Structure–activity relationship of piperine and its synthetic amide analogs for therapeutic potential to prevent experimentally induced ER stress in vitro

Article abstract of DOI:10.1007/s12192-017-0786-9

Endoplasmic reticulum (ER) is the key organelle involved in protein folding and maturation. Emerging studies implicate the role of ER stress in the development of chronic kidney disease. Thus, there is an urgent need for compounds that could ameliorate ER

Full text of DOI:10.1007/s12192-017-0786-9

Cell Stress and Chaperones
DOI 10.1007/s12192-017-0786-9
ORIGINAL PAPER
Structure–activity relationship of piperine and its synthetic amide
analogs for therapeutic potential to prevent experimentally
induced ER stress in vitro
Ayat S. Hammad1 & Sreenithya Ravindran1 & Ashraf Khalil1 & Shankar Munusamy1
Received: 10 January 2016 /Accepted: 13 March 2017
#
Cell Stress Society International 2017
Abstract Endoplasmic reticulum (ER) is the key organelle
involved in protein folding and maturation. Emerging studies
implicate the role of ER stress in the development of chronic
kidney disease. Thus, there is an urgent need for compounds
that could ameliorate ER stress and prevent CKD. Piperine
and its analogs have been reported to exhibit multiple
pharmacological activities; however, their efficacy against
ER stress in kidney cells has not been studied yet. Hence,
the goal of this study was to synthesize amide-substituted
piperine analogs and screen them for pharmacological activity
to relieve ER stress using an in vitro model of tunicamycin-
induced ER stress using normal rat kidney (NRK-52E) cells.
Five amide-substituted piperine analogs were synthesized and
their chemical structures were elucidated by pertinent spectro-
scopic techniques. An in vitro model of ER stress was devel-
oped using tunicamycin, and the compounds of interest were
screened for their effect on cell viability, and the expression of
ER chaperone GRP78, the pro-apoptotic ER stress marker
CHOP, and apoptotic caspases 3 and 12 (via western blotting).
Our findings indicate that exposure to tunicamycin (0.5 μg/
mL) for 2 h induces the expression of GRP78 and CHOP, and
apoptotic markers (caspase-3 and caspase-12) and causes a
significant reduction in renal cell viability. Pre-treatment of
cells with piperine and its cyclohexylamino analog decreased
the tunicamycin-induced upregulation of GRP78 and CHOP
and cell death. Taken together, our findings demonstrate that
piperine and its analogs differentially regulate ER stress, and
thus represent potential therapeutic agents to treat ER stress-
related renal disorders.
.
.
.
Keywords Piperine Amide piperine analogs ER stress
.
.
NRK-52E Tunicamycin Chronic kidney disease
Introduction
Endoplasmic reticulum (ER) is one of the most versatile and
adaptable organelles in eukaryotic cells (Brandizzi et al. 2014;
Staehelin 1997). It plays a principal role in controlling the
synthesis, folding and maturation of luminal, secreted and
transmembrane proteins (Brandizzi et al. 2014; Staehelin
1997; Zhuang and Forbes 2014). During protein synthesis,
only those proteins folded in proper configuration and
underwent specific post-translational modifications will be
processed through the ER secretory pathway and translocated
to Golgi bodies (Zhuang and Forbes 2014). If cells cannot
mitigate the changes caused by unfolded or misfolded proteins
and restore homeostasis, it results in ER stress and activation
of unfolded protein response (UPR) (Walter and Ron 2011).
The UPR is a collection of phylogenetically conserved sig-
naling pathways that attempts to re-establish homeostasis
through inducing several chaperones. It is composed of three
principal pathways: PERK (PKR-like endoplasmic reticulum
kinase), IRE1 (inositol-requiring enzyme 1), and ATF6 (acti-
vating transcription factor 6) (Nagelkerke et al. 2014; Walter
and Ron 2011). Under normal conditions, a chaperone named
glucose-regulated protein 78 (GRP78) is connected to the
three transducers of UPR (i.e., PERK and IRE1 in the
amino-terminal and ATF6 in the carboxy-terminal in the
intraluminal domain), and retain them inactive. Dissociation
of GRP78 from PERK, IRE1, or ATF6 results in the activation
Electronic supplementary material The online version of this article
(doi:10.1007/s12192-017-0786-9) contains supplementary material,
which is available to authorized users.
* Shankar Munusamy
shankar.munusamy@qu.edu.qa
1
College of Pharmacy, Qatar University, PO Box 2713, Doha, Qatar

A. S. Hammad et al.
of UPR signaling (Nagelkerke et al. 2014). If the ER stress is
severe and prolonged, and homeostasis is not restored by the
adaptive mechanism of the UPR, then the cell initiates a pro-
apoptotic response rather than a pro-survival response
(Sovolyova et al. 2014).
Yaffe et al. attempted to investigate the mechanisms by which
piperine mediates cell cycle arrest and apoptosis in colon can-
cer revealed that its pro-apoptotic effects are mediated through
increased expression of CHOP and GRP78 in colon cancer
cells (Yaffe et al. 2015). Paradoxical to these findings, a study
using a high fat diet (HFD)-induced model of hepatic steatosis
indicated that piperine decreases the messenger RNA
(mRNA) expression of GRP78 in the liver tissues of mice
fed an HFD (Jwa et al. 2012). To the best of our knowledge,
the effect of piperine on ER stress in kidney has not yet been
studied. Furthermore, although piperine and its amide piperine
analogs have been reported to exhibit diverse pharmacological
activities in various disease models (Faas et al. 2008; Ferreira
et al. 2011; Greenshields et al. 2015; Kumar et al. 2015;
Meghwal and Goswami 2013; Wattanathorn et al. 2008), none
of those studies were targeted to elucidate the impact of pip-
erine and its amide piperine analogs on ER stress markers in
kidney cells.
We hypothesize that piperine (and potentially its analogs)
would attenuate ER stress and protect renal cells against ER
stress-induced cell death. Hence, the objectives of this study
were as follows: (1) to synthesize an array of amide-
substituted piperine analogs and characterize the prepared an-
alogs using pertinent spectroscopic techniques (2) to establish
an in vitro model of ER stress-induced cell injury using
tunicamycin in normal rat kidney (NRK-52E) cells, and (3)
to evaluate the pharmacological activity of piperine and the
prepared piperine analogs to relieve ER stress and associated
cell death in the established in vitro model.
Numerous studies have provided evidence for the involve-
ment of ER stress in chronic kidney disease (CKD)
(Henriquez et al. 2005; Inagi 2010; Markan et al. 2009;
Peyrou and Cribb 2007). Currently, CKD is considered as
one of the most significant global health issues, which not
only endangers the health of our society but also places a huge
burden on our health-care cost (Drawz et al. 2015; Henriquez
et al. 2005). Studies have found that once the diagnosis of
CKD is established, the rate of decline in kidney function
was found to be 2.3 to 4.5 mL/min/year (Drawz et al. 2015).
Thus, a prophylaxis to hinder the deterioration of kidney func-
tion is a better strategy than treatment, especially in high-risk
populations such as patients with diabetes and/or hypertension
(Drawz et al. 2015; Markan et al. 2009; Rogers et al. 2013).
Findings from a study conducted on kidney biopsies taken
from patients with primary glomerulonephritis, one of the pri-
mary glomerular diseases (PGD), revealed increased expres-
sion of GRP78 and GADD153/CHOP, and decrease in the
expression of anti-apoptotic Bcl-2 proteins in PGD (Markan
et al. 2009). Several other studies also confirmed the involve-
ment of ER stress in a wide variety of kidney diseases
(Cybulsky et al. 2005; Cybulsky et al. 2009; Fujii et al.
2006; Inagi et al. 2005; Schonthal 2012). Thus, prevention
or treatment of ER stress could serve as a therapeutic strategy
to combat kidney disease and prevent the onset of CKD in
patients.
Natural products have served as a valuable source of drugs
and are considered as potential leads for drug development
(Doucette et al. 2015). Black pepper (Piper nigrum) is com-
monly used in conventional medicine especially in Chinese
and Indian medicine to treat several ailments (Doucette et al.
2015; Yoon et al. 2015). The medicinal properties of black
pepper are mainly attributed to its major phytoconstituent pip-
erine. Piperine [1-[5-[1,3-benzodioxol-5-yl]-1-oxo-2,4-
pentadienyl] piperidine] (PIP) is one of the four dia-
stereomeric geometric isomers isolated from black pepper
(Greenshields et al. 2015). It is also found in Piper longum
plants, which belongs to the family Piperaceae (Meghwal and
Goswami 2013). The chemical structure of piperine is com-
posed of three essential components: piperidine moiety linked
through carbonylamide linkage to the side chain,
methylenedioxyphenyl ring and conjugated double bond
chain.
Materials and methods
Materials used
All chemicals and reagents used for synthesis of piperine an-
alogs were of analytical grade and obtained from Sigma-
Aldrich, Germany. All reactions were monitored by thin layer
chromatography (TLC) and the spots were visualized using
ultraviolet (UV) transilluminator. TLC was conducted on pre-
coated silica gel aluminum plates (Merck, USA). Melting
points of the synthesized compounds were measured as range
using Stuart SMP40 automatic melting point apparatus. The
infrared (IR) spectra were recorded on Perkin Elmer Spotlight
400 Fourier transform-infrared (FT-IR) spectrophotometer.
The spectra were acquired using a universal attenuated total
reflectance (UATR) sensor to allow the application of the solid
samples.
Many recent studies have confirmed the medical properties
of piperine and further demonstrated its efficacy as anticarci-
nogenic, hepatoprotective, anti-inflammatory, anti-arthritic,
antidepressant, and antimicrobial (Kumar et al. 2015;
Meghwal and Goswami 2013). More recently, a study by
The prepared compounds were analyzed for carbon (C),
hydrogen (H), and nitrogen (N) (i.e., elemental analysis) using
Thermo Scientific Flash 2000 in the Central Laboratory Unit
at Qatar University. Mass spectra (MS) were recorded on an
Agilent 6460 Triple Quadrupole LC/MS system using

Structure–activity relationship of piperine
electrospray ionization (ESI) by direct injection technique and
reported as [M+1]+. Proton (1H) and carbon (13C) NMR spec-
tra were recorded using Bruker Avance III 400 MHz appara-
tus, and the chemical shifts were expressed in δ (ppm) with
reference to DMSO-d6 peak.
humidified incubator at 37 °C. The stock solution of
tunicamycin was diluted in media to a final concentration of
0.5 μg/mL. 4PBA was diluted in media and piperine analogs
were dissolved in DMSO.
Drug treatments
Synthesis of piperic acid (PA1) from piperine (PIP)
Twenty-four hours after seeding of cells on to 48-well
plates, piperine or its analogs were added to cells at either
250 or 500 nM concentrations for 24 h. After 24 h of pre-
treatment, the old media was removed and the cells were
washed with PBS and another fresh media containing
0.5 μg/mL tunicamycin was added and cells were incu-
bated for 2 h. After the completion of tunicamycin treat-
ment, the media was removed and cells were washed
twice with PBS and new fresh complete media was added
for another 22 h. To compare the potency and efficacy of
piperine and its analogs to inhibit ER stress and ER stress-
induced cell death, we used 4-phenylbutyrate (4PBA), a
well-known chemical chaperone, at 1 and 2 mM concen-
trations in the model.
Piperine (2 g, 7 mmol) was refluxed with 100 mL of 2 M
ethanolic potassium hydroxide for 25 h, and the ethanol was
evaporated under reduced pressure. The separated solid (potas-
sium piperate) was filtered and washed with cold ethanol, and
then dissolved in warm water and gradually acidified with
diluted HCl. The obtained yellow precipitate (piperic acid)
was filtered and washed with cold water. The crude product
(PA1) was recrystallized from ethanol to yield 1.4 g (6.4 mmol)
of yellow crystalline piperic acid (90% yield).
Synthesis of piperine analogs (PA2 to PA4)
The selected amido-piperine analogs (PA2 to PA4) were pre-
pared as described previously (Koul et al. 2000). Briefly, to a
solution of piperic acid (10 mmol) in 25 mL dichloromethane
(DCM), 2.0 mL (27.6 mmol) of thionyl chloride was added.
The mixture was kept under reflux for 1 h. Excess thionyl
chloride was removed under reduced pressure using rotary
evaporator. The obtained residue (piperoyl chloride) was dis-
solved in 20 mL DCM and the amine (10 mmol) in 20 mL
DCM was added dropwise. The mixture was stirred for 1 h at
room temperature. The solvent was evaporated and the residue
was crystallized from ethyl acetate. The yield of the final
products (PA2 to PA4) was in the range of 40–65%.
Cell viability assay
The effect of piperine and its analogs (PA1 to PA4) on
cell viability in the developed in vitro model of ER stress
was assessed by MTT assay, which is based on the enzy-
matic conversion of yellow tetrazolium salt 3-(4,5-dimeth-
ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide by mito-
chondrial dehydrogenases into purple formazan. After the
completion of the treatment, the media was removed and
replaced with fresh media containing 25 μL MTT
(0.5 mg/mL) for 3–4 h. Next, the media was removed
carefully and 100 μL of DMSO was added to dissolve
the formazan crystals. The absorbance was read at
570 nm using SpectraMax M2 multimode plate reader
(Molecular Devices, USA).
Cell culture
NRK-52E (rat renal proximal tubular cell line) was purchased
from Health Protection Agency, UK. Dimethyl sulfoxide, pip-
erine, thiazolyl blue tetrazolium bromide (MTT), 4-
pheynylbutryric acid (4PBA), and tunicamycin were pur-
chased from Sigma-Aldrich, Germany. Bicinchoninic acid
(BCA) protein assay reagent, Dulbecco’s modified Eagle me-
dium (DMEM), fetal bovine serum (FBS), L-glutamine,
penicillin-streptomycin (pen-strep), and phosphate-buffered
saline (PBS) were purchased from Thermo, UK. All primary
and secondary antibodies were purchased from Abcam, UK,
except for CHOP (GADD153) and cleaved caspase-3 primary
antibodies, which were obtained from Santa Cruz
Biotechnology, Germany, and Cell Signaling Technology
(CST), Netherlands, respectively.
Quantification of expression of ER stress markers
and apoptotic caspases
Total protein lysates from cell culture were normalized by
bicinchoninic acid (BCA) protein assay. Proteins were
separated using SDS-PAGE with an acrylamide concentra-
tion 15%. Equal concentrations (25 μg) of sample protein
were mixed with 4× sample loading buffer and electropho-
resed for 20 min at 70 V followed by 90 min at 140 V
(Bio-Rad, USA). Blots were incubated with rabbit anti-
GRP-78 (ab21685, 1:2000), mouse anti-CHOP (ab11419,
1:500), rabbit anti-caspase-12 (ab62484; 1: 1000), and
rabbit anti-cleaved caspase-3 (CST 9664; 1:1000) primary
antibodies overnight at 4 °C and subsequently with
a horseradish peroxidase (HRP)-labeled-goat anti-
NRK-52E cells were maintained in DMEM supplemented
with 10% FBS and 1% penicillin/streptomycin and 200 mM
L-glutamine (complete cell culture media). Cells were grown
in 100-mm tissue culture dish and kept in a 5% CO2

A. S. Hammad et al.
mouse IgG (1:10,000; Abcam, UK) or HRP-conjugated
goat anti-rabbit IgG (1:20,000; Abcam, UK) secondary
antibodies respectively for 1 h at room temperature.
Bands were visualized using enhanced chemiluminesence
(ECL) detection kit (Abcam, UK) and the band intensities
were quantified using a FluorChem-M imaging system
(Protein Simple, USA). The densitometry values were nor-
malized to beta-actin (used as loading control) and
expressed as percentage of DMSO-treated control.
in cell viability) as demonstrated by decreased reduction
of MTT (Fig. 1a). Cells were also subjected to western
blot analysis, at which there was a significant increase in
the expression of GRP78 and CHOP upon exposure to
tunicamycin. These results evidenced that using
tunicamycin at a concentration of 0.5 μg/mL for 2 h
caused induction of ER stress and consequent activation
of UPR in NRK-52E cells (Fig. 1b–d).
Dose-response studies (tolerability) for piperine and its
analogs in NRK-52E cells
Statistical analysis
All statistical analyses were performed using GraphPad Prism
7. Data were expressed as mean SEM relative to vehicle-
treated control. One-way analysis of variance (ANOVA) test
followed by Tukey’s post hoc test was used to determine sta-
tistical differences between the mean values of the different
experimental groups. A P value less than 0.05 was considered
statistically significant.
We compared the potency and efficacy of piperine and its
analogs against 4-phenylbutyrate (4PBA), a well-known
chemical chaperone, used at 1 and 2 mM concentrations
(Carlisle et al. 2014) (Fig. 2a–d). NRK-52E cells were treated
with piperine or its analogs at two concentrations—250 and
500 nM—for 24 h and the vehicle-control group received the
highest vehicle concentration (0.1% DMSO) used in the study.
Our results demonstrate that single doses of piperine or any of
its analogs at up to 500 nM were well tolerated by NRK-52E
cells (Figs. 3a, 4a, 5a, 6a, and 7a). The tolerability of NRK-
52E cells was measured by MTT assay. Based on the tolera-
bility studies, we chose 250 and 500 nM as optimal concen-
trations for the prepared analogs.
Results
Synthesis and characterization of amide-substituted
piperine analogs
The spectral and analytical data of the synthesized piperine
analogs were consistent with the previously reported data
in the literature (Koul et al. 2000; Sangwan et al. 2008;
Venkatasamy et al. 2004). Piperic acid (PA1) was obtained
by alkaline hydrolysis of piperine (PIP). Amide-substituted
analogs (PA2 to PA4) were prepared by conversion of PA1
to the corresponding acyl chloride using thionyl chloride
followed by the reaction with the appropriate amine as
described by Koul et al. (2000). The physical properties
and molecular formulae of the synthesized analogs and
the parent compound piperine are shown in Table 1.
All the IR spectra conducted on the solid sample using UATR
confirmed the presence of C=O stretching band, in the range of
1650 cm−1. From the mass spectra, it was possible to obtain the
mass of the molecular ion. The mass spectrum of the products
was recorded on an Agilent 6460 Triple Quadrupole LC/MS
system using electrospray ionization (ESI) by direct injection
technique and reported as [M+1]+. To further confirm the struc-
tures of the synthesized compound elemental analysis, 13C
NMR and 1H NMR spectra were analyzed.
Effect of piperine and its analogs on tunicamycin-induced
loss of renal cell viability
NRK-52E cells were pre-treated with the reference stan-
dard 4PBA, piperine (PIP), or synthesized analog (PA1 to
PA4) at the aforementioned concentrations for 24 h and
then exposed to tunicamycin (0.5 μg/mL for 2 h) to in-
duce cell injury. Upon measuring the cell viability (using
MTT assay) at 24 h post-tunicamycin exposure, the results
have demonstrated the ability of PIP, PA2, and PA4 to
protect against TM-induced loss of viability in NRK-
52E cells as seen in Table 2. While the rest of the
screened compounds including the reference standard re-
vealed no significant changes in cell viability as measured
by the MTT assay (Fig. 3a).
Effect of piperine and its analogs on tunicamycin-induced
expression of ER stress markers
To investigate the protective effect of pharmacologically eval-
uated compounds against TM-induced ER stress, NRK-52E
cells were seeded on 6-well plates and grown to sub-
confluency were treated with piperine for 24 h. After 24-h
treatment with piperine (PIP), cells were exposed to 0.5 μg/
mLTM for 2 h and evaluated for the expression of GRP78 and
CHOP at 24 h post-tunicamycin exposure. Densitometry anal-
ysis were consistent with the results obtained from the MTT
Effect of tunicamycin on cell viability and the expression
of ER stress markers in NRK-52E cells
Incubation of subconfluent cultures of NRK-52E cells
with 0.5 μg/mL tunicamycin for 2 h resulted in a signif-
icant loss of cell viability (evidenced by ~64% reduction

Structure–activity relationship of piperine
Table 1 Physical properties and molecular formulae of the synthesized analogs and the parent compound piperine
Melting
point
Molecular
Molecular
weight
Name and chemical structure
formulae
Piperine [(2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-1-
(piperidin-1-yl)penta-2,4-dien-1-one] (PIP)
128 131
C17H19NO3
285.14
218.06
299.15
−
Piperic acid [(2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)penta-
2,4-dienoic acid] (PA1)
220
202
C12H10O4
−
−
Cyclohexylamino analog [(2E,4E)-5-(benzo[d][1,3]dioxol-
5-yl)-N-cyclohexylpenta-2,4-dienamide] (PA2)
C18H21NO3
Diethylamino analog [(2E,4E)-5-(benzo[d][1,3]dioxol-5-
yl)-N,N-diethylpenta-2,4-dienamide] (PA3)
94
C16H19NO3
273.14
271.12
−
−
Pyrrolidinyl analog [(2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-
1-(pyrrolidin-1-yl)penta-2,4-dien-1-one] (PA4)
143
C16H17NO3

A. S. Hammad et al.
a
a
150
100
50
0
b
c
b
c
d
d
Fig. 1 a Dose-response studies with tunicamycin (TM) in NRK-52E
cells. Cell viability was determined by MTT assay. Values were
expressed as percentage of DMSO-treated control (mean SEM;
n = 3). b Effect of tunicamycin (TM) on GRP78 and CHOP expression
in NRK-52E cells as determined by western blotting and quantified using
densitometry (c, d). Values were normalized using β-actin and expressed
as percentage of DMSO-treated control (mean SEM; n = 10). #P < 0.05
compared to DMSO-treated control group
Fig. 2 a Effect of 4PBA on tunicamycin (TM)-induced loss of cell
viability (measured by MTT assay) in NRK-52E cells. Values were
normalized to TM-treated control and expressed as (mean SEM;
n = 3). b Effect of 4PBA on tunicamycin (TM)-induced expression of
GRP78 and CHOP in NRK-52E cells as determined by western blotting
and quantified using densitometry (c, d). Values were normalized to β-
actin and expressed as percentage of DMSO-treated control for GRP78
and percentage of tunicamycin (TM)-treated group for CHOP
(mean SEM; n = 3). #P < 0.05 compared to DMSO-treated control
group; *P < 0.05 compared to TM-treated group
assay for both PIP and PA2 as demonstrated by decreased
protein levels of GRP78 and CHOP (Figs. 3 and 5). The av-
erage percentage of decrease in GRP78 was 33% by using PIP
at 500 nM concentration. PIP also diminished the ability of
TM to induce CHOP by about 25% in comparison to TM-
treated group. Similar to PIP, cells treated with PA2 showed
decreased expression of GRP78 (by about 25%) and CHOP
(by about 30%) in comparison to TM-treated group. The
reference standard, 4PBA, decreased the expression of
GRP78 (~15%), and CHOP (~70%) compared to TM-
treated group (Fig. 2). PA1 and PA4 did not affect the expres-
sion of proteins tested (Table 2; Figs. 4 and 7). Intriguingly,

Structure–activity relationship of piperine
a
a
b
c
b
c
d
d
Fig. 4 a Effect of piperic acid (PA1) on tunicamycin (TM)-induced loss
of cell viability (measured by MTTassay) in NRK-52E cells. Values were
normalized to TM-treated control and expressed as mean SEM (n = 3).
#P < 0.05 compared to DMSO-treated control group; *P < 0.05
compared to TM-treated group. b Effect of piperic acid (PA1) on
tunicamycin (TM)-induced expression of GRP78 and CHOP in NRK-
52E cells as determined by western blotting and quantified using
densitometry (c, d). Values were expressed as percentage of DMSO-
treated control for GRP78 and percentage of TM-treated group for
CHOP (mean SEM; n = 3)
Fig. 3 a Effect of piperine (PIP) on tunicamycin (TM)-induced loss of
cell viability (measured by MTT assay) in NRK-52E cells. Values were
normalized to DMSO-treated control and expressed as mean SEM
(n = 3). b Effect of piperine (PIP) on tunicamycin (TM)-induced
expression of GRP78 and CHOP expression in NRK-52E cells as
determined by western blotting and quantified using densitometry (c,
d). Values were normalized to β-actin and expressed as percentage of
DMSO-treated control for GRP78 and percentage of tunicamycin
(TM)-treated group for CHOP (mean SEM; n = 3). #P < 0.05
compared to DMSO-treated control group; *P < 0.05 compared to TM-
treated group
Effect of piperine on tunicamycin-induced expression
of apoptotic markers
the PA3 caused a paradoxical increase in the expression of
CHOP by almost 69% using the concentration of 250 nM
(Fig. 6).
Todeterminethemechanism(s)thatunderlietheprotectiveeffect
of piperine against tunicamycin-induced ER stress and loss of

A. S. Hammad et al.
a
a
b
c
b
c
d
d
Fig. 5 a Effect of cyclohexylamino analog (PA2) on tunicamycin (TM)-
induced loss of cell viability (measured by MTTassay) in NRK-52E cells.
Values were normalized to DMSO-treated control and expressed as
mean SEM (n = 3). b Effect of cyclohexylamino analog (PA2) on
tunicamycin (TM)-induced expression of GRP78 and CHOP as
determined by western blotting and quantified using densitometry (c,
d). Values were normalized using β-actin and expressed as percentage
of DMSO-treated control for GRP78 or percentage of TM-treated group
for CHOP (mean SEM; n = 3). #P < 0.05 compared to DMSO-treated
control group; *P < 0.05 compared to TM-treated group
Fig. 6 a Effect of diethylamino analog (PA3) on tunicamycin (TM)-
induced loss of cell viability (measured by MTT assay) in NRK-52E
cells. Values were normalized to DMSO-treated control and expressed
as mean SEM (n = 3). b Effect of diethylamino analog (PA3) on
tunicamycin (TM)-induced expression of GRP78 and CHOP in NRK-
52E cells as determined by western blotting and quantified using
densitometry (c, d). Values were normalized using β-actin and
expressed as percentage of DMSO-treated control for GRP78 or
percentage of TM-treated group for CHOP (mean SEM; n = 3).
#P < 0.05 compared to DMSO-treated control group; *P < 0.05
compared to TM-treated group
cell viability, we evaluated its effect on the expression of apopto-
tic caspase-12 and caspase-3 at 24 h post-tunicamycin exposure.
IncorrelationtoourfindingsfromMTTassay,pre-treatmentwith
PIP at 500 nM caused a significant reduction (P < 0.05) in the
protein levels of both apoptotic caspases induced in renal cells
following exposure to tunicamycin (Fig. 8).

Structure–activity relationship of piperine
a
and anti-inflammatory effects (Zhang et al. 2015). Chemically,
piperine is composed of a basic piperidine moiety that is connect-
ed to side chain through carbonylamide, a side chain with conju-
gated double bonds, and a methylenedioxyphenyl (MDP) ring.
Basedonitsstructure,anyofitsthreecomponentscanbemodified
to evaluate their impact on the efficacy and potency of the parent
compound piperine. A previous study conducted by Koul et al.
has revealed that the piperidine moiety possess differential sensi-
tivity for inhibition of CYP450 (Koul et al. 2000). On the other
hand, the MDP ring in piperine, which could contribute to its
activity, is a common component in many natural compounds.
Therefore, in the current study, we focused on the modification of
the piperidine moiety and evaluated its effect on the pharmaco-
logical activity against ER stress.
In this study, piperic acid (PA1) was prepared by the alka-
line hydrolysis of piperine (PIP). Three amide piperine ana-
logs (PA1 to PA4) were synthesized from PA1 and character-
ized as described in the BMaterials and methods^ section. The
analogs were designed and prepared based on their similarity
to the parent compound piperine and screened to evaluate their
pharmacological activity against TM-induced ER stress and
cell death in renal cells.
b
c
To establish an in vitro model of ER stress in renal cells, we
chose tunicamycin (TM), an N-glycosylation inhibitor and a
chemical inducer of ER stress (Schonthal 2012). Our choice to
utilize TM was based on a previous study conducted by
Peyrou et al., which compared the efficacy of TM with other
chemical inducers of ER stress such as thapsigargin and oxi-
dized dithiothreitol (ox-DTT) in four different renal cell
lines—the porcine LLC-PK1, rat NRK-52E, canine MDCK,
and human HEK-293 cells (Peyrou and Cribb 2007). Results
from their study have indicated that tunicamycin is the most
potent inducer of ER stress among other chemical inducers
tested in the model.
d
We assessed the major hallmark proteins induced during
ER stress response such as the ER chaperone GRP78 and the
pro-apoptotic growth arrest and DNA damage-inducible pro-
tein 153 (CHOP/GADD153). GRP78 is an important resident
chaperone that maintains cellular homeostasis by ensuring the
proper folding of protein in the ER (Rovetta et al. 2012),
whereas, CHOP is a pro-apoptotic protein, which is induced
by all the three arms of UPR—ATF6, IRE1, and PERK path-
ways (Katsoulieris et al. 2010). Our results confirmed the
ability of TM (at 0.5 μg/mL for 2 h) to induce ER stress
(marked by the induction of GRP78 and CHOP), and cell
death (evidenced by ~60% reduction in cell viability) in
NRK-52E cells.
Fig. 7 a Effect of pyrrolidinyl analog (PA4) on tunicamycin (TM)-
induced loss of cell viability (measured by MTT assay) in NRK-52E
cells. Values were normalized to DMSO-treated control and expressed
as mean SEM (n = 3). #P < 0.05 compared to DMSO-treated control
group; *P < 0.05 compared to TM-treated group. b Effect of pyrrolidinyl
analog (PA4) on tunicamycin (TM)-induced expression of GRP78 and
CHOP in NRK-52E cells as determined by western blotting and
quantified using densitometry (c, d). Values were normalized using β-
actin and expressed as percentage of DMSO-treated control for GRP78 or
percentage of TM-treated group for CHOP (mean SEM; n = 3)
To compare the efficacy of piperine and its analogs to a
known reference standard, we chose to use 4PBA, a well-
known chemical chaperone. 4PBA is a nontoxic butyrate an-
alog, which has the ability to enhance the protein folding
capacity of ER and improve the trafficking of mutant proteins
(Humeres et al. 2014). In our study, we found that 4PBA
Discussion
Piperine, the major ingredient of pepper species, is reported to
possessmultipleactivitiesincludingantioxidant,neuroprotective,

A. S. Hammad et al.
Table 2 Summary of the effect of piperine and its analogs against tunicamycin (TM)-induced ER stress and cell death in NRK-52E cells
Compound
Concentration tested
Cell viability
Western blotting
GRP78
CHOP
4PBA
(reference standard)
Piperine
(PIP)
Piperic acid
(PA1)
Cyclohexylamino analog
(PA2)
Diethylamino analog
(PA3)
Pyrrolidinyl analog
(PA4)
1 and 2 mM
N.C
▼ (~15%)
▼ (~33%)
N.C
▼ (~70%)
▼ (~25%)
N.C
250 and 500 nM
250 and 500 nM
250 and 500 nM
250 and 500 nM
250 and 500 nM
▲ (~13%)
N.C
▲ (~11%)
N.C
▼ (~25%)
N.C
▼ (~30%)
▲ (~69%)
N.C
▲ (~11%)
N.C
The black upward and downward pointing triangles indicate an increase and a decrease respectively in the parameter measured
a
b
protects against TM-induced expression of both GRP78 and
CHOP. However, a higher magnitude of reduction was ob-
served on CHOP expression (Carlisle et al. 2014). Our find-
ings are in line with a previous study in HK-2 cells by Carlisle
et al., where 4PBA has been shown to prevent TM-induced
CHOP expression without affecting the expression of GRP78
(Carlisle et al. 2014).
In this study, four piperine analogs (piperic acid and 3
amide piperine analogs) were synthesized and their structures
were elucidated by pertinent spectroscopic techniques. Each
analog was tested at two different concentrations (250 and
500 nM) for its pharmacological activity to relieve ER stress
induced by TM in renal cells and compared against its parent
compound piperine and reference standard 4PBA.
Based on the literature, all the prepared analogs were used
in nanomolar (250 and 500 nM) concentrations, whereas the
reference standard 4PBAwas used in millimolar (1 and 2 mM)
concentration. Although the effects of piperine were similar to
that of 4PBA, its effects were mainly mediated through reduc-
tion in the expression of GRP78. In contrast, 4PBA appears to
mediate its effects mainly through reduction in CHOP expres-
sion. Thus, future studies could examine the synergistic ef-
fects expected out of combination of 4PBA and piperine as a
strategy to prevent ER stress in renal cells. Moreover, a pre-
vious study revealed the ability of piperine to downregulate
the mRNA expression of GRP78 and the protein expression
on XBP1 and ATF6 in the livers of HFD-fed mice (Jwa et al.
2012). Thus, future studies could also examine the effect of
piperine and its analogs on the expression of the XBP-1 and
ATF6 in NRK-52E cells exposed to TM.
c
The most potent compound in our study was piperine
followed by the cyclohexylamino analog (PA2). Similar to
piperine, PA2 decreased the expression of GRP78 and
CHOP in NRK-52E cells. Thus, its effects could be potentially
mediated through either PERK and/or ATF6 arms of the UPR.
Further studies to investigate the effect of this analog on the
Fig. 8 Effect of piperine (PIP) on tunicamycin (TM)-induced expression
of cleaved caspase-12 and caspase-3 in NRK-52E cells as determined by
western blotting (a) and quantified using densitometry (b, c). Values were
normalized to β-actin and expressed as percentage of DMSO-treated
control (mean SEM; n = 3). #P < 0.05 compared to DMSO-treated
control group; *P < 0.05 compared to TM-treated group

Structure–activity relationship of piperine
expression of XBP1 and JNK (downstream targets of IRE1
arm) would help to unravel the mechanism(s) behind its pro-
tective effects against ER stress. Our studies with piperine in a
model of DTT-induced ER stress in NRK-52E cells recapitu-
lated the findings obtained from the model of tunicamycin-
induced ER stress on GRP78 and CHOP expression (shown in
ESM 2). Consistent with our findings, topical application of
piperine and its cyclohexylamino analog for 4 weeks was
shown to ameliorate vitiligo (Faas et al. 2008), a condition
that is strongly associated with ER stress (Jeong et al. 2010;
Toosi et al. 2012). Together, these findings indicate the thera-
peutic potential of piperine and its cyclohexylamino analog to
act as Bchemical chaperones^ under the conditions of ER
stress.
Unlike piperine, piperic acid (PA1) did not affect the ex-
pression of CHOP and GRP78. Intriguingly, treatment with
diethylamino analog (PA3) caused a paradoxical increase in
the expression of CHOP in renal cells. Thus, further studies
are required to understand their impact on the CHOP expres-
sion and to determine whether its effects on CHOP are tran-
sient or permanent. Although treatment with pyrrolidinyl an-
alog (PA4) did not alter the induction of ER stress markers
GRP78 and CHOP in renal cells, it showed improvements in
cell viability against tunicamycin-induced cytotoxicity.
Our studies to unravel the mechanisms underlying the
chemical chaperone activity and cytoprotective effects of pip-
erine indicate that pre-treatment with piperine decreases the
induction of ER-associated caspase-12 and the executioner
caspase-3 in renal cells subjected to tunicamycin. Although
studies in cancer lines (Lin et al. 2013; Samykutty et al. 2013;
Yaffe et al. 2015) have demonstrated piperine’s ability to in-
duce apoptosis especially at high concentrations (in micromo-
lar range), to our knowledge, this is the first study to demon-
strate the anti-apoptotic effects of piperine (at nanomolar con-
centrations) in normal cells. The discrepancy in the pro-
apoptotic vs. anti-apoptotic effects of piperine could be attrib-
uted to the neoplastic state of the cells (cancerous vs. non-
cancerous) and the dose tested, i.e., anti-apoptotic at low
(nanomolar) concentrations and pro-apoptotic at high
(micromolar) concentrations.
increase in CHOP expression, which might aggravate
ER stress.
4. Removal of the piperidine ring by hydrolysis of piperine
(PIP) or replacement of six-membered ring with a five-
membered ring (pyrrolidinyl analog (PA4)) appears to re-
sult in loss of pharmacological activity against ER stress.
5. CompoundsPIP,PA2,andPA4alsopossesscytoprotective
properties against ER stress-induced cell death.
6. In terms of potency, piperine and its analogs (used in
nanomolar concentrations) are about 4000 times more
potent when compared to the reference standard 4PBA
(used in millimolar concentration).
In conclusion, considering their high potency (used in nM),
efficacy, and a wide margin of safety, piperine (PIP) and its
cyclohexylamino analog (PA2) appear to be promising drug
candidates for further investigation in vivo for prevention of
ER stress and potential application to treat ER stress-related
renal disorders in patients.
Acknowledgments This study was supported by the intramural grants
(QUUG-CPH-CPH-13/14-2; QUST-CPH-SPR-12/13-5; QUSTCPH-
FALL-13/14-6; QUST-CPH-SPR-13/14-8; QUST-CPH-FALL-14/15-5;
QUST-CPH-SPR-14/15-14) awarded by the Office of Academic
Research, Qatar University, Doha, Qatar.
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