Prion-derived tetrapeptide stabilizes thermolabile insulin via ...

Page 1

llOPEN ACCESS

iScience

Article

Prion-derived tetrapeptide stabilizes thermolabileinsulin via conformational trapping

Meghomukta

Mukherjee,

Debajyoti Das, Jit

Sarkar, ..., Samit

Chattopadhyay,

Subhrangsu

Chatterjee, Partha

Chakrabarti

[email protected],

[email protected] (S.C.)

[email protected] (P.C.)

HighlightsPrion-derived consensus

tetrapeptide motif V(X)YR

protracts insulin

fibrillation

IS1 (VYYR) preserves

bioactive insulin under

varying physicochemical

perturbations

IS1 serves as nontoxic,

cell-impermeable peptide

excipient for insulin

IS1 conformationally traps

dimeric insulin during

fibrillation

Mukherjee et al., iScience 24,102573June 25, 2021 ª 2021 TheAuthors.

https://doi.org/10.1016/

j.isci.2021.102573

Page 2

llOPEN ACCESS

iScience

Article

Prion-derived tetrapeptide stabilizes thermolabileinsulin via conformational trapping

Meghomukta Mukherjee,1,6 Debajyoti Das,2,6 Jit Sarkar,2,3,7 Nilanjan Banerjee,1,7 Jagannath Jana,1

Jyotsna Bhat,1 Jithender Reddy G,4 Jagadeesh Bharatam,4 Samit Chattopadhyay,5,8 Subhrangsu Chatterjee,1,*

and Partha Chakrabarti2,3,9,*

1Department of Biophysics,Bose Institute, Kolkata, India

2Division of Cell Biology andPhysiology, CSIR-IndianInstitute of Chemical Biology,4 Raja SC Mullick Road,Kolkata 700032, India

3Academy of Innovative andScientific Research,Ghaziabad 201002, India

4Centre for NMR andStructural Chemistry,CSIR-Indian Institute ofChemical Technology, UppalRoad, Tarnaka, Hyderabad,India

5Division of Cancer Biology &Inflammatory Disorder,CSIR-Indian Institute ofChemical Biology, Kolkata,India

6These author contributedequally

7These author contributedequally

8Present address:Department of BiologicalSciences, BITS-Pilani, KK BirlaGoa Campus, NH 17B BypassRoad, Zuarinagar, Goa403726, India

9Lead contact

*Correspondence:[email protected],[email protected](S.C.),[email protected](P.C.)

https://doi.org/10.1016/j.isci.2021.102573

SUMMARY

Unfolding followed by fibrillation of insulin even in the presence of various excip-ients grappled with restricted clinical application. Thus, there is an unmet needfor better thermostable, nontoxic molecules to preserve bioactive insulin undervarying physiochemical perturbations. In search of cross-amyloid inhibitors,prion-derived tetrapeptide library screening reveals a consensus V(X)YR motiffor potential inhibition of insulin fibrillation. A tetrapeptide VYYR, isosequentialto the b2-strand of prion, effectively suppresses heat- and storage-induced insu-lin fibrillation and maintains insulin in a thermostable bioactive form conferringadequate glycemic control in mousemodels of diabetes and impedes insulin amy-loidoma formation. Besides elucidating the critical insulin-IS1 interaction (R4 ofIS1 to the N24 insulin B-chain) by nuclear magnetic resonance spectroscopy, wefurther demonstrated non-canonical dimer-mediated conformational trappingmechanism for insulin stabilization. In this study, structural characterization andpreclinical validation introduce a class of tetrapeptide toward developing ther-mostable therapeutically relevant insulin formulations.

INTRODUCTION

Availability of injectable insulin formulation has been a breakthrough in diabetes management in achieving

long-term glycemic control and preventing complications (Baram et al., 2018; Heller et al., 2007; Moroder

and Musiol, 2017; Owens et al., 2001; Xiong et al., 2019; Zaykov et al., 2016); it still, however, suffers from

certain disadvantages including temperature-sensitive fibrillation in solution and development of subcu-

taneous tumor-like mass designated as ‘‘amyloidoma’’ at the site of injection (Hua andWeiss, 2004; Ivanova

et al., 2009; Nilsson, 2016; Woods et al., 2012; Yumlu et al., 2009). Worldwide efforts were thus made to

develop thermostable insulin either by making recombinant insulin species with mutations or stabilizing

native insulin with salts, Zn2+ ions, and small molecules such as meta-cresol (Frankær et al., 2017; Gong

et al., 2014; Han et al., 2017; Kachooei et al., 2014; Lee et al., 2014; Patel et al., 2018; Saithong et al.,

2018; Wang et al., 2011; Zheng and Lazo, 2018). However, small molecules are found to be toxic in long-

term usage and are inefficient in optimally preventing fibrillation (Teska et al., 2014; Weber et al., 2015).

Thus, the unmet need for better nontoxic insulin stabilizers is highly warranted and nonimmunogenic pep-

tides as stabilizers would have no or very low toxicity (Banerjee et al., 2013; Neddenriep et al., 2012; Wu,

2019). To date, an array of peptide-based stabilizers was designed and validated through in silico or bio-

physical investigations, but the exploration of their therapeutic potential in in vitro, in cellulo, and in vivo

preclinical models is mostly lacking (Das and Bhattacharyya, 2017; Mishra et al., 2013; Ratha et al., 2016;

Seidler et al., 2018; Wallin et al., 2018). Recent advances suggest screening and designing of cross-amyloid

inhibitors can either protracts amyloid self-assembly or inhibit potential cross-seeding of interacting amy-

loidogenic proteins (Armiento et al., 2020).

In the search for endogenous peptide motifs that could potentially hinder insulin fibrillation and stabilize

insulin in its stable conformation, we looked at misfolded human prion conformers because of its colocal-

ization at insulin-secreting pancreatic b-cells while having a converse functional association with glucose

homeostasis (Amselgruber et al., 2006; Ashok and Singh, 2018). Three conserved tetrapeptide motifs

(RYYR/VYYR/AYY(D/Q)) found in PrPc and the conformation-selective surface exposure of ‘‘YYR’’ associ-

ated with the b-sheet structure in misfolded PrPSc prompted us to consider these peptide motifs as the

iScience 24, 102573, June 25, 2021 ª 2021 The Authors.This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1

Page 3

Figure 1. Prion-derived peptide screening for antiamyloid activity

(A) Schematic representation of antiamyloid peptide screening.

(B) Heatmap of hierarchical clustering of fibrillated insulin in the presence of equimolar ratio of 77 tetrapeptide variants. Darker shades represent higher

fluorescence values.

(C) Amyloid activity as an inversely proportional function of PROTEOSTAT fluorescence distributed in 4 tetrapeptide sequence.

(D) Four independent clusters of peptides obtained from k-means clustering.

(E and F) ThT assay and DLS for heat-induced fibrillation of insulin in the presence of indicated peptides.

(G) AFM for heat-induced fibrillation of insulin (scale bars, 1 mm).

llOPEN ACCESS

iScienceArticle

seeding platform to design cross-amyloid inhibitors for insulin amyloidosis (Julien et al., 2009; Paramithiotis

et al., 2003).

RESULTS

Peptide screening reveals insulin stabilizing prion-derived tetrapeptide sequence

We designed a library of 77 tetrapeptides based on two conserved tetrapeptide motifs (RYYR/VYYR), each

having a single amino acid substitution at a time by the rest 19 natural amino acids for each position (Table

S1). Peptides were screened for the heat-induced insulin fibrillation inhibition using thioflavin T (ThT) and

PROTEOSTAT fluorescence (Figure 1A; S1A, r = 0.59,p % 0.001). Heatmap of fluorescence data revealed

that peptides having the formula of V-(X)-Y-R have the highest antiamyloid potential (�81.4%), whereas V-

Y-Y-(X) peptides showed the lowest impact (44.24% inhibition), where X is any amino acid (Figures 1B and

1C). Even unsupervised K-means clustering showed that cluster-1 containing 27 peptides had the lowest

fluorescence values (5.5% of native insulin) and highest antiamyloid potential (Figure S1C) with significant

correlation (R = 0.46, p = 0.017) (Figure 1D). Cluster-1 peptides were further analyzed for percent allowable

substitution [(substitution observed/total possible substitution i.e. 19) * 100] at each position that depicts

2 iScience 24, 102573, June 25, 2021

Page 4

llOPEN ACCESS

iScienceArticle

marked conservation of amino acids at the third and fourth positions by allowing only 21.05% and 15.5%

substitutions, respectively, making the Y(3)-R(4) sequence indispensable for the anti-fibrillating property.

We thereby propose a generalized tetrapeptide sequence V-(X)-Y-R that would potently prevent insulin

fibrillation.

To test the position-dependent importance of the core Y-Y sequence, we used five peptides (V-Y-Y-R, V-P-

P-R, V-Y-P-R, V-P-Y-R, and V-T-T-R) for heat-induced insulin fibrillation assay. Only V-Y-Y-R showed signif-

icant antiamyloid activity and preserved insulin’s glucose-lowering capacity during an insulin tolerance test

(ITT) in mice (Figures S2A–S2D). We further designed variant peptides by adding amino acid at N- and

C-terminal of VYYR (VVYYR and VYYRR), deleting one tyrosine from the core sequence (VYR) and reversing

the sequence to RYYV. VVYYR, VYYRR, and RVVY did not inhibit insulin fibrillation as confirmed by atomic

force microscopy (AFM) studies and corroborating these results with corresponding ThT and dynamic light

scattering (DLS) data, whereas VYR showed modest inhibitory effects than the rest of the peptide variants

as depicted by the ThT assay (Figures 1E–1G). Altogether, these results prompted us to select V-Y-Y-R (now

termed IS1) as a candidate for insulin stabilization and further validate its therapeutic potential in preclinical

mice models and commercial insulin formulations.

IS1 protects insulin from both heat- and storage-induced fibrillation

The kinetics of heat-induced insulin fibrillation with varying molar ratios of insulin:IS1 by revealing dose-

dependent inhibition insulin fibrillation maximally at the equimolar concentration is shown by ThT

fluorescence and AFM (Figure 2A). The interaction of insulin and IS1 determined by isothermal titration

calorimetry (ITC) by fitting the titration curve in the one-site binding model (Figure S3A) was thermodynam-

ically favorable (DG = �4.8 kcal.mol�1, DH = �18.73kcal.mol�1, DS = �46.5 kcal.mol�1; KA = 3.46 3

10�3M�1). IS1 remarkably restored the hydrodynamic radius of insulin monomers, otherwise which was

increased to 1000 nm at 150 min of heating (Figure 2B). Circular dichroism (CD) spectroscopy showed grad-

ually decay in dual negative ellipticities at 208 nm and 222 nm, which indicates the loss of a-helical confor-

mation for native insulin owing to heat-induced fibrillation, whereas the presence of IS1 retained its con-

formations even after 5 h (Figure 2C and Table S2). To test its therapeutic relevance, we have tested its

antiamyloid potential in commercial human insulin formulation (Actrapid) both in the presence and

absence of commercial excipients for heat-induced and storage-induced (37�C, 30 days) fibrillation.

AFM data showed that IS1 inhibits Actrapid fibrillation by trapping insulin in lower molecular weight olig-

omeric conformations, which was further corroborated with ThT and DLS data (Figure 2D, 2E, S3B, and

S3C).

IS1 maintains insulin in a thermostable bioactive conformation

To find whether IS1 confers insulin stabilization in physiologically active conformers, we performed an ITT in

mice. IS1-treated bovine insulin followed similar glucose-lowering kinetics to that of native insulin during

the ITT even after heat-induced fibrillation induction, while fibrillated insulin revealed amarked impairment

in glucose-lowering potential (Figure 3A). Next, we developed two independent disease mouse models,

streptozotocin-induced type 1 diabetes and high-fat-diet-fed type 2 diabetes. Expectedly, the addition

of IS1 significantly restored the glucose-lowering activity of insulin, effects that were comparable with

native insulin in both the disease models (Figures 3B and 3C). IS1 also preserves the bioactivity of Actrapid

even after 37�C storage for 30 days as depicted by ITT (Figure 3D). For further affirmation, we investigated

the induction of prototypical signaling cascade for the IS1-insulin complex. We observed a marked induc-

tion of Akt phosphorylation in IS1-treated insulin compared with that of fibrillated insulin both in cultured

hepatocytes (Figure S3D) and in the mouse liver (Figure 3E). Insulin-derived amyloidosis is defined as a sub-

cutaneous amyloid mass at the site of insulin injections owing to intrinsic fibrillation of commercial insulin

owing to long-term storage. To this end, we carried out amyloidoma formation assay (Figure 3F) where

repeated subcutaneous injection of fibrillated insulin forms a mass of extracellular amyloid fibrils in mice.

IS1 has limited cytotoxicity with no membrane permeability

Toward further characterization of IS1 peptide, we checked for its membrane permeability using fluores-

cein isothiocyanate (FITC)-labeled IS1 in HepG2 cells. FITC fluorescence was observed in the extracellular

space even after 24 h of incubation, whereas FITC-SLRP (Jana et al., 2018), a cell-penetrating tetrapeptide,

was detected intracellularly (Figure 4A). The spatial distribution (FITC) IS1-insulin complex showed

preferential accumulation on the cell membrane suggesting its receptor binding potential (Figure 4B).

Cytotoxicity has often been implicated in short peptides even for the cell-impermeable peptides. Both

iScience 24, 102573, June 25, 2021 3

Page 5

Figure 2. IS1 prevents intrinsic fibrillation of insulin

(A) ThT assay of insulin fibrillation with increasing concentrations of IS1(VYYR). Inset: AFM images of heat-induced fibrillation of insulin in the presence of IS1

(scale bars, 1 mm).

(B) DLS analysis as a function of incubation time of insulin (left panel) and IS1-insulin complex (right panel).

(C) CD spectra of insulin in the absence (left panel) and the presence (right panel) of IS1.

(D) AFM images of heat-induced and storage-induced fibrillation of Actrapid with IS1 in the presence and absence of excipients (scale bars, 1 mm).

(E) ThT and DLS assay for storage-induced fibrillation of Actrapid and Actrapid-IS1 in the presence and absence of commercial excipients. All experiments

were performed in triplicates, and values are presented as mean G SEM.

llOPEN ACCESS

iScienceArticle

dose-dependent and time-course cytotoxicity assays showed limited cytotoxicity of IS1 toward HepG2

cells using live-dead and MTT assays (Figures 4C–4E). Consistently, IS1 leads to only �0.5% hemolysis

compared with 1% Triton X-100 (Figure 4F).

Nuclear magnetic resonance spectroscopy reveals the structure and dynamics of IS1 and

insulin interaction

For structural characterization and deciphering atomic level interaction of the insulin-IS1 complex, we per-

formed a series of nuclear magnetic resonance (NMR) spectroscopy experiments. One-dimensional STD

NMR experiment showed that aromatic protons of tyrosine Y2 (2.6 H), Y3(2.6 H); side chain of arginine

R4 QD; and the b-proton of tyrosine Y2 Hb1 and Y3 Hb2 interacting with insulin which was also found to

be interacting with Actrapid (Figures S4A–S4D). To reveal the atomic-level dynamics of free IS1 and IS1-in-

sulin complex, one-dimensional longitudinal (R1 = 1/T1) and transverse relaxation (R2 = 1/T2) experiments

were performed. We observed a significant decrease in longitudinal relaxation rate (R1) for the IS1-insulin

complex compared with free IS1 for all amide protons. Similarly, the transverse relaxation rates (R2) for all

the residues of IS1 were increased in the presence of insulin stating spin-spin energy exchange in the

4 iScience 24, 102573, June 25, 2021

Page 6

Figure 3. IS1 maintains insulin in a thermostable bioactive conformation

(A–C) ITT of heat-induced fibrillated insulin with IS1 in BALB/c mice (A), preclinical models of type 1 diabetes (B), and type 2 diabetes (C) [n = 5/group].

(D) ITT of storage-induced fibrillated Actrapid in BALB/c mice [n = 5/group].

(E) Western blot of Akt (Ser 473, Thr 308) and p70S6K phosphorylation in BALB/c mice liver. pan AKT and b-actin served as a loading control.

(F) Schematics of generating local amyloidosis in mice by subcutaneous injection of insulin amyloid fibrils.

(G) Visualization of amyloid deposition for both fibrillated insulin (left panel) and fibrillated insulin with IS1 (right panel) in skin biopsies stained with

hematoxylin and eosin (H&E) and Congo Red using light and polarized microscopy (scale bars, 100 mm) [n = 3/group]. Values are presented as meanG SEM.

ns, not significant. *p < 0.05, **p < 0.01, ***p < 0.001.

llOPEN ACCESS

iScienceArticle

horizontal plane which confirms complexation (Figure S5A). Amide exchange of insulin in presence of IS1 at

a varying temperature ranging from 25�C to 65�C showed almost no exchange dictating that IS1 prevents

unfolding of monomeric globular insulin (Figure S5B).

The 1H–1H 2-dimensional (2D) NOESY experiment has traced the NOESY contacts [Y2(peptide):Y19(insu-

lin)], [R4(peptide):N24(insulin)], [Y3(peptide):S12(insulin)], [V1(peptide):E17(insulin)], [Y3(peptide):V10(insu-

lin)], thereby identifying the atomic interactions between IS1 and monomeric insulin (Figure 5A). A

3-dimensional (3D) (1H-1H-15N) NOESY-HSQC experiment further confirmed the interaction of terminal

R4 of IS1 with ASN24 of the insulin B-chain (Figure 5B).

Based on 2D and 3DNMR spectroscopy, inter-residual NOESY distance restraints were made using a semi-

quantitative method keeping the ASN24-R4 contact strong (derived from precision dock analysis in Schro-

dinger’s suite), and the distance-restrained NMR-derived structure calculation of insulin-IS1 complex was

performed. From the final 10 restrained MD structures, the insulin-IS1 inter-residual contacts were found

and compared with the evolved 3D and 2D NOESY data set (Figure 5C). Molecular dynamics simulation

iScience 24, 102573, June 25, 2021 5

Page 7

Figure 4. IS1 serves as a potent nontoxic membrane-impermeable stabilizer for insulin

(A) Confocal microscopy images showing time-dependent cell membrane permeability using FITC-tagged tetrapeptide SLRP and IS1 (VYYR), respectively,

at 10 mM concentration for 24 h in HepG2 cell line (scale bars, 20 mm).

(B) Representative confocal images showing localization of FITC-IS1 independently and in the presence of native bovine insulin heated at 62�C for 3 h,

treated in HepG2 cells (scale bars, 20 mm). Inset represents a 5X digitally magnified image of respective samples (scale bars, 10 mm).

(C) Dose-dependent live/dead cell viability assay showing live cells stained with Calcein-AM (green) and dead cells with EthD-1 (red) treated for 12 h with

both SLRP and IS1 in HepG2 cells (scale bars, 100 mm).

(D) Live/Dead cell viability assay of HepG2 cells in the presence of both IS1 and SLRP independently at 10 mM concentration for 24 h in HepG2 cell line

(upper panel: scale bars 100 mm; lower panel: scale bars 20 mm).

(E) Top: quantification of percent dead cells at each concentration of respective tetrapeptide IS1 and SLRP treatment. Quantified using threshold and

analyze particle function using Fiji (ImageJ) software (5 fields for each concentration of samples). Bottom: MTT assay showing the percent of viable cells at

each concentration of respective tetrapeptide IS1 and SLRP treatment.

(F) Hemolytic assay results in negligible hemolysis compared with 1% Triton X-100. Values are presented as mean G SEM. ns, not significant. *p < 0.05,

**p < 0.01, ***p < 0.001.

llOPEN ACCESS

iScienceArticle

for free insulin and IS1-insulin complex has revealed the interactions between Y2 (peptide) and E34 (B-chain

of insulin), V1 (peptide), and H26 (B-chain of insulin), Y3 (peptide), and Q25. The R4 of V-Y-Y-R forms a triad

H-bond network among Val10 (A chain of insulin) and Asn24 (B chain of insulin) (Figure 5D i-v). The adaptive

Poisson-Boltzmann surface area (APBS) was calculated for the insulin-IS1 complex. The blue and red sur-

faces designate positive and negative electronic charge surfaces in APBS, respectively (Figure S5D i-iii).

Previous studies suggested that B-chain N-terminal residues (Phe22-Gly29) of insulin are critical for

dimer-dimer interactions that essentially induce R6 hexameric conformation in the presence of Zn+

and phenolic excipients. The exchange of Asn24 for lysine affects monomerhexamer equilibria that allow

6 iScience 24, 102573, June 25, 2021

Page 8

Figure 5. NMR spectroscopy and restrained molecular simulation model of the insulin-IS1 complex

(A) 2D NOESY NMR peak of IS1 interacting with insulin and forming cross-peaks.

(B) 3D 15N-HSQC-NOESY spectrum of insulin in presence of IS1.

(C) The ensemble structure of restrained simulation of insulin-IS1 of the last 10 ps.

(D,i-v) Residue specific atomic interactions depicting a triad hydrogen bond network; molecular simulation showing the interactions between Y2 (peptide)

and E34 (B-chain of insulin), V1 (peptide) and H26 (B-chain of insulin), Y3 (peptide) and Q25.

llOPEN ACCESS

iScienceArticle

dimerization but predominantly prevent conformational transition toward R6 hexamers, even in the pres-

ence of zinc. But, protein engineering studies revealed that the removal or exchange of amino acids at B-

chain C-terminus (Tyr47-Thr51) drastically impairs the self-association of the insulin monomers (dimeriza-

tion). Interestingly, short-acting insulin glulisine bearing mutation at N24 and N50 favors monomeric

structure and is less amenable to fibrillation (Becker, 2007; Woods et al., 2012). Moreover, the N-terminal

of B chain is necessary for lateral aggregation so that the protofibrils can form fibrils (Jimenez et al.,

2002). Atomic traces from NMR and simulation studies of the IS1-insulin complex suggest that the bind-

ing of IS1 restricts insulin from having sufficient degrees of freedom to misfold under biophysical pertur-

bations while restricting conformational transitions by forming IS1(R4)-(V10-N24)INS triad hydrogen bond

network.

Dimer stability is crucial for IS1-mediated insulin stabilization

To investigate the mechanism of IS1-mediated insulin stabilization under physicochemical perturbations,

size exclusion chromatography was performed for fibrillated human insulin in the presence and absence of

IS1, while native insulin serves as a control. A broad peak was observed within the void volume for heat-

induced human insulin-depicting heterogeneous populations of large fibrillar aggregates, whereas native

insulin elutes at 118.5 mL. Interestingly, heat-induced insulin in the presence of IS1 showed 4 independent

iScience 24, 102573, June 25, 2021 7

Page 9

Figure 6. IS1 stabilizes human insulin majorly in dimeric conformation

(A) Size-exclusion profiles of heat-induced recombinant human insulin in the presence and absence of IS1; native human insulin (without heat) serves as a

negative control. [Inset] DLS assay were performed for the eluent corresponding to major chromatographic peaks, designated by downsize arrow in size

exclusion profiles.

(B) AFM images depicting the heat-induced (62�C, 3 hours) fibrillation of excipients-depleted Lantus and Lispro in the presence and absence of IS1

(scale bars, 1 mm).

(C) AFM images depicting the prolonged storage-induced (37�C, 30 days) fibrillation of excipients-depleted Lantus and Lispro in the presence and absence

of IS1 (scale bars, 1 mm).

(D) ThT data for storage-induced fibrillation of excipient-depleted Lantus and Lispro in the presence and absence of IS1.

(E) DLS analysis for storage-induced fibrillation of excipient-depleted Lantus and Lispro in the presence and absence of IS1.

(F) ITT of storage-induced fibrillation of Lantus and lispro (excipient-depleted) in the presence and absence of IS1in BALB/c mice [n = 5 mice/group]. Values

are presented as mean G SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

llOPEN ACCESS

iScienceArticle

peaks, while major fraction elutes at 114 mL (Figure 6A). To validate the hydrodynamic properties of all

three samples, we have collected the major eluent peaks and performed DLS to reveal the hydrodynamic

radius of eluent particles. While native insulin revealed an average hydrodynamic radius of 1.358 nm, heat-

induced fibrillated insulin showing a much larger radius ranging from 531 to 3580 nm. Interestingly, fibril-

lation in the presence of IS1 not only prevents the majority of insulin monomers from getting fibrillated but

also the major fraction of eluent particles having the hydrodynamic radius ranging from 3.1 to 4.8 nm (Fig-

ure 6A; inset), which overlaps with the dimeric insulin conformers (Pease et al., 2010).

The fibrillation of insulin and its inhibition in the presence of IS1 was next determined by diffusion ordered

spectroscopy (DOSY). The larger size (high molecular weight) molecules diffuse at a slower rate than that of

smaller size molecules (low molecular weight). The diffusion coefficient obtained from DOSY at 25�Cfor heated insulin was found to be lower (D = 9.4 3 10�5cm2sec�1) than that of the insulin-IS1 complex

8 iScience 24, 102573, June 25, 2021

Page 10

llOPEN ACCESS

iScienceArticle

(D = 2.2 3 10�4 cm2sec�1). Besides, the molecular weight of insulin without treatment of IS1 after 4 h incu-

bation at 62�C was approximately 57.0 kDa, supposedly the soluble oligomeric insulin and the insulin-IS1

complex showed much lesser molecular weight approaching 10 kDa representing dimeric insulin

conformer (Tables S4A and S4B). These data indicate that the free-insulin undergoes heat-induced oligo-

merization and conformational switch before its fibrillation, while IS1 protracts insulin oligomerization and

hence later aggregation events.

These results prompted us to hypothesize the existence of an IS1-mediated conformation trap between

stable hexamer and partially unfolded monomers toward fibrillation. To this end, we have checked the

antiamyloid potential of IS1 for two commercially available insulin analogs, Lantus (long-acting) and Lis-

pro (fast-acting). These two analogs were strategically developed majorly to favor hexameric and mono-

meric conformation, respectively (Figure S6). We removed the existing commercial excipients from both

the formulations (Figure S7) for avoiding excipient-induced conformational bias. Interestingly, IS1 signif-

icantly protracts fibrillation of Lantus by forming a smaller oligomeric complex in both the storage- and

heat-induced condition as depicted by AFM, ThT, and DLS data, whereas it fails to do so for Lispro (Fig-

ures 6B–6E). ITT for both Lantus and Lispro in storage-induced fibrillation conditions showed no

glucose-lowering potential for Lispro while markedly preserving the physiological role for Lantus

(Figure 6F).

We observed a similar trend in the presence of commercial excipients for both Lantus and Lispro (Figures

S8 and S9). The one-dimensional STD NMR experiment validated that aromatic protons of tyrosine Y2

Hb2, Y3Ha, Y3 side chain protons, R4 Ha, and V1 Ha of IS1 interacting with Lantus, while no significantly

interacting protons were observed with Lispro (Figure S10A). Even prototypical insulin signaling in

HepG2 cells suggests IS1-mediated insulin stabilization leads to biologically active conformers for

Actrapid and Lantus but not for Lispro (Figure S10B). Lispro was strategically developed by switching po-

sitions of 49Pro-50Lys to 49Lys-50Pro, which greatly reduces its dimerization potential, making it predom-

inantly monomeric fast-acting insulin analog. Hence, dimerization is a crucial determining factor for the

antiamyloid potential of IS1, thereby failing to preserve Lispro from physicochemical perturbation-

induced fibrillation.

DISCUSSION

The current advocacy of intensive insulin therapy regimens in patients with diabetes is met with several clin-

ical problems – insulin is proamyloidogenic and forms insoluble aggregates resulting in excess insulin

requirement; amyloidoma formation at the site of repeated insulin injection; gradual loss of excipients

and deposition of fibrils in the catheter system of insulin pumps; and temperature-sensitive insulin fibrilla-

tion entails storage and maintenance of cold chain. Even mild agitation of insulin during its storage and

transport has been reported to denature the protein through fibrillation, resulting in its inactivation. These

issues thereby call for a more stable form of formulations that would cater to the increasing global demand.

Taking cues from the conformation-selective surface exposure of ‘‘YYR’’ motif associated with the b-sheet

structure in misfolded prion protein PrPSc, we discovered a consensus amino acid sequence VXYR as a

potent inhibitor for both purified and commercial insulin formulations. Specifically, the endogenous prion

sequence VXYR (termed here IS1), a plasma-membrane-impermeable, nontoxic tetrapeptide showed

remarkable protraction of insulin fibrillation on heating and during prolonged storage, maintained hypo-

glycemic effects in vivo, and prevented subcutaneous amyloidoma formation. These effects weremediated

via direct binding of IS1 to insulin monomer through a triad hydrogen bond network that leads to nonca-

nonical dimer-mediated conformational ‘‘trapping’’ of insulin.

Toward development of insulin formulations with lower amyloidogenicity, newer excipients as well as

chemically modified insulin moieties are being reported. Recent studies depicted generation of ther-

mostable insulin by adding polysialic acid and proline-based homopolymer at specific amino acid

residues (Ghosh et al., 2020; Kabotso et al., 2020). Aromatic small molecules such as resveratrol and

rosmarinic acid were shown to enhance the biophysical stability of native insulin while preserving its

physiological activity by stabilizing the hexameric form or preventing dimer dissociation, thereby re-

stricting the thermal unfolding of monomers (Pathak et al., 2020; Zheng and Lazo, 2018). In the present

study, we have identified IS1 or VYYR, isosequential to the b-2 strand of human PrPC, as one small

endogenous tetrapeptide motif that prevents both heat- and storage-induced insulin fibrillation both

iScience 24, 102573, June 25, 2021 9

Page 11

llOPEN ACCESS

iScienceArticle

in vitro and in vivo and described a noncanonical molecular mechanism for inhibiting insulin fibrillation.

Peptide screening assay further highlights the position-specific sequence conservation of IS1 in preser-

ving its antiamyloid potential with a consensus amino acid sequence V-X-Y-R having a potent antiamy-

loid activity.

Mechanistically, IS1 predominantly binds to the N-terminal of B chain of insulin, forming a triad

hydrogen bond network (R4 (peptide)-V10(insulin)-N24(insulin)). Interestingly, short-acting insulin gluli-

sine bearing mutation at N24 and N50 favors monomeric structure and is less amenable to fibrillation

(Becker, 2007; Woods et al., 2012; Zhou et al., 2016). Direct interaction of IS1 to N24 may thus confer

its conformational stability. The residues from 31 to 41 of B chain form the hydrophobic core in insulin

fibril, while residues of 13–18 of A chain are susceptible to conversion from a-helix to beta strands

when misfolded. Moreover, the N-terminal of B chain is necessary for lateral aggregation so that

the protofibrils can form fibrils (Jimenez et al., 2002). STD NMR deciphered Y2 (2.6 H), Y3 (2.6 H),

Y2 (Hb1), and Y3 (Hb2) protons of IS1 interact with insulin monomer. Insulin in solution confers bioac-

tive monomer to zinc-coordinated less-active hexameric forms transitioning through intermediated

dimeric states. While monomeric forms are more susceptible to fibrillate, hexamers are somewhat im-

mune to fibrillation. DLS data of the size-exclusion chromatography profile suggested that the binding

of IS1 to insulin preferentially ‘‘trap’’ insulin in the dimeric conformation (Figure 6A), which not only

limits the degrees of freedom to misfold under biophysical perturbations but allows the release of

active monomers as needed.

IS1-stabilized insulin showed glucose-lowering ability and signaling potential that were comparable with

native insulin. Experiments in type 1 and type 2 diabetes mellitus mice models and different commercially

available insulin formulations in the presence and absence of excipients for both heat- and prolonged stor-

age-induced insulin fibrillation further suggests its therapeutic potential. Peptide solubility holds the major

concern restricting the potential use in commercial formulations. We find IS1 is highly soluble in citrate

phosphate buffer (pH 2.6) up to 10 mM concentration (the maximum concentration that we have checked)

and in distilled water. Moreover, the tetrapeptide is soluble in various commercial insulin formulations

(Actrapid, pH 7; Lispro, pH 7–7.8; and Lantus, pH 4) both in the presence and absence of variable salts

and nonpolar excipients for at least 1 mM concentration. IS1 is cell-impermeable and nontoxic and has pre-

viously been shown to be nonimmunogenic (Caughey, 2003; Paramithiotis et al., 2003; Taschuk et al., 2014),

further attributing to its future potential as a thermostable insulin stabilizer.

Limitations of the study

The present study has few potential limitations which we believe could create future research opportu-

nities. Although we have generated compelling NMR data for elucidating mechanistic underpinnings of

IS1’s antiamyloid potential, it still remains incomplete. X-ray crystallography of the IS1-insulin complex

would better clarify the molecular mechanism. Moreover, cellular toxicity data are preliminary and further

in vivo studies including pharmacokinetic and pharmacodynamic profiles would be required for possible

future development of therapeutics.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by

the lead contact: Partha Chakrabarti (Email: [email protected]).

Materials availability

This study did not generate new unique reagents.

Data and code availability

The accession number of the NMR derived structure is available with IDs i.e. PDB ID 7ELJ, BMRB ID 36417.

SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2021.102573.

10 iScience 24, 102573, June 25, 2021

Page 12

llOPEN ACCESS

iScienceArticle

ACKNOWLEDGMENTS

SC is thankful to the Bose Institute’s NMR facility of 700 MHz Bruker. SC thanks Prof. Uday Bandyopadhyay

for his generous support and DST, Govt of India for funding. We would also like to thank Dr. Surajit Ghosh

for providing FITC-SLRP peptide. We thank T. Muruganandan for his help with AFM experiments at IICB.

SC is thankful to Bose Institute’s intramural fund. PC acknowledges intramural funding from CSIR-IICB.

AUTHOR CONTRIBUTIONS

SC initiated the research and designed the prion derived peptide. PC (IICB) and SC (BI) conceived the idea

to proceed with peptide screening. Biophysical experiments such as all the fibrillation assays, ITC, DLS, and

CD spectroscopy were designed, analyzed by SC, MM, and executed by MM. Some peptides were synthe-

sized by MM. Peptide screening was performed by DD andMM, whereas analyzed by DD, JS, and PC. NMR

data acquisition and analysis were performed by MM, NB, JRG and SC. NMR derived structure calculations

were performed by NB and SC in AMBER 14.0. Size-exclusion chromatography coupled with DLS experi-

ments were performed by NB and analyzed by NB and SC. All the in-cellulo and in vivo assays were per-

formed by DD and analyzed by DD and PC. AFM data execution was performed by DD and analyzed by

DD and PC. 3D NMR data execution was performed by JB and analyzed by MM, NB. The manuscript

was written by DD, MM, SC, and PC.

DECLARATION OF INTERESTS

There is no conflict of interest.

Received: October 19, 2020

Revised: March 8, 2021

Accepted: May 18, 2021

Published: June 25, 2021

REFERENCES

Amselgruber, W.M., Buttnerbuttner, A.M.,Schlegel, A.T., Schweiger, M., and Pfaff, A.E.(2006). The normal cellular prion protein (PrPc) isstrongly expressed in bovine endocrine pancreas.Histochem. Cell Biol. 125, 441–448.

Armiento, V., Hille, K., Naltsas, D., Lin, J.S.,Barron, A.E., and Kapurniotu, A. (2020). Thehuman host-defense peptide cathelicidin LL-37 isa nanomolar inhibitor of amyloid self-assembly ofislet amyloid polypeptide (IAPP). Angew. Chem.Int. Ed. 59, 12837–12841.

Ashok, A., and Singh, N. (2018). Prion proteinmodulates glucose homeostasis by alteringintracellular iron. OPEN 8, 6556.

Banerjee, V., Kar, R.K., Datta, A., Parthasarathi, K.,Chatterjee, S., Das, K.P., and Bhunia, A. (2013).Use of a small peptide fragment as an inhibitor ofinsulin fibrillation process: a study by high and lowresolution spectroscopy. PLoS One 8, e72318.

Baram, M., Gilead, S., Gazit, E., and Miller, Y.(2018). Mechanistic perspective and functionalactivity of insulin in amylin aggregation. Chem.Sci. 9, 4244–4252.

Becker, R.H.A. (2007). Insulin glulisinecomplementing basal insulins: a review ofstructure and activity. Diabetes Technol. Ther. 9,109–121.

Caughey, B. (2003). Probing for prions:recognizing misfolded PrP. Nat. Med. 9, 819–820.

Das, S., and Bhattacharyya, D. (2017).Destabilization of human insulin fibrils bypeptides of fruit bromelain derived from Ananascomosus (pineapple). J. Cell. Biochem. 118,4881–4896.

Frankær, C.G., Sønderby, P., Bang, M.B., Mateiu,R.V., Groenning, M., Bukrinski, J., and Harris, P.(2017). Insulin fibrillation: the influence andcoordination of Zn 2+. J. Struct. Biol. 199, 27–38.

Ghosh, P., Bera, A., Ghosh, A., Bhadury, P., andDe, P. (2020). Side-chain proline-based polymersas effective inhibitors for in vitro aggregation ofinsulin3, 8th (ACS Applied Bio Materials),pp. 5407–5419.

Gong, H., He, Z., Peng, A., Zhang, X., Cheng, B.,Sun, Y., Zheng, L., and Huang, K. (2014). Effects ofseveral quinones on insulin aggregation. Sci. Rep.4, 1–8.

Han, X., Park, J., Wu, W., Malagon, A., Wang, L.,Vargas, E., Wikramanayake, A., Houk, K.N., andLeblanc, R.M. (2017). A resorcinarene forinhibition of Ab fibrillation. Chem. Sci. 8, 2003–2009.

Heller, S., Kozlovski, P., and Kurtzhals, P. (2007).Insulin’s 85th anniversary-An enduring medicalmiracle. Diabetes Res. Clin. Pract. 78, 149–158.

Hua, Q.X., and Weiss, M.A. (2004). Mechanism ofinsulin fibrillation: the structure of insulin underamyloidogenic conditions resembles a protein-folding intermediate. J. Biol. Chem. 279, 21449–21460.

Ivanova, M.I., Sievers, S.A., Sawaya, M.R., Wall,J.S., and Eisenberg, D. (2009). Molecular basis forinsulin fibril assembly. Proc. Natl. Acad. Sci. U. S.A. 106, 18990–18995.

Jana, B., Mondal, P., Saha, A., Adak, A., Das, G.,Mohapatra, S., Kurkute, P., and Ghosh, S. (2018).Designed tetrapeptide interacts with tubulin andmicrotubule. Langmuir 34, 1123–1132.

Jimenez, J.L., Nettleton, E.J., Bouchard, M.,Robinson, C.V., Dobson, C.M., and Saibil, H.R.(2002). The protofilament structure of insulinamyloid fibrils. Proc. Natl. Acad. Sci. U. S. A. 99,9196–9201.

Julien, O., Chatterjee, S., Thiessen, A., Graether,S.P., and Sykes, B.D. (2009). Differential stability ofthe bovine prion protein upon urea unfolding.Protein Sci. 18, 2172–2182.

Kabotso, D.E.K., Smiley, D.,Mayer, J.P., Gelfanov,V.M., Perez-Tilve, D., Dimarchi, R.D., Pohl, N.L.B.,and Liu, F. (2020). Addition of sialic acid to insulinconfers superior physical properties andbioequivalence. Cite This J.Med. Chem. 63, 6143.

Kachooei, E., Moosavi-Movahedi, A.A.,Khodagholi, F., Mozaffarian, F., Sadeghi, P., Hadi-Alijanvand, H., Ghasemi, A., Saboury, A.A.,Farhadi, M., and Sheibani, N. (2014). Inhibitionstudy on insulin fibrillation and cytotoxicity bypaclitaxel. J. Biochem. 155, 361–373.

Lee, H.H., Choi, T.S., Lee, S.J.C., Lee, J.W., Park,J., Ko, Y.H., Kim, W.J., Kim, K., and Kim, H.I.(2014). Supramolecular inhibition of amyloid

iScience 24, 102573, June 25, 2021 11

Page 13

llOPEN ACCESS

iScienceArticle

fibrillation by cucurbit[7]uril. Angew. Chemie - Int.Ed. 53, 7461–7465.

Mishra, N.K., Joshi, K.B., and Verma, S. (2013).Inhibition of human and bovine insulin fibrilformation by designed peptide conjugates. Mol.Pharm. 10, 3903–3912.

Moroder, L., and Musiol, H.J. (2017). Insulin—from its discovery to the industrial synthesis ofmodern insulin analogues. Angew. Chem. - Int.Ed. 56, 10656–10669.

Neddenriep, B., Calciano, A., Conti, D., Sauve, E.,Paterson, M., Bruno, E., and Moffet, A.D. (2012).Short peptides as inhibitors of amyloidaggregation. Open Biotechnol. J. 5, 39–46.

Nilsson, M.R. (2016). Insulin amyloid at injectionsites of patients with diabetes. Amyloid 23,139–147.

Owens, D.R., Zinman, B., and Bolli, G.B. (2001).Insulins today and beyond. Lancet 358,739–746.

Paramithiotis, E., Pinard, M., Lawton, T.,LaBoissiere, S., Leathers, V.L., Zou, W.Q., Estey,L.A., Lamontagne, J., Lehto, M.T., Kondejewski,L.H., et al. (2003). A prion protein epitopeselective for the pathologically misfoldedconformation. Nat. Med. 9, 893–899.

Patel, P., Parmar, K., and Das, M. (2018). Inhibitionof insulin amyloid fibrillation by Morin hydrate.Int. J. Biol. Macromol. 108, 225–239.

Pathak, B.K., Das, D., Bhakta, S., Chakrabarti, P.,and Sengupta, J. (2020). Resveratrol as a nontoxicexcipient stabilizes insulin in a bioactivehexameric form. J. Comput. Aided. Mol. Des. 34,915–927.

Pease, L.F., Iii, Sorci, M., Guha, S., Tsai, D.-H.,Zachariah, M.R., Tarlov, M.J., and Belfort, G.(2010). Probing the nucleus model for oligomer

12 iScience 24, 102573, June 25, 2021

formation during insulin amyloid fibrillogenesis.Biophys J. 99, 3979–3985.

Ratha, B.N., Ghosh, A., Brender, J.R., Gayen, N.,Ilyas, H., Neeraja, C., Das, K.P., Mandal, A.K., andBhunia, A. (2016). Inhibition of insulin amyloidfibrillation by a novel amphipathic heptapeptide:mechanistic details studied by spectroscopy incombination with microscopy. J. Biol. Chem. 291,23545–23556.

Saithong, T., Thilavech, T., and Adisakwattana, S.(2018). Cyanidin-3-rutinoside reduces insulinfibrillation and attenuates insulin fibrils-inducedoxidative hemolysis of human erythrocytes. Int. J.Biol. Macromol. 113, 259–268.

Seidler, P.M., Boyer, D.R., Rodriguez, J.A.,Sawaya, M.R., Cascio, D., Murray, K., Gonen, T.,and Eisenberg, D.S. (2018). Structure-basedinhibitors of tau aggregation. Nat. Chem. 10,170–176.

Taschuk, R., Marciniuk, K., Maattanen, P.,Madampage, C., Hedlin, P., Potter, A., Lee, J.,Cashman, N.R., Griebel, P., andNapper, S. (2014).Safety, specificity and immunogenicity of a PrPSc-specific prion vaccine based on the YYR diseasespecific epitope. Prion 8, 51–59.

Teska, B.M., Alarcon, J., Pettis, R.J., Randolph,T.W., and Carpenter, J.F. (2014). Effects of phenoland meta-cresol depletion on insulin analogstability at physiological temperature. J. Pharm.Sci. 103, 2255–2267.

Wallin, C., Hiruma, Y., Warmlander, S.K.T.S.,Huvent, I., Jarvet, J., Abrahams, J.P., Graslund, A.,Lippens, G., and Luo, J. (2018). The neuronal tauprotein blocks in vitro fibrillation of the amyloid-b(Ab) peptide at the oligomeric stage. J. Am.Chem. Soc. 140, 8138–8146.

Wang, J.B., Wang, Y.M., and Zeng, C.M. (2011).Quercetin inhibits amyloid fibrillation of bovineinsulin and destabilizes preformed fibrils.Biochem. Biophys. Res. Commun. 415, 675–679.

Weber, C., Kammerer, D., Streit, B., and Licht,A.H. (2015). Phenolic excipients of insulinformulations induce cell death, pro-inflammatorysignaling and MCP-1 release. Toxicol. Rep. 2,194–202.

Woods, R.J., Alarco�n, J., McVey, E., and Pettis,R.J. (2012). Intrinsic fibrillation of fast-actinginsulin analogs. J. Diabetes Sci. Technol. 6,265–276.

Wu, L.C. (2019). Regulatory Considerations forPeptide Therapeutics. Peptide Therapeutics:Strategy and Tactics for Chemistry,Manufacturing, and Controls (Royal Society ofChemistry), pp. 1–30.

Xiong, X., Blakely, A., Karra, P., Vandenberg,M.A., Ghabash, G., Whitby, F., Zhang, Y.W.,Webber, M.J., Holland, W.L., Hill, C.P., and Chou,D.H.C. (2019). Novel four-disulfide insulin analogwith high aggregation stability and potency.Chem. Sci. 11, 195–200.

Yumlu, S., Barany, R., Eriksson, M., and Rocken, C.(2009). Localized insulin-derived amyloidosis inpatients with diabetes mellitus: a case report.Hum. Pathol. 40, 1655–1660.

Zaykov, A.N., Mayer, J.P., and Dimarchi, R.D.(2016). Pursuit of a perfect insulin. Nat. Rev. DrugDiscov. 15, 425–439.

Zheng, Q., and Lazo, N.D. (2018). Mechanisticstudies of the inhibition of insulin fibril formationby rosmarinic acid. J. Phys. Chem. B 122, 2323–2331.

Zhou, C., Qi, W., Lewis, E.N., and Carpenter,J.F. (2016). Characterization of sizes ofaggregates of insulin analogs and theconformations of the constituent proteinmolecules: a concomitant dynamic lightscattering and Raman spectroscopy study.J. Pharm. Sci. 105, 551–558.

Page 14

iScience, Volume 24

Supplemental information

Prion-derived tetrapeptide stabilizes thermolabile

insulin via conformational trapping

Meghomukta Mukherjee, Debajyoti Das, Jit Sarkar, Nilanjan Banerjee, JagannathJana, Jyotsna Bhat, Jithender Reddy G, Jagadeesh Bharatam, SamitChattopadhyay, Subhrangsu Chatterjee, and Partha Chakrabarti

Page 15

Figure S1. Related to Figure 1

Figure S1:

(A) Scatter plot showing 2D-distribution of ThT (x-axis) and PROTEOSTAT (y-axis)

fluorescence from heat-induced bovine insulin fibrillation in the presence of 77

peptide variants (including IS1). (B) Scree plot obtained from kmeans clustering of

ThT and PROTEOSTAT values of heat-induced bovine insulin fibrillation in the

presence of 77 peptide variants (including IS1). The within-group sum of square (y-

axis) plotted against number of clusters (x-axis). (C) Dot plot of 77 peptides

(including IS1) distributed in 4 independent clusters (x-axis) obtained from kmeans

clustering plotted against % Anti-amyloid potential (left y-axis) and % ThT/

PROTEOSTAT fluorescence (right y-axis); Bovine insulin with no heat served as the

negative control, bovine insulin heated with no peptide served as positive control. (D)

Percent allowed substitution of cluster-1 peptides plotted against amino acid

substitution position. (E) Graphical representation of the anti-amyloid peptide

screening for insulin, explaining the results obtained from kmeans clustering. All

experiments were done in triplicates and Values are presented as mean SEM. *P <

0.05, **P < 0.01, ***P < 0.001.

Page 16

Figure S2, Related to Figure 1

Figure S2:

(A) ThT assay performed for heat-induced insulin fibrillation in the presence and

absence of VPYR, VYPR, VPPR, VTTR and VYYR. (B,C) ThT-assay and DLS of

heat-induced insulin fibrillation in the presence of VPYR, VYPR, VPPR and VTTR.

The insulin and peptides ratio was (1:1) with a concentration of 350mM. All the

samples were heat incubated at 62˚C for 400mins. All the samples were prepared at

50mM citrate-phosphate buffer with a pH of 2.6. (D) Insulin Tolerance Test (ITT) of

fibrillated bovine insulin in the presence of different peptides [n = 5 mice/ group].

Page 17

Figure S3, Related to Figure 2

Figure S3:

(A) ITC graph describing the binding affinity of IS1 towards insulin. insulin and IS1

concentration used to obtain the ITC graph were 80uM and 2.4mM respectively. The

experiment was carried out at 25˚C. (B) Contour plot (2D-kernel density estimation)

of commercial insulin fibrils (Actrapid) both in the presence and absence of IS1 (5

fields/sample; 16 measurements/field) for heat and storage-induced fibrillation. (C)

Time-dependent ThT-assay of heat-induced fibrillation of Actrapid (with excipients) in

the presence and absence of IS1. (D) Western blot showing Akt (Ser 473)

phosphorylation of Citrate Phosphate Buffer, IS1, native Insulin, native insulin with

IS1, and fibrillated insulin both in the presence and absence of IS1 in HepG2 cell

line; β-actin served as the loading control.

Page 18

Figure S4, Related to Figure 5

Figure S4:

(A) Comparative study of STD NMR of reference, IS1-insulin complex, and IS1. IS1

and insulin ratio was 100:1. The proton of IS1 interacting with insulin was marked

‘red star’ sign and the protons which are not interacting marked as ‘red cross’ sign.

(B) NMR-restrained molecular simulated ensemble structure model of insulin in

binding with IS1. (C) Comparative study of STD NMR of reference, reverse IS1

(RYYV)-insulin complex, and RYYV. RYYV and insulin ratio was 100:1. (D)

Comparative study of STD NMR of reference, IS1- Actrapid complex, and IS1. IS1

and Actrapid ratio was 100:1.

Page 19

Figure S5, Related to Figure 5

Figure S5:

(A) Residue-wise relaxation of freshly prepared IS1 in presence of insulin.

Longitudinal relaxation (R1) and transverse relaxation (R2) are plotted for each

residue of IS1.(B) A temperature-dependent 1D-H NMR of insulin in the presence

and absence of IS1 respectively.(C) Overlay the spectrum of 2D-NOESY of insulin

and insulin-IS1 complex. (D) (i-iii), surface-charge distribution of IS1-insulin

interaction. (i) The red surface shows the A chain and Sky Blue surface shows B

chain of Insulin (ii) APBS surface of Insulin-IS1 complex (red-APBS negative charge

surface, blue-positive charge surface) (iii) APBS surface of Insulin-IS1 complex with

transparency (red-APBS negative charge surface, blue-positive charge surface, IS1

in ball and stick model).

Page 20

Figure S6, Related to Figure 5

Figure S6:

Schematics explaining the IS1-mediated stabilization pathway for hexameric

(Lantus) and monomeric (Lispro) insulin analogs.

Page 21

Figure S7, Related to Figure 6

Figure S7:

HPLC chromatogram showing peaks for m-cresol for three insulin analogs (Actrapid,

Lantus and Lispro) with and without excipients.

Page 22

Figure S8, Related to Figure 6

Figure S8:

(A) Atomic force microscopy images showing heat-induced (620C, 3 h) fibrillation of

conformation-selective commercial insulin analogs (Lantus and Lispro) with

excipients both in the presence and absence of IS1 (scale bars, 1 µm). (B) Time-

dependent ThT-assay of commercial insulin with excipients, Lantus and Lispro in

presence and absence of IS1. All the samples were heat incubated at 62˚C for 8 h.

The IS1 and commercial insulin molar ratio were (1:1).

Page 23

Figure S9, Related to Figure 6

Figure S9:

(A) Atomic force microscopy images showing storage-induced (370C, 30 days)

fibrillation of excipients containing commercial insulin analogs (Lantus, and Lispro)

both in the presence and absence of IS1 (scale bars, 1 µm). (B) Time-dependent

ThT-assay of conformation-selective commercial insulin analogs (Lantus and Lispro)

with excipients in the presence or absence of IS1. (C) DLS analysis of conformation-

selective commercial insulin (Lantus and Lispro) in presence and absence of IS1. All

the samples were kept at 37˚C for 30 days. The IS1 and commercial insulin ratio

were (1:1). The data points were collected at different days of incubation.

Page 24

Figure S10, Related to Figure 6

Figure S10:

(A) Comparative study of STD NMR of reference, IS1- commercial insulin complex,

and IS1. IS1 and commercial insulin ratio was 100:1. (i) IS1 in presence of Lantus

and (ii) IS1 in presence of Lispro. All were prepared in citrate phosphate buffer pH

2.6. Proton of IS1 interacting with insulin were marked ‘red star’ sign and those

proton which are not interacting marked as ‘red cross’ sign. (B) Western blot

showing Akt (Ser 473, Thr 308) and p70S6K phosphorylation of excipients-removed

commercial insulin analogs (Actrapid, Lantus and Lispro) in the presence of absence

of IS1 under storage-induced fibrillation condition (370C, 30 days) in HepG2 cell line;

pan AKT and β-actin served as the loading control.

Page 25

Table S1. Peptide sequences used in screening, Related to Figure 1

Serial No.

PeptideSequence

1 YYVR

2 AYYR

3 CYYR

4 DYYR

5 EYYR

6 FYYR

7 GYYR

8 HYYR

9 IYYR

10 KYYR

11 LYYR

12 MYYR

13 NYYR

14 PYYR

15 QYYR

16 RYYR

17 SYYR

18 TYYR

19 YYYR

20 WYYR

21 VYYA

22 VYYC

23 VYYD

24 VYYE

25 VYYF

26 VYYG

27 VYYH

28 VYYI

29 VYYK

30 VYYL

31 VYYM

32 VYYN

33 VYYP

34 VYYQ

35 VYYV

36 VYYS

37 VYYT

38 VYYY

39 VYYW

40 VYAR

41 VYCR

42 VYDR

43 VYER

44 VYFR

45 VYGR

46 VYHR

47 VYIR

48 VYKR

49 VYLR

50 VYMR

51 VYNR

52 VYPR

53 VYQR

54 VYVR

55 VYSR

56 VYTR

57 VYRR

58 VYWR

59 VAYR

60 VCYR

61 VDYR

62 VEYR

63 VFYR

64 VGYR

65 VHYR

66 VIYR

67 VKYR

68 VLYR

69 VMYR

70 VNYR

71 VPYR

72 VQYR

73 VVYR

74 VSYR

75 VTYR

76 VRYR

77 VWYR

78 VYYR

Page 26

Table S2. CD deconvoluted data from CDNN software, Related to Figure 2

Free -Insulin % of α-helix

% of β-sheets

Random coils

Insulin, 0 min 53.9 21.5 20.5

Insulin, 300 min 21.8 35.5 40.7

IS1-Insulin

IS1-insulin, 0 min 66 18.5 16.5

IS1-insulin, 300 min 60.2 20.9 16.2

Page 27

Table S3. NMR and refinement statistics for protein structures, Related to Figure 5

NMR Distance and Dihedral

constraints

Distance Constraints Total Intra residual NOE 16 Strong (|i-į|≤3) 9 Medium (|i-į|≤4) 6 Weak (|i-į|>5) 1

Dihedral Angle Restraint Phi (φ) 4 Psi (ψ) 4 Omega (ω) 4

Average RMS deviation From ideal bond 0.0297 From Ideal Angles 4.304

Page 28

Table S4. Molecular weight Calculation from Dosy NMR spectroscopy, Related to

Figure 5

A

IS1-Insulin complex Oligomeric Insulin

Diffusion co-efficient (d2 IS1- insulin) = 2.2×10-4

Diffusion co-efficient (d2 free insulin) = 9.4×10-5

Molecular weight = 10kD Molecular weight = 57kD

B

Calculation of molecular weight of IS1-insulin

Calculation of molecular weight of oligomeric insulin

Logd1/d2=1/3 logmw2/mw1

Log1.12×10-3/2.2×10-4=1/3 logmw2/82.03

Mw2=10kD

Logd1/d2=1/3 logmw2/mw1

Log1.12×10-3/9.45×10-5=1/3 logmw2/82.03

Mw2=57kD

Page 29

TRANSPARENT METHODS

Protein and peptide

Bovine insulin (I6634) and Thioflavin T were purchased from Sigma Aldrich. Peptides

were chemically synthesized from Aapptech 90 peptide synthesizers whereas peptide

array of 77 variant peptides of IS1were purchased from Genscript. Commercial insulin

analogs (Actrapid, Lantus and Lispro) were purchased from a local pharmacy.

Insulin stock preparation

Bovine insulin concentration was measured by UV-Vis spectroscopy at 280 nm. The

extinction coefficient of insulin was considered as 0.91(mg/ml)-1.cm-1.1mM working

stock for the peptides and bovine insulin in citrate-phosphate (CP) buffer (pH 2.6) were

prepared. Human Actrapid (Novo Nordisk India Pvt Ltd, Bagsværd, Denmark), Insulin

Lantus (Deutschland GmbH, Germany) and Insulin Lispro (HumalogTM, Lilly,

Indianapolis) had greater than 1 year of shelf life remaining at the time of use and were

stored at the manufacturers’ recommended storage conditions. Insulin formulation

buffers were exchanged for CP buffer (pH 2.6) using desalting spin columns for

excipients removal. Desalting spin columns (Zeba 7K MWCO, Pierce, Rockford, IL)

were pre-equilibrated by washing with three column volumes of CP buffer. Insulin

analogs were loaded onto the columns (2.2 ml insulin per column) followed by a spin

elution for 3 min at 1500 g. Eluted insulin was collected and pooled to prepare

commercial insulin excipient removed stocks. Insulin stock concentration was

determined using both Bradford assay and absorbance at 277.5 nm using the extinction

coefficient of 0.9521 ml/mg-1/cm-1. The final concentration of each insulin analogs was

adjusted to 350 µM for most studies except as indicated. Buffer exchange on all the

fresh analog samples was performed immediately prior to initiation of all fibrillation

studies, and no buffer-exchanged samples were also used for fibrillation assay in the

presence and absence of IS1 respectively. Examination of post-column eluates of the

analog formulations was performed on representative samples using reversed-phase

high-performance liquid chromatography (HPLC).

Page 30

Fibrillation assay

Bovine insulin concentration was adjusted to 350 µM in CP buffer (pH 2.6) and insulin:

IS1 equimolar complex was incubated at 620 C hot water bath in a sealed

microcentrifuge tube for a required time period to induce insulin fibril formation.

Thioflavin-T (ThT) assay

ThT concentration was measured by UV-Vis spectroscopy at 420 nm. The extinction

coefficient of ThT was considered as 36000 M-1.cm-1. ThT was dissolved in distilled

water in a concentration of 2mM and the final concentration was 20µM. ThT assay of

insulin was performed using the Hitachi spectrophotometer (F-700 FL) at 62˚C. An

aliquot of insulin from the stock solution and ThT was added to it and the fluorescence

was measured (excitation at 440 nm and emission at 480 nm). Slit for the entire

experiment was kept as 2.5. CP buffer’s spectrum was substituted from the final

spectrum of the insulin. Each spectrum was the average of three replications.

Peptide Screening

A custom-designed peptide library of 77 peptides based on IS1 was purchased from

Genscript (USA). We have performed fibrillation assay of bovine insulin in the presence

and absence of all 77 peptide variants under identical Physico-chemical conditions (

350µM, pH 2.6, 620C). We have used both ThT and PROTEOSTAT assay for

determining the fibrillation inhibition potential. The above experiment was replicated

thrice and Statistical analysis was performed in RStudio (Version 1.1.447). K-means

clustering was done on fluorescence intensities for Proteostat and ThT for all the

peptides using the kmeans function. Hierarchical clustering was done on 77 peptides for

Proteostat and ThT separately based on the position of the amino-acid substitution for

all the 20 amino-acids using hclust function and the dendrogram was plotted in the form

of a heatmap using heatmap function. Clusters obtained were plotted for their intensities

using ggplot2 package in R.

Page 31

Circular Dichroism (CD)

Secondary structural change of insulin over time was analyzed using (Jasco 815

spectrometer). The scanning wavelength was from 195nm to 260nm at room

temperature with scan speed at 100nm s-1 and bandwidth as 1nm. The results of each

spectrum were the average of 3 independent scans. Spectra obtained from the buffer

were subtracted from the spectra of bovine insulin. All the spectra were acquired at

different time intervals.

Atomic Force Microscopy (AFM)

The aggregate insulin suspensions were diluted 300 times using freshly filtered Milli-Q

water, and5l of the sample was deposited onto freshly cleaved MUSCOVITE MICA-V1

( Electron Microscopy Sciences) and dried under mild vacuum for 15-20 min. AAC

mode AFM was performed using a Pico plus 5500 ILM AFM (Agilent Technologies,

USA) with a piezo scanner maximum range of 9m. Microfabricated silicon cantilevers

of 225m in length with a nominal spring force constant of 21-98 N/m were used

(Nanosensors, USA). The cantilever oscillation frequency was tuned into resonance

frequency. The cantilever resonance frequency was 150-300 kHz. The images (256 by

256 pixels) were captured with a scan size of between 0.1 and 8 m at the scan speed

rate of 0.5lines/S. Images were processed by flattening using Pico view1.1 version

software (Agilent Technologies, USA). Image correction has been done through Pico

Image Advanced version software (Agilent Technologies, USA). Height and width

measurement has also been done in Pico view 1.1 version software for approximately

80 data points from 5 independent fields for each sample.2-dimensional kernel density

estimation was done for height and width of insulin fibrils with and without the IS1 for all

3 different types of insulin (Actrapid, Lantus and Lispro) and the results were displayed

with contours over the scatter plots using MASS and ggplot2 package in R.

Dynamic Light Scattering (DLS)

DLS studies were performed on Malvern nano dynamic light scattering equipment. The

insulin sample was taken at a concentration of 350µM in the presence and absence of

IS1 for analysis. All the samples were filtered through micro-filter containing a pore size

Page 32

of 0.2µM. The Measured size was presented as the average value of 36 runs. Dynamics

7.10.0.10 software was used for data analysis.

Gel filtration chromatography coupled with Dynamic Light Scattering

Recombinant Human Insulin (Sigma) adjusted to 350 M in CP Buffer (pH 2.6), and

Insulin: IS1 equimolar complex was incubated at 620 C in a prewarmed water bath in a

sealed microcentrifuge tube for 4 h to induce insulin fibrillation. Native human insulin

served as a negative control. The sample was centrifuged and loaded on

HiLoadSuperdex 75pg Column (GE healthcare). Steep selectivity curves of this column

give resolving power for proteins in the molecular weight range~ 3000 to 70000 Dalton.

Loading was followed by the elution at the flow rate of 1ml/min and a pressure of 0.3

Pa. The eluent corresponding to the major chromatographic peak was collected and

then run on Malvern nano dynamic light scattering instrument to get the idea of the

eluent size. Dynamics 7.10.0.10 software was used for data analysis.

Isothermal calorimetric titration (ITC)

ITC experiment was carried out in 250C in VP ITC Micro Calorimeter equipment. The

insulin solution was taken in the cell and IS1 was taken in the syringe. The experiment

was performed in water (pH 2.6). After the equilibration was reached, then only the

titration was started. This titration of insulin with IS1 was total of 14 injections at 180 s

time intervals with 3µl IS1 per injection. All the raw data was analyzed in origin software.

The data were fitted in the ‘one site binding’ model.

NMR- T1 and T2 relaxation, STD NMR, and DOSY NMR

All spectra of NMR experiments were recorded using a Bruker AVANCE III 500 MHz

spectrometer equipped with a 5mm SMART probe at 298K. Data acquisition and

processing were performed using Topspin 3.1 software. All NMR samples were

prepared in 50mM citrate phosphate buffer for insulin and H2O for peptides, containing

10% D2O and using TSP as an internal standard (0.0 ppm). By performing Diffusion

Order NMR spectroscopy (DOSY), the hydrodynamic radius and molecular weight of

free insulin and IS1-insulin complex were determined. 1 mM Sodium acetate was added

Page 33

into the solution of free insulin and IS1-insulin complex as an internal reference. Total

24 one-dimensional 1H NMR spectra were recorded with increasing gradient strength

from 5% to 95% for both cases (free and complex insulin) using Bruker Pulse program

“stebpgp1s191d” (a diffusion measurement using stimulated echo) having a spectral

width of 20 ppm and 128 scans with 4 dummy scans. The intensity decay of 1H signals

from insulin was plotted using equation

I(g) = Ae(-dg2)

where I, g, and d represent the intensity of the peak, gradient strength, and decay rates,

respectively. The hydrodynamic radius was measured using the equation

Rh = dref/dRhref

where Rh is the hydrodynamic radius of insulin (free or complex), dref is the decay rate of

reference, and d is the decay rate of insulin (free or complex). Rh (ref) was taken to be 2.3

Å. The molecular weight of free insulin and IS1-insulin complex were taken from the

equation

log(d1/d2) = (1/3) log(MW2/MW1)

To perform saturation transfer difference (STD) NMR experiments, insulin and IS1 were

prepared in a buffer containing 99.9% D2O. The pH was adjusted to 2.6. The STD-NMR

spectra of insulin: IS1 (1:100) were acquired with 3 K scans and selective saturation of

insulin resonances either at 20.5 or at 7.3 ppm (reference spectra were at 40 ppm)

using a series of 40 Gaussian-shaped pulses (49 ms, 1 ms delay between pulses). The

free peptide, VR4 do not show any STD effects saturating at the same frequency as the

IS1-insulin complex. Data were processed using the TOPSPIN program suite. Atomic-

level dynamics were obtained from one dimensional longitudinal (T1) and transverse

(T2) relaxation experiments. T1 experiments were performed using previously reported

protocols with different inversion recovery delays ranging from 0 to 3 s. The T2

measurements were achieved from CPMG sequence and the delays ranging from 0 to

0.7 s.

Page 34

The 2D-1H,1H plane of 3D- 15N- edited NOESY HSQC, 2D-1H,15N –HSQC, and 2D-

1H,1H -NOESY spectra of NMR experiments were recorded using a Bruker AVANCE III

700 MHz spectrometer equipped with a 5mm Cryogenically cooled Quadruple-

resonance probe at 283K. Data acquisition and processing were performed using

Topspin 3.1 software. All NMR samples were prepared in 50 mM citrate phosphate

buffer for insulin and IS1, containing 10% D2O. Insulin and IS1 were taken in 1:1 ratio

with 500 M concentration each. Noesy mixing time was 100 ms.

Docking and Simulation

The co-ordinate file of the X-ray Crystallographic structure of insulin (PDB ID:2ZP6),

was downloaded from RSCB PDB site and further processed in PyMOL. Structure of

IS1 was deduced by NMR and solved using CYANA. Insulin and IS1 were docked in

HDOCK server, a protein-protein based docking server based on a hybrid algorithm of

template-based modeling and ab initio free docking and structures were prepared using

protein preparation wizard module of schrodinger. The ligand (IS1) was first minimised

using MacroModel, and the minimised output structure was processed using protein

preparation wizard with default parameters in it. Next in BioLuminate the Peptide

docking module was used for docking. The binding site was specified by selecting the

centroid of residues derived from 2D and 3D NMR. IS1 was specified from project table

selected entry and 100 ligand poses were saved. The best pose among all were

selected and further refined in AMBER. First AMBER parameter/topology files were

developed and then NMR restraint files were generated using protocols as mentioned in

AMBER tutorials. Finally, molecular dynamics simulation of the structure was performed

for 20 ps in AMBER14 using General Amber Force Field (GAFF) which uses simple

harmonic function and ff14SB force-field for parameterization of peptide-insulin

complex.

Cell lysis and Immunoblotting

Cells were rinsed with ice-cold PBS and total cellular protein was prepared with lysis

buffer containing 50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM EDTA, 1 mM EGTA and

1% Triton X-100 with protease and phosphatase inhibitor cocktail both from (Millipore,

Page 35

Billierica, MA, USA). The soluble fractions of cell lysates were isolated by centrifugation

at 15000g for 15 minutes at 40 C. Protein samples (40 μg/lane) were resolved by 10%

SDS-PAGE and transferred to Immobilon-P membranes (Millipore, Billierica, MA, USA)

by wet transfer (Trans-Blot, Bio-Rad) at 90V for 3 h. For immunodetection membrane

was blocked with 5% non-fat milk in PBS with 0.1% Tween 20 for 1 h followed by

incubation with specific primary antibody (1:1000 dilution) at 40 C overnight and with

horseradish peroxidase-labeled secondary antibodies (1:1000 dilution) for 1h at room

temperature. Signals were detected by chemiluminescence using LuminataClassico

Western HRP substrate (Millipore, Billierica, MA, USA) and scanned using a

ChemiDocMP System (Bio-Rad Laboratories, Hercules, CA). Primary antibodies used:

Anti--Actin, Anti-Akt (Pan), Anti- phospho-Akt (SER 473), Anti- phospho-Akt (Thr 308),

Anti-phospho-p70 S6 Kinase (Thr 389). All the antibodies were purchased from Cell

Signaling Technology (Boston, MA, USA).

Animal Study

Experiments with animals were performed under the approved Institutional Animal

Ethics Committee (Approved by CPCSEA, India) protocol. 6-8 weeks old wild-type male

BALB/c and C57BL/6 mice were kept at ambient temperature (22 ± 1°C) with 12:12 h

light-dark cycles and fed with standard chow diet (4.3% lipid and 70% carbohydrate)

and high-fat diet (45% kcal fat, MP Biomedical) for respective experiments. In order to

study insulin signaling in vivo, BALB/c mice (n=5/group) fasted for 6 h and a single dose

intraperitoneal insulin injection (1 U/kg of body weight) were administered. The same

dose of fibrillated insulin and fibrillated insulin + IS1 were administered to the respective

group of animals. An equal amount of IS1 was also administered as a negative control.

30 minutes post-injection, all the animals were sacrificed, livers were dissected and

homogenized in lysis buffer. In order to generate a type 1 diabetic preclinical model,

following fasting for 6 h, a single intraperitoneal injection of 180 mg/ kg Streptozotocin

(STZ) was administered to BALB/c mice. STZ was reconstituted in Na-Citrate buffer (pH

4.5) immediately before injection to avoid degradation. All mice were tested for weight

loss and hyperglycemia at 4 weeks post-injection. For generating type 2 diabetes

model, weight and age-matched male C57BL/6 mice were fed with a high-fat diet for 16

Page 36

weeks. We have tested for gradual weight gain and hyperglycemia at each 4 weeks

interval up to 16 weeks. BALB/c and C57BL/6 mice were fasted for 6 h and fasting

blood glucose was measured using a calibrated glucometer by taking one drop of blood

from the tail tip cut. Intraperitoneal injection of insulin (0.5 U/kg of body weight ) was

given to each mouse with 27 G needle and blood samples were taken from the initial tail

cut at 30, 60 and 120 minutes after intraperitoneal insulin injection. Insulin Tolerance

Test (ITT) has been replicated in five mice per group.8 weeks old BALB/c mice were

taken for generating subcutaneous amyloidoma (n=3/group). Each animal was

administered subcutaneously with 1µg of fibrillated insulin and fibrillated insulin + IS1

daily at the left and right flank in the ventral side of the body for 28 consecutive days.

The injection volume was 103.3 µl for both the samples. All groups received a normal

diet during the experimental period. After 28 days, the biopsies were collected from both

spots of each animal and tissue sections were embedded in paraffin and processed for

hematoxylin and eosin (H&E) as well as congo red staining. Both light microscopy and

polarized images were taken with Leica DFC450c camera using LAS EZ software

(v.4.6).

Live Dead Assay

HepG2 cells harvested in DMEM and 0.5 ml of 0.4% Trypan Blue solution (w/v) was

added to the suspension of cells. 50µl of Trypan Blue-cell suspension mixture was

placed on the hemocytometer for counting. Cells in all the chambers were counted and

five replicate readings were taken. Approximately 2.5 × 105 cells were counted and

plated in each well. The next day, cells were treated with variable doses (10, 20, 40 and

80 μM) of IS1 and SLRP peptides under serum-starved conditions. After 16 h of

treatment, cell viability for respective treatments was assessed using the Live/Dead

viability kit (Invitrogen, Renfrew). Images were captured with the confocal microscope

FluoView (FV10i; Olympus, Tokyo, Japan). We have also studied the cytotoxic potential

of both IS1 and SLRP for 24 hours using 10 μM concentration. Percent viable and dead

cells (Number of dead cells per field / total number of cells per field) * 100) were

calculated from 5 independent fields per group using threshold and analyze particle

Page 37

function by applying both red (PI) and green (FITC) channel independently using image

J (Fiji) software.

Cytotoxicity assay

Approximately105 HepG2 cells/100 μl of medium per well in 96 well polystyrene plates

following Trypan Blue exclusion. Cells were treated with IS1 and SLRP with varying

concentrations of 10, 20, 40 and 80 μM for 24 h. Cell viability was determined with the

addition of 10 μl of 5 mg/ml MTT to each well. After incubation for 4 hrs at 37 °C, the

medium was aspirated from each well and 100 μl of DMSO was added per well. Plates

were agitated at 25 °C for 10 min and absorbance was recorded at 590 nm by a multi-

well plate reader (Biotek-Epoch; Biotech Instruments). Cells were treated with citrate-

phosphate buffer and no test samples but MTT served as the positive control. Cells

without MTT served as blank. Percent viability [ % viability = ((Abssample – Absblank) /

(Abscontrol – Absblank)) * 100 ] has been calculated using three replicate values for each

concentrations.

Hemolytic Assay

5ml fresh blood was collected from volunteer in EDTA containing vial, centrifuged at

6000rpm for 10min. The cell pellet was resuspended in 1ml 1X PBS and washed

3times. Finally 50l of the peptide in PBS and 50µl cell suspension were mixed and

incubated at 37˚C for 1h with continuous shaking at 200rpm. Samples were centrifuged

at 6000 rpm for 10 min and supernatant were collected for spectroscopic measurements

at 540 nm. 1% Triton X100 serves as a positive control. The percentage of hemolysis

was calculated.

Statistics

Statistical values are presented as the mean ± SD. A two-tailed Student’s t-test was

used to calculate the p-values. Pearson’s correlation coefficient ‘r’ has been calculated

to explore the association between variables .p<0.05 was considered as significant.