Blended polar/nonpolar peptide conjugate interferes with human insulin amyloid-mediated cytotoxicity

Article abstract of DOI:10.1016/j.bioorg.2021.104899

Insulin, a peptide hormone and a key regulator of blood glucose level, is routinely administered to type-I diabetic patients to achieve the required glycemic control. Insulin aggregation and ensuing amyloidosis has been observed at repeated insulin injection sites and in injectable formulations. The latter occurs due to insulin agglomeration during shipping and storage. Such insulin amyloid leads to enhanced immunogenicity and allow potential attachment to cell membranes leading to cell permeability and apoptosis. Small molecule inhibitors provide useful interruption of this process and inhibit protein misfolding as well as amyloid formation. In this context, we report the propensity of a palmitoylated peptide conjugate to inhibit insulin aggregation and amyloid-mediated cytotoxicity, via designed interference with polypeptide interfacial interactions.

Full text of DOI:10.1016/j.bioorg.2021.104899

Bioorganic Chemistry 111 (2021) 104899
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Blended polar/nonpolar peptide conjugate interferes with human insulin
amyloid-mediated cytotoxicity
a
b
a,
1
b,*
Shantanu Sen , Prerana Singh , Narendra Kumar Mishra , Subramaniam Ganesh
,
c
,
d
,
e
,
f
,
*
a,d,e,*
Sri Sivakumar
, Sandeep Verma
a
Department of Chemistry, Indian Institute of Technology Kanpur, Kanpur 208016, UP, India
b
c
d
e
f
Department of Biological Sciences & Bioengineering, Indian Institute of Technology Kanpur, Kanpur 208016, UP, India
Department of Chemical Engineering, Indian Institute of Technology Kanpur, Kanpur 208016, UP, India
Centre for Nanoscience, Indian Institute of Technology Kanpur, Kanpur 208016, UP, India
Centre for Environmental Science & Engineering, Indian Institute of Technology Kanpur, Kanpur 208016, UP, India
Material Science Programme, Indian Institute of Technology Kanpur, Kanpur 208016, UP, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
Insulin, a peptide hormone and a key regulator of blood glucose level, is routinely administered to type-I diabetic
patients to achieve the required glycemic control. Insulin aggregation and ensuing amyloidosis has been
observed at repeated insulin injection sites and in injectable formulations. The latter occurs due to insulin
agglomeration during shipping and storage. Such insulin amyloid leads to enhanced immunogenicity and allow
potential attachment to cell membranes leading to cell permeability and apoptosis. Small molecule inhibitors
provide useful interruption of this process and inhibit protein misfolding as well as amyloid formation. In this
context, we report the propensity of a palmitoylated peptide conjugate to inhibit insulin aggregation and
amyloid-mediated cytotoxicity, via designed interference with polypeptide interfacial interactions.
Insulin
Aggregation
Inhibition
Peptide inhibitor
Palmitoylated-peptide conjugate
Hydrophobicity
Amyloid-mediated cytotoxicity
Cytoprotective
1
. Introduction
Protein aggregation and amyloidosis is a major physical trans-
amyloidosis’ [2,3]. Aggregates present in insulin injectable may also
lead to enhanced immunogenicity in patients [4]. With time insulin
aggregation leads to its shorter shelf-life, reduced bioavailability and
undesired deposition. Different factors of the exposed environment such
as pH, ionic strength of the environment, agitation, temperature, storage
conditions, properties of contact surfaces etc. may destabilize insulin
and further promote its aggregation. Hence, the ability to modulate or
inhibit aggregation is not only important for therapeutic reasons, but is
also needed to safeguard storage and suppress adventitious side-
reactions.
formation leading to debilitating pathophysiological conditions with
far-reaching clinical consequences. Till now, many diseases such as
Huntington’s disease, Alzheimer’s disease, Parkinson’s disease, Prion-
associated encephalopathies, to name a few, are known to be associ-
ated with protein aggregation [1]. Agglomeration in therapeutic poly-
peptides and proteins can also interfere with their (bio)processing
during shipping and storage. Latter necessitates deeper understanding of
the process for therapeutic proteins/polypeptides and its ramifications
due to adverse responses in patients. Immunogenic nature of such
aggregated therapeutic proteins could curtail synthesis processes,
product development and present undesired health risk issues.
Misfolded protein monomers aggregate due to synergistic coopera-
tion from intermolecular forces including hydrophobic and aromatic-
aromatic interactions, causing loss of native three-dimensional struc-
ture leading to thermodynamically stable amyloidogenic protein en-
sembles with the ability to trigger cytotoxicity [5,6]. In many instances,
protein aggregates have been reported for cellular damage via initiation
of apoptotic events. Although, definite mechanisms involved in toxic
amyloid triggered cellular apoptosis yet to be explored further.
Pharmaceutical insulin formulations availed for diabetic patients
also encounter similar problems for its aggregating tendency and known
to initiate formation of lobules due to increase in aggregate accumula-
tion at repeated insulin injection sites causing ‘injection-localized
*
Corresponding authors at: Department of Chemistry, Indian Institute of Technology Kanpur, Kanpur 208016, UP, India (S. Verma). Department of Chemical
Engineering, Indian Institute of Technology Kanpur, Kanpur 208016, UP, India (S. Sivakumar).
E-mail addresses: sganesh@iitk.ac.in (S. Ganesh), srisiva@iitk.ac.in (S. Sivakumar), sverma@iitk.ac.in (S. Verma).
1
Current address: Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen Ø, Denmark.
https://doi.org/10.1016/j.bioorg.2021.104899
Received 14 December 2020; Received in revised form 4 April 2021; Accepted 5 April 2021
Available online 9 April 2021
0
045-2068/© 2021 Elsevier Inc. All rights reserved.

S. Sen et al.
Bioorganic Chemistry 111 (2021) 104899
However, apoptotic events are cumulatively contributed by protein
aggregate mediated inflammatory responses, destabilization as well as
permeabilization of cellular membrane. Elevated cellular stress through
reactive oxygen species overproduction, cytokine overproduction and
activation of caspases etc were correlated as some of the resultant
apoptatic events [7]. Apoptotic cells can be distinguished from their
nuclear fading caused due to dissolution of chromatin by DNase and
RNase, nuclear shrinkage due to chromatin condensation, rupturing of
nuclear membrane and nuclear DNA fragmentation [8]. Insulin
amyloid-mediated cell apoptosis has already been reported in many
instances [9–11].
Previous studies have shown significant implications of design stra-
tegies in formulating such peptide-based inhibitors composed of aro-
matic amino acid residues, having significant inhibitory efficacy of
protein aggregation [12–15]. Further approaches including hybrid
peptides, bio-inspired peptides, aromatic compounds, nanoparticles,
metal complexes, trehalose, polycyclic compounds etc. have also been
demonstrated for delaying or inhibiting insulin aggregation process
delivery. Besides, Trp-Trp conjugation is also vital as it provides
π π
-
interactions in addition to terminal polarity of taurine residue as well as
non-polarity of palmitic acid. We hypothesized that precisely orches-
trated amphiphilic character of 1 will induce self-assembled nano-
structures. Indeed, merged polarity and non-polarity of such
nanostructures will result in insulin aggregation inhibition by altering
the pathways for amyloidosis as well as solubilizing any traceable toxic
insulin aggregates and thus hinder harmful interactions of aggregates
with critical cellular components [27,28]. Similar advantage of blended
polarity/non-polarity was reported for a surfactant (a myristoylated
phenylalanine conjugate with a polyether hydrophilic group) stabilizing
model protein drugs during heat-aging aggregation [15]. The following
study has been carried out to demonstrate the favourable outcome
regarding improved inhibitory efficacy after adapting the above
mentioned strategy.
2. Materials and methods
[
16–21].
Cytotoxic nature of insulin fibril was linked up to their detergent like
2.1. General information
capability of disaggregating cellular membranes in contact. Exposed
hydrophobic patches of misfolded inactive insulin monomers are known
to be involved in generation of insoluble toxic fibrillar aggregates
Human insulin (HI), FITC-phalloidin, trypsin
-
ethyl-
enediaminetetraacetic acid (trypsin-EDTA), Dulbecco’s modified eagle
medium (DMEM), Triton X-100, 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) were purchased from Sigma
Aldrich and Thioflavin T from Fluka. Penicillin-streptomycin antibiotic
solution was purchased from HiMedia. Dimethyl sulfoxide (DMSO) was
obtained from Merck Chemicals, India. Foetal Bovine Serum (FBS) was
purchased from Gibco Life Technologies. HeLa (Carcinoma) cells were
procured from National Centre for Cell Science (NCCS), Pune. DAPI and
Hoechst were purchased from Thermo fischer. Acridine Orange (AO)
and Ethidium bromide (EtBr) were procured from Sigma Aldrich. The
chemicals used for in vitro cell culture experiments were used as
received. All solvents used for these experiments were distilled prior to
use following standard procedures. All chemicals and resins were pur-
chased from Spectrochem, Mumbai, India and used without further
purification. 1H NMR spectra were recorded on JEOL-ECS 400 model
operating at 400 MHz and 13C NMR spectra were recorded on JEOL-
DELTA2 500 model operating at 500 MHz. High resolution mass
spectra (HRMS) were recorded at IIT Kanpur, India, on Waters, Q-Tof
Premier micromass HAB 213 mass spectrometer using a capillary
voltage of 2.6–3.2 kV.
[
22–24]. Hence, hydrophobic character driven engagement of such
patches would curtail or inhibit the aggregation process. Sufficient ev-
idences of hydrophobicity mediated fibrillation inhibition phenomena
encourage us to probe the inhibitory efficacy of the inspired newly
designed construct 1 (Fig. 1) [25].
Compound 1 is constructed from the hydrophobic chain of palmitic
acid conjugated at N-terminal, polar taurine residue at C-terminal and
sandwiched aromatic tryptophan residues (Trp-Trp). We have reported
on the potential of Trp-Trp-Taurine sequence interacting with the aro-
matic tripeptide stretch of Phe24Phe Tyr , C-terminal hydrophobic
sequence of insulin B chain important for its native dimerization, in
modulating hydrophobic interactions between insulin molecules and
preventing their fibrillation [12,13]. We anticipated that the introduc-
tion of a palmitoyl chain would provide additional hydrophobic char-
acter enabling the new construct to further interact with exposed
hydrophobic residues of monomeric insulin, under applied stress con-
ditions for self-aggregation.
25
26
This whole conjugate itself making micelle kind of structures in so-
lution phase due to its amphiphilic built. Critical micelle concentration
(
CMC) of compound 1 was estimated to be ~10 µM (Fig. S9). Whereas,
2.2. Synthesis of Palmitoylated Trp-Trp-Tau peptide (1)
no such micelle formation was observed for Trp-Trp-Tau alone at cor-
responding concentrations (Fig. S10). Apart from providing hydropho-
bicity, palmitic acid is also highly biocompatible in terms of its
utilization as a part of any drug candidate. As palmitic acid is highly
abundant in our body and found in approximately 5% of our total body
weight [26]. Some of the existing excipients were also employed in
combination with palmitic acid in several formulations used for drug
Palmitoylated Trp-Trp-Tau was synthesized and characterized as
1
13
shown in the supporting information (Scheme S1). H NMR spectra,
C
NMR spectra and HRMS-ESI data for the final synthesized compound
were also provided (Figure S12-S14).
2.3. Thioflavin T (ThT) binding assay
Freshly prepared insulin and insulin mixed with different peptide
◦
conjugates, were incubated at 65 C in 0.1 N HCl water (pH 1.6) with an
additional salt concentration of 25 mM NaCl. ThT dye was added to
reaction volume to attain a final concentration of 20 µM. Fluorescence
intensity was measured at room temperature with an excitation wave-
length (λex) of 410 nm and observed emission wavelength (λem) at 488
nm. The aggregation behavior of insulin alone and in presence of the
peptide conjugates were studied for the insulin concentration of 0.5 mg/
mL (~86
μ
M). Fluorescence spectra were recorded with a 10 mm quartz
◦
cells at 65 ± 0.1 C using a Varian Luminescence Cary Eclipse. Three
consecutive scans were taken for each sample and the average spectra
was plotted by Origin 9.1 software (Fig. 2). All the observations were
carried out under a fixed bandpass of 5 nm in addition to a magnetic
stirrer maintaining sample homogeneity at 800 rpm. AR-grade hydro-
chloric acid and HPLC-grade water were used for every preparation
Fig. 1. Palmitoylated Trp-Trp-Tau peptide (1): blended polar and
nonpolar patches.
2

S. Sen et al.
Bioorganic Chemistry 111 (2021) 104899
five folds dilution (17
μ
M) of incubated samples of insulin, procured at
different time intervals in the absence and presence of compound 1 (100
M) (Fig. 4).
μ
2
.6. Native Gel-electrophoresis
Native (non-denaturing) polyacrylamide gel electrophoresis was
performed at a constant voltage at 30 mA with a Bio-Rad mini-PROTEIN
II electrophoresis system using 15% Tris-HCl polyacrylamide gel. Ali-
quots of equal volumes of human insulin samples (~86
collected at different time intervals incubated in absence and presence of
(100 M) and directly loaded to the gel without any processing. The
μM) were
1
μ
gels were stained with coomassie blue stain and further destained
overnight (Fig. 5).
2
.7. Atomic force microscopy (AFM)
Fig. 2. Thioflavin T fluorescence spectra of human insulin (~86 M) aggre-
μ
gation as well as its inhibition by Trp-Trp-Tau (100
100 M).
μ
M) and compound 1
Freshly prepared and aged incubated samples of insulin (~86 M)
μ
(
μ
alone and mixed with compound 1 (100 M) were imaged with an
μ
atomic force microscope (Molecular Imaging, USA). The machine was
operating under the Acoustic AC mode (AAC) with 0.6 N/m force con-
stant and 150 kHz of resonant frequency, with the aid of a cantilever
involved in this study.
[
NSC 12(c) from MikroMasch]. A portion of 10
samples was diluted by 90 L of solution containing water (pH 1.6) and
5 mM NaCl. 5 L of sample from the final cocktail was deposited uni-
formly using a spin coater operating at speed of 200–500 rpm (PRS-
000), onto a freshly cleaved glass surface at room temperature. The
μ
L of fresh and incubated
2
.4. Circular dichroism (CD) spectroscopy
μ
2
μ
Freshly prepared solution of insulin (~86
μ
M) was either incubated
◦
alone or with 1 (100
μ
M) at 65 C in water (pH 1.6) with 25 mM NaCl.
4
All experiments were carried out at room temperature. Spectra were
collected for a diluted incubated samples of insulin (17 M) procured at
sample-coated glass was dried at room temperature for overnight
duration without any contact of dust particles and followed by AFM
imaging. Final images of the AFM micrographs were acquired by Pico
scan 5 software and extensive analysis of data was done by using visual
SPM (Fig. 6).
μ
different time points from the incubated reaction mixture, using JASCO
J-815 CD spectrometer and a quartz cuvette of 1 mm path length. CD
spectra were collected between 195 and 300 nm and each spectrum
represents the average of three scans (Fig. 3). To avoid any instrumental
baseline drift contributed by the working solution, the background
values were subtracted from each individual sample measurement.
2
.8. MTT assay
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bro-
2
.5. Fourier transform infrared (FTIR) spectroscopy
mide) reduction assay was used to analyze the biocompatibility of
compound 1 by determining the cytotoxicity of HeLa cells treated with
it. Varying concentrations of compound 1 were added to different sets of
cultured HeLa cells in 96-well plate. A set of untreated HeLa cells was
used as control. Only viable cells are capable of reducing MTT into blue
formazan crystals. Hence, reduction in formazan crystals can directly be
correlated with the reduction in cellular viability due to compound’s
cytotoxicity. Initially cells were seeded at 5000 cells/well in a 96 well
plate and incubated for 7 h for cells to adhere before the compound
treatment. Stock solution of 1 was prepared by diluting in DMEM cell
ATR-FTIR spectra were recorded with a PerkinElmer Spectrum Two
Fourier transform spectrometer equipped with peltier stabilized detec-
tor. To avoid any instrumental baseline drift contributed by the working
solution (water of pH 1.6 with 25 mM NaCl), the background value was
subtracted for each individual sample measurement. All experiments
were carried out at room temperature, and spectra were collected after
culture media and added to cells to attain final compound 1 concen-
◦
trations of 50, 100 and 200
humidified atmosphere with 5% CO
free media (0.5 mg/ml) was added to the wells and incubated for 4 h in
the CO incubator. After carefully removing the supernatant, 200 L of
μ
M, following incubation for 24 h [37 C,
2
]. Further, MTT solution in serum
2
μ
DMSO was added and incubated for 20 min to dissolve the formazan
crystals. The absorbance at 570 nm was analysed using Thermo Scien-
tific Multiskan Spectrum (UV ꢀ vis spectrometer) and percentage of cell
viability was measured (Fig. 7).
2
.9. Hoechst 33342 staining
Hoechst 33342 (Invitrogen, H3570) staining was carried out for
detection of apoptotic cells. HeLa cells were grown on gelatin coated
4
coverslips in 24 well plates at a cellular density of 5 × 10 per well and
◦
incubated at 37 C in a humidified atmosphere with 5% CO
2
. Compound
M) in
PBS (pH 7.4) were mixed with HeLa cells to attain final protein con-
centration of 40 M. A set of untreated HeLa cells as control were also
cultured which were devoid of any exposure to insulin. After 24 h of
1
(100
μ
M) treated or untreated, 48 h aged insulin samples (~86
μ
Fig. 3. Far UV-CD spectra during human insulin (~86
interference by compound 1 (100 M).
μM) aggregation and its
μ
μ
3

S. Sen et al.
Bioorganic Chemistry 111 (2021) 104899
Fig. 4. Time dependent ATR – FTIR spectra of HI (~86
μ
M) aggregation and its interference by compound 1 (100
μ
M).
formaldehyde and stained with Hoechst 33342 dye (1
μ
g/mL) for 20 min
at room temperature. Finally, the cells were washed with PBS thrice to
ensure complete removal of unbound stain. The samples were then
mounted and images were visualized using confocal microscope (Figs. 8
&
S7).
2
.10. Cytotoxicity and cellular integrity
HeLa cells were stained with FITC-phalloidin and DAPI to detect any
significant changes in cytoskeletal and nuclear morphology respectively.
Insulin (~86
μ
M) with or without compound 1 (100
μ
M) were first
◦
incubated at 65 C in PBS buffer (pH 7.4) for 48 hrs. HeLa cells were
grown on gelatin coated coverslips in 24 well plates at a cellular density
Fig. 5. Native polyacrylamide gel-electrophoresis of (M) Protein molecular
weight marker; (a) 0 h aged HI (~86 M); (b) 0 h aged HI (~86 M) co-
incubated with 1 (100 M); (c) 3 h aged HI (~86 M); (d) 3 h aged HI (~86
M) co-incubated with 1 (100 M); (e) 8 h aged HI (~86 M); (f) 8 h aged HI
~86 M) co-incubated with 1 (100 M), (g) 30 h aged HI (~86 M); (h) 30 h
aged HI (~86 M) co-incubated with 1 (100 M).
4
◦
μ
μ
of 5 x10 cells/well and incubated at 37 C in a humidified atmosphere
with 5% CO . Compound 1 treated or untreated insulin samples in PBS
buffer were directly added to attain a final concentration of 40 M. A set
μ
μ
2
μ
μ
μ
μ
(
μ
μ
μ
of untreated HeLa cells was used as negative control. The cells were
treated with the compounds for 24 h followed by fixation with 4%
formaldehyde solution for 20 min. Further, the cells were washed with
PBS and permeabilized with 0.1% Triton-X 100 for 10 min. The cells
μ
μ
incubation, the cells were washed with PBS, fixed with 4%
Fig. 6. Time-dependent AFM micrographs (scale bar ꢀ 200 nm) of HI (~86
incubated with 1 (100 M) for (D) 0 h and (E) 30 h.
μ
M) incubated for (A) 0 h, (B) 10 h and (C) 30 h. AFM micrographs of HI (~86 M) co-
μ
μ
4

S. Sen et al.
Bioorganic Chemistry 111 (2021) 104899
3
. Results and discussion
.1. Insulin fibrillation kinetics
Thioflavin T (ThT) dye binding was used to study effect of 1 and a
3
control tripeptide Trp-Trp-Tau on insulin aggregation. Amyloidogenic
incubation conditions were used in these assays consisting of elevated
◦
temperature (65 C), acidic environment (pH 1.6) and higher salt con-
centration (25 mM NaCl) [12]. Compound 1 (100
μ
M) and Trp-Trp-Tau
M) were co-incubated with human insulin (0.5 mg/mL or ~ 86
M) separately and their effect on aggregation were assessed. Notably,
(
100
μ
μ
ThT fluorescence was negligible for human insulin throughout the early
incubation period in absence or presence of these peptide ligands.
Whereas, a sharp exponential rise in ThT fluorescence was observed
after a lag phase of 8 h for only insulin. Although Trp-Trp-Tau was able
to suppress ThT fluorescence for a shorter duration, but a remarkable
anti-amyloidogenic effect was observed for compound 1 with a longer
lag phase and significant decrease in ThT fluorescence afterwards
Fig. 7. MTT assay in HeLa cells treated with varied concentrations of com-
pound 1 for 24 h. After 24 h of treatment cell survival values of treated or
untreated cells were represented as percentage of cell proliferation.
(
Fig. 2). Native secondary structure stabilization of insulin in presence of
was also inferred by both fourier-transform infrared (FTIR) spectros-
1
copy and circular dichroism (CD) spectroscopy.
were later blocked in 1% Bovine serum albumin and stained with FITC-
Phalloidin and DAPI solution. The samples were washed with PBS and
mounted on glass slides. Further, the cellular morphology was visualized
using Confocal Laser Scanning Microscopy (CLSM) using appropriate
lasers (Figs. 9 & S8).
3.2. Protein conformation analysis
CD spectra pattern of native insulin is characterized by two negative
minima around 208 nm and 222 nm reflecting the -helix rich structure,
α
whereas, insulin fibrils consisting of cross-β sheet like structure can be
identified with a negative peak around 218 nm [12]. Transformation of
2
.11. Apoptosis assay
α
-helix rich native human insulin towards β-sheet rich fibrillar bodies
was observed after 8 h of incubation under amyloidogenic conditions.
Notably, co-incubation with compound 1 (100 M) prevented the loss of
the -helix rich native structure of human insulin (~86 M) for a sig-
nificant duration (Fig. 3).
Although this compound was able to maintain
Insulin (~86
μ
M) with or without compound 1 (100
μ
M) were first
◦
incubated at 65 C in PBS buffer (pH 7.4) for 48 hrs. HeLa cells in log
μ
4
0
phase (5 × 10 cells/well) were cultured in 24 well plate at 37 C in a
humidified incubator containing 5% CO (v/v). Subsequently, the cells
α
μ
2
were treated with incubated human insulin and compound 1 co-
incubated human insulin for 24 h. Untreated HeLa cells were taken as
negative control in this assay. After 24-hour treatment, the cells were
washed thrice with PBS and stained with acridine orange (AO) (100 µg/
ml in PBS) and ethidium bromide (EtBr) (100 µg/ml in PBS) at room
temperature for 10 min. The stained samples were analysed by confocal
microscopy (Fig. 10). Cell apoptosis percentage was quantified as the
number of apoptotic cells divided by the total number of cells (Fig. 11).
Quantification of cells were carried out by ImageJ software (n = 3).
α
-helix content of
human insulin in spite of harsh incubating conditions, there is slight
decrease in protein helicity. This can be correlated with the slight in-
crease of ThT fluorescence in the later phase of incubation of insulin in
presence of compound 1. In order to gain knowledge about secondary
structure of insulin, signature amide I band pattern of different FTIR
spectra have been analyzed (Fig. 4) [9]. Amide I band is associated to the
–
–
C
O stretching and represents
α-helix structure with a peak around ~
ꢀ 1
1652 cm . While in case of transformed β sheet structure the amide I
band appears around ~ 1630 cm . Under amyloidogenic condition a
ꢀ 1
ꢀ 1
peak around ~ 1630 cm can be seen after 8 h of incubation along with
Fig. 8. Morphology of HeLa cells after 24-
hour treatment with insulin fibrils stained
with Hoechst 33342. Fluorescence micro-
scopy images of (A) Cells treated with 48 h
aged insulin (~86
sample of 48 h aged insulin (~86
incubated with 1 (100 M). Nuclear frag-
μ
M) (B) Cells treated with
μ
M) co-
μ
mentations were marked with yellow arrows
showing fragmented condensed nuclei as
well as nuclear blebbing. (Scale bar – 50
μ
m).
(
For interpretation of the references to colour
in this figure legend, the reader is referred to
the web version of this article.)
5

S. Sen et al.
Bioorganic Chemistry 111 (2021) 104899
Fig. 9. Confocal laser scanning microscopy images of HeLa cells stained with FITC-phalloidin (Green) and DAPI (Blue) for evaluation of cytoskeletal and nuclear
morphology (scale bar – 100 m). The representative images for (A) HeLa cells treated with 48 h aged human insulin (~86 M) and (B) HeLa cells treated with 48 h
aged human insulin (~86 M) co-incubated with 1 (100 M), both incubated for 24 h. Blue dashed circle in panel A shows the presence of amorphous insulin fibrils
around the HeLa cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
μ
μ
μ
μ
Fig. 10. Detection of apoptosis by AO/
EtBr staining in HeLa cells incubated
with human insulin and compound 1 for
2
4 h. The representative images for (A)
Untreated HeLa cells, (B) HeLa cells
treated with 48 h aged human insulin
(
~86
with 48 h aged human insulin (~86
co-incubated with 1 (100 M). [Blue
μ
M) and (C) HeLa cells treated
μ
M)
μ
arrow- viable cells, White arrow- early
apoptotic cells, Yellow arrow- late
apoptotic cells ] (Scale bar: 100 µm).
(
For interpretation of the references to
colour in this figure legend, the reader is
referred to the web version of this
article.)
Oligomerization status of incubated insulin at different time points
were assessed by native polyacrylamide gel electrophoresis (Native
PAGE) [29,30]. Initially, dimeric (Molecular weight ~ 11.6 kDa) form of
human insulin in either presence or absence of compound 1 can be
observed without any incubation (Fig. 5a & b). Whereas, on heating
both monomer (Molecular weight ~ 5.8 kDa) and dimer (Molecular
weight ~ 11.6 kDa) populations can be seen (Fig. 5c & d). On further
heating, in the absence of compound 1 appearance of smear can be
observed due to the generation of smaller insulin protofibrils formed
during insulin aggregation (Fig. 5e & g). However, for the corresponding
time points, insulin co-incubated with compound 1 was able to maintain
intact protein bands (Fig. 5f & h). Hence, from this result it can be
inferred that in presence of compound 1 human insulin maintains its
soluble non-aggregated form even under extreme amyloidogenic
conditions.
3
.3. Morphological changes using AFM analysis
Fig. 11. Quantification of viable and apoptotic cells treated with 48 h aged
human insulin (~86 M) and cells treated with 48 h aged human insulin (~86
M) co-incubated with 1 (100 M). Untreated Hela cells are taken as control.
μ
μ
μ
To visualize the extent of insulin aggregation in absence or presence
of this compound, we use atomic force microscopy (AFM) technique
[13]. AFM micrographs were collected for samples representing
different time points of incubation ranging from 0 to 30 h (Fig. 6). Under
amyloidogenic conditions human insulin forms fibrillar bodies of
micrometer range even after 10 h of incubation. Significant increase in
fibrillar complexity of these insulin aggregates can be observed in AFM
the amide I bond of
sulin into cross β sheet rich fibrils. However, insulin (~86
incubated with compound 1 (100 M) was able to sustain its amide I
signifying sustained helical structure of
human insulin for prolong duration.
α
-helix, indicating the transformation of native in-
μ
M) co-
μ
ꢀ 1
band around ~ 1652 cm
as time progresses. But in presence of 1 (100 M), human insulin (~86
μ
6

S. Sen et al.
Bioorganic Chemistry 111 (2021) 104899
μ
M) exhibits tiny spot like morphologies for a significant duration,
analyse the morphological changes quantitatively and qualitatively in
HeLa cells [38,39]. The two fluorescent probes i.e., AO and EtBr effi-
ciently bind to nucleic acids by intercalation and are utilized to visualize
apoptosis and aberrant nuclear changes. Acridine orange is a vital dye
which stains green both live and dead cells. Ethidium bromide will stain
red only those cells which have lost their membrane integrity. Viable,
early apoptotic and late apoptotic cells are stained in green, yellow and
orange respectively. Briefly, HeLa cells were treated with aged human
insulin and human insulin co-incubated with compound 1 for 24 h and
compared with untreated control cells (Fig. 10). In control, uniformly
green live cells with normal and intact nucleus were observed (Fig. 10
A). The cells treated with aged human insulin are seen mostly in orange
colour and showed significant aberrant morphological changes such as
nuclear fragmentation and cell shrinkage indicating late apoptotic stage
(Fig. 10 B). Whereas, treatment of cells with compound 1 co-incubated
insulin showed presence of viable green cells similar to untreated con-
trol cells (Fig. 10 C). However, it is to be noted that a few early apoptotic
cells are also observed. These results confirm that compound 1 signifi-
cantly reduces insulin fibril mediated apoptosis in HeLa cells. Also, cell
apoptosis percentage was quantified (n = 3) as the number of apoptotic
cells divided by the total number of cells (Fig. 11).
representing its non-fibrillar state even in amyloidogenic conditions.
3
.4. MTT assay
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bro-
mide) reduction assay was used to analyze cytotoxicity following com-
pound 1 treatment with HeLa cells. Varying concentrations of 1 were
added to different sets of cultured HeLa cells in 96-well plate. A set of
untreated HeLa cells was used as control. Even after 24 h treatment with
different concentrations of compound, overall cell viability was main-
tained at ~90% compared to control condition (Fig. 7). This study infers
the non-cytotoxic nature of compound 1 and opens up further scope to
comprehend about its anti-amyloidogenic efficacy in biological
conditions.
3
.5. Insulin amyloid-mediated apoptotic events and its inhibition
Insulin amyloid mediated cytotoxicity has been reported so far
mentioning disruptive inflammatory nature of such fibrils present in
injectable insulin dosages. Shielding effect of compound 1 can be
appreciated from its maintenance of nearly native secondary structure of
insulin for a prolong duration under harsh conditions. Inhibitor assisted
reduction in protein aggregate mediated cytotoxicity has already drawn
attentions so far [31–34]. Hence, anti-amyloidogenic activity of 1 might
also be admired further by screening its potential of reducing insulin
fibril mediated cytotoxicity.
Previously we had also done similar studies with different types of
conjugates that provide a kind of evidence that Trp-Trp combination is
essential to inhibit insulin fibrillation process. However, the conjugate 1
having an additional palmitic acid with Trp-Trp dipeptide and taurine
possesses a detergent like property with an intrinsic tendency to self-
assemble and in turn plays a pivotal role in insulin amyloidosis inhibi-
tion. Given the high value of insulin integrity in injectables, such an
approach may lead to a potential breakthrough in insulin amyloid
interference and for storage.
Hoechst 33342 dye flawlessly stains condensed DNA of apoptotic
cells in greater extent as compared to less condensed nuclei of healthy
cells [9]. After treatment with aged insulin (~86
μM) co-incubated with
compound 1 (100 M) for 24 h, HeLa cells were stained with Hoechst
μ
dye to detect changes in nuclei. Apoptotic nuclei seem to present an
extremely bright nuclear fluorescence of blue colour (Figure S7A) in
case of insulin amyloid treated cells. However, treatment of the cells
with compound 1 co-incubated insulin, resulting nuclear fluorescence
intensity was less as compared to its earlier counterpart (Figure S7B).
Thus, the presence of negligible amount of brighter blue cells affirms
protective role of 1 by producing negligible and less toxic insulin
amyloids.
4. Conclusions
In conclusion, we have proposed the designing of an inhibitor
through fine tuning between its intrinsic polarity and non-polarity in a
single platform. Anti-amyloidogenic nature of this compound can also
be further acknowledged due to its efficiency in reducing insulin amy-
loid mediated cytotoxicity. With the advent of its inhibitory as well as
cytoprotective role, efficacy of this compound can also be further
explored for the chances of being used as anti-aggregating agents in
pharmaceutical insulin formulations. Additionally, this compound may
also be useful for coating material of insulin injections and storage vials
rather than conventionally used silicon oil and teflon coatings reported
to promote insulin aggregation.
Cells treated with insulin fibrils also led to DNA condensation and
fragmentation leading to apoptotic membrane protrusions. Cell mem-
brane blebbing and fragmented nuclei are marked with yellow arrows
(
Fig. 8A). Nuclear condensation and DNA fragmentation lead to poly-
nucleated cells, is also a clear indication of apoptosis [35]. Such poly-
nucleated cells were noticed during the course of treatment of HeLa cells
with insulin amyloids (Fig. 9A and S8). However, polynucleated cells
were absent in case of HeLa cells treated with compound 1 co-incubated
insulin sample due to lack of traceable toxic insulin fibrils (Fig. 9B).
Protein aggregates are reported to disrupt native cellular morphology
through interactions with cell membrane and generate inflammatory
responses in cells leading to sequential apoptotic events in cells [36,37].
FITC-phalloidin and DAPI staining were carried out simultaneously for
evaluating cytoskeletal and nuclear integrity respectively of these
treated cells. The gross morphology of cytoskeleton and nuclei were
visualized using confocal fluorescence microscopy and detected for the
cytotoxicity of corresponding insulin samples (Figs. 9 and S8).
Author contributions
The manuscript was written through contributions of all authors. All
authors have given approval to the final version of the manuscript.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Treatment of HeLa cells with insulin fibrils shows disrupted and
shortened F-actin, which clearly indicates cell membrane rupturing
nature of these toxic fibrils present in background of HeLa cells treated
with aged human insulin. Existence of amorphous fibrils around HeLa
cells is shown by a blue circle in DIC microscopy image (Fig. 9A). No
traceable insulin fibrils were present around HeLa cells treated with
Acknowledgements
SV thanks SERB for a J C Bose Fellowship. SG thanks DBT for a Tata
Innovation Fellowship. This work is partly supported by SERB project
(SG, SV) and DST Nano Mission (SS, SV). S. Sen and P. Singh acknowl-
edge MHRD for pre-doctoral fellowship. NKM thanks CSIR-UGC for a
pre-doctoral research fellowship. We thank the Centre for Nanoscience
and Soft Nanotechnology and Advanced Centre for Materials Science
(ACMS), IIT Kanpur, for access to instrument facilities.
compound 1 (100
μ
M) co-incubated insulin (~86
μ
M) (Fig. 9B).
Furthermore, it was demonstrated that compound 1 treated insulin did
not show any evidence of cytoskeletal disruption.
Furthermore, AO / EtBr dual fluorescent staining was performed to
7

S. Sen et al.
Bioorganic Chemistry 111 (2021) 104899
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