Computational Biology and Chemistry 98 (2022) 107675

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Computational Biology and Chemistry

journal homepage: www.elsevier.com/locate/cbac

In silico design, synthesis and anti-HIV activity of quinoline derivatives as

non-nucleoside reverse transcriptase inhibitors (NNRTIs)
Vishal K. Singh a, Richa Mishra a, Priyanka Kumari b, Anup Som b, Aditya K. Yadav a,
Nand K. Ram a, Pradeep Kumar c, Dominique Schols d, Ramendra K. Singh a, *
a
Bioorganic Research Laboratory, Department of Chemistry, University of Allahabad, Prayagraj 211002, India
b
Centre of Bioinformatics, University of Allahabad, Prayagraj 211002, India
c
CSIR - Institute of Genomics and Integrative Biology, Mall Road, Delhi 110007, India
d
Rega Institute for Medical Research, KU Leuven, Leuven, Belgium

A R T I C L E I N F O A B S T R A C T

Keywords: A series of quinoline derivatives has been designed, synthesized and screened for their anti-HIV properties. The
NNRTIs drug-like properties of compounds were evaluated first and then molecular docking using DS v20.1.0.19295
HIV-RT software showed that the compounds behaved as non-nucleoside reverse transcriptase inhibitors (NNRTIs) while
TOPKAT
interacting at the allosteric site of target HIV-RT protein (PDB:3MEC). The docking results revealed that all
Docking
DFT
compounds formed hydrogen bonds with Lys101, Lys103, Val179, Tyr188, Gln190, Gly190, Pro225, Phe227,
Molecular dynamics and Tyr318, and showed π-interaction with Tyr188 and Tyr318. TOPKAT (Toxicity Prediction by Komputer
Assisted Technology) results confirmed that the compounds were found to be less toxic than the reference drugs.
Density functional theory (DFT) analysis was performed to assess the binding affinity of all compounds. Further,
molecular dynamics (MD) simulations were performed on compound 6 and delavirdine with HIV-RT enzyme.
Comprehensive MD analyses showed a similar pattern of conformational stability and flexibility in both the
complexes suggesting alike inhibitory action. The hydrogen-bonding interactions and the binding energy of
active-site residues for the compound 6 complex revealed strong inhibitory activity than the reference (dela­
virdine) complex. Thus, the compound 6 might act as a potential inhibitor against HIV-RT. Overall, this study
revealed that compound 6 (5-hydroxy-N-(4-methyl-2-oxo-1,2-dihydroquinolin-8-yl) thiophene-2-sulfonamide)
has prudent anti-HIV activity against both HIV-1 (SI = 2.65) and HIV-2 (SI = 2.32) that can further be uti­
lised in drug discovery against HIV virus.

1. Introduction 2010). Implication of combinatorial approach with multiple drugs

HIV/AIDS endures to be a major ecumenically hazardous epidemic
till date. Approximately 37.7 million people live with HIV and 0.37
million deaths have been reported so far June 15, 2021 (UNAIDS, 2019;
Vella et al., 2012). Regardless of ample efforts towards vaccine devel­
opment, currently none of the known FDA-approved vaccines are
available. A magnitude of endeavour has been subjected in scientific
community in the direction of management of HIV infection with potent
anti-HIV drugs (Ghosh et al., 2016). Albeit, drug regimens in highly
active antiretroviral therapy (HAART) are efficient enough to cope with
high HIV viral load in infected patients, the remediation is tentatively
limited because of several aversive side effects leading to a continuous
rise in drug resistance, high costs and global distribution issues (Clercq,
diminished the expeditious emergence of chemo-resistant viruses (Zhan
et al., 2016). Several antiviral drug targets in HIV life-cycle are known,
such as viral attachment, fusion, reverse transcription, integration and
protease activity. Amongst pre-mentioned drug targets, HIV reverse
transcriptase (RT) was pursued successfully with first and
second-generation non-nucleoside reverse transcriptase inhibitors
(NNRTIs), as exemplified by nevirapine introduced in 1996 and more
neoteric doravirine in 2018 (Zhan et al., 2016). HIV RT is crucial for
conversion of single-stranded viral RNA genome to double-stranded
DNA for subsequent integration into the host cell genome and multi­
plication via hijack of host cellular machinery. The heterodimeric
reverse transcriptase enzyme (p66/p51), a multifunctional protein, has
active sites for polymerase activity and also catalyses the ribonuclease H

* Corresponding author.
E-mail address: rksinghsrk@gmail.com (R.K. Singh).

https://doi.org/10.1016/j.compbiolchem.2022.107675
Received 19 February 2022; Received in revised form 23 March 2022; Accepted 29 March 2022
Available online 31 March 2022
1476-9271/© 2022 Published by Elsevier Ltd.
V.K. Singh et al.
Fig. 1. FDA approved NNRTI drugs.
activity (RNase H has recently been validated as a target for intervention
by small drug candidates) (Jonckheere et al., 2000; Yisma et al., 2014).
Thus, polymerase is responsible for the synthesis of DNA via RNA/DNA
hybrids by replication of viral RNA genome, and then degradation of
RNA by RNase H (Bhole et al., 2020). The active site of RNase H pos­
sesses two bivalent Mg2+ ions that enable the RNase H for chelation and
catalysis of RNA phosphate backbone cleavage by prompting hydrolysis
of phosphate linkage (Singh et al., 2000). This RNase ribonuclease H
activity can be halted by triggering conformational changes, either by
chelation or by blocking allosteric binding site of RT by employing
non-nucleoside reverse transcriptase inhibitors (Yisma et al., 2014).
Thus, novel therapeutics that efficiently target the vital steps of viral life
cycle are prerequisites to circumvent the onset of drug resistance and to
further ameliorate the current treatments (Boone, 2006). In order to
discern novel leads for drug discovery, one may focus on designing
compounds that have been either untapped so far or have fascinating
pharmaceutical and biological activity profiles. FDA approved NNRTIs
(Fig. 1) have a great focus on both WT (wild type) HIV-1 and mutant
strain, but their clinical uses are limited due to low solubility and poor
bioavailability. Therefore, there still exists a need to develop new
NNRTIs against HIV-1.
For the development of possible NNRTIs against HIV, we have
designed, synthesized and screened some novel quinoline derivatives 1-
8, which contain all pre-requisite structural features, viz. appropriate
number of rotatable bonds, donor sites, hydrogen-bond acceptors and
donors, sites of localized charge density, zones of steric encumbrance,
etc. The synthesis of these molecules as anti-SARS-CoV-2 agents has
already been reported in our previous work (Singh et al., 2021a). In this
article, we report the anti-HIV activity of the quinoline derivatives.
Quinoline derivatives bearing multiple aromatic rings and sulpho­
namide scaffolds have been screened as a privileged class of anti-HIV
candidates with minimum cytotoxicity. Compounds were analysed for
their anti-HIV activity as NNRTI’s via molecular docking and molecular
dynamics (MD) simulations. Other physicochemical parameters were
also evaluated by employing a series of computational methods.
Furthermore, all compounds were also screened for anti-HIV biological
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Computational Biology and Chemistry 98 (2022) 107675
activity under in-vivo conditions. The lead compounds, i.e., compound 6,
the thiophene derivative of quinoline sulphonamide, exhibited quite
satisfactory anti-HIV activity against both HIV-1 and HIV-2 (SI (Selec­
tivity Index) = 2.65 and 2.32, respectively), and the compound 5 dis­
played better anti-HIV activity only against HIV-2 (SI = 9.18), during
HIV-1 and HIV-2 single round infectivity assay.
2. Experimental
2.1. Physicochemical studies
Initially, a bunch of 50 quinoline derivatives had been designed as
the probable NNRTIs against HIV by using the pkCSM, Chemdraw and
Moleinspiration software. Out of these, eight best molecules were
selected for further studies on the basis of their in silico structure-based
analysis. The pharmaceutical descriptors such as hydrogen bond donor
(HBD), hydrogen bond acceptor (HBA), molecular weight (MW) and
lipophilicity (LogP) have been used to understand the drug-like prop­
erties of the molecules. ADMET prediction of all compounds was
calculated with the help of Swiss ADME software. All compounds
exhibited the drug-like properties just like the reference drugs (Singh
et al., 2020; Naaz et al., 2018; Srivastava et al., 2018; Ghosh et al.,
2020).
2.2. Molecular docking studies
In order to explore the binding interaction of the quinoline derivates
with HIV-RT protein, molecular docking studies were performed using
the BIOVIA/Discovery Studio 2020 Client (DS 20.1.0.19295 version)
protocol default parameters (Muegge and Martin, 1999; Lagos et al.,
2008; Singh et al., 2016, 2018). The 3D X-ray crystal structure of
docking receptor, i.e., allosteric site of HIV-RT protein (PDB ID:3MEC,
www.rcsb.org), the catalytic core of HIV-RT was retrieved as an adduct
from RCSB (www.rcsb.org) protein data bank (Rose et al., 2012). The
quinoline derivatives 1–8 were prepared accordingly and then molec­
ular docking was carried out, and the different docking alignments were
V.K. Singh et al.
chosen for further analysis. Standard protocol was employed for pre­
paring, scoring and docking of ligands with HIV-RT protein (Srivastava
et al., 2020).
2.2.1. Preparation of receptor
All the quinoline derivatives were docked with the HIV-RT protein
and missing hydrogen atoms were added via DS v20.1.0.19295. Posi­
tions of each atom were optimized by all-atom CHARMm forcefield with
Adopted Basis set Newton Raphson (ABNR) minimization algorithm till
the root mean square (RMS) gradient for potential energy became
< 0.05 kcal/mol/Å. Additionally, minimization of target protein (HIV-
RT) was done in DS v20.1.0.19295. The binding site enclosed by the
ligand has been decided in input site sphere with 5 Å radius on the re­
ceptor. The obtained receptor from above process was further mini­
mized and then used for molecular docking simulations (Srivastava
et al., 2020; Usman et al., 2017).
2.2.2. Ligand setup
3D structure of each compound was created in build-and-edit section
of DS v20.1.0.19295. Minimization of ligands was completed by
CHARMm forcefield using ABNR technique. Molecular dynamics tech­
nique was used for the conformational selection of the ligands.
Furthermore, the temperature of each ligand was increased up to 700 K
and then slowly decreased to 200 K for thirty times. After thirty cycles,
the conformation of ligand was taken and energy minimization was
done by ABNR technique. All minimized conformations were then
superimposed and the conformation with minimum energy was the
acceptable conformation.
2.2.3. Docking and scoring
Ligandfit protocol in combination with pose analysis filter and Monte
Carlo conformational quest, available in DS v20.1.0.19295, have been
employed while docking of designed ligands against targeted enzyme
receptor. Every single docked pose has been filtered out by rigid body
minimization of ligand, associated with grid-based prediction of inter­
action energy via dreiding forcefield. During the docking simulation, the
conformation of targeted protein receptor was kept inflexible. CHARMm
forcefield and smart minimization technique were employed for docked
poses until the RMS gradient for potential energy was < 0.05 kcal/mol/
Å. While minimization, the binding sites of ligands and protein were
concubine flexible employing simulation methods (Molecular dynamics,
energy minimization and Monte Carlo simulation). The scoring (Inter­
action energy, PLP, Ludi, Dock Score etc.) of ligands are explained in the
results and discussion section. All conformations have been refined via
LUDI scores. High LUDI scores were selected as the best conformation
and were taken for further analysis. Ligand-protein stability evaluated
by the analysis of binding energy, higher negative value of binding en­
ergy revealed more stability of complex formed between ligand and
target protein. Binding Energy protocol in DS v20.1.0.19295 using the
default settings was used to calculate the binding energy (Peele et al.,
2021).
2.2.4. Validation of docking protocol
The native crystallized ligand and molecules were docked against the
allosteric site of HIV-1 RT protein. The relative docking simulation
adverted that the used scoring function was suitable as the root mean
square deviation (RMSD) of compounds along with the native crystal­
lized ligand was under the justifiable limit, i.e., (RMSD) < 2 Å. Hence,
the result favoured the assumption that experimental binding modes
could be regenerated with accuracy using pre-mentioned protocol.
Binding energy of all the compounds was calculated through the
molecular docking studies. In order for the better prediction of drug-like
properties of all quinoline derivatives (1–8), their inhibition constant
(Ki) was evaluated through the following equation (Onawole et al.,
2018):
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Computational Biology and Chemistry 98 (2022) 107675
Ki = 10(BE/1⋅3666), BE = Binding energy
2.3. QSTR analysis
Quantitative Structure Toxicity Relationship (QSTR) of quinoline
derivatives 1–8 and reference drugs was performed to evaluate the
pharmaceutical potential. TOPKAT protocol present in DS
v20.1.0.19295 was used for the evaluation of QSTR (Bhole et al., 2020).
2.4. Density functional theory (DFT) analysis
DFT analyses of quinoline derivatives 1–8 and reference drugs (ne­
virapine and delavirdine) were performed by using default parameters
available in the simulation tool panel of BIOVIA/ Discovery Studio 2020
Client (DS v20.1.0.19295 version). Evaluation of HOMO and LUMO was
carried out in solvent free condition (Mishra et al., 2021, 2019; Hoque
et al., 2018; Singh et al., 2021a, 2021b).
2.5. Molecular dynamics simulations
The potential docked complexes (HIV-RT:Delavirdine complex and
HIV-RT:Compound 6 complex) were simulated using Gromacs-2019.6
package (Pronk et al., 2013) for comprehensive atomistic understand­
ing. The MD simulations were carried out using charmm36-jul2021
force field (Huang and MacKerell, 2013). The CgenFF web server was
used to generate parameters for ligand molecules (Vanommeslaeghe
et al., 2010). The X-ray crystallographic structure of HIV-RT in complex
with Etravirine (PDB Id: 3MEC) was used as starting structure for
Docking and MD simulations. The entire process was done in the
traditional way from file preparation to energy minimization, 2 phase
equilibration, and finally production phase. Both systems were solvated
using TIP3P model and electrically neutralized by adding counter ions
(Na+ and Cl-). Since at physiological pH, protein is positively charged,
both the systems (“HIV-RT: delavirdine” and “HIV-RT: compound 6”)
were neutralized with 4 Cl- ions. Then the systems were energy mini­
mized using steepest descent method to remove the bad contacts among
the atoms for 10000 steps. Subsequently, NVT equilibration of 100 ps
was run to maintain the system temperature at 300 K using the V-rescale
thermostat (Bussi et al., 2007) and 100 ps of NPT equilibration to
maintain a constant pressure of 1 bar for the system, using
Parinello-Rahman barostat (Parrinello and Rahman, 1981). The
time-constant for pressure coupling was set to 1 ps. It also maintained
the homogeneous density across the systems. Productive MD simula­
tions were performed in the isothermal-isobaric (NPT) ensemble for
100 ns. The LINCS (Hess et al., 1997) algorithm was used to constrain
bond lengths, allowing the use of 2 fs time step. Electrostatic interactions
were calculated using the Particle-mesh Ewald (PME) summation
scheme (Darden et al., 1993). Van der Waals and Coulomb interactions
were truncated at 1.0 nm. The coordinates were saved every 10 ps
during the production run for post MD analyses.
2.6. Cytotoxicity assay
Human embryonic kidney (HEK293) cell lines were preserved in
DMEM supplemented with 1% antibiotic and 10% FBS cocktail. These
cells were taken from the National Centre for Cell Science (NCCS), India
and preserved at 370 C in a humidified 5% CO2 incubator. Transfection
assay was performed with a plasmid encoding enhanced green fluores­
cent protein (EGFP) gene of 4.4Kbp under the cytomegalovirus imme­
diate early promoter (Kumar et al., 2010; Halawa et al., 2020).
MTT colorimetric assay was used in order to evaluate the cell
viability of pDNA complexes of all quinoline derivatives (1− 8). More
than forty samples of different concentrations (10, 20, 40, 50, 70, 80, 90,
V.K. Singh et al. Computational Biology and Chemistry 98 (2022) 107675

Table 1
Physicochemical properties of quinoline derivatives 1–8 and reference drugs.
Compound MW H-A H-D Log P TPSA Rotatable bonds noV

< 500 ≤ 10 ≤5 ≤5 ≤ 140 ≤ 10 ≤1

1 359.36 8 2 2.52 124.86 4 0
2 348.81 5 2 3.24 79.03 3 0
3 440.61 5 2 5.53 79.03 6 1
4 382.36 5 2 3.45 79.03 3 0
5 328.39 5 2 3.01 79.03 3 0
6 3336.39 6 3 2.41 99.26 3 0
7 278.31 4 2 2.68 61.96 2 0
8 212.25 3 2 1.81 44.89 2 0
Nevirapine 266.30 5 1 1.39 63.58 1 0
Delavirdine 456.57 9 3 2.88 110.43 6 0

MW: Molecular weight; H-A: number of H bond acceptors; H-D: number of H bond donors; Log P: predicted octanol-water partition coefficient; TPSA: total polar
surface area; noV: number of violations

Table 2
Bioactivity of quinoline derivatives 1–8 and reference drugs.
Compound GPCR ligand Ion channel modulator Kinase Inhibitor Nuclear receptor ligand Protease inhibitor Enzyme inhibitor

1 -0.32 -0.42 -0.40 -0.07 -0.30 -0.17

2 -0.20 -0.42 -0.29 -0.01 -0.25 -0.11
3 -0.10 -0.33 -0.21 0.05 -0.11 -0.03
4 -0.11 -0.29 -0.18 0.16 -0.12 -0.06
5 -0.21 -0.44 -0.30 -0.00 -0.22 -0.10
6 -0.11 -0.53 -0.18 -0.00 -0.05 0.05
7 -0.21 -0.33 -0.11 -0.26 -0.42 -0.11
8 -0.35 -0.40 -0.42 -0.45 -0.63 0.06
Nevirapine -0.12 -0.41 0.08 -0.72 -0.52 0.58
Delavirdine 0.33 0.16 0.34 -0.14 0.26 0.22

GPCR (G-protein coupled receptor-ligand): (− 0.62 to − 0.39, moderate activity); Ion channel modulator: (0.27–0.05, significant activity); Protein kinase in­
hibitors: (0.40–0.18, significant activity); Nuclear receptor: (− 0.63 to − 0.31, moderately active); Protease activated receptors: (− 0.15 to 0.00, moderate activity)
and Enzyme inhibitor: (0.61 to − 0.48, significant activity).

100 µg/mL in DMEM) were taken, and cells were incubated with these
samples for 24 h. After that, in each sample MTT reagent (100 μL, 1 mg/
mL) was added and incubated for 2 h in a humidified 5% CO2 atmo­
sphere at 37 ◦ C. The supernatant was then aspirated and the formazan
crystals, so formed, were suspended in 100 μL of isopropanol supple­
mented with 0.06 M HCl and 0.05% sodium dodecyl sulphate (SDS).
ELISA plate reader was used to evaluate the absorbance of samples at
540 nm. Fresh cells were taken as control with 100% viability. This
experiment was executed in triplicate, and cell viability (%) was eval­
uated (Abs-sample/Abs-control x 100).
luminescence obtained in experimental set divided by the luminescence
found from infected cells (lacking test compound), multiplied by hun­
dred. Second, by subtracting the above resultant value from hundred.
Moreover, the cytotoxicity assay was also performed on the same
TZM-bl cells, employing MTT [3-(4,5-dimethylthiazol-2-yl)− 2,5-
diphenyltetrazolium bromide; Sigma-Aldrich Inc.] assay.
3. Results and discussion
3.1. Physicochemical and ADMET properties of quinoline derivatives

2.7. Anti-HIV assay
TZM-bl cells and HIV-1 (strain NL4.3), at a 0.05 multiplicity of
infection (Mol), were used to access the anti-HIV activity of quinoline
derivatives. Standard drugs 9-[(R)− 2-(Phosphonomethoxy) propyl]
Adenine (PMPA) and AnorMeD3100 (AMD3100) have been employed
as positive reference controls and the cell without HIV was used as a
negative control.
For this, TZM-bl cells (4 ×104/well) were planted in 96-well culture
plate and incubated overnight at 37 0C in the presence of humidified 5%
CO2. Then, HIV-1 virus (strain NL4.3) get treated for 1 h with selected
test compounds in separate vials at 37 0C. Subsequently, pre-treated
viruses were also added in duplicates to TZM-bl cells, previously
cultured for 4 h. Afterward, the treated cells get washed with cold
50 mM PBS (pH 7.4) for the elimination of all untreated cell-free viruses,
followed by the addition of a fresh culture medium (with or without
tested compounds) for further incubation of cells up to 48 h. After in­
cubation, the cells were washed twice with PBS and 1X lysis buffer
(Promega Corporation, Madison, USA) was employed for cell lysis. The
obtained supernatant was then examined for their luciferase activity
using luciferase assay kit (Promega Corporation). Results presented as
percentage inhibition was evaluated in 2 steps: First, by taking the
The physicochemical data, bioactivity score and ADMET properties
of the quinoline derivatives 1–8 were found to be satisfactory. The
physicochemical data shown in Table 1, revealed that all compounds,
except 3, followed the Lipinski rule of five. The molecules had lower
value of LogP (lipophilicity) and this indicated that they could easily
pass through the cell membrane (except compound 3). Further, the
lower value of TPSA (except compound 3) referred to the good cell
internalization, just like the reference drugs. The bioactivity scores, viz.
ion channel modulator, G-protein coupled receptor (GPCR) ligand, nu­
clear receptor ligand, and kinase inhibitor were also estimated to assess
the significant drug-like properties. The molecules with bioactivity
scores greater than zero are expected to be pharmaceutically active,
with score between 0.00 to − 0.50 are expected to have moderate ac­
tivity and with score less than − 0.50 are expected to be inactive. The
bioactivity scores of quinoline derivatives 1–8 and reference drugs are
summarised in Table 2, which revealed good to moderate pharmaceu­
tical properties of the molecules studied.
3.2. Docking analysis of quinoline derivatives
In order to know the binding interaction of all compounds with
target protein (PDB ID: 3MEC), docking study was executed using

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V.K. Singh et al. Computational Biology and Chemistry 98 (2022) 107675

Table 3
Docking interaction of quinoline derivatives 1–8 with HIV-RT in ligand - receptor docked complexes.
Compound No. of Amino acid in H-B Type D D-A A-A No. of π/ Amino acid in π/ ‘ π-π/ π + ’ monitor
H-B H-B (Aº) π + -B π + -B
Bond D End1 End2
(Aº)

1 2 Pro225 1:O12 -Pro225:O 3.18 O12 O – – – – – –

Tyr318 Tyr318:OH -1 3.56 OH 1
2 3 Lys101 2:O12 -Lys101:O 2.58 O12 O – – – – – –
Lys103 Lys103:N - 2:O12 2.81 N O12
Tyr318 Tyr318:OH - 2 3.99 OH 2
3 2 Gln190 Gly190:N-3:O15 3.17 N O15 – – – – – –
Tyr188 3:N13-Tyr188:O 2.83 N13 O
4 4 Lys103 Lys103:N - 4:F26 2.82 N F26 – – – – – –
Gln190 Gly190:N - 4:O12 2.78 N O12
Val179 4:O12 - Val179:O 2.56 O12 O
Tyr188 4:O12 - Tyr188:O 2.66 O12 O
5 4 Gln190 Gly190:N − 5: 3.05 N O11 1(π-π) Tyr318 Tyr318 - 5 4.88 Tyr318 5
Tyr188 O11 3.13 O11 O
Val189 5:O11 -:Tyr188:O 3.01 O11 O
Phe227 5:O11 - Val189:O 3.34 N13
5:N13 - Phe227
6 5 Lys101 Lys101:N -6:O12 2.96 N O12 – – – – – –
Tyr318 Tyr318:OH -6: 2.63 OH O15
Lys101 O15 2.68 O12 O
Lys104 6:O12 - LYS101:O 2.62 O22 O
Ser105 6:O22 - A:Lys104: 3.41 C O22
O
Ser105:C -6:O22
7 2 Lys103 Lys103:N − 7: 3.00 N O15 1(π-π) Tyr188 Tyr188 - 7 4.85 7 Tyr188
Phe227 O15 3.73
7:O12 - Phe227
8 2 Val179 8:O12-Val179:O 3.11 O12 O – – – – – –
Gly190 Gly190:N-8:O12 3.21 N O12
Ref. 1 – – – – – – 2 (π-π) Tyr318 Tyr188 - 5.1 Ref.1 Tyr188
Tyr181 Ref.1 6.0 Ref.1 Tyr181
Tyr181 -
Ref.1
Ref. 2 4 Lys103 Lys103:N -:Ref.2: 2.83 N O18 – – – – – –
Pro236 O18 3.12 C13 O
His235 Ref.2:C13 - 3.58 C15 O
Tyr318 Pro236:O 3.49 C15 OH
Ref.2:C15 -
His235:O
Ref.2:C15
-Tyr318:OH

H-B: Hydrogen bond; D: Distance (Å); D-A: Donor Atom; A-A: Acceptor Atom; Ref. 1: Nevirapine; Ref. 2: Delavirdine

Discovery Studio v20.1.0.19295 software. The compounds showed
interaction with Lys101, Lys103, Gln190, Tyr188, Val179, Pro225,
Tyr318, Phe227, Ser105 and Gly190 amino acid residues through the
hydrogen bond and π-interaction at the allosteric site of HIV-RT protein.
H-bonds, π-π (hydrophobic), and non-polar π-cation (non-covalent)
interactions were used to explain the docking results. The existence of
these non-covalent interactions implied that the inter-atomic distances
were in the range of less than 6 Å. The docking results are summarised in
Table 3.
Molecular docking analysis showed that Compound 1 with 4-nitro­
benzenesulfonyl group at N position of 8-amino-4-methylquinolin-2
(1 H)-one interacted with HIV-RT and formed two hydrogen bonds with
amino acids Pro225 (3.18 Å) and Tyr318 (3.56 Å) and Compound 2 with
4-chlorobenzenesulfonyl group at N position formed three hydrogen
bonds with Lys101 (2.58 Å), Lys103 (2.81 Å) and Tyr318 (3.99 Å).
Compound 3 with isopropyl group at 1, 2 and 3 positions of the aryl
group of 8-amino-4-methylquinolin-2(1 H)-one formed two hydrogen
bonds with amino acids Gln190 (3.17 Å) and Tyr188 (2.83 Å), and
Compound 4 with 4-trifluoromethylbenzenesulfonyl group at N position
formed four H-bonds with amino acids Lys103 (2.82 Å), Gln190
(2.78 Å), Val179 (2.56 Å) and Tyr188 (2.66 Å).
Compound 5, with 4-methylbenzenesulfonyl group at N position of
8-amino-4-methylquinolin-2(1 H)-one, formed four H-bonds with
amino acids Gln190 (3.05 Å), Tyr188 (3.13 Å), Val189 (3.01 Å) and
Phe227 (3.34 Å) of HIV-RT protein. Additionally, it also interacted with
Tyr318 residue to form a π-interaction at a distance of 4.88 Å.
Compound 6 with 5-hydroxy thiophene-2-sulfonyl group at N posi­
tion of 8-amino-4-methylquinolin-2(1 H)-one formed five H-bonds, i.e.,
two with Lys101 (2.96 Å and 2.68 Å), one with Tyr318 (2.63 Å), one
with amino acid Lys104 (2.62 Å) and the fifth one with amino acid
Ser105 (3.41).
Compound 7 with benzoyl group at N position of 8-amino-4-meth­
ylquinolin-2(1 H)-one formed two H-bonds with Lys103 (3.00 Å) and
Phe227 (3.73 Å). Additionally, compound 7 also interacted with Tyr188
residue to form a π-bond at a distance of 4.85 Å, and Compound 8
formed two H-bonds with amino acids Val179 (3.11 Å) and Gly190
(3.21 Å).
Binding mode, hydrophilic and hydrophobic interaction of quinoline
derivatives 1–8 with certain amino acids of HIV-RT are displayed in
Fig. 2. The binding mode of all compounds with HIV-RT protein revealed
their behaviour as NNRTIs, just like the reference drug.
Analysis of docking results clearly displayed that all compounds
exhibited good interaction with amino acid residues of allosteric site of
HIV-RT protein, hence all of them may behave as plausible lead candi­
dates against HIV-RT. Compound 6 showed the highest extent of sta­
bility and safety profile.
Some scoring functions, like PLP1, PLP2, DS, LIG, Ludi values,
binding energy and predicted EC50 were generated from the molecular
docking simulation which helped in prediction of the stability of the
complexes formed between Compounds 1–8 and HIV RT protein. Their

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V.K. Singh et al. Computational Biology and Chemistry 98 (2022) 107675

Fig. 2. Docking interactions of quinoline derivatives within NNIBP of allosteric site of wild type HIV-1 RT.

6
V.K. Singh et al. Computational Biology and Chemistry 98 (2022) 107675

Fig. 2. (continued).

7
V.K. Singh et al. Computational Biology and Chemistry 98 (2022) 107675

Fig. 2. (continued).

8
V.K. Singh et al. Computational Biology and Chemistry 98 (2022) 107675

Fig. 2. (continued).

Table 4
Docking scores of quinoline derivatives 1–8 in ligand-receptor docked complexes.
S. No. PLP1 PLP2 Binding Energy PMF04 Lib-DS LigScore2 Dreiding Ludi2 Ludi3 PE ΔG Predicted EC50
8
1 91.05 80.92 -3.56 55.65 77.26 2.47 406 760 24.34 -5.75 2.51 × 10−
8
2 95.35 95.10 -45.15 77.24 107.93 5.33 441 788 11.19 -6.25 1.31 × 10−
9
3 99.26 102.97 -17.38 46.97 117.92 3.59 539 889 8.79 -7.64 1.28 × 10−
9
4 77.71 89.72 -27.73 58.14 94.45 4.24 407 810 10.77 -5.77 7.94 × 10−
8
5 95.42 93.86 -41.66 74.25 95.71 5.40 424 757 5.50 -6.01 2.69 × 10−
8
6 72.87 77.31 -13.41 46.62 72.06 3.67 399 749 4.66 -5.65 3.23 × 10−
9
7 79.57 78.99 -10.14 61.13 95.48 3.51 430 831 12.03 -6.09 4.89 × 10−
7
8 77.13 74.84 -24.77 39.13 90.87 4.19 382 681 3.33 -5.41 1.54 × 10−
10
Delavirdine 130.07 125.94 -49.61 62.17 132.20 5.58 547 1000 38.80 -7.75 1.00 × 10−
8
Nevirapine 66.92 69.08 -32.30 57.11 86.57 3.03 425 728 20.93 -6.02 5.24 × 10−

PLP: Piecewise Linear Potential; PMF: Potential of Mean Force; DS: Dock Score; Ludi2 and Ludi3: Empirical scoring functions derived from the Ludi algorithm; ΔG:
Predicted binding free energy (kcal/mol); Predicted EC50: Predicted 50% effective concentration of given compounds essential to reduce HIV-1 replication (µM)

Table 5
Inhibition constant (Ki) of the quinoline derivatives (1–8), and reference drugs.
S. No. 1 2 3 4 5 6 7 8 Delavirdine Nevirapine

Ki 6.14 2.56 2.55 5.97 3.98 7.30 3.48 0.001 2.12 3.92

results are presented in Table 4.
Strength of binding of the ligand with target protein was evaluated
with the help of Piecewise Linear Potential (PLP) functions. High nu­
merical value of PLP scores showed excellent binding with the receptor
protein. All the quinoline derivatives (1–8), revealed promising binding
affinity to the receptor protein (HIV-RT) via PLP1 and PLP2 scores
varying from 77.13 (Compound 8) to 99.26 (Compound 3) and 74.84
(Compound 8) to 102.97 (Compound 3), respectively. PLP1 scores of all
the quinoline derivatives were greater than the reference drug nevira­
pine and comparable to the reference drug delavirdine.
Binding efficiency of the quinoline derivatives 1–8 to the target
protein was evaluated by the Ligand Internal energy (LIG) involving van
der Waals and electrostatic interactions. All molecules had much better
LIG values than the reference drug nevirapine and were comparable to
the reference drug delavirdine, and thus, exhibited higher binding effi­
ciency than the reference drug.
Lib-Dock Score (DS), another important scoring function obtained
through the molecular docking simulation, once again supported the
interaction of the ligands with the HIV-RT protein in a better way. All
molecules showed significant values of dock score in comparison to the
reference drug nevirapine and delavirdine (Table 4).
The exact conformation of the complexes formed between quinoline
derivatives 1–8 and HIV-RT protein during docking process was evalu­
ated by empirical factors Ludi2 and Ludi3, derived from the Ludi al­
gorithm. Binding energy (ΔG) of the compounds was calculated by the
Ludi2 (lies between − 5.41 and − 7.64) value and predicted EC50 values
of compounds were calculated by the Ludi3 value (lies between 1.54 ×
10− 7 to 4.89 × 10− 9). The predicted EC50 values and binding energy of
all docked compounds were found to be comparable with known drug
nevirapine and delavirdine as shown in Table 4.
The results obtained from the docking studies showed that all com­
pounds showed promising interactions with the HIV-RT protein just like
the reference drugs.
After, performing the molecular docking studies. Ki was evaluated to
predict the bioactivity of all quinoline derivatives (1–8), along with the
reference drugs delavirdine and nevirapine. Ki represents the concen­
tration needed for the production of half maximal inhibition (Mondal
et al., 2020). Generally, good drug molecules have Ki value in the range
of 1–40 µM (Naidoo et al., 2020). The result of inhibition constant of all
the quinoline derivatives (1–8) with HIV-RT protein is summarized in
Table 5. Results showed that all the quinoline derivatives exhibited a
significant value of Ki (range between 2.552 and 7.308 µM), except

9
V.K. Singh et al. Computational Biology and Chemistry 98 (2022) 107675

Table 6
TOPKAT analysis of compounds 1-8.
Ligand Molecular DTP LD50 Log LC50 Log ALogP EC50 Log Rotatable Carcinogenicity/
formula bonds Mutagenicity
Predicted Confidence Predicted Confidence Predicted Confidence
(<10) (Discriminant
Value limit Value limit Value limit
score)

1 C16H15N3O5S 0.000 10 g/kg 10 g/kg 2.20 10 2.34 5.10 36.7 4 -3.65

2 C16H13ClN2O3S 0.970 1.5 g/kg 8.7 g/kg 3.21 2.8 2.85 4.84 23.6 3 -18.48
3 C25H32N2O3S 1.000 3.1 g/kg 10 g/kg 3.27 4.3 5.76 6.39 1.0 6 -30.80
4 C17H13F3N2O3S 0.954 67.0 mg/ 409 mg/kg 2.50 10 3.12 4.85 25.9 4 -12.57
kg
5 C17H16N2O3S 0.977 2.0 g/kg 10 g/kg 3.50 1.5 2.67 4.76 26.9 3 -22.64
6 C14H12N2O4S2 1.000 2.7 g/kg 10 g/kg 2.73 8.6 1.85 5.41 8.6 3 -10.85
7 C17H14N2O2 1.000 2.2 g/kg 10 g/kg 3.83 668.4 2.48 3.75 241.2 2 -7.56
8 C13H12N2O 0.983 1.8 g/kg 10 g/kg 3.71 740.4 2.69 4.80 17.9 2 -1.55
Nevirapine C15H14N4O 0.000 6.4 g/kg 10 g/kg 2.69 4.5 2.29 1.27 85800 1 -33.28
Delavirdine C23H29N5O3S 1.000 10 g/kg 10 g/kg 4.72 163.4 2.28 2.42 12800 6 -11.98

DTP: Developmental Toxicity Potential (< 0.70 score shows less toxicity); LD50: 50% Lethal Dose of a chemical that kills 50% of a sample population; LC50: 50% Lethal
Concentration (1/mol/h); EC50: 50% effective Concentration (1/mol); ALogP: Lipophilicity (<5 value shows good lipophilicity); Carcinogenicity: -ve discriminant
score shows no carcinogenicity

compound 8 against the HIV-RT protein.
Therefore, on the basis of inhibition constant, we can conclude that
all quinoline derivatives (1–8), exhibit significant binding affinity to­
wards the HIV-RT protein.
3.3. QSTR analysis
All the quinoline derivatives 1–8 were subjected to in silico toxicity
prediction by using the TOPKAT protocol in discovery studio
v20.1.0.19295 software. The results of TOPKAT analysis are summar­
ised in Table 6. The carcinogenicity of all molecules and reference drugs
(nevirapine and delavirdine) was evaluated by using NTP carcinoge­
nicity (Male rat) and NTP carcinogenicity (Female mouse) model in
TOPKAT module. TOPKAT analysis revealed that the molecules did not
show carcinogenicity, like the reference drug (nevirapine and dela­
virdine) because of their negative discriminant score.
calculate factors responsible for stability and biological activity of
compounds, like electron affinity (A), ionization potential (I), electro­
negativity (χ), chemical potential (μ) used to evaluate the releasing
tendency of an electron from equilibrium, global hardness (η) used to
evaluate the obstructions to charge transfer, global softness (S) which is
the contradictory of global hardness, global electrophilicity (ω) used to
illustrate the stabilization energy of the system on saturation, electron
donating power (ω-) used to study the ease of a molecule to release
fraction of charge, electro accepting power (ω + ) used to study the ease
of a molecule to accept a fraction of charge, net electrophilicity (Δω ± )
used to predict the favorable reactions and back-donation (ΔEback-d)
exhibiting the chemical reactivity via charge transfer. Results are pre­
sented in Table 8.
Where, I = - EHOMO and A = -ELUMO
ΔEGap = ELUMO - EHOMO and μ = - χ

Following the finite difference approximation and Koopman’s theo­

3.4. DFT analysis of quinoline derivatives rem, the values can be calculated as:

A smaller band gap energy obtained between the highest occupied μ = - ½ (I + A) = ½ (ELUMO + EHOMO)
molecular orbital (HOMO) and lowest unoccupied molecular orbital
η = - ½ (I - A) = ½ (ELUMO - EHOMO)
(LUMO), clearly indicates the higher intramolecular charge transfer
interactions of compounds with the targeted biological system that leads S = 1/ η and ω = μ2/2 η
to higher stability and greater biological activity of compounds with
target receptor enzyme. ω- = (3I + A)2 / 16 (I – A)
In DFT analysis, compound 1 showed HOMO-LUMO energy gap of ω+ = (I + 3 A)2 / 16 (I – A)
0.0399 eV, compound 2 of 0.1086 eV, compound 3 of 0.1138 eV,
compound 4 of 0.0847 eV, compound 5 of 0.1082 eV, compound 6 of Δω± = {ω+ - (ω-)} and ΔEback -d = - η / 4
0.1014 eV, compound 7 of 0.1183 eV, compound 8 of 0.1118 eV,
reference drugs delavirdine and nevirapine of 0.0856 eV and 0.1033 eV,
respectively.
3.5. Molecular dynamics trajectory analysis
From the DFT analysis, it was found that all test compounds had a
similar energy gap as that of the reference drugs delavirdine and nevi­
MD simulation presents an appropriate way to study atomic level
rapine. Thus, all molecules were found to be comparable to the reference
information about binding of ligands to target molecules (proteins/
drug and showed optimal binding affinity required for good biological
DNA/RNA) (Hollingsworth and Dror, 2018; Kumari et al., 2021). On the
activity (Singh et al., 2021a, 2021b; Halawa et al., 2020; Larsen et al.,
basis of MD trajectories, the RMSD, root-mean square fluctuation
2017). DFT analysis results of quinoline derivatives (1-8) and reference
(RMSF), radius of gyration (RoG), number of hydrogen bonds and
drug delavirdine are shown in Fig. 3. The results obtained from the DFT
binding free energy of the complexes were computed to analyse and get
analysis are summarised in Tables 7 and 8. Molecular electrostatic po­
insight into their structural stabilities, binding modes and binding
tential (MEP) of all the quinoline derivatives (1–8) and reference drugs
strengths of the designed molecules.
are presented in Fig. 3. In the MEP map blue, red and green colour
All quinoline derivatives (1–8) and Delavirdine belong to the first
represents negative (nucleophilic), positive (electrophilic) and inter­
generation NNRTIs, and also due to the structure similarity with quin­
mediate electrostatic potential respectively. MEP map explores that the
oline derivatives, delavirdine was taken as the reference dugs for mo­
binding affinity of all the quinoline derivatives (1–8) with HIV-RT
lecular dynamics simulation.
protein is similar to the reference drugs.
Energies obtained from DFT analysis were further employed to

10
V.K. Singh et al. Computational Biology and Chemistry 98 (2022) 107675

Fig. 3. DFT Analysis of compounds 1–8 and reference drug delavirdine; MEP map (rainbow surface) - blue, red and green surface represents positive, negative and
intermediate electrostatic potential sites, respectively (regions subject for nucleophilic or electrophilic attack). (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)

11
V.K. Singh et al. Computational Biology and Chemistry 98 (2022) 107675

Fig. 3. (continued).

3.5.1. Root-mean square deviation (RMSD) analysis

To evaluate the conformational/structural stability of the complexes,
Table 7
the RMSD was plotted (Fig. 4). This quantitative measurement was
DFT analysis of compounds 1–8 and reference drugs: calculation of binding
determined by aligning the backbone atom of the initial structure as a
energy.
reference to the 100 ns trajectory. The complexes started showing
S. No. Total Binding HOMO LUMO Dipole decreased fluctuations after 25 ns time and hence, they displayed sta­
Energy Energy energy Energy mag
bility. The average RMSD of compound 6 complex (0.63 nm or 6.3 Å)
1 -1546.12 -7.77 -0.1955 -0.1556 2.33 was relatively higher than that of the reference complex (0.55 nm or
2 -1800.05 -7.17 -0.2102 -0.1016 2.99
5.5 Å). This observation demonstrated that the HIV-RT: compound 6
3 -1692.54 -12.05 -0.1942 -0.0804 2.94
4 -1676.81 -7.80 -0.2026 -0.1179 2.51 complex exhibited comparable stability to the reference complex (i.e.,
5 -1380.87 -7.75 -0.1961 -0.0879 3.13 HIV-RT: delavirdine). Also, in case of compound 6 complex, after 25 ns
6 -1736.67 -6.61 -0.1959 -0.0945 3.45 lower and constant deviations were observed stating stable conforma­
7 -908.38 -7.56 -0.2024 -0.0884 2.88 tion for compound 6 complex.
8 -681.32 -5.82 -0.1791 -0.0673 1.77
Delavirdine -1778.13 -11.60 -0.1671 -0.0856 4.68
Nevirapine -866.71 -7.07 -0.1854 -0.0821 1.03 3.5.2. Root-mean square fluctuation (RMSF) analysis
The RMSF analysis was performed to get insight into the residue

Table 8
DFT analysis of compounds 1–8 and reference drugs: Calculation of various parameters.
Compounds 1 2 3 4 5 6 7 8 Delavirdine Nevirapine

EHOMO (eV) -0.1955 -0.2102 -0.1942 -0.2026 -0.1961 -0.1959 -0.2024 -0.1791 -0.1671 -0.1854
ELUMO (eV) -0.1556 -0.1016 -0.0804 -0.1179 -0.0879 -0.0945 -0.0884 -0.0673 -0.0856 -0.08205
I (eV) 0.1955 0.2102 0.1942 0.2026 0.1961 0.1959 0.2024 0.1791 0.1671 0.1854
A (eV) 0.1556 0.1016 0.0804 0.1179 0.0879 0.0945 0.0884 0.0673 0.0856 0.08205
Chemical potential (µ) -0.1755 -0.1555 -0.1373 -0.1602 -0.1420 -0.1452 -0.1454 -0.1232 -0.1263 -0.1337
Electronegativity (χ) 0.1755 0.1555 0.1373 0.1602 0.1420 0.1452 0.1454 0.1232 0.1263 0.1337
Chemical hardness (η) 0.0199 0.0543 0.0569 0.0423 0.0541 0.0537 0.0570 0.0559 0.0407 0.1033
Chemical softness (S) 50.25 18.41 17.57 23.64 18.48 18.62 17.54 17.88 24.57 9.68
Electrophilicity index (ω) 0.7897 0.2226 0.1652 0.3026 0.1857 0.1955 0.1850 0.1360 0.1953 0.08615
Electrodonating (ω-) 0.8600 0.3000 0.2414 0.3900 0.2642 0.2872 0.2658 0.2053 0.2486 0.3891
Electroaccepting (ω + ) 0.6870 0.1532 0.1041 0.2291 0.1221 0.1418 0.1201 0.0815 0.1381 0.1134
Net electrophilicity (Δω ± ) -0.17 -0.1468 -0.1373 -0.1609 -0.3863 -0.1424 -0.1457 -0.1238 -0.1105 -0.2757
Back-donation (ΔEback-d) -0.0049 -0.0135 -0.0142 -0.0105 -0.0135 -0.0134 -0.0142 -0.0139 -0.0101 -0.0258

12
V.K. Singh et al. Computational Biology and Chemistry 98 (2022) 107675

Fig. 4. RMSD of backbone atoms as a function of time for "HIV-RT: Dela­

virdine" (black) and "HIV-RT: Compound 6" (red). (For interpretation of the Fig. 7. SASA as a function of time for "HIV-RT: Delavirdine" (black) and "HIV-
references to colour in this figure legend, the reader is referred to the web RT: Compound 6" (red). (For interpretation of the references to colour in this
version of this article.) figure legend, the reader is referred to the web version of this article.)

Fig. 5. RMSF graph as a function of residues for "HIV-RT: Delavirdine" (black)

and "HIV-RT: Compound 6" (red). (For interpretation of the references to colour Fig. 8. Intermolecular hydrogen bonds for "HIV-RT: Delavirdine" (black) and
in this figure legend, the reader is referred to the web version of this article.) "HIV-RT: Compound 6" (red). (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.)

Table 9
Intermolecular interaction energies of the selected protein-ligand complexes.
Energy components (kJ/mol) HIV-RT: Delavirdine HIV-RT: Compound 6

Van der Waal energy (ΔEvdW) -136.236 -102.437

Electrostatic energy (ΔEelec) -18.728 -14.671
Polar solvation energy 96.126 56.360
SASA energy -15.075 -9.863
Binding energy (ΔGbind) -73.822 -70.594

Fig. 6. Rog graph as a function of time for "HIV-RT: Delavirdine" (black) and
"HIV-RT: Compound 6" (red). (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.)

flexibility of both the complexes (Aier et al., 2016). The RMSF graph of
HIV-RT: delavirdine and HIV-RT: compound 6 complexes were plotted
for 100 ns trajectory (Fig. 5). This analysis illustrated that the secondary
structures such as sheets and helices show low fluctuations while the
loop, coils and terminal ends show comparatively high fluctuations.
RMSF value of both the complexes showed the same pattern in the graph
emphasizing similar motion of atoms as well as the flexibility of the
structures. Average RMSF value of HIV-RT: compound 6 (0.213 nm) was Fig. 9. Binding energy contribution from individual residues of HIV-RT for
lower than that of HIV-RT: delavirdine (0.229 nm). Overall, the analysis Delavirdine (black), Compound 6 (red). (For interpretation of the references to
suggested that the binding of compound 6 as well as delavirdine with colour in this figure legend, the reader is referred to the web version of
HIV RT residues showed almost equivalent alteration in the conforma­ this article.)
tion, indicating that both complexes displayed similar inhibitory action
and a balance between flexibility and stability.

13
V.K. Singh et al.
Fig. 10. Binding energy of binding pocket residues of HIV-RT for Delavirdine
(black), Compound 6 (red). (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.)
3.5.3. Radius of gyration (RoG) analysis
Compactness and folding properties elucidate the functional and
dynamic behaviour of proteins/complexes that may be affected due to
the presence of a ligand (Ali et al., 2019; Khan et al., 2020). A higher
value of radius of gyration (RoG) implies the loose packing of pro­
tein/complex and vice-versa. The RoG plots for both the complexes are
shown in Fig. 6. Initially, compound 6 complex illustrated a relatively
less compact structure till 40 ns and then compactness of both the
complexes was nearly the same. The average RoG of compound 6
complex (3.575 nm) was similar to that of the reference complex
(3.520 nm) showing alike compactness in both structures.
3.5.4. Solvent accessible surface area (SASA) analysis
SASA represents the quantitative measurement of the surface area of
amino acids exposed to solvent. An increase or decrease in SASA reflects
changes in solvent accessibility of amino acid residues that may be
caused due ligand binding (Uba and Yelekçi, 2018). The SASA plots for
both the complexes are shown in Fig. 7. The mean values of SASA of the
HIV-RT: delavirdine and HIV-RT: compound 6 complexes were
315.11 nm2 and 316.05 nm2, respectively. The SASA value of the
HIV-RT: compound 6 complex is slightly higher than that of HIV-RT:
delavirdine. This result showed a similar solvent interaction for both
Scheme 1. Synthesis of compound 8-amino-4-methylquinolin-2(1 H)-one Reagents & condition: (i) Ortho-phenyl diamine, H2SO4 (1% mmol), Temp 140 – 150 0C
for 3–4 h.
14
Computational Biology and Chemistry 98 (2022) 107675
complexes and validated the residue flexibility analysis (RMFS) and
compactness (RoG) results.
3.5.5. Hydrogen bonding analysis
The structural stability and interaction specificity of protein-ligand
complex can be determined by hydrogen bonds (Rathod, 2019; Pace
et al., 2014). The intermolecular hydrogen bond formation between the
protein and ligand was analysed using a 100 ns trajectory for the
HIV-RT: compound 6 and HIV-RT: delavirdine complexes, Fig. 8. The
consistent and large number of hydrogen bonds formed between HIV RT
and compound 6 implies a stable target-inhibitor complex. The number
of hydrogen bonds for compound 6 complex (average hydrogen bond
number per frame 0.124) was found to be higher than that of delavirdine
complex (average hydrogen bond number per frame 0.05). This clearly
depicted a stronger binding affinity between compound 6 and HIV-RT
which indicates the stronger inhibitory activity of compound 6 than
delavirdine.
3.5.6. Binding free energy and residue interaction energy
The binding free energy is the sum of the total "non-bonded inter­
action energy" which reconfirms the inhibitory behaviour of the docked
candidate predicted by molecular docking analysis (Zhang et al., 2020).
The binding free energy was calculated using the MMPBSA tool (Gen­
heden and Ryde, 2015; Kumari et al., 2014). To calculate the binding
free energy of the system, the trajectory after 25 ns was taken at an
interval of 100 ps which had a total of 700 frames. The contributions of
different interaction energies for both complexes have been summarized
in Table 9. The interaction energy analysis revealed that the van der
Waals, electrostatic and non-polar solvation energy terms contributed
favourably to the binding of the inhibitor. Specifically, van der Waals
energy showed the most favourable contribution towards the negative
binding free energy of both complexes. Besides these interaction energy
components, binding energy per residue actively participating in the
receptor-ligand binding was evaluated as shown in Fig. 9. The residue
having binding affinity greater than 1.5 kJ/mol was considered as a
hotspot residue. Most of the residues showing high affinity of binding
with the ligand belonged to the catalytic/binding pocket residues and
are represented in Fig. 10. The number of these active-site residues
participating in the receptor-ligand binding was slightly higher for
delavirdine (Leu100, Val103, Val106, Arg172, Lys173, Val179, Tyr181,
V.K. Singh et al. Computational Biology and Chemistry 98 (2022) 107675

Scheme 2. Reagents & conditions; A: (i) – (vi) substituted-benzenesulfonyl chloride, Et3N, Dry DCM, Temp 30–36 0C for 8–10 h; B: (vii) – (viii) benzoyl chloride/
propargyl bromide, Et3N, Dry DCM, Temp 30–36 0C for 8–10 h.

Table 10
Cytotoxicity of compounds 1–8 against Human embryonic kidney (HEK293) cell lines (data showing cell viability at different concentrations of compounds).
Compound Concentration in µg/mL

10 20 40 50 70 80 90 100

% cell viability

1 96.35 98.35 77.45 76.21 75.69 71.61 70.92 70.11

2 97.06 97.63 93.95 84.03 83.76 82.55 79.01 75.68
3 98.73 80.12 78.94 75.52 75.23 73.50 73.57 69.74
4 96.47 88.61 80.71 74.91 73.03 72.41 70.55 69.24
5 97.02 96.25 92.77 90.15 88.02 84.99 82.81 79.47
6 98.79 94.44 91.72 87.01 81.47 79.20 76.97 70.08
7 97.31 93.42 90.68 89.40 87.29 82.55 80.79 77.85
8 95.56 83.49 80.84 73.36 72.59 71.27 71.12 70.32

Tyr188, Leu234, Tyr318) than compound 6 (Leu100, Val103, Val106,
Tyr181, Tyr 188, Val189, Trp229, Leu234, Tyr318). Some residues
negatively favoured the binding of inhibitors (Glu169 for delavirdine;
Asp237, Asp177, His235 for compound 6). The most favourably
contributing residue for compound 6 complex was Tyr188, with a
binding energy of − 5.50 kJ/mol, while that for reference complex was
Try181, with a binding energy of − 8.50 kJ/mol. Overall MD analyses
manifested that HIV RT: compound 6 complex showed properties
comparable to HIV RT: delavirdine complex and both were quite stable
complexes with nearly equivalent inhibitory action.
The result of MD simulation analyses based on the RMSD, RMSF,
RoG, SASA, Hydrogen bonds and energy plots convey the similar type of
motional behaviour of both the complexes and a stronger inhibitory
action of compound 6 complex.
3.6. Chemistry
The quinoline derivatives, 1–8, have been synthesized from the re­
action of 8-amino-4-methyl-1 H-quinoline-2-one (Scheme 1) with
different types of sulphonyl chloride/benzoyl chloride/propargylic
bromide as outlined in Scheme 2. Purification and characterization data
of final quinoline derivatives have been provided in supporting infor­
mation section.
3.7. Cytotoxicity evaluation
Cytotoxicity screening of quinoline derivatives was done using
HEK293 (Human embryonic kidney) cell lines maintained in DMEM
supplemented with 10% FBS and 1% antibiotic cocktail. Cytotoxicity
evaluation of all compounds (1–8) against HEK293 showed that all
compounds were much less toxic. Cytotoxicity at different

15
V.K. Singh et al. Computational Biology and Chemistry 98 (2022) 107675

Fig. 11. Cytotoxicity of quinoline derivatives against HEK293 (Human Embryonic Kidney) cells.

concentrations is displayed in Table 10. 3.8. Anti-HIV assay and cytotoxicity

The cytotoxicity evaluation displayed that all compounds (1–8) were
found to be less toxic against HEK293 cell lines at different concentra­
tions as shown in Fig. 11.
Quinoline derivatives 1–8 were screened for their biological inhibi­
tory activity against HIV-1 (NL4–3) and HIV-2 (ROD) using CD4 + cells
and the results are shown in Table 11. 9-[(R)− 2-(Phosphonomethoxy)
propyl] Adenine (PMPA) and AnorMeD3100 (AMD3100) were used as

16
V.K. Singh et al.
Table 11
HIV-1 and HIV-2 activity of compounds 1–8 tested in CD4 + cells.
Compound Concentration Unit Cellular Toxicity
CC50
1 µg/mL 74.81
2 µg/mL > 100
3 µg/mL > 100
4 µg/mL > 100
5 µg/mL > 100
6 µg/mL > 100
7 µg/mL > 100
8 µg/mL 52.02
PMPA µg/mL > 100
AMD3100 µg/mL > 100
CC50: 50% cytotoxic concentration of compound evaluated in this cell line
EC50: 50% effective concentration required to inhibit HIV-induced cytopathogenic effect in cell culture
SI: Selective Index (ratio of CC50 to EC50)
the reference for anti-HIV screening.
Anti-HIV screening showed that the compound 6 was active against
both HIV-1 and HIV-2 strains with significant inhibition. The selectivity
index (SI) of compound 6 against HIV-1 was 2.65 and against HIV-2 was
2.32. Compound 5 was active against only HIV-2 and its selectivity
index was 9.18. The anti-HIV activity of these two compounds may be
attributed to the methyl group present at the para-position of the ben­
zene ring, presence of thiophene ring and the presence of sulphonamide
linkage.
In summary, in silico study results of compounds 5 and 6 were quite
comparable to that of known reference drugs, which supported the
favourable inhibitory activity.
4. Conclusion
Considering the in-silico results, a novel series of quinoline scaffolds,
1–8, containing sulphonamide and amide linkages were developed as
the probable NNRTIs against HIV. All the compounds showed significant
drug-like properties and some of the compounds showed better drug-like
properties than the reference drugs. Molecular docking studies also
showed that the binding interaction of all the compounds into NNIBP of
HIV-RT was similar to that of the standard drug, and hence compounds
were supposed to be possible effective NNRTIs. All the compounds were
subjected to their anti-HIV activity against both HIV-1 and HIV-2 strains
using TZM-bl cells. The compound 6 was found to be active against both
HIV-1 (SI = 2.65) and HIV-2 (2.32), and it was interesting to note that
the compound 5 was found to be active only against HIV-2 (SI = 9.18).
The MD studies revealed that compound 6 complex had a stable struc­
ture with better inhibition potential that the reference complex. These
results paved the way for the development of novel and potent mole­
cules with promising anti-HIV activity in future.
CRediT authorship contribution statement
Vishal K. Singh synthesized and purified all the compounds along
with drafting of the manuscript. Priyanka Kumari and Anup Som
performed MD simulations and drafted the MD write-up. Richa Mishra,
Aditya K. Yadav and Nand K. Ram did computational study (Docking
and DFT analysis) of the compounds. Dominique Schols performed anti-
HIV screening of all compounds. Pradeep Kumar performed cytotox­
icity screening. Ramendra K. Singh Conceptualization, review, draft­
ing, editing and supervision.
Conflict of interest
The authors report no conflicts of interest.
17
Computational Biology and Chemistry 98 (2022) 107675
HIV-1 NL4.3 SI (HIV-1) HIV-2 ROD SI (HIV-2)
EC50 EC50
74.81 1 > 74.81 1
> 100 1 > 100 1
> 100 1 > 100 1
> 100 1 > 100 1
> 100 1 10.89 9.18
37.66 2.65 43.05 2.32
> 100 1 > 100 1
> 52.02 1 52.02 1
2.9 34.48 1.24 80.64
24.27 4.16 15.53 6.43
Acknowledgement
Financial assistance to Vishal K Singh in the form of Junior Research
Fellowship (Ref No: 349/CSIR-UGC NET DEC. 2017) by University
Grants Commission, New Delhi, India is sincerely acknowledged.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.compbiolchem.2022.107675.
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