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Online Journal of Bioinformatics ©

Volume 14 (3): 293-301, 2013

In silico inhibitors for falcipain-3 in Plasmodium falciparum

Madhu Sudhana Saddala, P. Gayathri, J. Obaiah, C. Vallamma and A. Usha Rani*

Dept. of Zoology, DBT-Bioinformatics Center, Sri Venkateswara University, Tirupati, A.P., India.

ABSTRACT

Saddala MS, Gayathri P, Obaiah J, Vallamma C, Usha Rani A., In silico inhibitors for falcipain-3 in
Plasmodium falciparum, Onl J Bioinform., 14 (3): 293-301, 2013.Inhibition of falcipain-3
prevents maturation of Plasmodium falciparum, suggesting that the protein could be a target
for antimalarial activity. Falcipain-3was energy minimized and subjected to molecular dynamics
simulations using NAMD 2.9 software with CHARMM27 force field in water and the receptor
structure was minimized with 25,000 steps for 500ps and simulated 10,000 steps for 2ns. 2500
compounds were screened from PubChem database through structure based Virtual screening
by referencing Mefloquine. The screened compounds were docked into the active site of the
protein using Autodock Vina in PyRx Virtual Screening tool. Results showed that CID3000506,
CID40468067, CID65330, CID40692 and CID4046had a highest binding energyof-9.4, -8.9, -8.4,
-7.9 and -7.2 kcal/mol, respectively. Lead hit compounds were tested for toxicity and
bioavailability with Osiris and Molinspiration online servers. Active site amino acidsHis18,
Asp44, Tyr63, Gln110, Tyr115, Asp168, His196, Glu199, Gly453 and Met434could play a role in
binding and catalytic activity.

KEYWORDS: Falcipain-3, simulations, docking, PubChem database, Autodock Vina

INTRODUCTION

Malaria is one of the most important infectious diseases in the world wide and affects about
40% of the world’s population (Breman, 2001).It is caused by different species of Plasmodium.
Four Plasmodium species have been well known to cause human malaria, namely,

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P. falciparum, P. vivax, P. ovale, and P. malariae. A fifth one, P. knowlesi, has been recently
documented to cause human infections in many countries of Southeast Asia. Most severe
diseases and deaths from malaria are caused by P. falciparum. It is responsible for about 80% of
all malaria cases and is also responsible for about 90% of deaths from malaria (Stanley et al.,
1991).

In P. falciparum, three papain-like cysteine proteases have been identified, characterized, and
isolated thus far. Falcipain-1 was identified in erythrocytic parasites and found to hydrolyze
hemoglobin (Salas et al., 1995). However, its low abundance and difficulties in developing
expression systems have limited its study. Recently, two closely related cysteine proteases were
identified and expressed. Falcipain-2 was shown to be one of the principal trophozoite cysteine
proteases and hemoglobinases (Shenai et al., 2000), and more recently, falcipain-3 was
identified in the acidic food vacuole of the parasite (Sijwali et al., 2001). Both proteases require
a reducing environment and acidic pH for optimal activity. They differ, however, in that
falcipain- 3 undergoes efficient transformation into an active enzyme only at acidic pH. It is
more active and stable at this pH with greater activity against native hemoglobin. Thus,
falcipain-3 is the second P. falciparum hemoglobinase well suited for the hydrolysis of native
hemoglobin in the food vacuole. It has been estimated that the concentration of falcipain-2 in
trophozoites is 1.8 times that of falcipain-3; however, the latter appears to cleave native
hemoglobin about twice as rapidly as the former. Thus, the relative contribution of the two
enzymes to the hydrolysis of native hemoglobin is essentially equivalent, making falcipain-2 and
falcipain-3 equally important targets for inhibition of hemoglobin degradation (Sijwali et al.,
2001).

A variety of antimalarial medications are available. In the last 5years, treatment of P. falciparum
infections in endemic countries has been transformed by the use of combinations of drugs
containing an artemisinin derivative (Govindarajan et al., 2007). Severe malaria is treated with
intravenous or intramuscular quinine or, increasingly, the artemisinin derivative artesunate
which is superior to quinine for both children and adults (Philip et al., 1998). The parasite has
developed resistance to several antimalarial drugs, most notably chloroquine. Computer Aided
Drug Design (CADD) is a specialized discipline that uses computational methods to simulate
drug-receptor interactions. Different methods CADD are profoundly dependent on
bioinformatics tools and applications. Therefore in this study we have performed molecular
docking studies of Mefloquine and its analogs with Falcipain-3 in order to find out the best one.
The main emphasis of this work was to identify the most appropriate drug molecule against the
malaria parasite. There are several parameters such as ADMET properties and Lipinski’s rule of
five (Christopher et al., 2000; Selick et al., 2002) that helps in identifying the best lead
compounds. In this study, we described inhibitors ofFalcipain-3using molecular docking,
molecular dynamics simulation and structure based Virtual screening.

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 MATERIALS AND METHODS

Protein preparation and MD simulations

The X-ray crystallographic structure of the Plasmodium falciparam Falcipain-3 was retrieved
from the Protein data bank (http://www.rcsb.org/pdb). The Accession no. of the Falcipain-3PDB
ID is 3BPM. The existing ligands and crystallographic water were removed. It was refined by
molecular dynamics in a solvated layer and equilibration methods using NAMD 2.9 (Nanoscale
Molecular Dynamics) software (Kale et al., 1999) using CHARMM27 (Schlick et al., 1999)
(Chemistry at Harvard Macromolecular Mechanics) force field for protein in water (Schlenkrich
et al., 1996). Protein was energy minimized with 2500 runs for 500ps and simulation with
1000000 steps for 2ns. Spherical periodic boundary conditions were included in this study.
Finally, the structure having the least RMSD of Cα trace was generated by employing the
molecular dynamics simulations which improves the quality of the target protein. The trajectory
analysis was analyzed by drawing the graph between Time in Ps on X-axis and RMSD (Å) on Y-
axis as shown in Figure1. The quality structure of Falcipain-3 was used for further analysis.

Figure1: Root mean square deviation (RMSD) during the molecular simulations of Falcipain-3.
Time (Ps) was taken in X-axis and RMSD was taken in Y-axis.

Simulation parameters
The MD simulations system was equilibrated at 250 k for 10 ps with Falcipain-3 atoms fixed,
followed by 20 ps MD without restraints. The system was subsequently simulated for 100 ps
at300 k with the following parameters. The classical equations of motion were integrated by a
leapfrog integrator using a time step at 1 fs. The impulse based ver let-I/r-RESPA method was
used perform multiple time stepping: 4 fs long-range electrostatic: 2fs for short range non-
bonded forces, and 1 fs for bonded force. The swift function was used to cutoff the Lennard-
Jones potential, with the first cut off at 10 Å and the second cutoff at 12 Å. Short range
interactions were calculated at intervals of 4 fs. All bonds involving hydrogen atoms were
constrained to their equilibrium bond parameters using the SHAKE along them. Langevin
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dynamics were employed to maintain the pressure at 1 atm, with a Langevin pisten period of
100 fs and oscillation decay time of 50fs.Trajectories were recorded every 200 fs. Subsequently
the dynamics behavior and structural changes of the receptor was analyzed by the calculation
of energy and the root mean square deviation (RMSD).

Active site Identification

Active site of 3BPM was identified using CASTp server (Computer Atlas of Surface Topology of
protein) (Dundas et al, 2006). A new program, CASTp, for automatically locating and measuring
protein pockets and cavities, is based on precise computational geometry methods, including
alpha shape and discrete flow theory. CAST identification and measurements of surface
accessible pockets as well as interior inaccessible cavities by locating, delineating and
measuring concave surface regions on three-dimensional structure of proteins. The
measurement includes the area and volume of pocket or void by solvent accessible surface
model (Richards’ surface) and by molecular surface model (Connolly’s surface), calculated
analytically. It can also be used to study surface features and functional regions of proteins.
Falcipain-3 secondary structure and active site are shown in Figures 2 and 3.

Figure2: Secondary structure of Falcipain-3. Figure3: Binding pocket of Falcipain-3

Ligand Preparation
The chemical structure of Mefloquine (CID40692), which display high potency for Falcipain-3
(IC50 = 0.2nm) is display in Figure4. It is a potent, reversible nonpeptidic biaryl inhibitor for
Falcipain-3. Therefore, our study used Mefloquine as a query for screening of compounds from
PubChem database through structure based virtual screening. Virtual screening has been
emerged as a complementary approach to high throughput screening and has become an
important in silico technique in the pharmaceutical industry (Lengauer et al., 2004). The
structure based virtual screening begins with the identification of potential ligand binding sites
on the target proteins. Usually, molecules that meet the criteria for biological activity fulfill

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characteristics contained in the Lipinski’s rule of five (Lipinski et al., 1997), or the more relaxed
rules revised by Veber et al., 2002.
In our work, we have selected 2500 docked ligands based on structure similarity with query
Mefloquine compound from PubChem database. The Autodock Vina in PyRx Virtual Screening
Tool URL http://pyrx.scripps.edu (Wolf, 2009; Trott and Olson, 2010) was used for the screening
of selected ligands from PubChem database and energy minimization.

Figure4: Structure of Mefloquine

Ligand-Protein docking studies

Docking is a computational method which predicts the preferred orientation of one molecule to
a second when bound to each other to form a stable complex. Docking has been widely used to
suggest the binding modes of protein inhibiters. Most docking algorithms are able to generate a
large number of possible structures, thus they also require a means to score each structure to
identify those that of greatest interest. Docking was performed using AutoDock Vina in PyRx
Virtual Screening tool (Wolf, 2009; Trott and Olson, 2010).

PubChem screened compounds were docked into active site of refined model. Lamarkian
genetic algorithm was used as number of individual population (150), max number of energy
evaluation (25000000), max number of generation (27000), Gene mutation rate(0.02),
crossover rate (0.8), Cauchy beta (1.0) and GA window size (10.0).The grid was set whole
protein due to the multi binding pocket at X=3.42, Y=-56.23, Z=98.32 and dimension Å) at
X=89.92, Y=98.56, Z=98.32 and exhaustiveness 8. The pose for a given ligands identified on the
basis of highest binding energy. Only ligand flexibility was taken into account and the proteins
were considered to be rigid bodies. The resulting complexes were clustered according to their
root mean square deviation (rmsd) values and binding energies, which were calculated using
the Autodock scoring function. Further characterization via MD simulations was conducted
using complexes that were selected according to their binding energy values and the
interactions made with the surrounding residues. The PyMol molecular viewer
(http://www.pymol.org/) was employed to analyze the docked structures.
RESULTS AND DISCUSSION

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The target protein (3BPM) was taken from the protein data bank, then the water molecules,
hetero atoms and ligands were removed and minimized and subjected to simulation by NAMD
tool. Active site of the protein was determined by using Castp server. The refined protein was
used for further docking of screened compounds. Two-dimensional structure of Mefloquine
was used to query for similar compounds in the PubChem database. Then, approximately 2500
compounds were screened, and the all compounds were saved for further molecular docking.
The ligands were taken and docked into target protein active site along with the template drug
molecule (Mefloquine) in Autodock Vina in PyRx Virtual Screening tool by which all the ligands
were embedded within the active site of the target protein, were observed forming hydrogen
bonds with same position as Mefloquine established active site of protein. Top 5 ligands were
found, to require lower energy as compared to query drug (Mefloquine), which are used as the
therapeutic agents against Falcipain-3 for malaria (Table1). Mefloquine have binding energy -
7.2kcal/mol where as CID08830211, CID13650942, CID65739916 and CID65748951 have -9.4, -
8.9, -8.4 and -7.9kcal/mol energies respectively. The interactions of ligands and protein
graphical view are shown in Figure5.

CID3000506 CID40468067 CID65330

CID40692 CID4046

Figure 5: Graphical representation of PubChem compounds and Falcipain-3 protein pocket.

The best binding affinity compounds were obtained through the molecular docking studies. The
compound CID3000506 was bound with the binding affinity -9.4kcal/mol by the formation of
one hydrogen bond interactions with Asp44 residue within the active site of Falcipain-3 protein.
The compound CID40468067 was bound with the binding affinity -8.9kcal/mol by the formation
of two hydrogen bond interactions with Gln110 and Tyr115 respectively within the active site of

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 Falcipain-3 protein. The compound CID65330 was bound with the binding affinity -8.4kcal/mol
by the formation of two hydrogen bond interactions with Gln110 and Gly453 respectively
within the active site of Falcipain-3. The compound CID40692 was bound with the binding
affinity -7.9kcal/mol by the formation of one hydrogen bond interactions with Met434within
the active site of Falcipain-3 protein. The compound CID4046 (Mefloquine)was bound with the
binding affinity -7.2kcal/mol by the hydrophobic, electrostatistic and Van der Waal bonds
within the active site of Falcipain-3 protein. The protein and ligand interactions, binding affinity
values and hydrogen bond lengths are represented in Table1.

Table 1: Binding affinity and bond lengths, of the best four ligands from PubChem database
with Falcipain-3, comparison with query drug (Mefloquine)
PubChem H-bond interactions Binding Affinity H-bond
S.No. H-bond
Compounds Structures (Kcal/mol) distance(Ǻ)
angle
Protein-----Ligand

1 CID3000506 -9.4 2.88

Asp44CO---NH 144.9

Gln110CO----NH 3.29
2 CID40468067 -8.9 110.04
Tyr115CN----OH 2.87
116.68

Gln110CN----OH 114.54 3.30

3 CID65330 -8.4
Gly453CO----OH 126.85 3.20

CID40692 Met434CO---OH -7.9 3.05

4 169.90

CID4046 Asp44, Gln110, Tyr115,

5 -7.2 ----
(Mefloquine) Gly453, Met434 ---

The lead hit compounds satisfied the Lipinski’s rule of five with zero violations and also the
octanol/water partition coefficient (miLogp), a useful parameter for predicting the drug
transport properties like absorption, bioavailability, permeability and penetration. As well as
topological molecular polar surface area (TPSA), number of atoms, their molecular weight
(MW), number of hydrogen donors and number of hydrogen acceptors. A topological
parameter is number of rotatable bonds and it describes the molecular flexibility of these
compounds represented in Tables 2 and 3. Our investigation revealed that the selected

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 compounds have exhibited significant binding affinity with in the active site of 3BPM protein,
when compared to query compound Mefloquine. Based upon this study, it is concluded that
these compounds may be used as leads for developing effective antimalarial drugs.

Table 2: Molecular physical-chemical properties and Lipinski properties of lead molecules with
OSIRIS

No. of
Drug Drug No. of H
S.No PubChem ID M I R T Solubility MW rotatable cLop
likeness score acceptors
bonds

1 CID3000506 - - - - 45.147 378.1 -3.63 0.04 9 4 4.242

2 CID40468067 - - - - 49.725 379.3 -4.64 0.07 8 4 1.227

3 CID65330 - - - - 45.147 378.6 -5.34 0.04 9 4 4.242

4 CID40692 - - - - 45.147 378.4 -5.21 0.03 9 4 4.242

CID4046
5 - - - - 45.147 378.2 -5.21 0.03 9 4 4.242
(Mefloquine)

Table 3: Parameters for predicting drug transport properties like absorption, bioavailability,
permeability and penetration using with Mollinspiration server.

Nuclear
Ion channel Kinase Protease Enzyme TPSA
PubChem ID GPCR ligand receptor
modulator inhibitor inhibitor inhibitor
ligand

CID3000506 0.45 0.21 -0.05 0.30 0.36 0.21 45.147

CID40468067 0.45 0.21 -0.05 0.30 0.36 0.21 49.725
CID65330 0.45 0.21 -0.05 0.30 0.36 0.21 45.147
CID40692 0.45 0.21 -0.05 0.30 0.36 0.21 45.147
CID4046 (Mefloquine) 0.45 0.21 -0.05 0.30 0.36 0.21 45.147

CONCLUSIONS
In the present work, we have searched for potent anti Falcipain-3 inhibitors through virtual
screening on PubChem database based on structure similarity of ligand (Mefloquine). The
newly identified cysteine protease enzyme Falcipain-3 is an important target in drug design part
therapeutic intervention of malaria disease. Inhibition of Falcipain-3 could result in reducing
heamoglobin degradation and further inhibiting the maturation of parasites. Among screened
compounds CID3000506, CID40468067, CID65330 and CID40692 have the highest binding
affinity and obey the drug properties compared to Mefloquine. Therefore the five compounds
were hopeful drug molecule like Mefloquine against malaria disease.

ACKNOWLEDGEMENTS
Author, Madhu Sudhana Saddala is grateful to the University Grants Commission, New Delhi for
the financial assistance with the award of BSR-Meritorious fellowship. This work was carried
out in DBT-Bioinformatics Infrastructure Facility (BIF), Department of Zoology, Sri Venkateswara
University, Tirupati (BT/BI/25/001/2006).

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