Rational Design, Synthesis, and In Vitro Neuroprotective Evaluation of Novel Glitazones for PGC-1α Activation via PPAR-γ: a New Therapeutic Strategy for Neurodegenerative Disorders

Article abstract of DOI:10.1007/s12640-019-00132-9

In the present study, two structurally diverse novel glitazones were designed and synthesized for activation of central PGC-1α signaling through stimulation of PPAR-γ receptor. The functional interaction between PGC-1α and PPAR-γ is a key interaction in the normal physiology of neuroprotective mechanism. Therefore, activation of PPAR-γ–dependent PGC-1α co-activator signaling could be an effective strategy to exhibit neuroprotection in several neurodegenerative disorders like Alzheimer’s disease, Parkinson’s disease, and cerebral ischemia. As part of rational design, analogs were designed manually based on principles of bioisosterism, followed by virtually screened using docking to predict the mode of interaction of compound towards the binding site and molecular dynamic simulation to observe the structural changes that occur during compound interaction with active site. The designed two glitazones (G1, G2) were synthesized and structurally analyzed. As part of evaluation, synthesized glitazones were subjected for preliminary neuroprotective evaluation in Lipopolysaccharide (LPS) intoxicated SH-SY5Y neuroblastoma cells. The results indicate that pre-treatment with synthesized glitazones have increased the percentage cell viability, protected the cell morphology, and decreased the release of pro-inflammatory cytokines (IL-1β, TNF-α), lipid peroxide (LPO), and nitric oxide (NO) level in LPS intoxicated SH-SY5Y cells. Interestingly, among the two glitazones, G2 has shown significant neuroprotection in comparison to G1 and neuroprotective effect exerted by G2 was similar and comparable with the standard pioglitazone. Altogether, neuroprotection exhibited by this non-thiazolidione–based glitazones during neuroinflammatory conditions may be attributed to the activation of central PGC-1α signaling via PPAR-γ receptor.

Full text of DOI:10.1007/s12640-019-00132-9

Neurotoxicity Research
https://doi.org/10.1007/s12640-019-00132-9
ORIGINAL ARTICLE
Rational Design, Synthesis, and In Vitro Neuroprotective Evaluation
of Novel Glitazones for PGC-1α Activation via PPAR-γ: a New
Therapeutic Strategy for Neurodegenerative Disorders
Antony Justin1 & Subhankar Mandal2 & P. Prabitha2 & S. Dhivya2 & S. Yuvaraj2 & Pradeep Kabadi3
&
Satheesh John Sekhar1 & C. H. Sandhya1 & Ashish D. Wadhwani1 & Selvaraj Divakar1 & Jeyabalan Jeyaram Bharathi1
&
Priya Durai2 & B. R. Prashantha Kumar2
Received: 27 February 2019 /Revised: 16 October 2019 /Accepted: 25 October 2019
#
Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
In the present study, two structurally diverse novel glitazones were designed and synthesized for activation of central PGC-1α
signaling through stimulation of PPAR-γ receptor. The functional interaction between PGC-1α and PPAR-γ is a key interaction
in the normal physiology of neuroprotective mechanism. Therefore, activation of PPAR-γ–dependent PGC-1α co-activator
signaling could be an effective strategy to exhibit neuroprotection in several neurodegenerative disorders like Alzheimer’s
disease, Parkinson’s disease, and cerebral ischemia. As part of rational design, analogs were designed manually based on
principles of bioisosterism, followed by virtually screened using docking to predict the mode of interaction of compound towards
the binding site and molecular dynamic simulation to observe the structural changes that occur during compound interaction with
active site. The designed two glitazones (G1, G2) were synthesized and structurally analyzed. As part of evaluation, synthesized
glitazones were subjected for preliminary neuroprotective evaluation in Lipopolysaccharide (LPS) intoxicated SH-SY5Y neu-
roblastoma cells. The results indicate that pre-treatment with synthesized glitazones have increased the percentage cell viability,
protected the cell morphology, and decreased the release of pro-inflammatory cytokines (IL-1β, TNF-α), lipid peroxide (LPO),
and nitric oxide (NO) level in LPS intoxicated SH-SY5Y cells. Interestingly, among the two glitazones, G2 has shown significant
neuroprotection in comparison to G1 and neuroprotective effect exerted by G2 was similar and comparable with the standard
pioglitazone. Altogether, neuroprotection exhibited by this non-thiazolidione–based glitazones during neuroinflammatory con-
ditions may be attributed to the activation of central PGC-1α signaling via PPAR-γ receptor.
.
.
.
.
.
.
Keywords PGC-1α PPAR-γ Glitazones Docking Molecular dynamic simulation Cytokines Free radicals
Introduction
Neuroinflammation refers to inflammation in the nervous
tissue and it is a host defensive mechanism in the brain
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s12640-019-00132-9) contains supplementary
material, which is available to authorized users.
tissue due to infection, direct cell injury, accumulation of
toxic metabolites, or autoimmunity leads to metabolic al-
terations within the central nervous system (CNS)
(DiSabato et al. 2016). Increasing evidences are
supporting the imperative role of neuroinflammation in
the pathogenesis of several neurodegenerative diseases
such as Alzheimer’s disease (AD), Multiple sclerosis
(MS), Parkinson’s disease (PD), amyotrophic lateral scle-
rosis (ALS) and cerebral ischemia (Zhao et al. 2019).
Neuroinflammation is characterized by rapid activation
of microglia followed by production of pro-inflammatory
* B. R. Prashantha Kumar
brprashanthkumar@jssuni.edu.in
1
Department of Pharmacology, JSS College of Pharmacy, Ooty, Tamil
Nadu 643 001, India
2
Department of Pharmaceutical Chemistry, JSS College of Pharmacy,
Mysuru, Karnataka 570015, India
3
Bioanalytical Division, Biocon Pvt. Ltd, Bengaluru 560100, India

Neurotox Res
cytokines, oxy-radicals, and infiltration of various periph-
eral immune cells into the brain tissue and these cellular
events will contribute to neurodegeneration (Jin et al.
2010). Activation of toll-like receptors-4 (TLR-4) is
expressed in the microglial membranes by pathogens or
h o s t - d e r i v e d m o l e c u l e s t h a t i n i t i a t e t h e
neuroinflammatory pathways through activation of nucle-
ar factor-kappa B (NF-κB) signal transduction pathway
(Rosenberger et al. 2014). The increased expression of
NF-κB will further trigger the release of pathophysiolog-
ical factors of neuroinflammation like interleukins-1β (IL-
1β), interleukins-6 (IL-6), tumor necrosis factor-α
(TNF-α), adhesion molecules, prostaglandins, and reac-
tive oxygen species (ROS) resulted in neurodegeneration
and neuronal death (Lattke et al. 2017).
Peroxisome proliferator-activated receptors (PPARs)
are members of the nuclear receptor superfamily, and they
regulate various gene expressions either through ligand-
dependent or independent molecular processes (Clarke
et al. 1999). There are three isoforms of PPAR receptor:
alpha (α), beta (β)/delta ( ), and gamma (γ), all of which
form obligate heterodimers with the retinoid-X-receptor
(RXR), which bind to the deoxyribonucleic acid (DNA)
response elements resulting in respective gene regulation
(Tyagi et al. 2011). Among the three isoforms, PPAR-γ is
mainly focused by several researchers for neuroprotection
due to its wide distribution in the different brain regions
(Moreno et al. 2004) and exhibits anti-neuroinflammatory
activity upon activation through modulating NF-kB sig-
naling pathway (Choi et al. 2017). The report has shown
that deficiency of neuronal PPAR-γ receptors increases
intensity of brain damage in response to cerebral ischemia
(Zhao et al. 2009) and activation of PPAR-γ by its agonist
exerted neuroprotection by attenuation of brain cytokine
levels in cerebral ischemic rats (Luo et al. 2006) and
Parkinson’s disease (Randy and Guoying 2007).
Recent evidences indicate that agonist activity at
PPAR-γ receptor has decreased the expression of
autophagy-related proteins, including microtubule-
associated protein 1 light chain 3 type II (LC3-II),
beclin-1, and cathepsin D (Li et al. 2017) and maintains
the acute mitochondrial integrity (Patel et al. 2017) in
traumatic spinal cord injury animal model. Activation of
PPAR-γ–dependent signaling in advanced glycation end
product treated human neural stem cells has shown neu-
roprotection suggesting that PPAR-γ ligands are promis-
ing agents in the therapeutic management of patients with
neurodegenerative diseases (Chiang et al. 2017). In gen-
eral, stimulation of PPAR-γ with ligands resulted in
heterodimerize with RXR and recruits the proliferator-
activated receptor gamma co-activator 1-alpha (PGC-1α)
to form a regulatory complex (Viswakarma et al. 2010). It
could regulate the expression of several target genes
involved in mitochondrial dysfunction, oxidative stress,
proteasomal dysfunction, autophagy, neuroinflammation,
and apoptosis lead to neuronal survival and neuroprotec-
tion (Katsouri et al. 2016).
PGC-1α is a metabolic co-activator contributes in the glu-
cose, lipid, and energy metabolism, also promotes mitochon-
drial biogenesis and exhibits neuroprotective effects against
several neurodegenerative diseases (Róna-Vörös and Weydt
2010). The reports implicated that expressions of PGC-1α
were remarkably decreased in the Alzheimer’s disease brain
(Qin et al. 2009) and up-regulation of PGC-1α protected the
neurons against amyloid β1–42–induced neurotoxicity (Zhu
et al. 2012; Katsouri et al. 2011). Impairment of the PGC-1α
triggers the degeneration of neurons by mitochondrial dys-
function (Johri et al. 2013) and induction of PGC-1α regulates
the gene expression of several ROS enzymes such as super-
oxide dismutase 1 and 2, catalase, and glutathione peroxidase-
1 which decreasing oxidative stress and increasing mitochon-
drial biosynthesis resulted in neuroprotection (St-Pierre et al.
2006). In this context, stimulation of PGC-1α signaling
through activation of PPAR-γ receptors could be a promising
strategy for neuroprotection in neurodegenerative conditions
(Hunter and Bing 2007).
Hence, we proposed to design and synthesize few
glitazones as novel glitazars which could activate the
PPAR-γ–dependent PGC-1α signaling in neurons that
may have more therapeutic impact on neurodegenerative
disorders. In the present study, structurally diverse two
novel glitazones were designed using in-silico ligand-
based drug design methods. Then binding affinity and
interaction analysis of designed glitazones with PPAR-γ
were performed through docking studies. The compounds
were subjected to molecular dynamics study using leap-
frog verlet dynamics integrator to find out the energy pa-
rameters such as potential energy, kinetic energy, and con-
formational changes of the docked protein-ligand com-
plexes (PPAR-γ and PGC-1α). The designed glitazones
were synthesized using appropriate synthetic schemes
and the synthesized glitazones were purified and analyzed
to confirm their molecular structures. Thereafter, com-
pounds were subjected to preliminary neuroprotective
analysis in lipopolysaccharide (LPS) intoxicated SH-
SY5Y neuroblastoma cell lines. The neuroprotective ac-
tivity was assessed by performing cell viability assay,
plotting dose response curve of test glitazones vs standard
glitazone and morphological observation in LPS-
intoxicated SH-SY5Y cell lines. The pathophysiological
parameters during neuroinflammation such as pro-
inflammatory cytokines (TNF-α, IL-1β) and ROS-like
lipid peroxide (LPO) and nitric oxide (NO) were estimat-
ed to assess the level of anti-neuroinflammatory and free
radical scavenging potential of novel glitazones in LPS-
mediated neuroinflammatory conditions.

Neurotox Res
Materials and Methods
Additionally, SHAKE algorithm was introduced to fix all
bond and angle constraints during the simulation process.
Finally, energy parameters such as potential energy, kinetic
energy, and conformational changes of the complexes and
apo-protein were studied (the PPAR-γ bound to PGC-1α
and rosiglitazone dynamics simulation movie is available as
supporting information).
Rational Design of Glitazones for PGC-1α Activation
via PPAR-γ Binding Using Molecular Docking
Docking is a lock and key method; it can be executed with
various algorithms. CDOCKER algorithm is basically a sim-
ulated annealing-based docking that utilizes CHARMm force
field for simulation (Ewing et al. 2001, Wu et al. 2003a, b).
Prior to docking, three-dimensional (3D) structure of the pro-
tein (PDB ID-3CS8) (Li et al. 2008) was downloaded from
Structural database PDB (http://www.rcsb.org). The X-ray
crystal structure of PPAR-γ bound to PGC-1α having 2.3 Å
resolution was processed using prepared protein protocol to
make sure that side chain, loops region, and other conformers
were removed. It was also ought to be free from water mole-
cules because in this case there is no role of water molecule to
bridge the connection between ligand and active site.
Subsequently, ligands were prepared to remove the duplicates
and to fix its chemical valences. The prepared protein and
ligands were subjected to docking. The binding site of the
protein was identified through receptor cavity tool using site
search and flood filling algorithm. The site-1 had the volume
of 1065 Ǻ and a point count of 8520 in equal grid spacing of 0.
5(X), 0.5(Y), and 0.5(Z) directions, respectively. The com-
pounds Glitazone 1 (G1), Glitazone 2 (G2), and standard
rosiglitazone were docked with the defined sphere site of 22.
91(X), 2.019(Y), and 25.46(Z) using random conformations
with 1000 steps of dynamics with default simulation anneal-
ing. The final best pose results were taken for interaction anal-
ysis and molecular dynamics (the PPAR-γ bound to PGC-1α
and G1 docked complex in the form of pdb file is available as
supporting information).
Synthesis and Characterization
Synthetic work was done by procuring available laboratory-
grade reagents and analytical grade solvents, Thin layer chro-
matography (TLC) was performed to monitor the reactions,
and all the reported compounds were purified by column chro-
matography. Infrared (IR) spectra of compounds were record-
ed on Shimadzu FT-IR 8400-S spectrophotometer by KBr
1
pellet technique and are expressed in cm−1. H-NMR and
13C-NMR spectra were recorded on Bruker 400 MHz FT-
NMR spectrophotometer using DMSO D6 and CDCl3 as the
solvents and TMS as internal standard (δ ppm). Mass spectra
were obtained using LC-MS ACQUITY UPLC mass spec-
trometer under ES ionization at 70 eV and time of flight de-
tector. Retention time (RT) was also observed on the same
UPLC instrument under optimized chromatographic condi-
tions; Column: C18 1.7 micron, flow rate 0.4 ml/min, run time
15 min, injection volume 10 μL, Detector: PDA Detector,
TOF, Elution: Gradient, Mobile phase: 0.1% FA in water
and Acetonitrile, Column temperature 60 °C.
General Procedure for the Synthesis of Sodium Salt of Vanillin
and p-Hydroxy Benzaldehyde
Sodium salt of vanillin and p-hydroxy benzaldehyde was pre-
pared by mixing 0.02 M equivalents of vanillin or p-hydroxy
benzaldehyde in 20 ml of water in a beaker with 0.02 M so-
dium hydroxide and the solution was mechanically stirred at
room temperature until whole solution became clear (Bala
et al. 2010).
Molecular Dynamics and Simulation
The best pose of a compound having maximum favorable
bonded interaction with protein and dock score was subjected
to 1000 ps dynamics with leapfrog verlet dynamics integrator
(Tuckerman et al. 1992). In this study, apo-protein (PPAR-γ
with PGC-1α) and other three receptor-ligand interaction
complexes such as PPAR-γ with PGC-1α and compound
G1; PPAR-γ with PGC-1α and compound G2; and PPAR-γ
with PGC-1α and standard rosiglitazone were considered.
Initially, complexes and apo-protein were geometrically opti-
mized using popular minimization techniques steepest descent
and conjugate gradient for 1000 steps individually.
Consequently, all the samples were gradually simulated and
heated for a temperature of 50 K followed by its ramping to
300 K and then equilibrated by 100 steps at 300 K for even
distribution of atoms in the system. At last, dynamics produc-
tion was processed for 1000 ps using CHARMm
(Vanommeslaeghe et al. 2010; Skeel et al. 1997).
General Procedure for Linking Sodium Salts of Vanillin
and p-Hydroxy Benzaldehyde with Chloroacetic Acid
It was prepared by modifying Williamson’s ether synthesis
protocol as described in Zubrys and Siebenmann 1954. The
prepared solution of sodium salt of vanillin and p-hydroxy
benzaldehyde was taken in a beaker, to that, 30 ml of chloro-
form was added and the solution was stirred for a period of
10 min at room temperature. To the above solution, 0.02 M
chloroacetic acid crystals dissolved in 15 ml of distilled water
and 0.02 M sodium hydroxide pellets dissolved in 15 ml of
distilled water were added. The reaction mixture was contin-
ued to stir for another 10 min. The resultant mixture was
allowed to settle, then the whole mixture was poured into a

Neurotox Res
separating funnel and the aqueous layer was taken in a round
bottom flask, and the solution was refluxed with stirring at a
temperature of 120–140 °C for 3 h. Reaction mixture was
allowed to cool and concentrated HCl was added dropwise
until the precipitation ceases, filtered, and 20 ml of chloroform
was added and suspended in a separating funnel. To this mix-
ture, saturated solution of sodium bicarbonate was added until
whole precipitate goes into the aqueous phase. Sequentially,
aqueous phase was acidified with concentrated HCl to precip-
itate out the product.
55.999, 65.826, 110.342, 112.990, 115.335, 122.620,
123.599, 130.231, 144.764, 149.507, 150.546, 157.338,
158.218, 164.489, 172.386. MS (m/z): M+1 peak found
287.4666, (M+1 peak calculated 287.10). Mass fragments
(m/z): 287.4666, 278.4613, 271.9437. HPLC (RT):0.50 min.
2-(4-((1E)-(2-(2,4-dinitrophenyl)hydrazin-1-
ylidene)methyl)phenoxy)acetic acid (G2)
Orange amorphous solid, yield 82%, mp 193–195 °C. IR
(KBr, cm−1):3618.58 (N-H), 3294.53 (O-H, Acid), 3109.35
(Ar-H), 1728.28 (C=O, Acid), 1604.83 (C=N, Imine),
1504.53 (NO2), 1334.78 (C-N), 1257.63 (C-O). 1H NMR (δ
ppm, DMSO D6): 4.552 (s, 2H, CH2), 6.870–6.893 (d, 2H,
ArH), 7.613–7.635 (d, 2H, ArH), 7.976–8.000 (d, 1H, ArH),
8.196 (s, 1H, CH=N), 8.203–8.226 (m, 1H, ArH), 8.956 (s,
1H, ArH), 11.327 (s, 1H, NH). 13C NMR: (δ ppm, DMSO
D6): 65.037, 77.460, 77.786, 77.983, 78.105, 115.047,
116.891, 123.311, 126.983, 129.108, 129.268, 129.662,
144.916, 148.550, 160.062, 170.155. MS (m/z): M+1 peak
found 361.1061, (M+1 peak calculated 361.07). Mass frag-
ments (m/z): 361.1061, 351.1051, 349.2133. HPLC (RT):8.56
min.
2-(4-formylphenoxy) acetic acid (1)
Pale brown solid, yield 75%, mp 185–187 °C. IR (KBr,
cm−1): 3448.84 (O-H, Acid), 3069.00 (Ar C-H), 1759.14
(C=O, Acid), 1651.12 (C=O, Aldehyde), 1427.37 (C-C),
1
1226.77 (C-O). H NMR (δ ppm, CDCl3):4.487 (s, 2H,
CH2), 6.837–7.655 (m, 4H, ArH), 9.694 (s, 1H, CHO). MS
(m/z): M+1 peak found 181.0835, (M+1 peak calculated
181.16). Mass fragments (m/z): 181.0835, 182.0845. HPLC
(RT) 3.19 min.
2-(4-formyl-2-methoxyphenoxy) acetic acid (2)
Off white solid, yield 60%, mp 145–147 °C. IR (KBr,
cm−1):3510.56 (O-H, Acid), 3091.99 (ArC-H), 1766.85
(C=O, Acid), 1643.41 (C=O, Aldehyde), 1411.94 (C-C),
In Vitro Neuroprotective Evaluation of Novel
Glitazones in LPS Intoxicated SH-SY5Y Cell Lines
1
1273.06 (C-O). H NMR (δ ppm, CDCl3):3.849 (s, 3H,
OCH3), 4.656(s, 2H, CH2), 6.814–7.347 (m, 3H, ArH),
9.758 (s, 1H, CHO). MS (m/z): M+1 peak found 211.0897,
(M+1 peak calculated 211.05). Mass fragments (m/z):
211.0897, 212.0941. HPLC (RT):3.33 min.
Cell Culture, IC50, and EC50 Determination of Synthesized
Glitazones
SH-SY5Y human neuroblastoma cells were obtained from
National Centre for Cell Science (NCCS), Pune, India and
maintained as per the standard protocol in Minimum
Essential Medium (MEM) supplemented with 10% fetal bo-
vine serum (FBS), amphotericin (3 μg/ml), gentamycin (400
μg/ml), streptomycin (250 μg/ml), and penicillin (250 units/
ml) in a carbon dioxide incubator at 5% CO2. The neuronal
viability in terms of mitochondrial metabolic function was
evaluated by MTT [(3-[4,5-dimethylthiazol-2-yl]-2,5
diphenyl tetrazolium bromide)] assay based on the principle
involving reduction of MTT to formazan. To find the cytotox-
icity of the compounds, SH-SY5Y cells were incubated with
seven different doses of novel glitazones between 1.953125
and 125 μg/ml for 24 h. Then SH-SY5Y cells were washed
with phosphate-buffered saline (PBS) and incubated with
MTT (5 mg/ml) in PBS for 3 h at 37 °C in 5% CO2. After
further washing, the formazan crystals were dissolved with
isopropanol and the absorbance was measured at 570 nm.
The standard formula was applied to calculate the % cytotox-
icity of the novel glitazones in SH-SY5Y cells and IC50 value
was calculated for further assays (Kaja et al. 2011). To assess
the protective effect, the SH-SY5Y cells were pre-treated with
eight different doses of novel glitazones between 1.953125
and 250 nM and each group was incubated with LPS 10
ng/ml for 24 h. The same procedure was carried out as above
General Procedure for the Synthesis of Schiff Base
The imine (-CH=N-) linkage between an amine and aldehyde
was done by reacting equivalent quantities of arylamine and
aldehyde in the presence of catalytic amount of glacial acetic
acid, stirred without heating according to the procedure report-
ed by Hugo Schiff (1864) (Shah and Baseer 2012). Equimolar
quantities of formylphenoxyacetic acid and substituted aro-
matic amine were dissolved in absolute ethanol and mixed
together, to that, few drops of glacial acetic acid and few
activated molecular sieves were added and finally stirred for
8–12 h. The reaction was monitored from time to time. The
formed precipitate was filtered, washed with minimal quanti-
ties of cold aqueous ethanol, and purified by column chroma-
tography using 25% ethyl acetate in pet ether as mobile phase.
2 - ( 2 - m e t h o x y - 4 - ( ( E ) - ( p y r i d i n - 2 - y l i m i n o )
methyl)phenoxy)acetic acid (G1)
White amorphous solid, yield 80%, mp 187–189 °C. IR
(KBr, cm−1): 3425.69 (O-H, Acid), 3078.20 (Ar-H), 1690.55
(C=O, Acid), 1604.83 (C=N, Imine), 1273.06 (C-N), 1218.05
(C-O). 1H NMR (δ ppm, CDCl3): 3.817 (s, 3H, OCH3), 4.404
(s, 2H, CH2), 6.419–6.433 (d, 2H, ArH), 6.921 (m, 1H, ArH),
7.258–7.330 (d, 2H, ArH), 7.652 (m, 1H, ArH), 8.122 (d, 1H,
ArH), 9.124 (s, 1H, CH=N). 13C NMR: (δ ppm, CDCl3):

Neurotox Res
and percentage viability of cells was calculated in terms of
percentage neuroprotection. Then the dose response curve of
new compounds vs known glitazones was plotted using Graph
Pad Prism software. The above different doses of compounds
were converted to Log dose and the dose response curve was
plotted with Log dose vs % protection to find out the EC50
value to compare the protective efficacy between test com-
pounds and standard Pioglitazone.
incubated for 15 min at room temperature, followed by addi-
tion of 100 μl stop–solution to all wells including blank wells.
The absorbance was determined at 410 nm using above-
mentioned ELISA reader (Roeske-Nielsen et al. 2004).
Lipid Peroxide (LPO) Assay
Lipid peroxidation was evaluated in cell lysates by measuring
the malondialdehyde content according to the TBA test de-
scribed by Ohkawa et al. (1979) with slight modification.
0.2 ml of the cell lysate was taken and to this, 0.8 ml saline,
0.5 ml of BHT, and 3.5 ml TBA reagent (0.8%) were added
and incubated at 60 °C. After cooling, the solution was cen-
trifuged at 2000 rpm for 10 min. The absorbance of the super-
natant was determined at 532 nm using spectrophotometer
against the blank.
Drug Pre-treatment and LPS Intoxication
The SH-SY5Y cell lines were maintained as per the standard
protocol in Minimum Essential Medium (MEM). 5000–
10,000 cells/well were seeded in 96 well plates and the via-
bility was tested using trypan blue dye with the help of a
hemocytometer and 95% of viability was confirmed. The cells
were then pre-treated with synthesized glitazones and standard
pioglitazone at IC50 dose. After 24 h of treatment with respec-
tive drugs, all the cells were incubated with LPS (10 ng/ml) to
induce the inflammation. After 24 h of LPS incubation, the
following evaluations were performed.
Nitric Oxide (NO) Assay
Nitric oxide was assayed by taking 0.2 ml of medium follow-
ed by the addition of 1.8 ml of saline and 0.4 ml of 35%
sulphosalicylic acid (SSA) for protein precipitation. The pre-
cipitate was removed by centrifugation at 4000 rpm for 10
min. To 1 ml of aliquot of supernatant, 2 ml Griess reagent
(1 g of sulphanilamide dissolved in small volume of water,
2 ml of orthophosphoric acid and 100 mg of naphthyl ethyl
diamine were added and the final volume was made up to
100 ml with distilled water). The mixture was allowed to stand
for 20 min under dark conditions. The color intensity of the
chromogen was read at 540 nm. The standard calibration
curve was plotted using sodium nitrite in the concentration
range 200–1000 ng (Green et al. 1982).
Cell Morphological Observation
The morphological changes in the SH-SY5Y cell lines after
respective treatments were observed by using phase contrast
microscopy (Motic).
Preparation of Cell Lysates
After the study period, the medium was aspirated and cells
were washed with the ice-cold phosphate buffer saline,
scraped, and were centrifuged at 5000 rpm for 5 min at 4
°C. The cell pellets were resuspended in 2000 μl of lysis
buffer (10 mM Tris–HCl, pH 7.5, 50 mM NaCl, 1% Triton
X-100, 5 mM EDTA, 50 mM NaF, 100 μM Na3VO4, 1 mM
PMSF, 10 μg/ml leupeptin, and 10 μg/ml aprotinin) and in-
cubated on ice for 30 min. The cell lysates were obtained by
centrifugation at 12,000 rpm for 20 min at 4 °C. Cell lysates
obtained were stored at − 20 °C until use.
Results and Discussion
Rational Design of Glitazones
Thiazolidinediones (TZDs), a class of glitazone family are
widely used as insulin sensitizers due to their characteristic
PPAR-γ agonism. Since PPAR-γ receptors are bound to
PGC-1α domain; the efforts are put forth to design novel
PPAR-γ agonists that can cause conformational changes in
the PPAR-γ and co-activators especially PGC-1α binding do-
main. The rationale here is, the conformational changes at
PGC-1α binding domain may up-regulate the mitochondrial
biogenesis process thereby offer advantage in the treatment of
neurodegenerative disorders. In this context, the structures of
glitazones are contrived to meet the pharmacotherapeutic re-
quirements. Based on bioisostere principle, the structural fea-
tures necessary for glitazone to interact with PPAR-γ recep-
tors such as acidic head group, aromatic trunk, heteroatom
spacer, and finally lipophilic tail were modified to build novel
Measurement of IL-1β & TNF-α (Pro-inflammatory Cytokines)
by ELISA
The levels of IL-1β and TNF-α were assessed in cell lysates
using respective ELISA kits (Invitrogen, R&D systems &
Alpha diagnostics, USA) as per the manufacturer’s protocol.
Briefly, 150 μl distilled water was added to the standard and
blank wells for standard calibration and 100 μl distilled water
and 50 μl of each supernatant were added in duplicate into the
wells. After incubation for 3 h at room temperature, the wells
were emptied and washed three times with 150 μl of wash
buffer. TMB substrate (100 μl) was added to each well and

Neurotox Res
glitazones (Kulkarni et al. 1999). Phenoxy acetic acid consti-
tuted acidic head instead of thiazolidinedione, substituted ben-
zene constituted trunk, and imine linkage constituted two car-
bon linker followed by pyridine and its isostere benzene as
lipophilic tail as shown in (Fig. 1a–c).
was connected to the lipophilic tail by condensation with 2-
amino pyridine (compound G1) and 2,4-dinitro
phenylhydrazine (compound G2) using absolute alcohol as
solvent in the presence of catalytic amount of acetic acid and
activated molecular sieves to form Schiff’s base imine link-
age. The products were purified by column chromatography
using ethyl acetate and n-hexane as mobile phase by gradient
elution technique. The method adopted to synthesize the final
product was very feasible and facile. The two synthesized
Schiff’s bases were checked for their possible aqueous hydro-
lysis of imine linkage. However, both the Schiff’s bases were
found to stable in water when studied for 48 h.
Chemistry and Synthesis
Rationally designed compounds were synthesized according
to the scheme described in Fig. 2 (Zubrys and Siebenmann
1954; Shah and Baseer 2012). Two aromatic aldehydes, viz.
4-hydroxy benzaldehyde and 4-hydroxy-3-methoxy benzal-
dehyde (vanillin) are selected as building blocks. Initially,
the hydroxyl group of aldehydes was converted to corre-
sponding phenoxy-acetic acids by adopting the modified
Williamson’s ether synthesis. Further, the formed intermediate
The structures of the synthesized glitazones confirmed via
IR, NMR, and Mass spectral interpretation. The appearance of
characteristic peak in the range of 1580.50–1604.90 cm−1 in
all IR spectra along with the absence of NH stretch proved the
Fig. 1 a General structural features of glitazones or TZDs. b Structural features of pioglitazone. c Structural features of designed glitazones

Neurotox Res
R
H
Fig. 2 Synthetic scheme of
glitazones
R
O
R
H
O
(a)
H
O
(b)
H
O
OH
OH
ONa
O
O
1, 2
R=H, 1
(c)
R= OCH3, 2
(c)
O
O
O
H
N
OH
H
N
OH
O
NO2
N
NO2
G1
G2
formation of imine bond (CH=N). All the compounds showed
characteristic C=O stretching of the carboxyl group in the
range of 1650.42–1743.35 cm−1 along with O-H stretching
in the range of 3200.25–3400.23 cm−1. From 1H-NMR spec-
tra it is observed that methylene protons (CH2), which are
bridge between phenoxy and carboxylic acid moiety appeared
as singlet in the range of δ 4.45 to δ 4.68 ppm and proton
attached to imine linkage (H–C=N) of Schiff base has reso-
nated between δ 8.23 and δ 8.98 ppm which in turn confirmed
the formation of imine. It is very interesting to notice that the
compounds G1 and G2 showed, –CH signal for imine (-
CH=N) at δ ppm 8.9, that indicates the –CH=N linkage is of
‘E’ configuration.
ligands into a receptor binding site (Wu et al. 2003a, b). In
order to validate the tool effectiveness, the native ligand and
the standard drug rosiglitazone re-docking study indicates that
the tool predicts the reliable results. The molecular docking
results provided significant information about the -
CDOCKER energy, -CDOCKER interaction energy, and
binding orientation of receptor-ligand interactions. The
docking results are summarized in Table 1. It has been found
that G1, G2, and rosiglitazone bound to the same active site as
reported in the RCSB crystallographic data. The standard drug
rosiglitazone showed docking and interaction energy of
32.3167 kcal/mol and 39.6259 kcal/mol, respectively. The
binding accuracy of reference standard, rosiglitazone was val-
idated by redocking with an RMSD value of 0.909 A°. The
binding interactions include one hydrogen bond with Tyr473
and several hydrophobic contacts with Ile281, Cys285,
Ile326, Ile341, Met348, Met364, and His449 from arm-I and
arm-II of the LBD of PPAR-gamma. Thus, possibly any
PPAR-γ binding with His323, Tyr327, His449, and Tyr473
from arm-I of the LBD of PPAR-γ has transactivation activity
(Cardin et al. 2012). Similarly, Compound G1 and Compound
G2 have shown docking and interaction energy of 16.1183
kcal/mol, 23.4904 kcal/mol and 45.1763 kcal/mol, 32.6616
kcal/mol, respectively. The common interacting amino acid
with hydrogen and hydrophobic interaction with arm-I, arm-
II, and arm-III or entrance (E) residues like Ile281, Cys285,
Arg288, Leu330, Ile341, Met348, Met364, and Lys261 are
shown in Fig. 3A–C. These interactions are typical for
PPAR-γ partial agonists (Guasch et al. 2012).
Reagents and Condition (a) NaOH, H2O, stir. (b)
ClCH2COOH, NaOH, H2O reflux at 120–140 °C for 3 h. (c)
Aromatic amine, gl. acetic acid, absolute ethanol, molecular
sieves, reflux with stirring for 8–12 h.
Binding Affinity and Interaction Analysis
Through Docking with PPAR-γ
The main objective of the molecular docking study was to
elucidate binding interactions between agonists/glitazones
and PPAR-γ receptor and also to identify conformational
changes towards the PGC-1α domain. There are various al-
gorithms existing for docking, one such best tool used for
docking via discovery studio is CDOCKER because it uses
CHARMm-based molecular dynamics (MD) scheme to dock
Table 1 CDOCKER Score for
the respective ligand protein
Compounds
CDOCKER energy (kcal/mol)
CDOCKER interaction energy (kcal/mol)
interaction
Glitazone 1 (G1)
− 16.1183
− 23.4904
− 32.3167
− 45.1763
− 32.6616
− 39.6259
Glitazone 2 (G2)
Standard-rosiglitazone

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Fig. 3 A 3D and 2D representation of Compound G1. B 3D and 2D representation of Compound G2. C 3D and 2D representation of standard drug
rosiglitazone
Activation and Molecular Mechanism of PPAR-γ
with PGC-1α
Duchen 2015). Apparently, the function of PPAR-γ agonist
with PGC-1α was addressed as a neuroprotectant in neurode-
generative disorders like Parkinson disease (PD) and
Alzheimer’s disease (Zheng et al. 2010; Jin et al. 2013) to
support the statement, administration of TZDs increases the
PGC-1α responsive genes and blocked the loss of dopaminer-
gic regions (Zheng et al. 2010). But the molecular level mech-
anism and the activation of the PGC-1α upon binding of
PPAR-γ agonist and its atomic level interaction were not
clearly reported.
The peroxisome proliferator-activated receptor gamma
(PPAR-γ) belongs to the nuclear hormone receptor superfam-
ily. They are broadly involved in the regulation of genes for
various physiological processes such as inflammation, glu-
cose metabolism, cellular differentiation and proliferation,
and lipid homeostasis (Delerive et al. 2001). Many studies
have reported peroxisome proliferator-activated receptor gam-
ma (PPAR) co-activator (PGC-1α) as a crucial potential ther-
apeutic target for neurological dysfunction (Corona and
This study is focused only on the role of how the confor-
mational changes in the secondary structure of the protein

Neurotox Res
activate the PGC-1α. Previously, it was reported that TZDs
bound PPAR-γ ligand-binding domain (LBD) has a strong
binding affinity with various LXXLL motif co-activator (Li
et al. 2008). In order to identify the structural contribution of
the PPAR-γ agonist in the ligand-binding domain (LBD) mo-
lecular docking was initially processed followed by Monte
Carlo simulation using CHARMm force filed. The outcome
has revealed that structure has undergone various conforma-
tional changes at helix and coil region. To support the argu-
ment, the realistic molecular dynamics and simulation were
conducted for four different complex apo-proteins (PPAR-γ
and PGC-1α), the best-docked complex of apo-protein with
various agonists like G1, G2, and standard drug.
transformation of the secondary structure of the protein.
Eventually, 1000 ps dynamics shows Compound G1 binding
to LBD affects the C-terminal helix and it transformed the coil
conformation while other helices moderately show changes in
secondary structure with the final potential energy of −
17,687.60 kcal/mol (Fig. 4C). Moreover, the residue Leu145
of PGC-1α 1D1 forms two direct hydrogen bonds with
Asn312 and one hydrogen bond interaction with Asp310.
Secondly, Ser142 and Leu143 form direction conventional
hydrogen bonding with Gly471 present at C-terminal of
PPAR-γ. The apo-protein complex energy − 17,407.10 kcal/
mol forms only one direct hydrogen bond interaction between
Leu145-Asn312, no bonding with Ser142 that implies least
stability and interaction between the PPAR-γ and PGC-1α,
furthermore weak conformation change was observed (Fig.
4A). Similarly, Compound G2 binding with LBD of
PPAR-γ had shown changes in intermolecular interactions
compared with Compound G1 and rosiglitazone. Evidently,
It was observed that without ligand binding to the LBD of
PPAR-γ, the structure of the adjacent helix, C-terminal helix
and PGC-1α doesn’t show any structural variation. On the
other hand, any agonist binding to the PPAR-γ LBD such as
compound G1, compound G2, and rosiglitazone shows the
Fig. 4 Conformational changes and interaction of PPAR-γ with PGC-
1α. A Apo-protein interaction (PPAR-γ-PGC-1α). B Compound G2
binding to PPAR-γ LBD and its associated conformation changes in
PGC-1α. C Compound G1 binding to PPAR-γ LBD and its associated
conformation changes in PGC-1α. D Standard drug rosiglitazone binding
to PPAR-γ LBD and its associated conformation changes in PGC-1α.
Pink color in the figure is LXXLL motif of PGC-1α

Neurotox Res
Table 2 The IC50 value of synthesized glitazones in SH-SY5Y neuro-
blastoma cell lines
Compound G2 binding to LDB region affects the binding of
A: LYS224: HZ3 - B: ALA152: OCT2(PPAR-γ-PGC-1α)
also the helix region (Asn308–Ile 325) shows more structural
deviation and transformation with a final potential energy of −
17,432.10 kcal/mol (Fig. 4B). Likewise, standard drug
rosiglitazone binding to LDB forms two direct hydrogen bond
between residue Leu145 of PGC-1α and Asn312 of PPAR-γ
(Li et al. 2008) and conformation changes are observed in
PGC-1α with an energy of − 17,627 kcal/mol (Fig. 4D).
All the complexes except apo-protein forms more than sev-
en intermolecular interactions. These intermolecular interac-
tions stabilize the helical structure of the PGC-1α 1D1 motif,
thus facilitating the hydrogen and hydrophobic docking of this
helix into PPAR-γ. Simultaneously, these unique interactions
and interface contacts serve as a basis for the high affinity,
specific binding of PPAR-γ-PGC-1α 1D1 motif. Also, the
binding of ligand to the PPAR-γ LBD is directly influencing
the conformational changes of PGC-1α 1D1 motif thereby
transcripts the various downstream genes involved in mito-
chondrial biogenesis and antioxidant defenses. In this context,
it is evident that PPAR-γ agonists have great influence on
PGC-1α activation. Thus, compound G1 shows equal in en-
ergy and binding as like as rosiglitazone even though with
50% of docking score. Perhaps, Compound G2 with 72% of
dock score with energy of − 17,432.10 kcal/mol can serve as a
better drug candidate. From this, it was clearly stated that only
the strong influential binding of drug to the LBD pocket will
influence the structural changes, thereby it activates the PGC-
1α. Also, PGC-1α and PPAR-γ agonists regulate the expres-
sion of several target genes involved in neuroprotection by
suppressing neuroinflammation, mitochondrial dysfunction,
and proteasomal dysfunction.
S. no.
Compound name
IC50 μg/ml
EC50a nM
1
2
3
Glitazone 1 (G1)
53.46 3.81
86.65 6.11
45.95 3.98
46. 04 4.10
47.88 2.60
> 250b
Glitazone 2 (G2)
Standard-Pioglitazone (P)
a EC50 was measured in the presence of LPS 10 ng/ml
b Pioglitazone exhibited a maximum neuroprotection of 44.78 1.51 % at
62.5 nM
neuroinflammatory conditions to evaluate the neuroprotective
activity of several agents (Rampe et al. 2004).
Effect of Glitazones on Cell Viability Assay
in LPS-intoxicated SH-SY5Y Cell Lines
The synthesized novel glitazones were subjected to MTT as-
say to evaluate the extent of toxicity (IC50) and neuroprotec-
tive (EC50) effects. The test glitazones G1 and G2 are less
toxic to SH-SY5Y cell lines because G1 (53.46 3.81) and
G2 (86.65 6.11) have greater IC50 than Pioglitazone (45.95
3.98). Interestingly, the test compound G2 has approximate-
ly twofold less cytotoxic than standard pioglitazone (Table 2).
The IC50 values of the each compound were taken for further
neuroprotective evaluation. The neuroprotection was ob-
served in LPS-induced inflammatory condition in SH-SY5Y
cells. The G1 and G2 had significant neuroprotective effect at
doses 31.25, 62.5, 125, and 250 nM (p < 0.001) when com-
pared to standard pioglitazone (Fig. 5). Pioglitazone exhibited
8 0
* * *
P
# # #
Neuroprotective Evaluation of Synthesized Glitazones
in LPS-intoxicated SH-SY5Y Neuroblastoma Cell Lines
* * *
* * *
# # #
# # #
G 1
G 2
6 0
4 0
2 0
0
The synthesized novel synthesized glitazones G1 & G2 were
subjected to preliminary in vitro neuroprotective activity
against LPS-induced inflammatory events in SH-SY5Y neu-
roblastoma cell lines. The neuroprotective activity was
assessed by performing cell viability assay, morphological
observation and measuring the intracellular pro-
inflammatory cytokines TNF-α, IL-1β using ELISA.
Additionally, LPO and NO assay were carried out to assess
the level of free radical scavenging properties of novel
glitazones. The neuroinflammation induced by incubation of
SH-SY5Y cell lines with LPS (10 ng/ml) in minimum essen-
tial medium at 37 °C for 24 h is one of the standard protocols.
In our laboratory, it has been observed that 24 h incubation
with 10 ng/ml of LPS is sufficient to stimulate inflammatory
reactions. The previous report suggests that LPS-treated neu-
ronal cell lines serve as a good in vitro model mimicking the
# # #
* * *
5
.5
9 5
9 1
8 1
6 3
2
2
1 2 5
2 5 0
1 .
3 .
7 .
6
1 5 .
3 1 .
D o s e in n M
Fig. 5 Neuroprotective effect of test glitazones and standard pioglitazone
in LPS-intoxicated SH-SY5Y cell lines. Statistical significance was de-
termined by one-way ANOVA followed by Dunnett-t test using Graph
pad prism Version 5.0. Values are expressed as mean SEM. Superscript
###
*** denotes P vs G1 and
Pioglitazone, G1—Glitazone 1 and G2—Glitazone 2
denotes P vs G2 at p < 0.001. P—

Neurotox Res
G1 + LPS 10ng/ml
G2 + LPS 10ng/ml
which is further witnessed in dose response curve of test
glitazones and standard pioglitazone (Fig. 6). Hence, we could
not calculate the EC50 of pioglitazone. The G1 and G2 exhib-
ited a maximum protection of 69.77% and 65.19% at 125 nM.
The IC50 and EC50 of synthesized glitazones in SH-SY5Y
neuroblastoma cell lines are given in Table 2.
60
P
+ LPS 10ng/ml
40
20
0
Effect of Glitazones on Morphological Changes
in LPS-intoxicated SH-SY5Y Cell Lines
0.0
0.5
1.0
1.5
2.0
2.5
Log dose [10-9mol/L]
The control SH-SY5Y cells are small, highly light retractile,
fibroblast-like or teardrop-shaped, cells, growing densely and
often forming focal accumulations. LPS (10 ng/ml) intoxica-
tion caused morphological changes with reduced cell density,
swollen, reduced proliferation, and differentiation of cells
when compared to the control cells. Pre-treatment with
glitazones protected the cells from the LPS toxicity which is
evidenced by increased cell density and proliferation.
Interestingly, the pre-treatment with G2 has shown remark-
able protective effect on LPS-intoxicated SH-SY5Y cells, this
Fig. 6 Dose response curve of test glitazones vs standard pioglitazone in
LPS-intoxicated SH-SY5Y cell lines. Values are expressed as mean
SEM. P—Pioglitazone, G1—Glitazone 1 and G2—Glitazone 2
a maximum of neuroprotection of 44.78 1.51 % at 62.5 nM.
After that, at 125 nM (43.71 2.55) and 250 nM there is a
decrease in the neuroprotective effect. Pioglitazone did not
achieve the 50% neuroprotection in the tested dose in com-
parison to the test glitazones indicate that the test glitazones
are having more neuroprotective efficacy than pioglitazone
Fig. 7 Effect of novel glitazones
on morphological changes in
LPS-intoxicated SH-SY5Y cell
lines. a Control cells. b LPS-
treated cells. c Standard pioglita-
zone. d Glitazone 1. e Glitazone 2
treated cells. C—Control, LPS—
Lipopolysaccharide, P—
Pioglitazone, G1—Glitazone 1
and G2—Glitazone 2
100µm
100µm
100µm
100µm
100µm

Neurotox Res
C
- C o n t r o l
Fig. 8 Effect of glitazones on IL-
1β level in LPS-intoxicated SH-
SY5Y cell lines. Statistical sig-
nificance was determined by one
way ANOVA followed by
Dunnett-t test using Graph pad
prism Version 5.0. Values are
expressed as mean SEM,
500
400
300
200
100
0
L P S -L ip o p o ly s a c c h a rid e
P
G
* * *
- P io g lita z o n e
- G lita z o n e s
Superscript *** denotes p < 0.001
vs control and ### denotes p <
0.001 vs LPS, respectively
# # #
# # #
# # #
2
P
S
C
G 1
G
L P
T r e a tm e n t s
effect was comparable with standard pioglitazone which is
summarized in Fig. 7a–e.
significant reduction (p < 0.05) of TNF-α level in comparison
to Glitazone 1 and standard pioglitazone in LPS-intoxicated
cell lines. Thus, the effect of glitazones was found to be sim-
ilar and comparable with that of the standard pioglitazone in
LPS-intoxicated SH-SY5Y cell lines.
The release of IL-1β plays a critical role in the effect of
microglial activation on motor neuron viability and IL-1β is
among a wide range of factors that up-regulate the expression
of COX-2 and the subsequent production of pro-inflammatory
cytokines. Previous studies reported that LPS increases the
secretions of IL-1β in microglial cells (Arai et al. 2004). The
data from the present study shows that glitazones inhibits the
level of IL-1β release in LPS-intoxicated SH-SY5Y cells.
TNF-α is a potent pro-inflammatory cytokine that plays an
important role in neuroinflammation. The present study
showed that LPS-stimulation increases TNF-α secretion from
Effect of Glitazones on Pro-inflammatory Cytokines
IL-1β & TNF-α Level in LPS-intoxicated SH-SY5Y Cell
Lines
In comparison to control SH-SY5Y cell lines, LPS treatment
has significantly (p < 0.001) increased the level of IL-1β &
TNF-α in LPS-intoxicated SH-SY5Y cell lines which show
the extent of inflammatory responses mediated by LPS toxic-
ity. In comparison to LPS-treated group, pre-treatment with
pioglitazone, G1, and G2 has remarkably (p < 0.001) de-
creased the level of IL-1β (Fig. 8) & TNF-α (Fig. 9) which
indicates the anti-inflammatory potential of novel glitazones.
Interestingly, among the two Glitazones, G2 has shown
C
- C o n t r o l
Fig. 9 Effect of glitazones on
TNF-α level in LPS-intoxicated
SH-SY5Y cell lines. Statistical
significance was determined by
one-way ANOVA followed by
Dunnett-t test using Graph pad
prism Version 5.0. Values are
expressed as mean SEM,
Superscript *** denotes p < 0.001
vs control, ### denotes p < 0.001
vs LPS, $ denotes p < 0.05 vs P,
@ denotes p < 0.05 vs G1,
respectively
800
L P S -L ip o p o ly s a c c h a rid e
P
G
- P io g lita z o n e
- G lita z o n e s
* * *
600
@
400
200
0
# # #
$
# # #
# # #
1
P
S
C
G
G 2
L P
T r e a tm e n t s

Neurotox Res
SH-SY5Y cell lines. We further demonstrated that synthetic
glitazones significantly downregulated the TNF-α secretion
which was triggered by LPS in neuronal cells. G2 compound
exhibited significant effect compared to G1 and the standard
pioglitazone. The results suggest that inhibition of TNF-α
secretion from the LPS-intoxicated neuronal cell lines proba-
bly participates in the neuroprotective effect of glitazones.
The attenuation of cytokine (IL-1β & TNF-α) level in
LPS-intoxicated SH-SY5Y cell lines endorses the anti-
neuroinflammatory activity exhibited by novel glitazones.
The mechanism might be attributed through agonist activity
at PPAR-γ receptors, because earlier report implicates that
pioglitazone has decreased the secretion of pro-inflammatory
cytokines in astrocytes stimulated with LPS through activa-
tion of PPAR-γ receptors (Swanson et al. 2011). In addition,
previous studies evidence that the stimulation of PPAR-γ re-
ceptors will recruit/activate its co-activator PGC-1α which
may further suppress the NF-kB expression mediated cyto-
kine release (Qiu and Li 2015). Hence, the novel glitazones
evaluated in this study might have ameliorated the IL-1β &
TNF-α level through activation of PGC-1α via PPAR-γ
agonism which is supported by our in silico molecular inter-
action study.
anti-oxidant potential of pioglitazone and synthesized
glitazones. Interestingly, G2 has shown significant reduction
(p < 0.05) of NO level than G1 and pioglitazone, the effect of
G2 is similar and comparable with pioglitazone in LPS-
intoxicated SH-SY5Y cell lines.
Earlier study revealed that PGC-1α is a master regulator of
ROS scavenging enzymes and PGC-1α decreases the oxida-
tive stress by elevating the expression of antioxidant enzymes
(Chen et al. 2011). On other hand, downregulation of PGC-1α
results in impairment of antioxidants expression and triggers
the reactive oxygen species (ROS) burst and augment the
damage to the mitochondrial proteins resulted in neurodegen-
eration (Marmolino et al. 2010). In the present study, the at-
tenuation of LPO and NO level by glitazones might be attrib-
uted through activation of PGC-1α signaling via PPAR-γ re-
ceptor activation, because induction of PGC-1α regulates the
gene expression of several reactive oxygen species (ROS)
with decreasing oxidative stress and increasing mitochondrial
biosynthesis resulted in neuroprotection (St-Pierre et al.
2006).
Previous findings implicate that the neuroinflammation-
mediated neurodegeneration play an imperative in the patho-
genesis of several neurodegenerative diseases, including
Alzheimer’s disease (AD), Parkinson’s disease (PD),
Huntington’s disease, multiple sclerosis (MS), and amyotro-
phic lateral sclerosis (ALS) (Zhao et al. 2019). In the present
study, we have incubated potent neuroinflammatory toxin li-
popolysaccharide (LPS) in SH-SY5Y cell lines to mimic the
inflammatory conditions in the neuronal cell lines. In general,
LPS will act on Toll-like receptor-4 (TLR-4) and activate the
respective cells to release the pro-inflammatory cytokines
such as IL-1α, IL-1β, IL-6, and TNF-α which are the key
mediators of neuroinflammation [Leow-Dyke et al. 2012].
The LPS-mediated cytokine release is predominantly
Effect of Glitazones on LPO & NO Levels
in LPS-intoxicated SH-SY5Y Cell Lines
Treatment with LPS in SH-SY5Y cell lines have significantly
(p < 0.001) increased the level of LPO & NO in comparison to
control cell lines which indicates the extent of oxidative stress-
mediated by LPS toxicity. In comparison to LPS-intoxicated
group, pre-treatment with pioglitazone (p < 0.001), G1 (p <
0.05), and G2 (p < 0.001) have remarkably decreased the
levels of LPO (Fig. 10) & NO (Fig. 11), which indicates the
C
- C o n t r o l
Fig. 10 Effect of glitazones on
60
40
20
0
LPO levels in LPS-intoxicated
SH-SY5Y cell lines. Statistical
significance was determined by
one-way ANOVA followed by
Dunnett-t test using Graph pad
prism Version 5.0. Values are
expressed as mean SEM,
Superscript *** denotes p < 0.001
vs control and ##, ### denotes p <
0.01, p < 0.001 vs LPS,
L P S -L ip o p o ly s a c c h a rid e
P
G
- P io g lita z o n e
- G lita z o n e s
* * *
# #
# # #
# # #
respectively
P
C
G 1
G 2
P S
L
T r e a tm e n t s

Neurotox Res
C
- C o n t r o l
Fig. 11 Effect of glitazones on
NO levels in LPS-intoxicated SH-
SY5Y cell lines. Statistical sig-
nificance was determined by one-
way ANOVA followed by
Dunnett-t test using Graph pad
prism Version 5.0. Values are
expressed as mean SEM,
Superscript *** denotes p < 0.001
vs control, ### denotes p < 0.001
vs LPS, $ denotes p < 0.05 vs P,
@ denotes p < 0.05 vs G1,
respectively
150
100
50
L P S -L ip o p o ly s a c c h a rid e
P
G
- P io g lita z o n e
- G lita z o n e s
* * *
@
$
# # #
# # #
# # #
0
P
C
G 1
G 2
P S
L
T r e a tm e n t s
occurring through stimulation of nuclear factor-kappa B
(NF-κB) signaling pathway. The activation of NF-κB trans-
duction pathway could increase the transcription of genes as-
sociated with cytokine production leads to pro-inflammatory
mediator’s release [Liu et al. 2017] resulted in neuroinflam-
mation and neuronal death.
LPS also enhances the production of ROS in the neuronal
cells (Zhao et al. 2014; Yu et al. 2015). Though several studies
are emphasized, the inflammatory mechanism of LPS, the
LPS-induced oxidative damage in the brain cells also plays a
major role in the several neurodegenerative disorders
(Noworyta-Sokołowska et al. 2013). LPS accelerates the
ROS formation which causes a significant alteration in NO,
malondialdehyde (MDA), reduced glutathione (GSH), super-
oxide dismutase (SOD), and catalase (CAT) levels in neurons.
Earlier experimental findings have shown that an increased
level of pro-inflammatory cytokines (e.g., TNF-α, INF-γ,
and IL-6) can be the basis of formation of ROS through acti-
vating NF-kB transcription (Sharma and Nehru 2015). In our
study, we have also observed that treatment with LPS in SH-
SY5Y cell lines has increased the LPO and NO level along
with pro-inflammatory cytokines. The LPS mainly triggers
the ROS through TLR-4 receptor activation followed by stim-
ulation of NF-kB–dependent expression of genes associated
with oxidative stress (Leow-Dyke et al. 2012).
The significant increase in the IL-1β and TNF-α level of
present experimental findings indicates the LPS accelerated
the NF-κB expression in SH-SY5Y cell lines through TLR-
4 receptors. It might be caused by the neuroinflammation
followed by neurodegeneration which is further evidenced in
Fig. 12 Proposed mechanism of
action of novel glitazones

Neurotox Res
morphological alterations in LPS-treated cell lines.
Interestingly, treatment with novel glitazones (designed as
PPAR-γ agonist) exhibited significant protective effect in
LPS-intoxicated cell lines through attenuation of pro-
inflammatory cytokine release and scavenging of oxy-radi-
cals. Research findings are evidenced that agonist activity at
PPAR-γ receptors elicits anti-neuroinflammatory effect
through controlling NF-kB signaling in activated microglia
(Choi et al. 2017). A study had shown that deficiency of neu-
ronal PPAR-γ receptors increased the intensity of brain dam-
age in response to cerebral ischemia (Zhao et al. 2009).
Whereas, agonism of PPAR-γ receptors by rosiglitazone
exerted neuroprotection through ameliorating the brain cyto-
kine levels in focal ischemic rats (Luo et al. 2006). Activation
of PPAR-γ–dependent signaling in advanced glycation end
toxin-treated human neural stem cells has shown neuroprotec-
tion, suggesting that PPAR-γ ligands are promising candi-
dates for the therapeutic management of several neurological
disorders (Chiang et al. 2017). Interestingly, the neuroprotec-
tive mechanism of PPAR-γ agonism is mediated through up-
regulating the expression of its co-factor, namely PGC-1α.
The activation of PPAR-γ results in heterodimerize with
RXR and recruits the PGC-1α to form a regulatory complex
(Viswakarma et al. 2010). It could inhibit the mitochondrial
and proteasomal dysfunction, oxidative stress, autophagy,
neuroinflammation, and apoptosis in neurons through regulat-
ing various target genes leading to neuroprotection and neu-
ronal survival.
sequentially adopting modified Williamson’s ether synthesis
and Schiff’s base formation. The structures of the synthesized
compounds were confirmed through the spectral data. The
desired neuroprotective activity of the synthesized com-
pounds was assessed in LPS-intoxicated SH-SY5Y cells.
The intracellular pro-inflammatory cytokine (TNF-α & IL-
1β) level was measured by ELISA to evaluate the anti-
neuroinflammatory ability of synthesized glitazones. The free
radical scavenging properties of novel glitazones were
assessed by LPO and NO assay. The in vitro results and in
silico result cross-correlate for the possibility of PGC-1α ac-
tivation thereby possibly enhancing mitochondrial biogenesis.
Cognizance study indicates that compounds G1 and G2 sat-
isfactorily fulfills the hypothesis, besides compound G2
showed better results in both in silico and in vitro studies.
Altogether, we have identified one candidate compound to
investigate further using animal models for its potency and
efficacy in neurodegenerative disorders.
Funding Information The authors sincerely thank the Department of
Science and Technology—Science and Engineering Research Board
(DST-SERB), New Delhi for extended financial support to carry out this
project (Grant Sanction order No. CRG/2018/002084).
Compliance with Ethical Standards
Conflict of Interest The authors declare that there is no conflict of
interest.
Therefore, the mechanism of neuroprotective effect of nov-
el glitazones might have attributed through activation of cen-
tral PPAR-γ receptors thereby it would activate the PGC-1α
signaling which was confirmed in the ligand interaction, mo-
lecular dynamics, and conformational analysis part of this
study. Further, the activation of PGC-1α signaling would have
attenuated the NF-kB expression in neurons resulting in de-
creased cytokines and free radicals released during
neuroinflammatory conditions lead to neuroprotection. The
proposed neuroprotective mechanism of synthesized novel
glitazones in LPS-induced neuroinflammatory conditions is
depicted in Fig. 12.
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