CHAPTER 7

DOCKING SIMULATION AND MODE OF ACTION STUDIES

7.1 Introduction

7.1.1 Docking simulation

7.1.1.1 Molecular docking

Molecular docking is a computational tool used extensively in Structure-Based

Drug Design (SBDD) for the determination of binding affinity and relative orientation

between a protein and a ligand when they are bound to each other. This technique

simulates the process of molecular recognition by predicting the binding free energy as

well as interaction geometry of a bound protein-ligand complex. Docking thus predicts

the preferred orientation (i.e., ‘best fit’ orientation in 3D space) of a ligand that binds

to a particular protein of interest. The aim of molecular docking is to achieve an

energetically favorable conformation for both the protein and ligand such that the

ligand molecule undergoes optimal interaction with the binding site of protein (or

receptor) molecule to obtain a stable protein-ligand complex1,2.

7.1.1.2 Docking components

The molecular docking is divided into two main components, viz., search algorithm

and scoring function.

Search algorithm determines all possible optimal conformations for a given

protein-ligand complex i.e., the position and orientation of both molecules relative to

each other. They also calculate the energy of the resulting complex and of each

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individual interaction. More clearly, a search algorithm generates ‘poses’ i.e.,

orientations of particular conformations of the ligand in the active (binding) site of

protein molecule. It is followed by finding the best conformation of the ligand

appropriate for optimal interaction with the protein structure which is measured by

scoring function. The different types of algorithms that can be used for docking

searches are molecular dynamics, Monte Carlo methods, genetic algorithms and

fragment-based methods3-5.

Scoring function is a mathematical method used to predict the strength of

intermolecular interaction (non-covalent) between protein and ligand after they have

been docked. The prediction of the binding free energy (binding affinity) is called

scoring. A molecule with a good docking score is potentially a good binder. The

scoring function assigns a priority order to a set of structurally diverse ligands docked

to the same protein and estimates binding (or interaction) energy. The ligands are

ranked according to the interaction energy based on scoring functions. There are

different types of scoring functions, namely, force field-based, empirical-based,

knowledge-based, and consensus scoring. More accurate a scoring functions, more

expensive it is. Most scoring functions are physics-based molecular mechanics force

fields that estimate the energy of binding interaction of ligand molecules. The

empirical scoring function of any docking program is expressed as follows (Equation

7.1):

Fitness = Elec + vdW + H-bond (7.1)

The binding energy refers to that change in free energy of the system as depicted

below (Equation 7.2):

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 Protein (in water) + Ligand (in water) -----> Protein-Ligand complex (in water) (7.2)

The free energy of binding is calculated as the sum of the electrostatic energy (due

to ionic interaction, Gelect), van der Waals energy (GvdW), solvation effects (due to

H-bonding, Gsol) and internal energy changes due to conformational (Gconf),

rotational (Grot) and torsional (Gtor) energies. It is represented as ΔG bind (kcalmol -1)

and is calculated as follows (Equation 7.3):

ΔG bind = ΔGelect + ΔGvdW + ΔGsol + ΔGconf + ΔGrot + Gtor (7.3)

The ΔG bind value has to be low for a structure to be stable. A low (negative)

binding energy indicates a stable complex and thus a more likely binding

interaction1,3,5.

7.1.1.3 Assessment of docking

The results of docking are analyzed by a statistical scoring function which converts

interacting energy into numerical values, called the docking score. The 3D pose

(binding mode) of the docked ligand can be visualized using different visualizing tools

like Chimera, Pymol, Rasmol etc. which could help in to identify the best fit

orientation of ligand considering both docking score (or binding energy) and various

non-covalent interactions. Docking methods are usually assessed by their ability to

reproduce the binding mode of experimentally resolved protein-ligand complexes. The

ligand is removed from the complex, a search area is defined around the actual binding

site, the ligand is redocked into the protein, and the binding mode generated is

compared with the experimental positions usually in terms of a root mean square

deviation (RMSD). RMSD refers to the distance between the initial and final position

of the ligand in the binding cavity of protein molecule. RMSD has often been used to

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measure the quality of reproduction of a known (i.e., crystallographic complex)

binding pose by a docking method. Lower the value of RMSD, higher is the accuracy

of docking. If the docking protocol is able to produce similar docking pose of a ligand

with respect to the biological configuration of the same ligand in the crystal structure

of complex protein then it means that the docking method is validated. If the RMSD is

below 2 Å, it is generally considered a successful prediction. This represents good

reproduction of the correct pose (or true binding pose) 6-9.

7.1.1.4 Docking methodology

Molecular docking studies are usually performed on free like AutoDock (Art Olsen,

David Goodsell, Scripps), and commercial software packages such as Discovery

Studio (Accelrys), Glide (Schrodinger), GOLD (CCDC) and FlexX (BiosolveIT). The

crystal structures of protein molecules of interest are retrieved from the RCSB Protein

Data Bank (https://www.rcsb.org)10. The methodology involved in molecular docking

is depicted in Figure 7.1.

7.1.1.5 Applications of docking

Docking has two important roles: the search for the conformation and configuration

of the ligand in the binding site, which rank poses for a given small molecule on a

given target (docking), and the evaluation of the interaction energy between the target

and ligand, which rank different ligands according to their relative affinity for a given

target (compounds selection). Molecular docking studies are thus frequently used to

predict the binding orientation (or binding mode/pose) of small molecules to their

protein targets (or receptor) in order to predict the affinity and activity of the small

molecules. A binding interaction between a small molecule ligand and an enzyme

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protein may result in activation or inhibition of the protein. If the protein is a receptor,

ligand binding may result in agonistic or antagontic effect.

Small molecule Target molecule

Ligand library Protein

Preparation Preparation

Drug-likeness filter, Binding site identification,

ADMET, Toxicity screen Reeptor grid generation

Molecular docking

Docking

Scoring

Post-process

Reranking (solvation corretion)

Consensus (inadequecy of single scoring)
Validation (Redocking & superimposition)
Evaluation (Functional geometry & shape complementarity)

Compound selection
Experimental
screening

In vitro assays

Figure 7.1: Methodology of molecular docking

Predicting the mode of protein-ligand interaction can assume the active site of the

protein or receptor molecule, and further help in protein annotation. Docking is also

used for virtual screening of new ligands on the basis of biological structures for

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identification of hits and generation of leads or optimization (potency/property) of

leads in drug discovery program. Hence, docking plays an important role in rational

design of new drug molecules1,3,5.

7.1.2 Study of mode of action (MOA)

7.1.2.1 Malaria and oxidative stress

Recent studies suggest that malaria infection induces the generation of reactive

oxygen and nitrogen species (ROS and RNS) which relate to the pathophysiology of

the disease, and thus play a crucial role in the development of systemic complications.

As a result of the high metabolic rate of the rapidly multiplying malaria parasites, large

quantities of ROS are generated (as toxic by-products) as a result of host’s

haemoglobin degradation. Apart from this metabolically derived oxidative stress (OS),

the production of ROS by the host’s immune system adds to the overall oxidative

burden in malaria. The generation of hydroxyl radicals in the liver is the main reason

for the induction of OS in malaria infection. Moreover, currently used antimalarial

drug therapies (for example, chloroquine and derivatives of artemisinin) are based on

the production of free radicals or oxidants and their lethal action to malaria parasites.

This effect may be due to the drug’s ability to promote direct production of free

radicals or by inhibiting molecules of metabolic pathway of parasite (molecules

essential for survival of parasite) with antioxidant activity11-14. Therefore, reduction of

cellular OS is necessary for the successful treatment of malaria.

7.1.2.2 Density functional theory (DFT) study

DFT method has been successfully used to elucidate the structure-radical

scavenging activity relationship (SAR) for antioxidants, especially phenolic

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compounds. In DFT, Chemical properties such as electronic and molecular properties

are studied which are of great importance to understand the mechanism of the

antioxidant activity of phenolic compounds. DFT study thus provides important clues

for antioxidant development. It is well known that the antioxidant activity of a given

compound is strongly related to its capabilities to scavenge free radicals. According to

one potential mechanism, antioxidants (phenolic) can play role in scavenging free

radicals via hydrogen-atom transfer (HAT), Equation 7.4:

ArOH + R• → ArO• + RH (7.4)

In HAT, the reactivity of an ArOH can be estimated by calculating the bond

dissociation enthalpy (BDE) of O-H bond. The BDE of O-H bond for a phenolic

compound bond can be calculated using the Equation 7.5.

BDE = Hf (ArO•) + Hf (H•) – Hf (ArOH) (7.5)

where, Hf (ArO•) is the enthalpy of the radical generated via H-atom abstraction, Hf

(H•) is the enthalpy of the H atom and Hf (ArOH) is the enthalpy of the neutral

molecule.

The BDE is determined as the difference in the heat of formation between the

molecule and corresponding radical, and thus relates to the O-H bond breaking energy.

Thus, the BDE is an important property for evaluating antioxidant activity of a given

phenolic compound using the HAT mechanism. BDE evaluates the capacity of the OH

groups of phenolic antioxidants to react by H-transfer. It is established that the weaker

the O-H bond, the higher is the antioxidant activity. DFT study was performed to

understand the relation between structure and radical scavenging activity, the chemical

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properties responsible for scavenging activity and the mechanism of scavenging

activity15-18.

7.2 Materials and methods

7.2.1 Docking simulation

The x-ray crystal structure of falcipain 2 - E-64 (PDB ID: 3BPF) was retrieved

from the RCSB Protein Data Bank (http://www.rcsb.org/pdb/), and Chain A of the

protein determined at a resolution of 2.9 Å was used in the study19. Prior to docking,

protein was prepared using protein preparation wizard tool of Biovia Discovery

Studion (DS) v 4.5 software. Polar hydrogen atoms were added to the proteins and

charges were assigned. All bound water molecules, other heteroatoms and ligands

were excluded from the crystal structure as they were not significant for the protein’s

function. Subsequently, the 3D structure of protein molecule was optimized by energy

minimization using CHARMM Force Field. It was done in two steps to remove the

bad steric clashes using Steepest Descent (SD) and Conjugate Gradient (CG) methods

for 5,000 steps at RMS gradients of 0.01 and 0.05 kcal/mol/Å, respectively10,20.

Molecular docking was carried out using Biovia DS v 4.5 (2015) and Molegro

Virtual Docker (MVD) v 6.1 (2014) software. After energy minimization, the Chain A

of falcipain 2 protein was defined as a receptor and the binding site sphere was

selected based on the ligand binding location of E-64. Binding site was determined

from the previous knowledge19 of the original ligand binding site of E-64.

A receptor grid was then generated around the binding cavity (active sites) of

protein by specifying the key amino acid residues (Cys 42, Gly 83 and His 174). In

DS, binding site sphere (receptor grid) was set with a radius of 20 Å and x, y, z

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dimensions of -52.25, -4.46,
4.46, -19.25, respectively, while in MVD, the binding site

sphere was set of radius 20 Å with a grid box size of -52.45, -4.10, -17.49
17.49 in x, y, and

z dimensions, respectively.

Prior to docking,, the protein model (3D crystal structure of falcipain 2 co-
co

crystallized withh the inhibitor E

E-64)
64) used for docking study was validated by

redocking of E-64
64 ligand into the binding cavity of falcipain 2 molecule. The protein

model used for docking study was validated as follows: the 3D crystal structure of

falcipain 2 co-crystalliz
crystallized with the inhibitor E-64 [trans-epoxysuccinyl
epoxysuccinyl-L-

leucylamido-(4-guanidino)
guanidino) butane] with active site
te as defined by Cys 42, Gln 36 and

His 174 residues was optimized and used for the study. The co-crystal
co crystal structure of

falcipain 2 - E-64
64 and the receptor gri
grid model
el used for docking study are presented in

Figure 7.2.

Figure 7.2 (a): Optimized co-crystal

co crystal structure of falcipain 2 (Chain A)-E-64,
A)
(b): Receptor grid for docking

Co-crystallized
crystallized ligand, E
E-64 was re-docked
docked using flexible docking simulations

(LibDock programme of DS) into the original structure of the falcipain

falcipain-22 molecule by

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non-covalent docking procedure. For this study, docking parameters were set to the

software’s default values19,20.

Flexible molecular docking was performed where the protein was held rigid, while

the ligands were allowed to be flexible. Accordingly, a molecular docking study was

performed for all synthesized compounds 3a-e, 3ʹa-n, 3ʹʹa-e using the falcipain 2

enzyme as protein molecule. During docking, the falcipain 2 - E-64 complex was

imported and E-64 molecule (co-crystal ligand) was removed, and ligands were placed

in the predicted binding site (grid box) and docking was performed using the ‘Dock

Ligands’ module of LibDock and MolDock genetic algorithm programs of DS and

MVD, respectively. The docking parameters were as follows: no. of hotspots- 100,

docking tolerance- 0.25, no. of runs- 100, no. of iterations- 1000, conformation

method- best and RMS gradient- 0.01. All other docking and consequent scoring

parameters used were kept at their default settings10,21.

The LibDock and MolDock scores of the docked ligands were calculated. Different

dock poses were studied to know the best binding mode of receptor-ligand complex in

terms of scoring function. All docked poses were scored, ranked and the best pose of

(best minding mode) having the highest score for each compound was selected, and

the same docked pose was later used for the receptor-ligand interaction analysis. The

strength of interaction of a ligand with the receptor molecule was studied to know the

best binding orientation of receptor-ligand complex having maximum dock score.

Binding modes of the best pose for each compound was also analyzed with the help of

3D receptor-ligand complex. Different non-bonding interactions (hydrogen bonding

and hydrophobic) were also analyzed with the help of 2D diagram of docked receptor-

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ligand complexes. Analysis of receptor-ligand interaction gave insight into the

molecular interactions between the binding site residues of receptor molecule and

complimentary groups/atoms of ligands involved in interactions.

7.2.3 MOA

7.2.3.1 Radical scavenging activity

The in vitro antioxidant activity of the two most potent trioxane compounds, 3ʹl and

3ʹʹd was carried out by the following three assay methods in accordance with

previously reported procedures with minor modifications.

Superoxide radical scavenging activity: It was determined by the nitro blue

tetrazolium (NBT) reduction method22. In this assay, the non-enzymatic phenazine

methosulfate/nicotinamide adenine dinucleotide (PMS/NADH) system generates

superoxide radicals, which reduce NBT to a purple color formazan. The reaction

mixture contained phosphate buffer (0.5 ml, 100 mM, pH 7.4), 1.0 ml of NADH (0.4

mM), 1.0 ml of NBT (0.156 mM), 0.1 ml of PMS (0.06 mM) and 3 ml of the

compound/standard drug (quercetin) of various concentrations (10-50 µg/ml, in 90%

ethanol). After incubation at 25 °C for 1 h, the absorbance of the reaction mixture is

measured at 560 nm against an appropriate blank to determine the quantity of

formazan formed.

Hydroxyl radical scavenging activity: This assay was performed by the

determination of the thiobarbituric acid (TBA) reacting substance (TBARS) generation

method23. Hydroxyl radicals were generated by the Fenton reaction using

Fe3+/ascorbate/EDTA/H2O2 system. The hydroxyl radical generated in the system

attacks deoxyribose eventually resulted in the formation TBARS which was estimated.

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The reaction mixture contained 0.1 ml of 2-deoxy-2-ribose (10 mM), 0.30 ml of

phosphate buffer (50 mM, pH 7.4), 0.1 ml of FeCl3 (0.1 mM), 0.1 ml ethylenediamine

tetra-acetic acid (EDTA) (0.1 mM), 0.1 ml of H2O2 (2 mM), 0.1 ml of ascorbic acid (1

mM) and 1.0 ml of various concentrations (10-50 µg/ml) of the test or standard

compound (quercetin). After incubation for 45 min at 37 oC, 1.0 ml of 2.8% (v/v)

TCA, and 1.0 ml of 0.5% (v/v) TBA in 0.025 mol/L NaOH solution containing 0.2%

(w/v) of butylated hydroxyl anisole (BHA) was added in the reaction mixture, and the

mixture was incubated at 95 °C for 15 min to develop the pink chromogen. After

cooling, the absorbance was measured at 532 nm against an appropriate blank

solution.

Lipid peroxidation scavenging activity: The lipid peroxide radical scavenging

activity was determined by the TBARS generation method24. The Fe3+/ascorbic acid

dependent non-enzymatic lipid peroxidation in the liver extract was performed as

follows. Reaction mixture (0.5 ml) containing rat liver homogenate (0.1 ml, 25% w/v)

in Tris-HCl buffer (40 mM, pH 7.0), KCl (30 mM), FeCl3 (0.15 mM) and ascorbic

acid (0.06 mM) was incubated for 1 h at 37 oC in the presence and absence of the test

compound/standard drug (quercetin) at various concentrations (10-50 µg/ml). The

lipid peroxide formed was measured by TBARS formation. For this 0.4 ml of

incubation mixture was treated with sodium dodecyl sulphate (SDS, 8.1%, 0.2 ml),

TBA (0.8%, 1.5 ml) and acetic acid (20%, 1.5 ml, pH 3.5). The total volume was then

made upto 4.0 ml by adding distilled water and kept in a water bath at 100 oC for 1 h.

After cooling, 1 ml of distilled water and 5.0 ml of a mixture of n-butanol and pyridine

(10:1 v/v) were added to the reaction mixture, shaken vigorously and centrifuged at

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4000 rpm for 10 min. The butanol-pyridine layer was removed and its absorbance at

532 nm was measured to quantify TBARS.

In all the above three methods, the compound/standard drug (quercetin) was tested

at the concentration range of 10-50 µg/ml and the percent inhibition of scavenging

activity was calculated using the following equation (Equation 7.4).

% ℎ = 100 (7.4)

where, Acontrol is the absorbance of the control and Atest represents the absorbance

of a test (compound/standard drug).

Values were obtained as mean ± SEM of three triplicate studies. Results were

evaluated by comparing the percent inhibition of activity of the test compound with

that of the standard drug (quercetin).

6.2.3.2 DFT study

In DFT, the scavenging activity of trioxane compounds, 3ʹl and 3ʹʹd was

investigated by HAT mechanism, the O-H/N-H BDE, electronic energy parameters

(HUMO and LUMO energies), and atomic charges were computed in the gas phase as

well as in solvent phase (water and ethanol). The geometry structure, radical and

electronic character were analyzed to explore the key factors that influence the radical

scavenging activity of the trioxane compounds. Single-point energy (SPE) calculations

were performed (after optimization of geometries of trioxane compounds and their

radical structures) using the 6-311++G(d,p) basis set at B3LYP (hybrid functional)

level, according to quantum chemical calculations25,26. The DFT calculations were

performed on an Intel (R), Pentium (R) Dual Core personal computer using the

Gaussian-03W program package without any constraint on the geometry.

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7.3 Results and discussion

7.3.1 Docking simulation

In our study, Plasmodium cysteine protease falcipain 2 protein was used as possible

antimalarial drug target for the newly reported 1,2,4-trioxane derivatives. Falcipain 2

enzyme plays an essential role in the degradation of host hemoglobin by P. falciparum

in malaria infection. Recent literature27 claims that endoperoxide antimalarials have

potential to inhibit falcipain 2 enzyme and therefore, drug targeting of this enzyme

would be an attractive avenue in the design and development of new antimalarial

drugs. Accordingly, a molecular docking study was performed to confirm the

antimalarial efficacy of 1,2,4-trioxane derivatives as possible falcipain 2 inhibitors.

Docking rationalizes finding novel bioactive molecules and also gives an insight into

SARs and mode of action based on scoring function,n and further analysis of binding

modes from protein-ligand interaction studies28. Docking simulation may therefore be

useful in identifying the bioactive binding orientation of trioxane compounds in the

active site of falcipain 2 protein for exploration of their mode of antimalarial action.

Co-crystal-ligand, E-64 was successfully re-docked to the predicted active sites of

falcipain 2 with an acceptable RMSD value of 1.124 Å. Further, in order to reproduce

an experimentally observed ligand-binding mode, the co-crystallized ligand, E-64 (a

selective falcipain 2 inhibitor) was used as reference ligand. Results confirmed

experimental binding conformations of E-64 in the binding pocket of receptor

molecule. The binding modes and protein-ligand interaction diagrams obtained in re-

docking of E-64 are given in Appendix 1.

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 Docking results (Table 7.1) revealed that LibDock program successfully docked all

1,2,4-trioxane derivatives into the binding pocket of falcipain 2 enzyme.

Table 7.1: Dock score and no. of hydrogen bonds

Comp. code LibDock score* MolDock score* No. of H-bond(s)

3a 93.077 -57.350 3
3b 96.153 -61.891 3
3c 101.69 -62.882 3
3d 111.343 -67.284 3
3e 108.143 -66.320 5
3ʹa 107.807 -67.793 4
3ʹb 126.390 -77.898 6
3ʹc 116.533 -74.744 4
3ʹd 110.908 -67.891 3
3ʹe 127.665 -73.369 4
3ʹf 118.391 -68.136 5
3ʹg 117.436 -68.127 2
3ʹh 110.983 -66.972 3
3ʹi 120.229 -71.189 3
3ʹj 118.985 -68.362 4
3ʹk 120.370 -76.479 1
3ʹl 128.609 -79.234 3
3ʹm 100.364 -72.298 1
3ʹn 122.864 -73.605 4
3ʹʹa 101.911 -72.298 3
3ʹʹb 105.097 -73.605 5
3ʹʹc 110.952 -73.605 6
3ʹʹd 124.747 -81.474 5
3ʹʹe 112.646 -72.063 6
E-64 104.364 -101.103 7
*
against P. falciparum cysteine protease falcipain-2 (PDB: 3BPF)
All trioxane compounds that were active in in vitro antimalarial screening could

bind with the active sites of falcipain-2 with high binding affinities. The Libdock and

MolDock scores were obtained in the range between 96.077 and 126.390, and -57.350

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and -81.474, respectively. Except compounds, 3a, 3b, 3c, 3ʹm and 3ʹʹa, all trioxane

derivatives exhibited more binding affinity (higher dock scores) than the co-crystal

ligand, E-64 (Libdock score = 100.364). On the other hand, the MolDock scores of

trioxane compounds were less than that of the co-crystal ligand, E-64 (MolDock score

= -101.103). Dock scores supports the results of in vitro antmalarial activity for the

trioxane compounds.

It is important to note here that docking results are generally analyzed by a

statistical scoring function which converts interacting energy (binding energy) into

numerical values called as the docking score, and therefore docking score is nothing

but an expression of binding affinity. Binding energy (ΔG) includes the sum of all

non-bonded interactions including hydrogen bonding, hydrophobic and Van der Walls

between protein residues and the bound ligand3,8.

Five compounds that were found most active in in vitro antimalarial screening also

ranked top in docking simulation with LibDock scores of 126.390, 116.533, 128.609,

124.747 and 112.646, and Moldock scores of -77.898, -74.744, -79.234, -81.474 and -

72.063 for 3ʹb, 3ʹc, 3ʹl, 3ʹʹd and 3ʹʹe, respectively. Dock scores of five most

compounds along with the no. of hydrogen bonds and their in vitro antimalarial

activity data are listed in Table 7.2.

Protein-ligand docking was performed to generate the bioactive binding poses of

designed inhibitors in the active site of falcipain 2 enzyme. LibDock score is useful to

assess the antimalarial activity of ligands as potential falcipain 2 inhibitors. MolDock

score obtained from a different algorithm validates the LibDock score. These two dock

scores are consistent with each other supporting the experimental activity of

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molecules. LibDock is a high throughput docking algorithm that finds various

conformations of the ligands in the protein active site based on polar interaction sites

(hotspots). The docking scoring function of MolDock is an extension of the piecewise

linear potential (PLP) including new hydrogen bonding and electrostatic terms 5,21.

Table 7.2: Dock score, no. of hydrogen bonds and in vitro antimalarial activity for five
potent compounds

Comp. Dock score No. of H-bonds In vitro IC50 (µM)

code
LibDock MolDock Pf 3D7 Pf RKL9

3ʹb 126.390 -77.898 6 5.43 5.68

3ʹc 116.533 -74.744 4 7.62 7.83

3ʹl 128.609 -79.234 3 1.24 1.06

3ʹʹd 124.747 -81.474 5 1.24 1.17

3ʹʹe 112.646 -72.063 6 7.68 6.52

The 3D poses of bound ligands were visualized using visualization tool of DS

which revealed the best orientation of ligands relative to the receptor as well as the

best fit conformation of the ligand with the receptor molecule. Analysis of 2D diagram

showed various non-bonded interactions mainly polar hydrogen bonding between

binding site residues (active site amino acids) and ligand moieties/atoms. Apart from

hydrogen bonding interactions other non-bonded interactions like hydrophobic

bonding were also observed to a limited extent.

Table 7.3 reveals hydrogen bonding interactions of five most potent compounds

(3ʹb, 3ʹc, 3ʹl, 3ʹʹd & 3ʹʹe). Higher the number of hydrogen bonds, higher is the binding

affinity. Compounds interacted with the active site residues (such as Asp 154, Arg 12,

Gln 36, Glu 14, Glu 15, Glu 138, Gly 13, Val 152 and His 19) of falcipain-2 through

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hydrogen bond formation. The 3D binding modes and 2D interaction diagrams of three

most potent compounds, 3ʹb, 3ʹl and 3ʹʹd are given in Appendix 1. The compound 3ʹb

formed six hydrogen bonds with amino acid residues like Asp 154 (O··H··N), His 19

(O··H··O), Arg 12 (O··H··O), Arg 12 (O··H··O), Gly 13 (O··H··O) and Val 154

(O··H··O) with bonding distances of 2.749, 2.811, 1.720, 2.393. 2.488 and 2.262 Å,

respectively. The second compound, 3ʹl formed three strong hydrogen bonds with

residues like Gln 36 (O··H··O), Asp 154 (O··H··N) and Arg 12 (O··H··O) with

bonding distances of 2.737, 2.985 and 1.834 Å, respectively. In case of third one, 3ʹʹd,

five bonds were observed with residues like Arg 12 (O··H··O), His 19 (O··H··H), Gly

13 (O··H··O), Glu 14 (O··H··O) and Glu 15 (O··H··O) with bonding distances of

1.868, 2.938, 2.308, 2.795 and 2.489 Å, respectively. The co-crystal ligand, E-64

formed 7 hydrogen bonds with amino acid residues like Gly 83 (O··H··O), Gln 171

(O··H··O), Leu 172 (O··H··O), Lys 37 (O··H··O), Cys 39 (2 O··H··O) and Gly 82

(O··H··O) with bonding distances of 1.894, 2.147, 2.991, 2.076, 2.564/2.764 and

2.425, respectively. Analysis of best docking poses of 3ʹb, 3ʹl and 3ʹʹd reveal that the

trioxane scaffold was oriented in the binding cavity (active site residues) of falcipain 2

receptor molecule. In 2D diagram, the trioxane moiety could occupy the binding sites

of falcipain-2 through strong H-bonding interactions. Such interactions afforded good

stability of receptor-ligand complexes. Trioxane ring interacted with multiple amino

acid residues such as Arg 12, Asp 154, Val 12, Glu 138 and Gly 13, whereas,

Arg12/Val152, Gln 36 and Glu 14 residues were found to interact with the 2-

hydroxyphenyl, 4-hydroxy-3-methoxyphenyl and idol-3-yl moieties of compounds

3ʹb, 3ʹl and 3ʹʹd, respectively.

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 Table 7.3: Details of hydrogen bonding for five most active compounds

Comp. H- H-binding ligand H-binding receptor H-bond

bond(s) distance (Å)
Element Type Residue Element Type
3ʹb 6 O A Asp 154 N D 2.749
O A His 19 H D 2.811
O A Arg 12 H D 1.720
O A Arg 12 H D 2.393
H D Gly 13 O A 2.488
H D Val 152 O A 2.262
3ʹc 4 O A Asp 154 N D 2.234
O A His 19 H D 2.534
H D Asp 154 O A 2.874
H D Glu 138 O A 3.016
3ʹl 3 O A Gln 36 H D 2.737
O A Asp 154 N D 2.985
O A Arg 12 H D 1.834
3ʹʹd 5 O A Arg 12 H D 1.868
O A His 19 H D 2.938
H D Gly 13 O A 2.308
H D Glu 15 O A 2.795
H D Glu 14 O A 2.489
3ʹʹe 6 O A Arg 12 H D 2.976
O A Arg 12 H D 2.107
H D Glu 14 O A 2.804
O A Asp 154 H D 2.515
H D Gly 13 O A 2.247
H D Glu 14 O A 2.735
E-64 7 O A Gly83 H A 1.894
O A Gln171 H A 2.147
H D Leu172 O A 2.991
H D Lys37 O A 2.076
H D Cys39 O A 2.564
H D Cys39 O A 2.764
O D Gly82 O A 2.425

197
 Strong H-bonding interactions between C=O group of the trioxane and amino acid

residues were also observed. The OH and NH groups were involved for H-bonding

interactions from the substituent moieties for compounds 3ʹb, 3ʹl and 3ʹʹd,

respectively. Further, upon analysis of docking interactions, it was found that trioxane

ring played a crucial role in protein-ligand binding. Substituents increased binding

strength by forming additional H-bonds that in turn facilitated much stronger

interaction of ligands with the receptor (falcipain-2 protein) molecule.

7.3.2 Radical scavenging activity

The trioxane compounds, 3ʹl and 3ʹʹd showed good in vitro radical scavenging

activity. The radical scavenging activity was found to be in a concentration dependant

manner. Compounds, 3ʹl and 3ʹʹd exhibited 78.34 ± 0.56% and 62.10 ± 0.36% of

superoxide radical scavenging effect, respectively at the highest concentration i.e., 50

µg/ml. The percent inhibition of hydroxyl radicals were 84.72 ± 0.27% and 75.64 ±

0.47% for the compound 3ʹl and compound 3ʹʹd, respectively at 50 µg/ml. A

considerable amount of lipid peroxidation inhibitory effect was also observed by 70.13

± 0.18% and 52.62 ± 0.13%, for compounds 3ʹl and 3ʹʹd, respectively. Quercetin

showed 82.38±0.54%, 88.40 ± 0.22% and 72.12 ± 0.12% inhibition of superoxide,

hydroxyl and lipid peroxidation scavenging activities, respectively at the concentration

of 50 µg/ml. Results are depicted in Figure 7.3, Figure 7.4 and Figure 7.5.

198
 100

80

60

% Inhibition
3'l
40
3''d
20 Quercetin

0
0 20 40 60
Conc. (µg/ml)

Figure 7.3: Superoxide radical scavenging activity

80
70
60
50
% Inhibition

40 3'l
30 3''d
20
Quercetin
10
0
0 20 40 60
Conc. (µg/ml)

Figure 7.4: Hydroxyl radical scavenging activity

80
70
60
50
% Inhibition

40 3'l
30 3''d
20
10 Quercetin
0
0 20 40 60
Conc. (µg/ml)

Figure 7.5: Lipid peroxidation radical scavenging activity

199
 The scavenging activity (%inhibition) produced by the compound 3ʹl is comparable

to the standard drug, quercetin. Results indicate potent antioxidant activity of the

trioxane compound, 3ʹl as compared to the other compound, 3ʹʹd, which might be due

to the phenolic nature of the compound.

6.3.3 DFT study

The optimized structures of most stable conformers of trioxane compounds, 3ʹl and

3ʹʹd used for quantum chemical calculations in DFT study are given in Figure 7.6
7.6.

OH NH
H H
O O O O
O
O O
O O

(a) (b)

Figure 7.6: Optimized structure of compound 3ʹl (a) and compound 3ʹʹd (b)
(b),
in gas phase

In DFT study, HAT mechanism was employed for assessing the radical scavenging

activity of the trioxane compounds. HAT is the most favored mechanism for

explaining the radical scavenging activity of phenolic antioxidant molecules in the gas

phase. In HAT, the reactivity of an ArOH can be estimated by calculating the O-H
O

BDE, where the lower the BDE value, the higher the expected activity15,16. The

200
calculated BDE depicted in Table 7.4 indicates high reactivity of the trioxane

compounds in scavenging free radicals. BDE values of both the test compounds are

consistent with the experimental scavenging activity and are comparable to that of the

standard compound, quercetin. BDE in water is most relevant for the biological

antioxidant activity as compared to non-polar and polar solvents. Both the trioxane

compounds exhibited high BDEs in water indicating their high level of antioxidant

activity. The influence of solvent was relatively weak on the BDEs, indicating that

similar reactivity of trioxane compounds in both polar solution and non-polar medium.

This finding implies that the trioxane compounds have both polar and non-polar

behavior in balance which is required for the antioxidant activity in vivo.

Table 7.4: Calculated BDE values (in kcalmol-1) of trioxane compounds

Comp. code BDE in gas BDE in water BDE in ethanol

3ʹl 76.6227 78.5850 78.4620

3ʹʹd 89.4792 91.7294 91.6604

BDE of quercetin (standard compound): 65.8-89.7 kcalmol -1 (in gas phase)

The atomic charges are very much dependent on how the atoms are defined. It also

plays an important role in the application of quantum chemical calculations to

molecular systems. The Mulliken and and Hirshfeld atomic charges (in a.u.) of

significant atoms are presented in Table 7.5. For compound 3ʹl, O11 (O-H group) has

more negative charge (-0.24548) and O10 (O-CH3 group) has little less negative

charge charge (-0.14864). On the other hand, for compound 3ʹʹd, N14 (N-H group) has

still less negative charge (-0.09234).

201
 Table 7.5: Mulliken charge (a.u.) and Hirshfeld charge (a.u.) of significant atoms

Comp. Gas (a.u.) Water (a.u.) Ethanol (a.u.)

code
Mulliken Hirshfeld Mulliken Hirshfeld Mulliken Hirshfeld
Charge Charge Charge Charge Charge Charge

O11 -0.24548 -0.18325 -0.28232 -0.19108 -0.28048 -0.19049

(O-H)

O10 -0.14864 -0.15993 -0.19153 -0.18041 -0.18912 -0.17926

(O-CH3)

N14 -0.09234 -0.08808 -0.07759 -0.07179 -0.07775 -0.07254

(N-H)

Results of HOMO and LUMO (Frontier Molecular Orbitals) energies (Table 7.6)

revealed the electronic property of the trioxane compounds. HOMO and LUMO play

an important role in determining the electronic property as well as molecular reactivity

of compounds. Electronic property further supports the radical scavenging activity of

trioxane compounds. The dipole moment was found to be 4.069 and 3.504 debye for

compounds 3ʹl and 3ʹʹd, respectively. High dipole moment also confirms the

electronic property (polar behavior) of molecules. The HOMO can act as an electron

donor, and the LUMO can act as an electron acceptor. A molecule with higher energy

of the HOMO orbital has stronger electron donating ability. A low value of HOMO

energy can be correlated to a low electron-donating ability, while a high value of

HOMO energy correlates with good electron-donor ability to empty orbitals of suitable

energy in an acceptor.

202
 Table 7.6: HOMO and LUMO energies (eV) of trioxane compounds

Gas (eV) Water (eV) Ethanol (eV)

Comp. code HOMO LUMO HOMO LUMO HOMO LUMO

3ʹl -6.53908 -2.25416 -6.61880 -2.33334 -6.61037 -2.33117

3ʹʹd -6.00628 -2.25470 -6.14642 -2.32001 -6.13907 -2.31674

While describing the radical scavenging activities of phenolic antioxidants,

parameters such as the energy and distribution of the HOMO orbital play an important

role16,29-32. Based on our calculations, compound 3ʹl provided more HOMO energy (-

6.53908 eV) than the other compound, 3ʹʹd (-6.00628 eV) in gas phase. This clearly

confirms that compound 3ʹl has the strongest electron-donating capability. LUMO

energies of these compounds were found to be -2.25416 and -2.25470 eV (in gas

phase), respectively. It was observed that the HOMO is centered about the phenolic

oxygen (O11 of compound 3ʹl) or the amino nitrogen (N14 of compound 3ʹʹd), while

LUMO is centered about the carbonyl carbon atom (C 32 of compound 3ʹl and C 31 of

compound 3ʹʹd). The HOMO-LUMO gaps (in gas phase) were found to be 4.28492

and 3.75158 eV for compounds 3ʹl and 3ʹʹd, respectively (Figure 7.7).

Phenolic hydroxyl (OH) or aryl amino (NH) group donates a hydrogen atom (as H +)

and thereby react with reactive oxygen or nitrogen species (ROS/RNS), which

subsequently stops the cyclic process of generation of free radicals. Thus, trioxane

compounds can act as antioxidant molecules through the formation of negatively

charged radical species stabilized by delocalization of the electron with the aromatic

ring.

203
 LUMO

3.75158 eV

HOMO

(a) (b)

Figure 7.7: HOMO-LUMO energy levels of (a) compound 3ʹl and (b) compound 3ʹʹd,
in gas phase

The antioxidant activity of the phenolic trioxane compound, 3ʹl and the other

heteroaryl amine i.e., compound¸ 3ʹʹd is attributed to be due to the above fact. In

biological system, the radical scavenging activity might also be brought about due to

their ability to chelate metal ions involved in the production of free radicals15. Finally,

the findings of DFT study suggest that being potential nucleophilic sites the phenolic

OH group of compound 3ʹl and indolyl NH group of compound 3ʹʹd are potential can

play an important role in scavenging free radicals (induced in oxidative stress) by

hydrogen abstraction or chelation of metal ions.

7.3.4 MOA

Based upon docking and radical scavenging activity (in vitro and DFT) studies, the

possible mode(s) of antimalarial action of the trioxane compounds can be suggested as

follows:

204
 Trioxane compounds may act either as a direct inhibitor of falcipain 2 receptor or as

a peroxide prodrug by interfering the process of haemoglobin degradation at

erythrocytic stages of P. falciparum. Electronic property of the substituent may play a

significant role for the bio-activation of trioxane molecule in generating highly

reactive oxyl radical (toxic radical species) through O-O cleavage, which subsequently

exerts lethal action to parasites by lipid peroxidation of parasitic cell membrane

(Figure 7.8).

Hb Heme-Fe(II)
Pf HDE

H O-O H
(1) O O R O O R (2)
Inhibitory cleavage Oxidative damage
activity O to parasite P/L
E/R O O LPO
inhibition CH3 O Fe(III)
CH3
Trioxane peroxide Oxyl radical

Figure 7.8: Proposed mode(s) of action of trioxane derivatives

(1): Inhibition of haemoglobin degrading enzyme (by inhibiting P. falciparum falcipain 2 receptor, as
falcipain 2 inhibitor), (2): Direct lethal action to parasite by LPO of biomolecules as peroxide prodrug.
(Hb- Haemoglobin, Pf HDE- P. falciparum haemoglobin degrading enzyme, LPO- Lipid peroxidation,
E/R- Enzyme/Receptor, P/L-Protein/Lipid)

Further, in view of having good radical scavenging activity, the antimalarial action

for the phenolic trioxane compound, 3ʹl is proposed. Being phenolic compound it

gives up a proton and forms stable phenoxide radical under cellular oxidative stress,

thereby scavenges reactive free radicals or reactive oxygen species in

pathophysiologic (inflammatory) conditions in malaria complications like cerebral

malaria. Another alternative mode of action proposed is flavonoid-like antimalarial

action where the trioxane phenolic compound (compound 3ʹl) acts as iron chelator

through radical scavenging mechanism (Figure 7.9) in hemoglobin degradation

pathway33,34. It is therefore suggested that as an iron chelator the trioxane phenolic

205
compound may be used as drug synergist in combination with artemisinin-based drugs

(in ACTs) for the treatment of resistant malaria. As phenolic antioxidant molecule, the

trioxane compound the compound may serve as ART synergist by reacting with heme

iron and converting Fe+3 to Fe+2, the latter being important for the bioactivity of ART,

leading to the release of toxic free radicals that destroys malaria parasites by alkylation

or lipid peroxidation of parasite biomolecules (proteins/lipids)35,36.

OFe(II) OH

Phenol iron +e- Phenolic trioxane

chelate
Fe+2 Fe+3

O - e- OFe(III)
CH3
O O CH3
O
O

ART-based drug Oxyl radical

Figure 7.9: Flavonoid-like antimalarial action of trioxane compound

7.4 Conclusion

Docking simulation validates the antimalarial activity of 1,2,4-trioxane derivatives

against P. falciparum. Docking study also supports the antimalarial potential of

trioxane molecules as novel falcipain 2 inhibitors. Along with virtual screening for

drug-likeness, this study would help for rational design of newer trioxane

molecules based on the target molecule to identify novel antimalarial inhibitors

206
with high affinity and specificity. Results of radical scavenging activity imply the

role of trioxane compounds in the reduction of oxidative stress associated with

inflammatory malaria conditions. DFT study confirms that the trioxane compounds

possess significant antioxidant potential. In view of antimalarial potential with good

target specificity and antioxidant activity, the trioxane compounds (particularly,

compounds with 4-hydroxy-3-methoxyphenyl, 3-indolyl and 2-hydroxyphenyl

substitutions) may act against malaria parasites as erythrocytic schizonticide either by

direct inhibition of P. falciparum falcipain 2 enzyme or by oxidative damage to

parasite cells through formation of toxic radical species. Another alternative

mechanism that the phenolic trioxane compound might possess is flavonoid-like

antimalarial action due to the radical scavenging activity as metal ion chelator. The

radical scavenging property of trioxane compounds is considered to have beneficial

effect in the treatment of malaria complications and also in resistant malaria, in the

form of single-drug or multi-drug therapy.

207
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