Blocking Oligomeric Insulin Amyloid Fibrillation via Perylenebisimides

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Blocking Oligomeric Insulin Amyloid Fibrillation via Perylenebisimides Containing Dipeptide Tentacles Sayan Roy Chowdhury, Subrata Mondal, and Parameswar Krishnan Iyer ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00927 • Publication Date (Web): 27 Sep 2018 Downloaded from http://pubs.acs.org on September 27, 2018

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ACS Biomaterials Science & Engineering

Blocking Oligomeric Insulin Amyloid Fibrillation via

Perylenebisimides

Containing

Dipeptide

Tentacles Sayan Roy Chowdhury,a Subrata Mondal,a Parameswar Krishnan Iyer*a,b a

Department of Chemistry,

b

Centre for Nanotechnology, Indian Institute of Technology

Guwahati, Guwahati–781039, India Email: [email protected] KEYWORDS Perylenebisimide; Insulin amyloid; Oligomers; Aggregation; Inhibition; Modulation.

ABSTRACT

Molecular motifs that could interfere with amyloid fibrillation via non covalent interactions are very vital towards aberrant protein aggregation and related human diseases. Mutual aggregation ensues in the presence of these structural motifs and nucleation on the particle surface leads to inhibition of the fibrillization process. This modular process generates a new generation of inhibitory reagents. Oligomers are the primary toxic species that initiate pathogenic aggregation leading to toxic β-sheet rich structures. To inhibit these toxic oligomers, two dipeptide linked perylenebisimide isomers (PAPAP and APPPA) are developed as selective modulators for insulin fibrillization. Early insulin aggregates are adsorbed into the modulator surface and stabilizes soluble oligomeric aggregates. Fibrillation and inhibition were examined by Thioflavin

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T (ThT) assay in the presence and the absence of both inhibitors, PAPAP and APPPA. Conformational modulation using far UV circular dichroism studies also high-lighted their role as an aggregation inhibitor via reduction of α-helix into β-sheet along with increased random coil contents. Moreover, the inhibitory effects were more pronounced due to the varying multiple non covalent interaction ability of these isomers, a performance well beyond all known modulators with respect to selectivity and efficiency, with the more aggregation prone derivative due to higher probability of a hydrophobic encounter between the protein and the molecular modulator. These results also lead to the elucidation of insulin fibril regulating mechanism via selective non covalent binding and provide fundamental insights into the chemistry of peptide-based probes as tools for developing next-generation therapeutics.

INTRODUCTION Protein misfolding leading to amyloid fibrillation is connected to several human diseases. 1-3 Despite intensive research, the involvement of the amylodogenic proteins in disease pathogenesis is yet to be understood.4 Insulin (~5.8 kDa) plays a major role in glucose metabolism and commonly used as an anti-diabetic drug.5 Insulin is not bioavailable in the amyloid form and amyloid formation leads to erratic delivery in insulin pumps and contributes to complications like diabetic ketoacidosis.6-8 To prevent this it is promoted as zinc bound hexameric form rather than monomeric form of two peptide chains-A and B of 21 and 30-residues respectively.9 Although this hexameric form is conformationally stable, it is most effective and required in monomeric form.10,11 Therefore, amyloidogenesis associated with insulin actually delays the insulin activity.12-14 A toolkit or an excipient that provide stability toward the monomeric form due to its fast action and eliminate aggregation, can represent a paradigm shift in designing


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modulators or inhibitors of medically important protein aggregates.15,16 There are almost 27 different human proteins which have been reported in systemic or localized amyloidosis forming toxic β-sheet rich oligomers via losing their native conformations.17 Insulin is cheap and apart from its clinical complications, it also provides in vitro system to monitor amyloid fibril formation.18 Despite these advantages, inhibitors of insulin aggregations are not explored in plenty in comparison to β-amyloid and α-synuclein. A few synthetic and natural compounds have been identified and explored to reduce or prevent the fibrillation process both in vitro and in vivo, for example, designed peptide conjugates,19-21 antigen or epitope specific antibodies22 and small compounds consisting anti-aggregation property.23-26 Among them small molecules have contributed significantly to the progression of various drug discovery programs. This molecular material has evolved as a leading aspect in terms of phenotypic and targeted drug delivery with recent advances in profiling assays and refined integration strategies in terms of differentiation, prioritization and mechanism of action.27 Herein, we have explored two water soluble dipeptide linked perylenebisimide isomers (PAPAP and APPPA) as inhibitors of insulin fibrillation. In this study, both the isomers were shown to interfere the insulin fibrillation under physiological conditions using a variety of physico-chemical studies. Interestingly, the isomer (PAPAP) which is more prone to aggregation showed improved inhibition towards insulin compared to the other derivative. This inhibition stratagem based on mutual aggregation was further validated by Thioflavin T (ThT), circular dichroism (CD), atomic force microscope (AFM) studies, and isothermal titration calorimetry (ITC). EXPERIMENTAL PROCEDURE Materials


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Insulin (recombinant human, expressed in yeast), Thioflavin-T were obtained from Merck. Stock solutions were prepared using Tris-HCl buffer adjusted at pH 7.4 using pH-meter from Eutech Instruments with 1N HCl prior to modulation studies. Synthesis of PAPAP and APPPA PAPAP and APPPA were synthesized and characterized as shown in the Supporting Information. 0.254 mmol (1 equivalent) PTCDA, 0.535 mmol (2 equivalent) Phe-Ala/Ala-Phe were stirred along with heating at 140°C for 12 h in presence of imidazole (1.5 g), and 0.005 mmol (catalytic) Zinc acetate followed by careful pouring into 2M HCl solution slowly to precipitate at 90 °C. Finally, the precipitates were obtained after washing with water and drying under vacuum (139 mg, 65% APPPA; 150 mg, 71% PAPAP). In Vitro Insulin Fibrillation Human insulin (HI) stock solution was prepared in Tris-HCl (25 mM) buffer at pH 7.4 containing MgCl2 (2 mM) and NaCl (125 mM). The final stock concentration was determined by measuring absorbance at 280 nm. Insulin fibrillation reaction was studied with 0.5 mg/mL insulin concentration (~86 µM) at 37 °C at 60 rpm. Reaction mixtures were collected at different time intervals for further aggregation and modulatory studies. Thioflavin-T (ThT) Fluorescence Kinetics ThT fluorescence was measured to investigate the kinetics associated with fibril formation of insulin with time. ThT fluorescence was monitored with incubated insulin solution at different time intervals, mixed with ThT solution in 1:2 ratio of protein. and ThT dye consisting 10 µM and 20 µM final concentration of insulin and ThT. Fluorescence measurements were recorded on


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a Tecan infinite M1000 multi-mode micro plate reader containing 10 µM ThT and 20 µM of insulin. The ThT fluorescence data were analyzed and fitted to the sigmoidal curve (solutions were excited at 440 nm and emission was noted at 482 nm).13 Y = y1 + m1x + (yf+mfx) ⁄ 1+e-[(x-x0)/τ] [1]………………………..(1) ThT fluorescence intensity is denoted by y, x denotes time and x0 is the time required to achieve 50% of maximum fluorescence intensity, the apparent rate constant is kapp, fibrillar growth is 1/τ and the time period in lag phase is derived by x0-2τ. Circular Dichroism (CD) Spectroscopy Jasco J-1500 spectrometer was used to record Circular dichroism (CD) spectra in 0.2 cm path length cuvette cell. In CD measurements, 10 µM insulin was taken in Tris-HCl buffer (5 mM) at pH 7.4. Far-UV regions (190-250 nm) were noted for the monomeric and fibrillar insulin at room temperature both in presence and absence of the modulators at a scan rate of 100 nm/min with a step size of 0.2 nm. The spectra were recorded by averaging 5 scans and corrected by subtracting the Tris-HCl buffer spectra. The percentage of α-helix can be obtained using the following formula.28 % Helix = ([θ]obs × 100)/{[θ]helix × (1 − 2.57/l)}…………………..(2) Where [θ]obs denotes mean residue ellipticity at 222 nm, [θ]helix is −39,500 deg.cm2/dmol, and l is the number of peptide bonds. MRE222 = Intensity of CD (mdeg) at 222 nm / 10 nlC……………..(3)


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Where n represents the total number of amino acid residues, l denotes the path length of the sample cell and C signifies the molar concentration of insulin. Secondary structure components were analyzed using CONTIN and SELCON3 program available in Dichroweb server. Isothermal Titration Calorimetry (ITC) PAPAP and APPPA binding with monomeric insulin was examined by ITC using MicroCal iTC-200. Both protein, and inhibitor stock solutions were taken in Tris-HCl buffer (20 mM) at pH 8.4 to minimize the contribution of dilution heat and degassed before ITC measurements. 0.5 mM of each inhibitor in the syringe was titrated with insulin (50 µM) at 37 °C. Briefly, 25 injections of 1.5 µL each were added with an interval of 90 seconds between successive injections. Prior to the binding experiment, solutions were at stirred at 450 rpm to make sure proper mixing of the inhibitors with the protein solution. Similar experimental conditions were maintained to perform control experiment, where inhibitors were titrated against buffer and subsequent heat change were subtracted from the initial titrations in presence of insulin. ∆G was derived using the equation ∆Gapp= ∆H- T∆S and other parameters such as n, Ka, ∆S, and ∆H were calculated from multi injection mode of ITC at a constant temperature. Zeta potential measurement Zeta potential (ξ) of inherent and aggregated insulin and their co-incubated samples with PAPAP and APPPA were measured using a Malvern Zetasizer Nano-ZS. A suspension of aboveincubated solutions of different concentrations was taken in Tris-HCl buffer (5 mM) at pH 7.4 and transferred into 1 mL zeta potential cuvette (DTS1060, Malvern). Apparent zeta potential (mV) of all protein solutions were analyzed with Zetasizer software (version 7.11, Malvern) both in absence and presence of inhibitors.


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Atomic Force Microscopy (AFM) studies To monitor morphological changes, pristine solutions of native and aggregated insulin, APPPA, PAPAP and their co-incubated insulin were taken in Tris-HCl buffer (10mM) at pH 7.4 and diluted 100 times. Prior to AFM experiments, 2-5 µL of each sample was mounted and dried under argon flow onto the freshly cleaned glass slide. Images were taken on a Bruker, Innova AFM in tapping mode with non-contact approach and analyzed with WSxM 5.0 Develop 8.0 software.

Scheme 1. Chemical structures of inhibitors, PAPAP and APPPA. RESULTS Insulin is considered as natively structured amyloidogenic protein which is commonly used as a model to probe fibrillization and related diseases. Importantly, amyloid oligomers and fibrils lead to cell death via nucleation dependent aggregation pathway. A number of reports based on amyloid inhibitors have been established over the past decade.27, 29-31 Herein we have studied the


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insulin fibril formation and their inhibition in the presence of two dipeptide linked perylenebisimide isomers PAPAP and APPPA (Scheme 1) via ThT assay, conformational modulation by CD spectrometer, binding kinetics by ITC measurements and finally the changes were visualized using AFM. Moreover, the modulation strategy and inhibition mechanism were discussed in detail and a structural relationship has been elucidated based on photophysical properties of the perylenebisimide isomers. Insulin fibrillation kinetics monitored by Thioflavin T assay Fibrillation process mechanism in various proteins are commonly investigated through ThT based fluorescence assay in which ThT delivers typical emission spectra at 482 nm after exciting buffer containing protein solutions at 440 nm. The onset and maximum intensity of ThT fluorescence signal represent the lag phase and fibril quantity respectively. Figure 1 demonstrated the time evolution of insulin fibrillation mechanism based on ThT fluorescence values after incubation of insulin (~86 µM) at 37 °C under physiological pH 7.4 with stirring at 60 rpm. The ThT fluorescence curve represented a typical sigmoidal curve comprising three distinctive phase, lag phase, a succeeding growth phase followed by a plateau. Lag phase duration and apparent rate constant (kapp) of fibrillation were derived from equation 1 (in experimental procedure). The resulting time duration of lag phase was found to be as 3.87±0.12 h. Apparent rate constant for insulin fibrillation was calculated as 0.687 h-1 and a plateau was reached within 12 h. The fibril formation was also established through AFM images that showed a mesh of insulin fibrils (in AFM analysis) in control. The effect of two synthetic inhibitors, PAPAP and APPPA were investigated on insulin fibrillation with a final concentration of 8.6 µM as PAPAP showed a maximum reduction in ThT fluorescence intensities with this concentration and highlighted the strong inhibitory effect (Figure 1). Native insulin was incubated with varying


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concentrations of these inhibitors from 0.86 µM to 43 µM under same in vitro conditions at 37 °C with stirring at 60 rpm but ThT fluorescence intensity is al-most reduced above 8.6 µM concentration of PAPAP and was not fitted to equation 1 for kinetic analysis, therefore, we investigated the fibrillation inhibition of insulin with 8.6 µM of both inhibitors.

Figure 1. (a) Insulin fibrillation kinetics mechanism monitored through ThT fluorescence intensity with insulin amyloid fibrils. (b) Maximum ThT fluorescence intensity during the fibrillization in presence and absence of PAPAP and APPPA. ThT data are normalized with the maximum fluorescence intensity; error bar depicts standard error of five different measurements. (c) Kinetic parameters lag phase and (d) apparent rate constant (kapp) during insulin fibrillation and inhibition (e) Residual protein kinetics of amyloid fibrillation in presence and absence of inhibitors. Figure 1 illustrated the ThT fluorescence curve of fibrillation in presence of these two inhibitors with 8.6 µM of final concentrations. Decrease in ThT fluorescence intensity illustrated that the quantitative reduction in the fibril formation in presence of both inhibitors. ThT reduction is more in presence of PAPAP as compared to APPPA and demonstrated its inhibitor efficiencies over APPPA. Kinetic analysis of insulin fibrillation has shown a maximum increase in the time


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duration of lag phase as 7.69±0.17 h in presence of PAPAP while it was approximately 6.58±0.13 h in case of APPPA. Extended lag phase in PAPAP mediated fibrillation highlighted its efficient inhibitory property and delayed the nucleation process as compared to insulin fibrillation in absence of inhibitors. The apparent rate (kapp values) for PAPAP and APPPA mediated inhibition of insulin fibrillation was calculated as 0.330 h-1 and 0.383 h-1 (Table 1) respectively which suggested their involvement in the suppression of fibrillation along with delaying the process. Subsequently, it was concluded that both isomers are potent inhibitors and perform the efficacy through both ways, delaying, and suppression of the process. Figure 1c explained the increase in lag phase in presence of inhibitors compared to the control and the apparent rate constant of fibrillation in figure 1d revealed that PAPAP mediated inhibition was superior to APPPA inhibition (for comparison see Table 1). Both the modulators helped to restore native insulin which was proved by a similar residual protein ThT assay (Figure 1e). Table 1. Lag Phase, Apparent rate and maximum intensity of insulin amyloid fibrillation in presence and absence of synthetic inhibitors, PAPAP and APPPA. Reaction

Lag phase (h)

Apparent rate (Kapp/h-1)

Max Intensity

Insulin

3.87±0.12

0.687

1.000

Insulin+PAPAP

7.69±0.17

0.330

0.139

Insulin+APPPA

6.58±0.13

0.383

0.410

Further, to check the effect of inhibitors on preformed β-rich aggregates, ThT assay was performed with the preformed insulin aggregates, similarly incubating with 8.6 µM of PAPAP and APPPA. PAPAP co-incubated aggregates resulted in a decrease in β-content compared to


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APPPA co-incubated and control insulin fibrillar aggregates as observed from ThT fluorescence profile (Figure S1) due to the better self-aggregation ability of PAPAP. Conformational restoration via CD analysis

(b)

40

20 10 0

0

4 8 Time (h)

5

35 30 25 20 15 10 5 0

12

Ins 0 h Ins 4 h Ins 8 h Ins 12 h

10

0 -5 -10

Secondary Structure (%)


30

0

4 8 Time (h)

10 Ellipticity (mdeg)


40 40

α−helix β−sheet β−turn

(c)

Ins+PAPAP 0 h Ins+PAPAP 4 h Ins+PAPAP 8 h Ins+PAPAP 12 h

5 0 -5 -10

random coil

35 30 25 20 15 10 5 0

12

0

4

8 Time (h)

12

Ins+APPPA 0 h Ins+APPPA 4 h Ins+APPPA 8 h Ins+APPPA 12 h

10 Ellipticity (mdeg)

(a)

Ellipticity (mdeg)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


5 0 -5 -10

-15 200

210

220 230 240 Wavelength (nm)

250

-15 200

210

220

230

240

250

Wavelength (nm)

200

210

220 230 240 Wavelength (nm)

250

Figure 2. CD spectra and changes (bar diagram) in insulin secondary structure in (a) control as native insulin, (b) co-incubated with PAPAP, and (c) co-incubated with APPPA with time (up to 12 h) in Tris-HCl buffer (5 mM) at pH 7.4. Insulin concentration was 10 µM. CD spectra were recorded in the far UV region in order to prove conformational modulation in the absence and presence of inhibitors (Figure 2). In CD spectra, bands at 208 nm and 222 nm are distinctive for α-helix while 218 nm bands indicate β-fraction in respective protein and highlights the change of α-helix into the β-sheet conformations (Figure 2a).29,30 Changes in the protein secondary structures could be analyzed in the presence of both the inhibitors (8.6 µM) (Figure 2b and 2c) and the changes in α and β fraction was carefully detected. In the present study, CD spectra of native insulin highlighted 37 and 22.2 % of α-helix and β-sheet fraction along with 14.7 and 26 % of β-turn and random coil respectively which is in good agreement


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with previous reports.31 PAPAP induced more reduction (~3.24 %) in α-helix of native insulin compared to APPPA molecule (~0.81 %) because of its ability to form multiple non covalent interactions with native insulin via the perylene backbone and dipeptide side chains with terminal phenyl. (Table 2 and Figure S2). Table 2. Changes in conformation of insulin (10 µM). α-helix, β-sheet, β-turn, and Random coil percentage in absence and presence of the inhibitors (PAPAP and APPPA, 8.6µM) in Tris-HCl buffer (5 mM) at pH 7.4. Time (h)

Reaction

α-helix (%)

β-sheet(%)

β-turn(%)

Random coil(%)

0

4

8

12

Insulin

37

22.3

14.7

26

Insulin+PAPAP

35.8

22

14.5

27.7

Insulin+APPPA

36.7

22

14.6

26.7

Insulin

26.9

23.5

22.9

26.7

Insulin+PAPAP

32.2

21.2

17

29.6

Insulin+APPPA

28.1

22.2

22

27.7

Insulin

3.6

40.6

22

33.8

Insulin+PAPAP

8.2

34.3

20.3

37.2

Insulin+APPPA

5.9

37.6

20.6

35.9

Insulin

2.9

44.2

22.4

30.5

Insulin+PAPAP

6.5

35.1

21.1

37.3


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Insulin+APPPA

5.2

36.9

22.2

35.7

Conformational modulation was also investigated in absence and presence of the inhibitors during the fibrillation that revealed ~92.2 %, ~82.4 % and ~85.9 % loss of α-helicity while ~98.2 %, ~57.4 % and ~65.5 % increase in a β-sheet fraction in control, PAPAP and APPPA mediated insulin fibrillation respectively. It was observed that presence of PAPAP at low concentration restored maximum α-helicity with a minimum β-sheet fraction in insulin during the fibrillation (Figure S3). PAPAP also enhanced total fraction of the random coil as compared to control and APPPA mediated fibrillation which highlighted its potential anti-aggregation property (Figure S4). Conclusively, CD analysis highlighted that both PAPAP and APPPA act as aggregation inhibitors and accomplished their modulating role through the reduction of α-helix into β-sheet along with increased random coil contents. Morphological changes using AFM analysis Further, to visualize the insulin aggregates in presence of modulators, fibrillization was confirmed by using AFM (Figure 3). Insulin was incubated with the modulators for 72 h to monitor fibril formation and the changes occurred due to modulation. Native insulin monomers formed mature fibrils after 72 h of incubation. The diameter and height of the native insulin monomer were 200 nm and 12-16 nm respectively in topological image (Figure 3a, S5a and S5b). After 72 h of incubation mature fibrils were found to form with diameter 300-400 nm and ∼25-35 nm in height (Figure 3b, S5c and S5d). PAPAP and APPPA co-incubated with insulin showed no fibril formation (Figure 3c and 3d). In presence of PAPAP, insulin oligomers (monomers and early aggregates) were blocked and thus restricted to come in contact with other


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protein counterparts thereby preventing formation of mature fibrils. PAPAP co-incubated insulin showed larger aggregates with diameter ∼1 µm and height of ∼40-60 nm (Figure 3c, S5e and S5f). As the attached phenyl ring of phenyl alanine was away from the conjugated perylene center in PAPAP, making it more prone to self-aggregation and caused the formation of larger aggregates compared to the other isomer, APPPA. On the other hand, APPPA co-incubated insulin resulted in aggregates with diameter of 450 nm and height of ∼20 nm (Figure 3d, S5g and S5h). From AFM studies, it was confirmed that PAPAP and APPPA successfully inhibited insulin aggregation and stopped the fibril formation. More self-aggregation prone PAPAP derivative resulted in larger aggregates and better blocking surface toward early insulin aggregates.

(a)

1.0µm

(c)

2.0µm

(b)

1.0µm

(d)

1.0µm

Figure 3. AFM images of control (a) native insulin monomers, (b) mature fibrils, and modulation (c) co-incubated with PAPAP, and (d) co-incubated with APPPA. All the stock solutions were


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incubated at 37 °C in Tris HCl buffer (10 mM) at pH 7.4. Images were taken after 72 h of incubation. PAPAP and APPPA concentrations were 8.6 µM and insulin concentration was 86 µM. Scale bar in (a, b, and d) are 1 µm and in (c) is 2 µm. Binding interaction analysis through ITC Binding kinetics of these inhibitors with native insulin is decisive to understand the mechanistic insights of fibrillation inhibition mediated with PAPAP and APPPA (Figure 4 and Table 3). Therefore, an ultrasensitive technique, ITC was applied for thermodynamic characterization of intermolecular interactions between native insulin and studied inhibitors. We calculated binding affinity (Ka), binding stoichiometry (n), total entropy change (∆S) and the total change in enthalpy (∆H) during the inhibition reaction. To evaluate the underlying association between insulin and the inhibitors as various potential non covalent interaction forces that dictate the compelling force of association reaction were considered. Both enthalpy change and entropy change were contemplated as driving factors for protein-ligand interaction. Negative ∆H and ∆S signifies hydrogen bonding and Van der Waal’s forces; positive ∆H and ∆S indicates hydrophobic encounter while negative ∆H and positive ∆S infers the dominance of electrostatic interaction as the predominant driving force. To rule out the electrostatic contribution at peptidemodulator interface, zeta potential of peptide aggregates co-incubated with the inhibitors in TrisHCl buffer (Figure S6) were measured. An increase in is seen in the apparent zeta potential in case of co-incubated sample compared to pristine modulators. Thus, ITC and zeta potential measurements revealed Van der Waal’s contribution as one of the predominant reason toward perylene-insulin interaction.


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(a)

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(b)

Figure 4. ITC thermogram of native insulin interaction with inhibitors PAPAP and APPPA at 37 °C. Table 3. Thermodynamic parameters of PAPAP and APPPA interaction with native insulin. Inhibitors

Stoichiometry (n)

kb (Mole-1)

PAPAP

0.658±0.01

1.92x104±148

-13.83±0.15

-18.8

-5.82

APPPA

0.512±0.076

2.79x103±119

-11.86±0.28

-25.35

-3.99

∆H (kcal.mole-1)

∆S (cal.mol-1k -1)

∆Gapp (kcal.mole-1)

DISCUSSION Soluble insulin oligomers formed in lag phase are crucial for the fibrillization process. As observed in far UV CD spectra, there was an increase in the β-sheet fraction when native insulin was incubated alone in buffer solution. The enhanced exposure of hydrophobic amino-acid residues, which are shielded in the native protein, helps the nucleation and the association reaction leading to final amyloid fibrils. The observed variations in presence of the modulators are due to inhibition of fibrillization caused by the trapping of early aggregates selectively which occurred during lag phase. The effect of modulators was not only observed in fibrillization but also on the prefibrillar aggregates as obtained in ThT profile of insulin fibril incubated with the modulators. This dual effect implies that the association with the perylene derivatives arises at


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the early aggregation stages, a performance well beyond all known modulators with respect to selectivity and efficiency for amyloid oligomer. Two factors mainly contribute to the aggregation of the peptide, first the surface chemistry of modulator and the interaction with the hydrophobic core of the peptide which is responsible for the formation of pathological aggregates. Therefore, a stronger modulator−monomer interactions compared to the self-association guide the oligomers to adsorb on the modulator surface and dissociate into oligomers of smaller sizes causing an increase in the lag phase of the peptide fibrillation (Figure 1).32 In general, perylenebisimides are hydrophobic in nature and insoluble in water due to highly favorable π-stacking. The imide capping using di-peptide made it soluble in aqueous media but still persists their unique nature of π-stacking leading to self-assembled structures. But less focused mutual aggregation33 plays an important role in steering and stabilizing protein aggregates as well as provides an inhibitory platform toward amyloid fibrillation. A different aggregation pattern for a particular concentration was seen with varying dipeptide substitution, A0→0/A0→1 changed from 1.5 to 0.7 and 1.5 to 1 in aqueous media of PAPAP and APPPA respectively (Figure S7). This ground state phenomenon was supported by their structural conformations optimized by Gaussian studies (Figure S8). PAPAP is more prone to aggregation as phenylalanines are not directly attached to the perylene core and thus hang away on one side of the perylene plane allowing another perylene molecule to come close and being available for π-stacking. But in case of APPPA, phenyl alanine is attached to perylene directly via amine end and thus to avoid steric hindrance phenyl rings are placed in a trans-fashion with respect to the perylene plane unlike in the case of PAPAP (Figure S8). As a result, both isomers show a distinct photo physical behavior in the aqueous environment. PAPAP is more aggregation-prone and thus hydrophobicity increases at the interface of small molecule-peptide interaction. For a smaller peptide viz., β


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amyloid, APPPA aggregation plays a key role in modulating early soluble protein aggregates due to their structural similarity and as it is less aggregating in water, it could actually pass through the blood brain barrier easily compared to PAPAP.33 However, in case of insulin, PAPAP dominates over APPPA due to favorable hydrophobic encounter as the nucleation progresses, the hydrophobicity of the peptide aggregates increases. To probe the electrostatic encounter, we measured the zeta potential of the peptide both in presence and absence of modulators. Both protein and the small molecule exhibit a negatively charged surface. Initially, both co-aggregates show a zeta potential lower than their pristine values of both insulin and modulators. Further incubation leads to a higher increase in case of APPPA compared to PAPAP due to more probable electrostatic affection. Although the probability of available electrostatic encounter in both the modulators is same, their individual self-aggregation creates a difference in hydrophobicity making PAPAP a selective modulator for insulin, unlike the other isomer which is selective toward β amyloid.33 Overall, lag phase increases due to the presence of modulator which helps the protein to adsorb and non covalent association with its hydrophobic aromatic core and available hydrophobic peptide tails of the modulators with the exposed hydrophobic residues responsible for fibrillation. The difference in their aggregation behavior makes them selective toward different structural amyloid via modifying the protein secondary structures and most importantly shifting the pathogenic equilibrium towards the monomeric side. Such dipeptide linked perylenebisimides isomers provide unique insights into toxic oligomers and natively un-structured amyloids and act as a functional skeleton entity contributing to the destabilization of intermolecular cross β strand hydrogen bonds and other noncovalent interactions between oligomers and early amyloid aggregates responsible for mature fibril formation. A proper peptidic trigger at the imide position (tuning aggregation) creates a key


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source for designing new anti-amyloid inhibitors or modulators in addition to making them water soluble. Apart from this, functionalizing the bay position tunes the optical and electronic properties and can further be extended toward diagnostic applications. Hence, multiple noncovalent interacting molecules opens an interesting extension of this re-search based on more complex perylenebisimides. CONCLUSION Selective modulation of insulin fibrillization as well as mechanism of preventing them from forming mature fibrils was achieved and delineated using two perylenebisimide isomers (PAPAP and APPPA). Two factors are vital for developing a selective amyloid modulator: first the compatible interface and second the inherent hydrophobicity of the protein upon aggregation. These two features regulate the association reaction between protein and the modulator which effects both self-aggregation events i.e., fibrillization of insulin and π-stacking of the perylenebisimide. These findings suggest that both the modulators interact with insulin at its monomeric and oligomeric stage selectively via specific non covalent interactions thereby stabilizing the lag phase and provide fundamental insights into the chemistry in peptide-based probes, therefore shifting the pathogenic aggregation to the opposite side. The more aggregation prone isomer produces a better hydrophobic encounter with insulin amyloid and thus acts as a better modulator compared to the other isomer. These results confirmed that insulin fibril regulation via selective noncovalent binding using peptide-based hydrophobic probes provide fundamental insights into the chemistry involved to understand amyloidosis mechanism and allow the design of versatile materials containing biomolecules tagged to supramolecular cores. ASSOCIATED CONTENT


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Supporting Information. ThT Assay of insulin fibrillation in presence of the inhibitors, PAPAP and APPPA, CD and AFM of native insulin monomers and mature fibrils, Zeta Potential of the co-incubated peptides, and Gaussian studies of PAPAP and APPPA. “This material is available free of charge via the Internet at http://pubs.acs.org.” AUTHOR INFORMATION Corresponding Author Parameswar Krishnan Iyer Department of Chemistry & Centre for Nanotechnology, Indian Institute of Technology Guwahati, Guwahati-781039, India. *Email: [email protected] ORCID Sayan Roy Chowdhury 0000-0003-1607-3805 Subrata Mondal 0000-0001-6638-7947 Parameswar Krishnan Iyer 0000-0003-4126-3774 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources We

are

thankful

to

Department

of

Science

and

Technology

(DST),

India

(DST/SERB/EMR/2014/000034), Department of Information Technology, DeitY Project no. 5(9)/2012-NANO

(Vol.II),

and

DST-Max

Planck

Society,

Germany

(IGSTC/MPG/PG(PKI)/2011A/48) for financial support.


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Notes The authors declare no competing financial interest. ASSOCIATED CONTENT Supporting Information. ThT fluorescence intensity modulation experiment, PAPAP and APPPA mediated conformational modulations, Conformational restoration, Changes of secondary structures of insulin, AFM images and height profiles of insulin and coaggregates, Apparent zeta potential of Insulin in presence and absence of modulators, Optimized chemical structures of PAPAP and APPPA, synthetic scheme, NMR and MALDI of modulators

ACKNOWLEDGMENT Centre for Nanotechnology, CIF, and Department of Chemistry, IIT Guwahati are acknowledged for instrument facilities. SRC thanks Mr. Ajeet Singh for his help in data analysis and improving this manuscript. ABBREVIATIONS CD, Circular Dichroism; AFM, Atomic Force Microscopy; ITC, Isothermal Titration Calorimetry. REFERENCES (1) Selkoe, D. J. (2003) Folding proteins in fatal ways. Nature 426, 900–904. DOI: 10.1038/nature02264 (2) Tycko, R.; Wickner, R. B. (2013) Molecular Structures of Amyloid and Prion Fibrils: Consensus versus Controversy. Acc. Chem. Res. 46, 1487–1496. DOI: 10.1021/ar300282r


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Table of Contents artwork

Blocking Oligomeric Insulin Amyloid Fibrillation via Perylenebisimides Containing Dipeptide Tentacles Sayan Roy Chowdhury, Subrata Mondal, Parameswar Krishnan Iyer*

APPPA

PAPAP


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Blocking Oligomeric Insulin Amyloid Fibrillation via Perylenebisimides

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