP Conf. Series: Earth and Environmental Science 977 (2022) 012017 doi:10.1088/1755-1315/977/1/012017

Figure 2. A. 3D interaction between pepstatin A and SAP5.

B. 3D interaction between oleic acid and SAP5. Arrows indicate the
location of binding sites as revealed in the shape of pocket-size region
of the enzyme.

Four class of antifungal agents have been implemented publicly for antifungal therapy since their
discovery in 1955 namely polyene, pyrimidine analogues, triazoles, and echinocandins. However,
antifungal resistance still develops and hampers the clinical use of its maximum potential. Natural
product is an ideal source for the discovery of drug candidates with least side effects in the brink of less
effective antifungals. The exploration of potential bioactive phytochemicals may involve a time-
consuming and laborious processes. Then, molecular docking analysis of known metabolites has been
promoted as one of the promising methods to search for new antifungal agents especially against C.
albicans [18].
Virulence factor of C. albicans as expressed into the SAPs, has been targeted for many bioactive
compounds such as endophytic fungal metabolites [19], propolis [20], and plant phytochemicals [14].
In this study, we evaluated the chemical composition in the extract of local A. chinense cultivated in
North Sumatra for gaining deeper understanding on its high antifungal activity against C. albicans based
on in vitro test [12]. The chemical compounds were known to exert inhibitory activities to SAP5 based
on the molecular docking result especially by oleic acid and anozol. Recently, Muthamil et al [21]
reported that plant-based oleic acid reduced the SAPs production in Candida spp both in culture test and
gene expression profiling assay (sap1, sap2, sap4). Other potential inhibitor, anozol or diethyl phthalate
has also been reported to induce superoxide production and cytoplasmic stress in bacterial cells and
displayed antifungal activity against C. albicans [22,23]. However, it may also be noteworthy that the
chemical compounds are in cocktails or crude plant extracts while there is still unknown information
upon the synergistic or antagonistic mechanism of each compound which may delay their potential for
drug design in the future.

4. Conclusion
The selected compounds (1-tetradecanol, anozol, hyacinthin, isosorbide, mannitan, oleic acid)
documented from the aqueous extract of A. chinense displayed binding properties with the certain
residues in SAP5 of C. albicans with a range of -4.9 – -5.8 kcal/mol. Oleic acid displayed the most
similar binding sites with pepstatin A thus promoted its use as a potential inhibitory agent to SAP5.

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The 5th International Conference on Agriculture, Environment, and Food Security IOP Publishing
IOP Conf. Series: Earth and Environmental Science 977 (2022) 012017 doi:10.1088/1755-1315/977/1/012017

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