Microbial Pathogenesis 118 (2018) 365–377

Contents lists available at ScienceDirect

Microbial Pathogenesis
journal homepage: www.elsevier.com/locate/micpath

Identification of inhibitor against H. pylori HtrA protease using structure- T

based virtual screening and molecular dynamics simulations approaches
Nivedita Rai, R. Muthukumaran, R. Amutha∗
Centre for Bioinformatics, Pondicherry University, Pondicherry, 605014, India

A R T I C LE I N FO A B S T R A C T

Keywords: The HtrA protease of Helicobacter pylori, which efficiently colonizes at the gastric epithelial of host cells, disrupts
HpHtrA the mucosal integrity of E-cadherin and spreads inflammatory diseases including gastric cancer by cleaving the
Homology modeling cell-cell adhesion of the host. The lack of knowledge on the molecular diversity, structural and functional be-
Molecular dynamics havior of HpHtrA necessitated the present study to explore its inhibition mechanism. At first, the similarity of
Virtual screening
HpHtrA with other gastro-intestinal pathogenic HtrA bacteria and its remote relationship with the Human HtrA
Binding energy
Hydroxyl-piperidine
homologs were ensured by the phylogenetic analysis and hence was identified as a novel therapeutic target for
4-aminopiperidine scaffold further design of inhibitors. The three dimensional structure of HpHtrA was modeled and simulated to achieve
its stable conformation and was used as a receptor to screen for the possible lead compound through virtual
screening (using ∼ 1.3 million compounds). Molecular dynamics simulations followed by the binding energy
analysis revealed the affinity of the compound 300040 in forming a stable complex with HpHtrA and thereby
revealed its potent role in inhibiting HpHtrA. It is also worthy to mention that, structurally, the ligand binding at
the catalytic site of HpHtrA is mainly facilitated by the significant dynamics of L2 loop. Based on the present
study, the hydroxyl-piperidine with 4-aminopiperidine scaffold is proposed to be one of the best possible lead
compounds for the inhibition of H. pylori.

1. Introduction
The beneficial bacterial flora influences the anatomy, physiology of
the host and also provides protection against pathogenic susceptibility.
Whereas, the pathogenic bacteria possesses inherent ability to breach
host defense mechanism and cause infection in specific area of the body
like skin, urinary tract, gastroenteritis, etc. [1,2]. The bacterial gas-
troenteritis, a gastrointestinal infection, is caused by Escherichia coli
pathogenic strains, Shigella dysenteriae, Salmonella typhimurium, Yersinia
pseudotuberculosis, Vibrio cholera, Clostridium perfringens, Bacillus cereus,
Staphylococcus aureus, Campylobacter jejuni, and Helicobacter pylori, etc.
According to World Health Organization (WHO), the H. pylori is rated
as the class-I carcinogen causing gastric cancer [5–8]. The H. pylori
efficiently colonizes in the gastric epithelial of host cells for more than a
decade, and disrupts the mucosal integrity and hence involves in sev-
eral inflammatory diseases such as chronic gastritis, ulceration of gas-
tric and duodenum, gastric malignancies and gastric cancer [3,4]. The
antibiotic resistant nature of H. pylori encourages finding novel in-
hibition mechanisms that control disease progression. Hence, it is very
important to identify the novel targets in H. pylori.
The host epithelial surface is the first protective barrier against most
of the toxins and pathogens. Similarly, in gastric epithelial cells, the E-
cadherin plays a very crucial role in cell-cell adhesion, and also pre-
vents bacterial invasions and metastasis of many tumor cells [5,6]. The
E-cadherin is considered as an attractive target by the bacterial or-
ganism like H. pylori which colonizes in the gastric epithelial lining and
involves in the metastasis of gastric cancer-related diseases. Recent
reports revealed that High-temperature requirement A (HtrA, a secre-
tary protease) [7,8] preferentially recognizes and cleaves the signature
motif, containing the [VITA]-[VITA]-x-x-D-[DN] consensus sequence of
E-cadherin, in which ‘x’ could be any amino acids [9]. Thus HtrA is
crucial for the pathogenic invasiveness of the host cells. The inhibition
of HtrA-mediated cleavage of E-cadherin significantly reduces the mi-
gration of H. pylori infections [10]. HtrA protease and their homologs
are evolutionarily conserved in all living organisms. E.coli contains
three differentially regulating HtrAs proteases such as DegP, DegQ, and
DegS. The DegP is well-characterized as a serine protease which de-
grades the unfolded protein in an ATP independent manner. It mainly
consists of a conserved N-terminal serine protease and two PDZ (Post-
synaptic density protein/Dlg1/Zo1) domains such as PDZ1 and PDZ2
[11]. Under various stress conditions, the bacterial cells would establish
an effective mechanism of protein quality control to monitor the folded

∗
Corresponding author.
E-mail addresses: amutha_ramu@yahoo.com, ramutha@bicpu.edu.in (R. Amutha).

https://doi.org/10.1016/j.micpath.2018.03.027
Received 16 October 2017; Received in revised form 21 January 2018; Accepted 15 March 2018
Available online 16 March 2018
0882-4010/ © 2018 Elsevier Ltd. All rights reserved.
N. Rai et al.
state of the proteins and support further protein folding, repair and
degradations in both cytoplasmic and periplasmic locations [12,13].
The HtrA protein is implicated as a virulence factor in several patho-
genic bacteria such as E. coli (Entero-pathogenic strain), S. typhimurium,
B. cereus, S. aureus, C. jejuni, H. pylori, etc [14,15]. In this study, the
HtrA from H. pylori is considered as a target for the inhibition of H.
pylori (and will be referred as HpHtrA throughout the text). The three-
dimensional crystal structure of HpHtrA remains to be solved.
In the drug discovery field, the High throughput screening (HTS)
method remains the most preferred choice by pharma industry, despite
having its drawbacks such as high cost, time consuming, and low spe-
cificity as well as sensitivity. Hence, an alternative in silico computa-
tional method named as Virtual screening (VS) has been developed for
faster screening process. In in silico drug design, small molecular in-
hibitors are identified through VS method [16]. The VS is a computa-
tional approach, in which a large collection of compounds is screened
against a particular target to identify a potential lead. For example,
rhodanine scaffold (a competitive inhibitor at low micromolar con-
centration) was identified by structure-based virtual screening method
for the inhibition of arylalkylamine N-acetyltransferase (AANAT) [17].
The Captopril (an anti-hypertensive) drug, which is a potent inhibitor of
Angiotensin-converting enzyme, was successfully identified by struc-
ture-based virtual screening. Similarly, Zanamivir is a neuraminidase
targeted anti-influenza drug designed by using the structure-based
virtual screening approach [18]. Similarly, some inhibitors are pro-
posed for inhibiting HpHtrA, but there is no “on-shelf” drug or vaccine
available so far that passed the clinical trials. Hoy, B. et al., have de-
signed inhibitor against HtrA (H. pylori HtrA inhibitor, HHI) using
virtual screening combined with comparative homology modeling [8].
In 2015, Perna, A.M et al. used fragment based approach and identified
a lead compound with a higher binding affinity towards HpHtrA [19].
With the advantages of virtual screening, the present work also adopts
virtual screening approach to identify lead compound against HpHtrA
(Helicobacter pylori secreted HtrA) inhibition. Even though HtrA pro-
tease is mostly conserved among all living organisms (particularly in
bacteria), the HpHtrA protease is yet targeted by considering the facts
such as: (i) accessibility in the extracellular region of the host epithelial
cell, (ii) poor homology (∼33% sequence identity) at protein level with
the human homolog of HtrA (like HtrA1, HtrA2, HtrA3, and HtrA4) and
also (iii) the different site specificity due to the active site polarity and
sub-pocket shape of human HtrA which differs with HpHtrA [8]. Hence
the HpHtrA showing no cross-reactivity with the human HtrA, is con-
sidered as a novel therapeutic target [20].
2. Materials and methods
2.1. Structure prediction of HpHtrA
2.1.1. Sequence, alignment and phylogenetic analysis of HpHtrA
The gastrointestinal bacterial HtrAs (such as E. coli strains, S. dys-
enteriae, S. typhimurium, Y. pseudotuberculosis, V. cholera, C. perfringens,
B. cereus, S. aureus, C. jejuni, and H. pylori) and human homologs of HtrA
sequences (HtrA1, HtrA2, HtrA3 and HtrA4) were retrieved from
UniProt. The conserved regions among HtrA of the selected pathogenic
bacteria are identified through multiple sequence alignment (MSA).
The MSA was performed by ClustalW [21] algorithm using the default
parameters. The evolutionary analysis was performed by using MEGA7
tool [22] to identify the evolutionary relationship of HpHtrA with other
gastrointestinal pathogenic bacteria as well as human HtrA homologs
(Fig. 1). The phylogenetic tree was constructed for these selected se-
quences using maximum likelihood statistical method based on JTT
(Jones-Taylor-Thornton) matrix [23]. The phylogenetic tree topology
was obtained by Neighbor-joining algorithm [24]. In tree topology, all
positions containing gaps or missing data were completely eliminated.
Overall 315 positions were found in the final dataset. The initial tree
was obtained automatically by applying default Neighbor-Join and
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Microbial Pathogenesis 118 (2018) 365–377
Fig. 1. Phylogenetic tree diagram of Helicobacter pylori HtrA (HpHtrA).
BioNJ algorithms to a pairwise distance estimation matrix.
2.1.2. Homology modeling and validation of HpHtrA
The three-dimensional structure of HpHtrA is not yet solved and
hence the homology modeling approach is employed to generate
structure of HpHtrA. The template for homology modeling was identi-
fied by performing BLAST (blastp) for HpHtrA protein sequence (target)
against protein data bank (PDB) with default parameters [25]. The
sequence (UniProt ID: G2J5T2) showing highest similarity with the
target is selected as template for homology modeling using Modeller
9v11 software [26]. The alignment of both target and template se-
quences generated about ten optimized structures. From the generated
structures, the most reliable model was screened based on the least
Discrete Optimized Protein Energy (DOPE) score. The modeled struc-
ture of HpHtrA was validated using SAVES server [27] in which the
stereochemical properties as well as the model quality were evaluated
by PROCHECK and ERRAT programs, respectively. The significance of
consistency between modeled and template HtrA was evaluated using
ProSA (Protein Structure Analysis) server [28]. Further, the secondary
structure information of HpHtrA was predicted from the aligned se-
quences using ESPript (Easy Sequencing in PostScript) server [29].
2.2. Screening of novel inhibitors against HpHtrA
2.2.1. Receptor preparation
Modeled structure of the receptor, HpHtrA was simulated for a
period of 50 ns and the stable structure corresponding to the least en-
ergy was selected for further virtual screening studies. The receptor
structure was prepared using the protein preparation wizard of Maestro
v9.2, Schrödinger, in which the structure of HpHtrA was corrected to
have appropriate bond orders. Sufficient hydrogen atoms were added to
reproduce the pH of 7.0. Finally, the structure was minimized using the
OPLS-2005 force field to void off the steric clashes.
2.2.1.1. Binding site identification and receptor grid
generation. Identification of hot spots is the most important step in
the design of inhibitor against protein-ligand interactions. Here, the
binding site of HpHtrA was predicted using FTMap server [30].The
missing atoms in the structure/receptor were added prior to the
calculation of Poisson–Boltzmann potential. To identify the hot spot,
the receptor is assumed to be wrapped by a dense grid over its surface
and small molecular probes such as organic solvents are allowed to
generate bound poses as much as possible within the receptor surface.
This is achieved using the rigid body docking with fast Fourier
transform correlation approach.
Each probe samples millions of docked poses and the generated
bound conformations were minimized using Chemistry at Harvard
Macromolecular Mechanics (CHARMM) potential. Finally, the cluster
N. Rai et al.
was ranked based on the average Boltzmann energy. The predicted
binding site residues are used by the receptor grid generation panel of
Glide module. A grid box of dimension 10 × 10 × 10 Å was generated
by fixing the predicted binding site residues at the centroid of the grid
box. The non-polar part of the receptor atom is scaled using the van der
Waals radii of 1.0 Å and a partial charge cutoff value of 0.25 Å.
2.2.1.2. Ligand preparation and virtual screening. The drug-like small
molecule library of ∼1.3 million compounds was prepared by
retrieving compounds from various databases such as Specs, Zinc
(clear Drug-Like), Asinex (BioDesign and Antibacterial), and ChemDiv
(peptidomimetic). The LigPrep module implemented in Schrödinger
package was used for ligand preparation. The LigPrep module will filter
the ligands based on several descriptors such as force field,
stereochemical information, ionization states, tautomer, low energy
ring conformations, and also by retaining their native chirality. The
ionization states at the physiological pH (7.0 ± 2.0) were set for all
ligands using Epik state penalty. The structure of filtered ligands was
minimized using OPLS-2005 force field.
After completing both receptor and ligand preparations and grid
generation, the ligands were screened using the Glide docking program
implemented in Maestro v9.2 suite [31]. For docking, a semi-flexible
docking method, which applies some flexibilities to the bonds in the
ligand, was adopted. The ligands were systematically screened based on
a hierarchal screening approach to find the best possible binding poses
in the receptor grid space. Based on scoring function and sampling
criteria, a three-stage filter process using (i) High Throughput Virtual
Screening (HTVS), (ii) Standard Precision (SP), and (iii) Extra precision
(XP) with 50, 30 and 10% of cutoff criteria has been used for docking.
The ring conformers and nitrogen inversions were sampled during
flexible docking. The best poses were generated by Glide XP and ranked
according to both Glide score and energy values [32]. A set of top-
ranked three ligands was subjected to molecular dynamics simulations
for a period of 50 ns, to study the behavior of HpHtrA protein in the
presence of inhibitor.
2.2.2. ADME properties
In-silico study of the absorption, distribution, metabolism and ex-
cretion (ADME) properties of all selected compounds were predicted
using QikProp Version3.4 module. Qikprop provides a rapid prediction
of ADME properties for drug candidate molecules. For each successfully
processed molecule, the Qikprop predicts the pharmaceutically relevant
descriptors. It evaluates the acceptability of the filtered compounds
based on Lipinski’ rule of 5, log IC50, logP values, etc. [33].
2.3. Molecular dynamics simulations
At first, the modeled structure of HpHtrA was subjected to mole-
cular dynamics simulations to analyze its conformational stability. The
stable conformation of HpHtrA was used in virtual screening (VS) to
identify the lead compounds. From a set of 10 ligands, screened from
VS, the top three ligands (compound IDs: 402721, 402751 and 300040,
which are listed in Table 2) and a previously reported lead compound
by Perna, A.M. et al., showing a ligand efficiency constant, KD value of
13 ± 2 μm, which is considered as reference compound [19], were used
for further simulations. The general protocol adopted in MD simulation
is briefed below.
The molecular dynamic simulation was carried out using GROMACS
v4.5 tool for a period of 50 ns by applying GROMOS43a1 force field
[34]. The ligand topology was generated using PRODRG server [35].
The steepest descent algorithm is used to remove the steric clashes
present in the modeled structure. The modeled HpHtrA structure was
solvated explicitly using SPC water model in a cubic cell of dimension
12 × 12 × 12 Å. The solvated system was neutralized by adding suffi-
cient counter-ions and was subjected to energy minimization (using
steepest descent algorithm), which will automatically be truncated
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Microbial Pathogenesis 118 (2018) 365–377
when the force value is lesser than 1000.0 kJ/mol/nm. The long-range
electrostatic interactions were evaluated using Particle Mesh Ewald
method [36] with a cut-off of 10 Å. All non-bonded interactions were
treated using Lennard–Jones interaction with a cut-off of 14 Å. The
LINCS algorithm was used to constraint the bonds involving hydrogen
atoms [37]. The constant temperature was maintained using V-rescale
thermostat for NVT ensemble with a coupling constant of 0.1 ps. The
Parrinello–Rahman Barostat for NPT ensemble was used at a constant
pressure of 1 bar, coupling constant of 2.0 ps and compressibility of
4.5e−5/bar. The entire system was equilibrated using NVT and NPT
ensembles for 500 and 500 ps, respectively [38,39]. The final produc-
tion run for all equilibrated systems was performed for a period of 50 ns
and the trajectories were saved at every 2 ps. The stable trajectory has
been used to explore the free energy landscape as well as binding free
energy analysis. All binding interactions were visualized using Dis-
covery Studio 3.1 (Biovia, http://www.accelrys.com).
2.4. Principal component analysis and free energy landscape
The principal component analysis (PCA) reduces the dimensionality
of the data obtained from the stable MD trajectory. PC represents the
collective motion of the molecule based on their mass weighted cov-
ariance matrix [40]. The covariance matrix (Cij ) is calculated from the
mass-weighted Cartesian coordinates (i and j) of the N-particle system
sampled over the simulated trajectory and is given
Cij = (x i − x i )(x j − x j ) (1.1)
First two PC modes i.e., PC1 and PC2 are taken further for con-
formational sampling.
The free energy landscape (FEL) is a conformational sampling pro-
cedure used to explore all possible conformational changes of HpHtrA
after ligand binding [41,42]. The principal modes PC1 and PC2, gen-
erated using the stable trajectory (between 30 and 50 ns), were used to
calculate the free energy of HpHtrA in different complex states. The
two-dimensional FEL plot shows the joint probability of finding HpHtrA
complex in given conformational states during dynamics. The FEL is
defined as:
G (PC1, PC 2) = −kb T ln P(PC1, PC 2) (1.2)
Where kb is Boltzmann constant, T is temperature, and P(PC1, PC2) is the
normalized joint probability distribution.
2.5. Binding free energy calculation
The MM/PBSA approach was used to compute the binding free
energy of HpHtrA complexes, which quantify the molecular interaction
strength. A total of 100 frames from stable MD trajectory (between 30
and 50 ns) were extracted and subjected to GMXPBSA tool [43]. In
MM/PBSA calculation, the binding energy of ligand in binding cavity of
top three HpHtrA complexes was computed as:
ΔGbinding = Gcomplex − (Gprotein + Gligand ) (1.3)
Where, Gcomplex is the total binding free energy of the HpHtrA-ligand
complex. Gprotein and Gligand are the individual binding free energy of
HpHtrA and ligand molecule, respectively. The free energy per residue
contribution was calculated as
Gx = ΔEMM − TΔS + ΔGsolvation (1.4)
Where, x denotes the HpHtrA, ligand and complex forms as well. EMM is
the average molecular mechanics potential energy in vacuum. T and S
are temperature and entropy, respectively and Gsolvation is a free energy
of solvation. The EMM is calculated by
ΔEMM = ΔEbonded + ΔEnonbonded (1.5)
ΔEMM = ΔEbonded + EElectrostatic + Evdw (1.6)
N. Rai et al. Microbial Pathogenesis 118 (2018) 365–377

EElectrostatic and Evdw were obtained using Coulomb and Lennard-jones

(LJ) potential functions, respectively. For single trajectory approach,
Ebonded was set to zero. In an implicit solvent model, the free energy of
solvation was derived from Gpolar and Gnonpolar contributions using
Adaptive Poisson-Boltzmann (APBS) program [44].The polar con-
tribution refers to the energy required to transfer the solute from a low
dielectric continuum medium (ε = 1.5) to a solvent having the di-
electric constant for water (ε = 80).
The non-electrostatic term Gnonpolar was calculated by SASA-only
non-polar model such as:
Gnonpolar = γA + β (1.7)
−1 −2
Where, γ = 0.0227 kJ mol Å is a surface tension coefficient of
the solvent, A is the SASA and b is the fitting parameter. For non-polar
solvation energy, the dielectric boundary is defined by the probe of
radius 1.4 Å.

3. Results and discussion Fig. 2. Superimposition of the modeled (blue) and template (grey) structures. The pro-
tease, PDZ1 and PDZ2 domains of HpHtrA are labeled and the catalytic triad residues are
represented in stick model. (For interpretation of the references to color in this figure
3.1. Sequence and phylogenetic analysis of HpHtrA legend, the reader is referred to the Web version of this article.)

The HtrA sequences of gastro pathogenic bacteria like H. pylori

(UniProt ID: G2J5T2), E. coli (UniProt ID: P0C0V0), S. dysenteriae (UniProt
ID: Q32JU7), S. typhimurium (UniProt ID: P26982), Y. pseudotuberculosis
(UniProt ID: Q66EE4), V.cholera (UniProt ID: C3LS56), C. perfringens
(UniProt ID: A0A0N7BTU8), B. cereus (UniProt ID: B7IRP7), S. aureus
(UniProt ID: Q2FZP2), and human homologs of HtrA1 (UniProt ID:
Q92743), HtrA2 (UniProt ID: O43464), HtrA3 (UniProt ID: P83110), and
HtrA4 (UniProt ID: P83105) were retrieved from UniProt database. The
conserved regions were identified using MSA and the evolutionary re-
lationship between HpHtrA and other selected pathogens as well as human
homologs was studied using phylogenetic analysis.
The molecular evolution and sequence diversity between the gastro
pathogenic bacteria and human HtrAs were explored using the seed tree
with a highest log-likelihood score of −7234.85. All nodes in the
phylogenetic tree, showing high bootstrap scores, reveal the reliability
of the observed phylogenetic relation. The phylogenetic tree consists of
three major taxons (group 1, II and III encircled in Fig. 1) with boot-
strap values 92, 100 and 82, respectively. The target sequence of
HpHtrA (UniProt ID: G2J5T2 which is underlined in Fig. 1) is classified
in group I. This group 1 is characterized by two subgroups in which the
target HpHtrA and C. jejuni share a common ancestor with a bootstrap
value of 100, whereas the remaining pathogens such as E. coli, S. ty-
phimurium, Y. pseudotuberculosis, V. cholera, form the major sub group.
In group II, the closely related human homologs such as HtrA1, HtrA3
and HtrA4 are grouped together with a bootstrap value of 92 and the
HtrA2 shares a common ancestor (a bootstrap value of 100) with other
group II members. The members of group III also ensure their evolu-
tionarily distant relationship with human HtrAs. The HpHtrA shares
similarity with E.coli (Fig. 1), and is distantly related to human HtrA.
The separation of human homologs with other HtrA homologs clearly
depicts the diversion of HpHtrA with all other human homologs. It
indicates that even though all these sequences belong to the same
protein family (HtrA), they clearly disclose a separate evolutionary
lineage from others. Hence, the HpHtrA can be used as a novel ther-
apeutic target.
3.2. Structure of HpHtrA
modeled structure superimposes well over the template with a least root
mean square deviation (rmsd) of 0.39 Å (Fig. 2). The HpHtrA structure
consists of Protease, PDZ1 and PDZ2 domains and in overall it is formed
by eight α-helices, three 310-helices, and twenty four β-sheets (Fig. S1).
The MSA performed on the pathogens classified under group I reveals
that the protease domain consists of highly conserved motifs formed by
residues Leu109-Ile115, Glu207-Ala214, Gly219-Gly225, Gly248-
Met257 and the active site residues His116, Asp147, Ser221 (forming
the catalytic triad) and is shown in Fig. S1.
Details on the evaluation of modeled HpHtrA structure is given in
Table 1. The modeled structure of HpHtrA, validated using Ra-
machandran plot, reveals the presence of about 99.7% of amino acid
residues in favored as well as allowed regions and only 0.3% of residues
in the disallowed region (Fig. S2a). In addition, the Verify3D and
ERRAT scores (79.44 and 74.94%, respectively), reflect the reliability of
the generated model (Figs. S2b and c). The protein structure analysis
performed using ProSA server reveals the Z-score values −7.21 and
−8.86 for the modeled HpHtrA and template (PDB ID: 3CS0) struc-
tures, respectively (Fig. S2d). All these analyses confirm the reliability
of the modeled HpHtrA structure.
3.3. Structure dynamics of HpHtrA
The modeled HpHtrA structure was simulated for a period of 50 ns.
During simulation, the backbone rmsd of HpHtrA is stabilized after 20
ns with an average value of 5.51 ± 0.3 Å (Fig. 4a). The residual fluc-
tuations (rmsf) calculated using Cα atoms of HpHtrA is shown in Fig. 5.
In HpHtrA, mainly the loop regions of protease and PDZ domains
show higher fluctuations while the rest of the residues are significantly
stable. In particular, the protease domain residues such as (i) Gly68-
Phe83 (from LA loop), (ii) Pro129-Ser131 (loop in between β4-β5
sheets), (iii) Ser166-Asp168 (310-helix) and (iv) Ser241-Gly245, (in L2
loop region) display higher fluctuations above 3.9 Å. Similarly, (i) the
Table 1
Evaluation of modeled HpHtrA.

DOPE score −38902.08 kcal/mol

The HpHtrA discloses about 43% of sequence similarity as well as PROCHECK 87.5% in core
82% of query coverage (e-value of 2e−80) with HtrA of E. coli (PDB ID: 10.6% in allowed
3CS0). As the crystal structure of HpHtrA is not yet reported experi- 1.6% generously allowed
mentally, a comparative homology modeling is performed by Modeller 0.3% in disallowed
Verify3D 79.44%
software to model the structure of HpHtrA using 3CS0 as a template.
ERRAT 74.94%
Among the generated 10 models, the structure with the least DOPE Z-score −7.21
score (−38902.08 kcal/mol) is selected for further analysis. The

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Table 2
Top ten compounds with Glide score and Glide energy (kcal/mol).

No Database Comp. ID Glide score Glide energy Interacting residues

1 402721 −8.13 −48.65 Asn114, His116, Ser144, Glu145, Ser146, Asp147, Ile203, Asn204, Thr237 Ser241,
Thr243, Lys242

2 402751 −7.32 −46.51 His116, Ser144, Glu145, Asp147, Ile203, Asn204, Ser205, Ile239, Thr243, Arg334

3 300040 −6.95 −41.43 Lys62, Glu96, Asn114, Asn115, His116, Asp119, Ser144, Asp147, Ile203, Asn204,
Thr237, Ile239, Thr243, Gln382

4 180786 −6.92 −35.60 Lys62, Glu96, His116, Asp119, Asp147, Asn204, Ile239

5 110 −6.91 −58.06 Asn114, Asn115, His116, Asp119, Ser144, Asp147, Asn204, Ser221, Thr237, Ile239,
Ala381, Gln382, Gln389, Asp392, Gln414, Asn416

6 3056 −6.89 −48.17 Lys62, Asn115, His116, Ser144, Glu145, Asp147, Ile203, Asn204, Thr237, Ile239,
Thr243, Gly244

7 92950 −6.87 −41.3 Arg97, Asn114, His116, Asp147, Ile203, Asn204, Thr237, Thr243, Gly244

8 18760 −6.87 −47.23 Lys62, Glu96, Asn114, His116, Asp119, Asp147, Ile203, Asn204, Ser221, Thr237

9 177468 −6.85 −45.75 Lys62, Asn114, His116, Asn115, Ser144, Glu145, Ser146, Asp147, Ile203, Ser204,
Ala381, Gly244, Thr237, Ile239, Thr243

10 4871 −6.83 −38.99 Lys62, Glu96, Arg97, Asn114, His116, Asp147, Asn204, Ser221, Ile239, Gly244,
Thr243

11 reference −4.5 −32.37 His 116, Asp147, Glu145, Ile203, Asn204, Ser205, Ala238, Ile239, Thr243, Gly244,
Arg334

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Fig. 3. Representation of the electrostatic surface of HpHtrA-ligand complex (a). The compounds 402721, 402751 300040 and reference in stick model are represented in magenta, green,
yellow and blue, respectively. The subsets b, c, d and e, depicts a close view of ligand bound at the binding pocket of HpHtrA in HpHtrA1, HpHtrA2, HpHtrA3 and HpHtrAref complexes,
respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

310-helix of PDZ1 (Leu283-Lys294), (ii) loop in between β19-β20 sheets
(Met401-Asp405) and (iii) α7-helix of PDZ2 (Pro420-Gln423) show
higher fluctuations above 3.6 Å. The compactness of HpHtrA, measured
from the radius of gyration (Rg), is stabilized after 20 ns with an
average value of 21.52 ± 0.10 Å (Fig. 4c). The least energy structure of
HpHtrA (extracted at 20.9 ns) is used further for virtual screening.
3.3.1. Active site prediction
The FTMap server is used to predict the active site of HpHtrA. It
employs the small molecules (which vary in size, shape and polarity) as
probes and performs a global search over a dense grid around HpHtrA,
and finds favorable positions as the possible binding pockets using
simple energy function.
The best ranked binding pocket, identified using FTMap server, is
located inside the protease domain. This binding pocket is formed by
Asn114-His116, Ser144-Asp147, Ile203-Tyr206, Ser221, and Thr237-
Gly244 residues and are considered for the VS studies. It is interesting
to observe that the binding pocket identified by FTMap includes the
conserved catalytic triad residues (His116, Asp147 and Ser221) of
serine protease family as well.

Fig. 4. Variation in the rmsd (a), Rg(c), and SASA (d) of HpHtrA in native (blue), HpHtrA1 (black), HpHtrA2 (red), HpHtrA3 (green), and HpHtrAref (yellow) structures. The subset b
depicts only the rmsd of the ligands present in the complex. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

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N. Rai et al.
Fig. 5. Variation in the rmsf (a) of HpHtrA in native (blue), HpHtrA1 (black), HpHtrA2
(red), HpHtrA3 (green) and HpHtrAref (yellow) structures. The regions of LA, L2 loops and
310-helix are labeled. (For interpretation of the references to color in this figure legend,
the reader is referred to the Web version of this article.)
3.3.2. Screening of potential lead molecule against HpHtrA
Structure-based virtual screening (SBVS) involves the screening of a
large collection of small molecules present in the database using
docking protocols. In this study, the compounds from Specs, Asinex
(BioDesign, Antibacterial), ChemDiv (peptidomimetic), and Zinc (clean
Drug-Like compound) databases are used to generate the library
(having ∼1.3 million compounds) to screen against HpHtrA.
As per the standard protocol, the screening begins with different
scoring system HTVS and followed by SP as well as XP, to narrow down
the compounds with desired features and also to select the lead com-
pounds, finally. After the completion of every screening stage, a certain
percentage of screened compounds is used as the input to the next
scoring scheme. In this study, about 50% of the best conformational
states screened by HTVS are fed as the input for SP and about 30% of
the best output from SP is provided to the XP scoring scheme. In XP
protocol, only 10% of compounds with best scoring states are filtered
out and are ranked based on the docking score (GLIDE score) shown in
Table 2. From Table 2, the first three compounds with IDs 402721,
402751, and 300040 (shown in Fig. 3, will be referred as HpHtrA1,
HpHtrA2, and HpHtrA3, respectively, in the text) show higher glide
score (varying from −8.13 to −6.95 kcal/mol) and hence are subjected
to molecular dynamics simulations. For comparison purpose, one of the
reported lead compound with good binding affinity in an in vitro assay,
is also docked to HpHtrA structure (HpHtrAref) and docking score is
shown in Table 2. This docked complex is further simulated for 50 ns to
understand the interaction pattern of this reported lead compound.
3.3.3. ADME properties of predicted compounds
The drug-like properties of these top three compounds are evaluated
for Absorption, Distribution, Metabolism, and Excretion (ADME)
properties using the Lipinski's rule of five such as, (i) molecular
weight < 500 Da, (ii) hydrogen bond donors < 5, (iii) hydrogen bond
acceptors < 10 and (iv) logP < 5. The physiochemical and pharma-
ceutical properties such as the partition coefficient (QPlogPo/w) and
water solubility (QPlogS) is predicted in the range of −1.066 to 2.666,
and −3.465 to −0.304, respectively.
The estimated number of H-bonds formed by the solute with the
solvent (H2O) in aqueous solution is shown in Table 3. The values for
the donors and acceptors are averaged over each configuration and are
observed to vary from 3.0 to 4.0, and 8.9–9.9, respectively. The polar
surface area ranges from ∼82.54 to 147.4 Å2. The bioavailability and
toxicity values range between −4.6 and −5.96. Overall, ∼34–80% of
oral absorption in human body is revealed by these compounds. All
these physiochemical parameters, predicted by Qikprop module [33],
are within the acceptable range for these selected compounds and
hence indicate their potential to act as drug-like molecules (Table 3).
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Microbial Pathogenesis 118 (2018) 365–377
3.4. Molecular dynamics of complexes
The nature of interactions between the ligand and HpHtrA is ana-
lyzed by performing molecular dynamics simulations on the docked
complexes for a period of 50 ns. The HpHtrA complexes are stabilized
well with respect to native HpHtrA. The average backbone rmsd of
HpHtrA1, HpHtrA2, HpHtrA3 and HpHtrAref decreases by about 30.6,
26.3, 42.3 and 51.8% respectively, when compared to the native
HpHtrA. Simulations of the docked complexes such as HpHtrA1,
HpHtrA2, HpHtrA3 and HpHtrAref reveal a highly stable dynamics after
20 ns with an average backbone rmsd value of 3.83 ± 0.3 Å,
4.06 ± 0.4 Å, 3.2 ± 0.2 Å and 2.65 ± 0.2 Å, respectively. Both HpHtrA1
and HpHtrA2 complexes show similar pattern of rmsd variation,
whereas the deviation is significantly lesser in HpHtrA3 and HpHtrAref
complexes. In general, the rmsd profile of all four complexes including
HpHtrAref reveals a favorable interaction with HpHtrA and hence are
stabilized during dynamics (Fig. 4a).
Similarly, the rmsd of ligands is also calculated separately (Fig. 4b).
The observed consistent rmsd pattern reflects the stability of ligand in
the binding cavity. It is interesting to observe that the ligands in
HpHtrA1, HpHtrA2, HpHtrA3 and HpHtrAref complexes show a stable
rmsd profile with marginal deviations 2.49 ± 0.25, 2.78 ± 0.11,
2.4 ± 0.44 and 1.3 ± 0.25 Å, respectively. The average Rg of HpHtrA1,
HpHtrA3 and HpHtrAref complexes are more compact with an average
of 21.86 ± 0.09, 21.72 ± 0.20 and 21.29 ± 0.1 Å, respectively, when
compared to the HpHtrA2 complex for which the Rg increases to
22.31 ± 0.16 Å (Fig. 4c). The slightly enhanced Rg values of the com-
plexes indicate the structural relaxation of the binding pocket, after the
ligand binding.
The solvent accessible surface area (SASA) is calculated to measure
the solvent exposure of HpHtrA in native and complex structures. The
native HpHtrA shows an average area of 10905 ± 248 Å2, whereas the
HpHtrA-ligand complexes namely HpHtrA1, HpHtrA2, HpHtrA3 and
HpHtrAref reveal a comparatively increased solvent exposure with
average values of 11323.4 ± 221, 10942.8 ± 232, 10993 ± 214 and
10987 ± 184 Å2, respectively (Fig. 4d). As the L2 loop is observed to
play a significant role in localizing the ligand inside the binding pocket
of HtrA, the SASA of the receptor binding pocket (formed by residues
such as Asn114-His116, Ser144-Asp147, Ile203-Tyr206, Ser221,
Thr237-Gly244) including the L2 loop (only the residues involving in
ligand binding such as Thr237-Gly244) has been examined. In Fig. S3,
the variation shown in Set 1 is the SASA calculated for the binding
pocket with L2 loop, which is increased from 912 ± 45 Å2 (native
structure) to 992.5 ± 37 Å2 (HtrA complexes) during the dynamics due
to ligand binding. Whereas this calculated SASA is dropped to
526 ± 22 Å2, when the L2 loop residues are not considered (Set 2 in Fig.
S3). Such increased solvent exposure of binding pocket with L2 loop
(from 526 to 992.5 Å2) discloses its active participation during complex
formation.
In order to identify the fluctuations of key residues, rmsf were cal-
culated using Cα atoms for all complexes and are shown in Fig. 5. All
these complexes show less amplitude fluctuations except the LA loop
(Ile64-Glu93) which is present in the protease domain [45] and the 310-
helix in PDZ1 domain. It is interesting to observe that the binding of
ligand in HpHtrA1 and HpHtrA3 increases the fluctuation of L2 loop
(region Ser241-Gly245) by 4.13 and 4.73 Å, respectively. This pro-
nounced fluctuation is presumed by the rotation of L2 loop that facil-
itates the passage of the ligand molecule into the catalytic site.
Whereas, the same L2 loop fluctuation is arrested in HpHtrA2 and as
well as HpHtrAref complex with rms fluctuation of 2.58 and 1.92 Å,
respectively. In addition, the observed free dynamics of 310-helix of the
PDZ1 domain with rmsf fluctuation of 4.4 Å in HpHtrA1 is gradually
decreasing in HpHtrA2, HpHtrA3 and HpHtrAref complexes with an
average rms fluctuation of 3.0, 2.7, and 2.3 Å, respectively. In com-
parison to all other complexes, the dynamic fluctuation of HpHtrA was
arrested in HpHtrAref complex.
N. Rai et al. Microbial Pathogenesis 118 (2018) 365–377

Table 3
ADME properties of top three compounds.

Compound ID Molecular Weighta QPlogPo/wb QPlogSc PSAd hERGe % of human Oral Absorptionf DonorHBg AcceptorHBh

402721 343.39 −1.06 −0.30 147.43 −4.605 34.02 4 9.9

402751 444.52 2.66 −3.46 101.40 −5.960 80.39 3 9.2
300040 395.93 1.44 −2.08 82.54 −5.677 65.09 3 8.9

a
Molecular Weight (Weight < 500).
b
Predicted octanol/water partition coefficient (from −2.0 to 6.5).
c
Predicted aqueous solubility; S in mol/L (acceptable range: −6.5 to 0.5).
d
Polar surface area.
e
Predicted blockage of hERG K + channel (reasonable value < −6).
f
% of human oral absorption (<25% is weak and >80% is strong).
g
H-bond Donor (range for 95% of drugs: 0–6).
h
H-bond Acceptor (range for 95% of drugs: 2–20).

3.4.1. Principal component analysis and free energy landscape
Principal component analysis (PCA) is performed on the stable MD
trajectories (between 30 and 50 ns) to explore the essential dynamics of
HpHtrA complexes over the two-dimensional free energy landscape.
The dynamics of HpHtrA1, HpHtrA2, HpHtrA3, and HpHtrAref complex
are mainly governed by the first 10 PCs as evidenced by their cumu-
lative contributions 64.38, 69.54, 70.37 and 62.7%, respectively (Fig.
S4).
The direction of conformational evolution of HpHtrA complexes in
the free energy landscape was derived using the first two PCs. The re-
gion colored in purple indicates the clustered least free energy (ΔG = 0)
states with the highest probability of occurrence in FEL. The con-
formation of HpHtrA2 and HpHtrA3 is observed to sample a wider area
of FEL with two prominent clusters of least energy basins (Fig. 6b–c).
Whereas the HpHtrA1 and HpHtrAref complex samples a comparatively
narrow FEL space with two clusters separated by high energy barriers.
Similarly, the HpHtrAref compound spans a single distinct low energy
cluster surrounded by high energy barriers (Fig. 6a, d). In HpHtrA2
complex, the two most prominent clusters are located significantly
closer to each other. The representative structures from the clusters of
least energy was extracted and superimposed over the initial structure
using modevectors module of PyMOL and are shown in Fig. 7a–d. In
HpHtrA complexes, the α5 helix in PDZ1 domain moves closer to the
protease domain and hence this α5 helix (Asn335) interacts with the L3
loop (Tyr206) of protease domain. The present analysis discloses the
interactions between PDZ1 and protease domains as reported in the

Fig. 6. Free Energy Landscape calculted using PC1 (nm) and PC2 (nm) modes of HpHtrA in HpHtrA1 (a), HpHtrA2 (b), HpHtrA3 (c), and HpHtrAref (d) complexes. The least energy basins
are indicated using yellow triangle. The FEL energy scale is indicated in kJ/mol. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version
of this article.)

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Fig. 7. Superimposition of the initial (grey) over the structures extracted from the two least energy basins of FEL (intermediate (blue) I, and final (green) structure II) in HpHtrA1 (a),
HpHtrA2 (b), HpHtrA3 (c), and HpHtrAref (d) complexes. The arrow direction indicates the change in position of lower energy structure over initial structure. The ligand motion are shown
for all the complexes. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

crystal structure (PDB ID: 3CS0). In addition, the LA loop of protease
domain reorients toward the PDZ2 domain, which enhances the ac-
cessibility of the ligand into the binding cavity.
In HpHtrA1, the 310-helix of PDZ1 domain moves in the direction of
L3 loop of protease. In addition, the upward motion of L2 loop provides
the accessibility for the ligand binding and hence, the benzenesulfonyl
group of ligand molecule is re-located towards the binding site
(Fig. 7aII). The benzenesulfonyl group is gradually translocated to-
wards the catalytic site with an angular rotation of 161.1° and a dis-
tance of 2.8 Å. Similarly, in case of HpHtrA3, the upward movement of
L2 loop results in the translocation of chlorophenyl group towards the
catalytic site with the angular rotation of 88.6° and distance of 3.2 Å
(Fig. 7cII). Due to this rotation of chlorophenyl group in HpHtrA3 and
subsequently the carbonyl group is forming interaction with catalytic
Ser221 residue. Whereas in HpHtrA2, the L2 loop is partially lifted
upwards. Hence, the hydrophobic benzyl group is not able to accom-
modate itself inside the binding cavity and thus results in a rotational
shift of 131.5° and moves away from the catalytic site by a distance of
3.3 Å (Fig. 7bII). Similar to HpHtrA2, the movement of L2 is restricted in
HpHtrAref which results in relocating the pyrrole group along with
propanoic group away from the binding site to a distance of 6 Å
(Fig. 7d). The amplitude of upward lifting of L2 loop is higher in
HpHtrA1 and HpHtrA3 when compared to HpHtrA2. Whereas in case of
HpHtrAref complex, the L2 loop motion is less pronounced and re-
inforces similar rmsf pattern (Fig. 7dI, dII). This could be one of the
facts of observing lesser affinity of these compounds (402751 and re-
ference compounds) towards HpHtrA when compared to the rest of the
402721 and 300040 compounds (Fig. 7b, d).
3.4.2. Interaction analysis of HpHtrA complexes
The top two hit compounds, such as 402721 and 402751 are
identified with pyrrolidine group (core moiety) in which, the two hy-
droxyl groups are attached in cis conformation. These hydroxyl groups
of pyrrolidine are responsible for the major interactions with the
HpHtrA binding site. The compound 300040 present in the HpHtrA3
complex, is the piperidine derivative and forms interactions with the
catalytic triad. Whereas, in reference compound, the carboxylic group
of pyrrole derivative interacts with one of the catalytic triad residue.
Overall, all simulated compounds are having nitrogen based

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heterocyclic group. The electrostatic surface for all complexes is shown
in Fig. 3a which clearly depict the nature of the binding cavity.
3.4.2.1. Compound 402721. The top hit compound 402721 possesses a
higher glide score of −8.134 kcal/mol and forms H-bonding
interactions with residues Ser144, Glu145, Asp147, Ile203 and
Asn204 of HpHtrA. One of the active site residues, Asp147 forms H-
bonding interactions with the hydroxyl group (–OH group) of
pyrrolidine and amine nitrogen (-NH2+) at distances 2.1 and 1.8 Å,
respectively (Fig. 8a). It is also forming charge interaction (2.8 Å) with
-NH2+ group of ligand. The hydroxyl group of pyrrolidine derivative
forms H-bonding interactions with the amide group of Asn204 and the
carbonyl oxygen of Ile203 at distances 2.1 and 1.7 Å, respectively. The
carbonyl oxygen of Ser144 and Glu145 interact with the amide group of
the compound (1.8 and 1.9 Å, respectively).
In addition to these H-bonding interactions, other residues such as
Ser205, Asn114, His116, Thr237-Ser241, Thr243, and Gly244, etc. are
involved in non-bonded interactions with the compound. Two lower
energy conformations (observed at 33.68 and 47.23 ns) were extracted
from the FEL generated using PC1 and PC2 modes. The superimposition
of these two FEL structures over the initial conformation of HpHtrA1
shows the dynamics of both LA and L2 loops of protease and also the
310-helix of the PDZ1 domain (Fig. 7a). The ligand binding is con-
sidered responsible for the cavity opening mediated by the upward
motion of L2 loop (shown by an arrow in Fig. 7aI, aII). The ligand is
translocated inside the binding cleft with a conformational tilt of ben-
zenesulfonyl group towards the cleavage site near Ser221 (by a distance
of 2.8 Å as shown in Fig. 7aII). This movement of the ligand releases the
interactions of hydroxyl pyrrolidine with Asp147 and Ile203 residues
and reforms interactions between Ser205 and carbonyl oxygen of li-
gand. The number of H-bonds, polar and nonpolar interactions in
HpHtrA1 complex before and after simulations are shown (Fig. 8a and
b). Fig. S5 depicts the number of H-bonds observed throughout the si-
mulation time and the specific atoms involving in H-bonds are tabu-
lated as well (Table S1).
3.4.2.2. Compound 402751. This ligand interacts well with the residues
present in the binding cavity of HpHtrA with a glide score of
−7.325 kcal/mol. The ligand benzene ring forms π-π and π-cation
interactions with the imidazole ring of His116 (in the catalytic triad)
and ε-amino group of residue Lys62, respectively. The amino group
(–NH (CH2)2+) forms H-bond (1.9 Å) with Asp147. The hydroxyl group
of pyrrolidine forms H-bonding interactions with the carbonyl oxygen
group of Ile203 (1.9 and 1.8 Å) and side chain -NH2 group of Asn204
(1.8 Å). The carbonyl group of Asp147 forms two H-bonds with -NH2+
group of the ligand with a distance of 2.6 and 1.7 Å, respectively. The
carbonyl oxygen of the ligand forms H-bond (1.9 Å) with amino group
of Ile239 residue (Fig. 8c). To examine the receptor-ligand interaction
stability during MD simulations, the representative FEL structures
extracted at 37.0 and 44.94 ns were superimposed on the initial
structure of HpHtrA2 (Fig. 7bI, bII). The rotational shift of benzyl
group towards the proximity of L2 loop forms various non bonded
interactions with Ile239-Gly244 residues. Due to which, the L2 loop
fluctuation is arrested and, the π-cation and π-π interactions are lost in
the HpHtrA2 complex. Another H-bond, between carbonyl oxygen and
Gly244 (L2 loop residue), is formed at a distance of 1.6 Å (Fig. 8d).
Overall, four H-bonds (Table S1) are formed in HpHtrA2 complex and
are consistent throughout the simulation (Fig. S5).
3.4.2.3. Compound 300040. Compound 300040 is bound well inside
the binding cavity of HpHtrA with glide score of −6.95 kcal/mol. In
HpHtrA3 complex, the benzyl group forms π-π and π-cation interactions
with His116 and Lys62 residues. The trans-OH group of piperidine
derivative forms H-bonding interactions with Asn204, Ile239, and
Ile203 residues, while the –NH2+ group of ligand forms H-bond
interaction with Asp147 (in the catalytic triad) at a distance of 1.8,
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Microbial Pathogenesis 118 (2018) 365–377
1.7, 1.7 and 2.6 Å, respectively. Apart from these H-bonding
interactions, the residues such as Glu96, Asn114, Asn115, His116,
Asp119, Glu145-Ser146, Ile203-Ser205 and Arg366 are involved in the
non-bonded interactions (Fig. 8e). During MD simulations, the
rotational shift of benzyl chloride towards the catalytic center is
responsible for the L2 loop uplift motion and hence both π-π and π-
cation interactions have vanished. This π interaction is replaced by two
π-cation interactions which are formed between piperidine nitrogen
and –NH2+ group of ligand with the imidazole ring of His116 (Fig. 8f).
The H-bond interaction of Ile203, Asn204 and Ile239 residues with the
trans-OH group of piperidine has been replaced by (i) Asp147
interacting with piperidine nitrogen and (ii) Asn204 interacting with
–NH2+ group of ligand (Fig. 8e and f).
Two lower energy conformations (at 37.37 and 48.31 ns) were ex-
tracted from the FEL and were superimposed over the initial con-
formation of HpHtrA3. Superimpositions of these conformations clearly
depict the conformational changes in both LA and L2 loops as well as
ligand molecule (Fig. 7c1, c2). The H-bonds formed in HpHtrA3-ligand
complex during simulation are consistent and similar to HpHtrA1 is
shown in Fig. S5. In the least energy structure, the ligand forms the H-
bond interactions (with the catalytic triad residues such as Ser221 and
Asp147) and π-cation interactions (with His116) with HpHtrA (Fig. 8f).
These interactions clearly depict that this compound is capable of in-
teracting with all the catalytic triad residues and hence form a sig-
nificantly stable complex.
3.4.2.4. Reference compound. To understand the binding affinity of the
identified compounds in comparison with the known inhibitor, a
previously known inhibitor with KD value of (13 ± 2) μM [19],
referred as HpHtrAref compound was used. This reported compound
was docked and simulated for 50 ns to understand the interacting
pattern. The reference compound interacts with the residues present in
the binding cavity of HpHtrA with a glide score of −4.5 kcal/mol. The
furan ring of the ligand forms π-cation interaction with the guanidine
moiety of Arg334 residue. The carboxyl group forms H-bond with
Asn204 and electrostatic interaction with Asp147 (with a distance of
1.8 and 3.7 Å, respectively). Apart from these interactions, some other
non-bonded interactions are formed by His116, Glu145, Ile203, Ser205,
Ala238, Ile239, Thr243 and Gly244 residues (Fig. 8g).
Superimposition of the lowest energy structures extracted at 37.9
and 46.5 ns over the initial structure clearly depicts the overall motion
of LA loop (protease domain) and 310 helix (PDZ1 domain). In addition,
the L2 loop residue, mainly Ile240 residue, moves toward the pyrrole
group of ligand (Fig. 7d). The representative structure extracted from
FEL shows that the outward translocation of the ligand releases the H-
bond and π-cation interactions due to which, the imidazole side chain
of His116 residue forms H-bond (2.2 Å) with the carbonyl group of li-
gand molecule (Fig. 8h). Fig. S5 clearly depicts the number of H-bonds
formed by the ligand inside the cavity in HpHtrAref, which is highly
flexible during simulation.
3.4.3. Binding free energy of docked complexes
The binding energy is computed to quantify the efficiency of ligand to
interact with the binding pocket of HpHtrA. The binding energy of each
complex is calculated by extracting the frames at an interval of 200 ps from
the trajectory recorded between 30 and 50 ns. The energy components such
as van der Waal, electrostatic, polar and nonpolar solvation and the cal-
culated contributions are listed in Table 4. The electrostatic energy for
HpHtrA1, HpHtrA2, HpHtrA3 and HpHtrAref complexes are −144.87 ± 1.3,
−112.37 ± 1.5, −233.84 ± 2.7 kcal/mol and −21.11 ± 1.71, respec-
tively. Among all these complexes, only the HpHtrA3 complex possesses
highly negative electrostatic energy and the favorable contribution of polar
energy. As a result, the overall binding free energy of HpHtrA3 is higher
(−57.3 ± 1.0 kcal/mol) when compared to HpHtrA2 (−49.66 ± 0.6 kcal/
mol), HpHtrA1 (−45.63 ± 0.4 kcal/mol) and HpHtrAref
(−15.84 ± 0.54 kcal/mol) complexes. Overall, the compound 300040
N. Rai et al. Microbial Pathogenesis 118 (2018) 365–377

Fig. 8. The 2-D representation of the HpHtrA-ligand interactions observed in the docked (I) and simulated (II) complexes such as HpHtrA1 (a, b), HpHtrA2 (c, d), HpHtrA3 (e, f) and
HpHtrAref (g, h).

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Table 4
Binding Energy (in kcal/mol) calculated for HpHtrA-complexes using MM/PBSA method.
Name of the complex vdW Energy Electrostatic Energy
HpHtrA1 −42.29 ± 0.4 −144.87 ± 1.3
HpHtrA2 −53.24 ± 0.4 −112.37 ± 1.5
HpHtrA3 −37.83 ± 0.43 −233.84 ± 2.7
HpHtrAref. −34.43 ± 0.38 −21.11 ± 1.71
Polarc: Polar contribution.
Nonopolarc: Nonpolar contribution.
expresses a significantly higher affinity towards HpHtrA and hence could be
considered as a potential lead molecule to inhibit HpHtrA.
4. Conclusion
The HtrA of Helicobacter pylori cleaves the extracellular domain of E-
cadherin present in the gastric epithelial of the host cell to break the
cell-cell adhesion and spread infections. As a result, it is associated with
several gastric diseases like mucosa-associated lymphoid tissue (MALT)
lymphoma, chronic gastritis, gastric cancer, etc. It is also up-regulated
during stress conditions caused by an oxidative agent, H2O2 [46] and
low pH in gastric epithelial. The inhibitor design for the active site of
HpHtrA is required to prevent the fragmentation of E-cadherin. This
study mainly focuses on the (i) molecular evolution of HpHtrA, (ii)
identification of lead molecules to inhibit HpHtrA protease and (iii) the
structural dynamics of HpHtrA in free and complex forms.
The phylogenetic analysis reveals the similarity of HpHtrA with E.
coli and other gastrointestinal bacteria. The distant relationship of
HpHtrA with human HtrA homologs clearly witnesses that the human
HtrA protease shares no cross-reactivity with other pathogenic HtrA.
The homology modeling and validation of HpHtrA revealed a good
agreement with the template structure of E. coli (PDB ID: 3CS0) with a
rmsd of 0.39 Å. It is also observed that both HpHtrA and E. coli share
common conserved catalytic triad residues. The modeled structure of
HpHtrA is simulated for a period of 50 ns and the least energy con-
formation was used for further screening process. Based on the struc-
ture-based virtual screening approach, complexes of HpHtrA with
402721, 402751 and 300040 compounds were simulated further to
identify the possible potential lead compound. For a comparative
analysis, a reported lead compound is also been simulated for 50 ns.
The dynamic LA, L2 loop regions of protease and 310-helix of PDZ1
domains of HpHtrA accommodate the ligand inside the binding pocket
and further stabilize the complexes. The interaction between HpHtrA
and ligand is monitored using both H-bond and binding energy ana-
lyses. The L2 loop motion is mainly regulating the ligand binding to-
wards the catalytic site. The higher amplitude fluctuation of an L2 loop
is significantly observed in complex HpHtrA1 and HpHtrA3, wherein
HpHtrA3, the ligand is interacting with the catalytic residue Ser221.
The complex of HpHtrA with ligand 300040 (HpHtrA3) is observed to
express a higher binding energy (−57.3 ± 1.0 kcal/mol) than other
complexes such as HpHtrA1 (−45.63 ± 0.4 kcal/mol) and HpHtrA2
(−49.66 ± 0.6 kcal/mol). Overall, the identified lead molecule, from
the present analysis, is having improved binding energy when com-
pared to the reference compound (−15.84 ± 0.54 kcal/mol). Hence,
the compound 300040 that expresses significantly higher affinity to-
wards HpHtrA could be considered as a potential lead molecule to in-
hibit HpHtrA. In other words, the ligand with hydroxyl-piperidine and
4-aminopiperidine scaffold could be considered for the designing and
development of novel therapeutic inhibitors against HpHtrA induced
diseases. It is also worth mentioning that the compound containing
hydroxyl-piperidine and 4-aminopiperidine scaffold identified from this
study is also known for its anti-cancerous property [47–49].
Microbial Pathogenesis 118 (2018) 365–377
Polar Energy Nonpolar Energy Polarc Nonpolarc Binding Energy
145.25 ± 1.0 −3.70 ± 0.01 0.37 −46.00 −45.63 ± 0.4
120.34 ± 1.1 −4.39 ± 0.01 7.97 −57.64 −49.66 ± 0.6
218.52 ± 2.1 −4.15 ± 0.0 −15.31 −41.98 −57.3 ± 1.0
42.78 ± 1.47 −3.07 ± 0.02 21.66 −37.5 −15.84 ± 0.54
Acknowledgement
One of the authors, Amutha Ramaswamy acknowledges the Science
and Engineering Research Board, India for providing computational
facilities under Fast Track Research Project for Young Scientists
Scheme. Nivedita Rai, and R. Muthukumaran acknowledges
Pondicherry University for providing research fellowship. The authors
declare no conflict of interest.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.
doi.org/10.1016/j.micpath.2018.03.027.
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