Sulfobutylether-Î²-Cyclodextrin for Inhibition and Rupture of Amyloid

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Sulfobutylether-#-Cyclodextrin for Inhibition and Rupture of Amyloid Fibrils Meenakshi N. Shinde, Raman Khurana, Nilotpal Barooah, Achikanath C. Bhasikuttan, and Jyotirmayee Mohanty J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.7b07286 • Publication Date (Web): 22 Aug 2017 Downloaded from http://pubs.acs.org on August 24, 2017

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The Journal of Physical Chemistry

Sulfobutylether-β-Cyclodextrin for Inhibition and Rupture of Amyloid Fibrils Meenakshi N. Shinde,#,†,$ Raman Khurana, #,‡,$ Nilotpal Barooah,# Achikanath C. Bhasikuttan,*,#,‡ and Jyotirmayee Mohanty*, #, ‡ #

Radiation & Photochemistry Division, Bhabha Atomic Research Centre, Mumbai 400 085,

India †

Student under BARC-SPPU PhD Program, Department of Chemistry, Savitribai Phule Pune

University, Pune, India ‡

Homi Bhabha National Institute, Training School Complex, Anushaktinagar, Mumbai 400094,

India

ACS Paragon Plus Environment

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ABSTRACT Anomalous aggregation of proteins into amyloid fibrils leads to various amyloidosis diseases including neurodegenerative disorders. Inhibition of fibrillation process and rupture of mature amyloid fibril/plaques using small organic molecules are the promising remedial strategies to combat neurodegenerative diseases. In this study, we present sulfobutylether-β-cyclodextrin (SBE7β-CD), a water soluble macrocycle, as an efficient additive to inhibit the fibril formation and also for the breakage of mature fibrils into non-toxic small particles. The steady-state and time-resolved fluorescence, circular dichroism measurements and fluorescence microscopic images collectively confirm the inhibition and rupture of the amyloid fibrils in the presence of SBE7β-CD. In one hand, the macrocyclic encapsulation of certain amino acid residues on the protein stabilizes the native form of Insulin and Lysozyme and prevents their transformation into the β-sheet conformers, resulting in the inhibition of fibrillation. On the other hand, the degeneration of the fibril strands became feasible due to the overall positive charge of the fibril surface and the negative portals of the SBE7β-CD host. Positively, the nontoxic SBE7β-CD additive mitigates the toxicity of the system and is highly promising as therapeutics for amyloidosis.


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Introduction Amyloid fibrils, the unusual aggregation of soluble proteins and peptides, are related to more than 100 amyloidosis including Alzheimer’s disease, Parkinson’s disease, prion disease, type II diabetes, etc.1,

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Despite the fact that most of these fibril forming proteins posses distinctly

differing amino acid sequences, the amyloid fibrils formed out of these proteins exhibit some common features in morphology and they all exhibit the secondary structure which is rich in the β-sheet content.3 Amyloid fibrils are typically straight and unbranched and are formed from an assembly of proto-filaments 2–5 nm wide. X-ray diffraction analysis has indicated a characteristic structure, the β-cross motif, in which the polypeptide chains form β-strands oriented perpendicular to the long axis of the fibril, while the direction of the inter chain hydrogen-bonds that stabilize the β-strands is parallel to the same axis and β-sheets propagating in the fibril direction.4-6 However, in some systems, the fibril formation does not involve alpha to beta conversion, viz. lysozyme undergoes fibril formation without involving alpha to beta conversion at physiological pH.7 Since amyloidosis is characterized by the extracellular accumulation of amyloid fibrils in body organs or tissues,6 a detailed knowledge of the mechanisms of fibril formation as well as its possible inhibition/disintegration pathways are of current interest.8-10 Human insulin (Ins) and lysozyme (Lyz) from hen egg white (Scheme 1A) are the two model proteins of immense importance, which show amyloid fibril formation in the lower pH region. Insulin is a 51-residue protein hormone having a largely α-helical structure, but its unsolicited conversion to β-sheet rich amyloid fibrillar form raises major concern and has been widely investigated for improving its storage and preparation of long acting insulin formulations for the treatment of diabetes.11, 12 Recently it has been shown that lysozyme (a ~130 amino acid residue


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protein) is involved in amyloid-related human disorders.13 In all the reported cases the disease is associated with single point mutations in the lysozyme gene, and fibrils appear to be deposited widely in tissue.13, 14 Lysozyme is a very well characterized bacteriolytic enzyme, synthesized by macrophages throughout the body.15, 16 The largest resident population of macrophages is in the liver, but lysozyme is also present at high concentrations in articular cartilage, milk, and saliva and in trace amounts in body fluids such as serum and cerebrospinal fluid.17 Depending upon the ambient conditions, say pH, salt etc, these proteins exist in different dynamic structural/folded state. For example, insulin exists in different forms,18 mainly as monomer, dimer or hexamer and the monomeric form (prevalent at PH 2) is very vulnerable to the formation of amyloid fibrils and can be followed by in vitro measurements when incubated at 60 °C. Therefore, inhibition of fibril formation and disintegration of mature fibril/plaques into smaller nontoxic oligomers are the promising strategies for therapeutic interventions.1, 19 At physiological pH i.e. 7.4, lysozyme forms amorphous aggregates rather than the fibrillar structures, hence, in our study we followed the fibrillation process of lysozyme at pH ~2.7 For several decades, numerous in vitro studies on the inhibition of protein aggregation focused on the screening small organic molecules,19 nanoparticles20-22 or macrocyclic hosts23, 24 as potential candidates for the clinical treatment of amyloid-related diseases.10, 19 In the studies by using macrocycles, cyclodextrin derivatives such as hydroxypropyl-β-cyclodextrin was found to delay the fibril formation.25-28 Recently, resistance to fibrillation through the interaction of CB7 host has been demonstrated.23 Following the recent demonstration of fluorescence lifetime assay of Thioflavin T probe for the early detection of the fibril formation by our group,29 very recently we further established the inhibition and disintegration of amyloid fibrils in the presence of p-sulfonatocalixarenes by using steady state and time-resolved fluorescence assay of ThT


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along with the DLS and AFM measurements.24 Essentially, the presence of keto, SO3- or -OH groups in the macrocyclic additive do interact and modify peptide/protein chains and arrest the amyloid fibril formation.23,

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In this context we investigate upon the possible use of large

water soluble cavitands additionally having such interactable hydroxyl, sulfonate or carbonyl groups to attain control over the fibrillation process. One such promising macromolecule among the β-cyclodextrin derivatives is sulfobutylether-β-cyclodextrin (SBE7β-CD) (Scheme 1A) which contains an extended hydrophobic cavity along with multiple hydroxyl groups and sulfonate groups at the portals. 31

(A)

(B)

Scheme 1: (A) Ribbon diagrams of the Lysozyme (Lyz) and Insulin (Ins) monomers and the chemical structure of the sulfobutylether β-cyclodextrin (SBE7β-CD) macrocycle. (B) The schematic representation of the fibril inhibition and disintegration pathways in Lyz and Ins proteins demonstrated with SBE7β-CD.

Sulfobutylether-β-cyclodextrin (SBE7β-CD, commercially available as Captisol) is a chemically modified β-CD with sulfobutyl ether chains as shown in Scheme 1A.32, 33 Essentially, the β-CD comparable hydrophobic cavity is laced with long sulfobutyl chains on either portals distinguishing it also as a cation receptor.32 The portals of SBE7β-CD can bind small guest molecules through electrostatic interactions and in the case of Ins/Lyz proteins, various amino acid units can interact with SBE7β-CD and stabilizes the α-helices of the protein, which in turn prevents its transformation to the β-sheets. Uehata et al. have reported that the presence of SBE7β-CD increased the solubility and bioavailability of a synthetic long-acting insulin product


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i.e. insulin glargine and sustained the blood-glucose lowering effect by inhibiting the enzymatic degradation.34 The derivatives of β-CD, such as hydroxypropyl β-CD, glucosyl β-CD and β-CD dimer inhibit the fibrillation process of insulin/insulin analogues and Aβ(1-40) to some extent through the encapsulation of hydrophobic amino acids in the hydrophobic β-CD cavity.26-28 In a recent study we have demonstrated that CB7 macrocycle forms nanoassembly with proteins such as bovine serum albumin, human serum albumin and lysozyme and stabilizes their native form.35 Among various approaches to reduce the health hazards from the amyloid fibril deposits, its degeneration in to small nontoxic particles through the interaction with certain macrocyclic host molecules have been attempted. As mentioned before, our studies with psulfonatocalix[4/6]arene hosts, have established the degeneration of the large fibrillar lumps into smaller oligomeric particles. In practical sense, this procedure did not introduce any additional cytotoxicity to the system.24 Earlier few attempts to achieve fibril disintegration have been made by using 100% DMSO or curcumin-functionalized gold nanoparticles.36,22 However, the application of sulfobutylether-β-cyclodextrin macrocycle to inhibit or disintegrate the amyloid fibrils has not been reported. Here, we establish complete retardation of fibrillation in two model proteins, i.e. human insulin and lysozyme in the presence of sulfobutylether-β-cyclodextrin (SBE7β-CD) and importantly, for the first time we also demonstrate, SBE7β-CD-assisted rupture of the mature insulin/lysozyme fibrils into smaller particles with reduced toxicity, a facile supramolecular therapeutic strategy to counteract amyloidosis.

Experimental Section Human insulin and lysozyme were obtained from Sigma-Aldrich. β-CD and SBE7β-CD were obtained from TCI Mark, Tokyo and Advent ChemBio Pvt. Ltd., India, respectively. Thioflavin


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T (ThT) obtained from Sigma-Aldrich was purified by column chromatography on a silica gel column with mildly acidic methanol (1 ml of 1 N HCl in 500 ml of methanol) as eluent. The purity was further confirmed by the 1H NMR.37 Nanopure water (Millipore Gradiant A10 System; conductivity of 0.06 µS cm–1) was used throughout for solution preparation. The sample solution was prepared by dissolving human insulin (1.5mg/ml) or lysozyme (2 mg/ml) in 25 mM of HCl, 0.1 M NaCl (pH ~2) and ~10 µM ThT was added to this solution, as reported in the literature.22,24,29,38 The net solution was incubated at 60 °C. For the fibril inhibition studies, SBE7β-CD was added to the insulin solution before incubation. Initially a turbid solution was obtained upon addition of SBE7β-CD which becomes clear after 30 minutes incubation at 60 °C. Small aliquots (~200 µl) of samples were drawn at regular time intervals, diluted judiciously with blank solvent and the extent of fibril formation was monitored by recording the absorption, fluorescence spectra and fluorescence decay traces of the sample solution. Steady-state

fluorescence

spectra

were

recorded

using

a

Hitachi

F-4500

spectrofluorimeter (Tokyo, Japan) and the samples were excited at 390 nm. Circular Dichroism (CD) studies were carried out on a Biologic spectrometer (MOS-500). The spectra were measured in the 200−400 nm range with a 1.0 mm path length. Fluorescence lifetime measurements were carried out using a time-correlated-single photon-counting (TCSPC) spectrometer (IBH, UK). 374 nm diode laser (~100 ps, 1 MHz) was used for excitation and a MCP PMT for detection. From the decay traces, the time constants were evaluated following a reconvolution procedure.39 The fluorescence decays, I(t) were analyzed using the following function;

I(t) = ∑ Bi exp( − t / τ i )

(1)


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where, Bi and τi are the pre-exponential factor and fluorescence lifetime for the ith component, respectively. The overall binding constant value between SBE7β-CD and Ins/Lyz has been estimated by following the fluorescence changes of DAPI-SBE7β-CD system31 at pH 2 with gradual addition of Ins/Lyz and found to be ~4.1x1011 M−3 with insulin and ~9.8x107 M−2 with lysozyme. Fluorescence images were recorded in Olympus fluorescence microscope (Model – BX53, Japan) attached to Progres® digital camera. The atomic force microscopic (AFM) images were recorded in semi-contact mode using a NT-MDT solver model P47 instrument with 50 µm scanner head and silicon nitride tip. The samples were prepared by drop casting a dilute solution on a mica sheet, and dried. Zeta potential measurements (ζ) were carried out with a Nanosizer Z (Malvern Instruments, Malvern, UK) by using He-Ne laser (633 nm, 4 mW) as the light source and the ζ values were calculated from the electrophoretic mobility data using Smoluchowsky approximation. The experiment was carried out using a quartz cuvette (universal ‘dip’ cell) with 10 mm light pathway. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay presents a quantitative colorimetric method for studying cytotoxic agents, where the amount of MTT reduced by cells to its blue formazan derivative is quantified spectroscopically at 570 nm and is equivalent to the number of viable cells. In our experiment we have used Chinese Hamster Ovary (CHO) cell and DMEM as the cell culture medium. MTT assay has been carried out for the fibril solutions in the absence and presence of pre- and post-added SBE7β-CD which were incubated at 37 °C for 48 hours in CHO cells. The CHO cell without sample has been used as the


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control/reference. The cell viability of the solutions has been determined by considering 100% cell viability for the control CHO cell.

Results and Discussion Aqueous solutions of the Insulin or lysozyme protein samples were prepared by dissolving 1.51.5 mg/ml of the proteins in the presence of 100 mM NaCl at pH 2 maintained by HCl.22, 38 Fibril formation of insulin was achieved in vitro on its incubation at 60-65 ºC for about 3 hrs. Since Thioflavin T (ThT) has been established as a convenient and sensitive probe for monitoring fibril evolution, we employed ThT dye40 as the extrinsic fluorescence probe41-43 to map the time evolution of the fibrillation process by using both the steady-state and time-resolved fluorescence measurements. Because of the highly feasible nonradiative torsional relaxation of the excited state, in solutions of normal viscosity the free ThT dye is apparently nonemissive. However, its specific interaction/binding with the cavities in the protein fibrils render the excited state more planar and rigid and thereby exhibiting huge enhancement in its fluorescence emission and increase in fluorescence lifetime. Using this information of enhancement in the ThT fluorescence, in the first step we followed the fibrillation of Insulin in presence of ThT at various time intervals during incubation of insulin solution at the specified solution conditions. In the small aliquots of solutions sampled out at different incubation time, the emission intensity increased sharply after ~90 min of incubation (Fig.1 A), in good agreement with earlier measurements.29 Similar procedure was followed for the fibrillation of Lys solutions (2 mg/ml) set at pH ~2 (25 mM HCl) containing ~100 mM NaCl. As shown in Fig.1 B, in this case the ThT emission displayed enhancement and saturation due to fibrillation at a much longer time of ~15 hrs and is in good agreement with that reported under similar conditions.22 In both the cases the fibrillation is also visible as the appearance of turbidity in the solution.


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In our attempt to explore the ability of sulfonato group rich SBE7β-CD host to affect/inhibit the fibrillation process, the above discussed fibrillation procedures were repeated in the presence of SBE7β-CD in both the cases of Ins/Lyz. The fluorescence intensity and the lifetime data measured from the aliquots at regular time intervals during incubation has been plotted and are presented in the respective plots of Fig.1. It is seen that the presence of ~2 mM of SBE7β-CD in insulin solution the fluorescence intensity or the lifetime value of ThT probe did not indicate any signature attributable to fibrillation up to ~ 50 hrs as evident from the trace in Fig. 1A. Similarly, the presence of ~280 µM of SBE7β-CD in Lyz (as higher concentration of SBE7β-CD results turbidity) also did not display any emission enhancement in ThT (Fig. 1B) nor the solution became turbid up to ~ 50 hrs of incubation, indicating inhibition of fibril formation in Lyz also.

Figure 1. (A) Emission intensity changes of ThT in Ins (250 µM) with and without SBE7β-CD having different mole ratio, (Ins: SBE7β-CD): no host (1:0) (a), 1:0.2 (b), 1:1 (c), 1:4 (d), 1:8 (e) and in the presence of β-CD (1:8) (f). (B) Fluorescence titration curve of ThT in lysozyme (140 µM) with different mole ratio of SBE7β-CD. (Lyz: SBE7β-CD): no host (1:0) (a), 1:0.2 (b), 1:1 (c), 1:2 (d) and in the presence of β-CD (1:1) (e).

The above observations of inhibition/retardation of fibrillation in Ins and Lyz proteins were quite promising. To adjudge an effective composition to achieve the fibrillation, the


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measurements were followed at different mole ratios of Ins/Lyz and SBE7β-CD and the resulting fibrillation curves are shown in (Fig. 1A and B). From the point of saturation, we confirmed a composition of 1:1 of Ins:SBE7β-CD for an effective inhibition of fibrillation. With further lower the composition of SBE7β-CD (1:0.2), insulin fibrillation occurred at a much longer incubation time, almost delayed by 6-8 hrs (Fig. 1A(b)). Similar inhibition pattern was also seen in the case of Lyz:SBE7β-CD system and are presented in Fig. 1B. In the light of these two data sets, we state that a 1:1 composition of Ins/Lyz with SBE7β-CD is adequate to arrest the fibril formation. In both the cases the observation was carried out to about 50 hrs. In a separate control experiment we have assessed and verified that any contribution in the emission intensity from ThT-SBE7β-CD interaction is much less (Fig. S1, SI) and are not considered for reporting the fluorescence intensity changes in the fibril system. Parallel to the steady state emission intensity measurements, the Protein-SBE7β-CD-ThT systems were also probed by time resolved fluorescence decay measurements with and without SBE7β-CD, before and after the incubation. At the start of incubation, the S1 state lifetime of ThT in Ins in the absence of SBE7β-CD is found to be ~ 0.09 ns (τav), which gradually became slower to ~1.9 ns along with fibrillation (Fig. 2A, traces a and b).43 But in the solutions having SBE7β-CD, the ThT lifetime traces remained more or less unchanged at both the conditions (Fig. 2A, traces a and c; Fig. S2, SI), supporting the contention of fibrillation inhibition when SBE7β-CD macrocycle is introduced into the proteins system.


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Figure 2. (A) Fluorescence lifetime traces of ThT in insulin (250 µM) prior to incubation (a) and after ~6 hrs, 60 °C, in the absence (b) and presence of 250 µM SBE7β-CD (c). Decay trace of ThT in mature insulin fibrils post treated with 0.25 mM (d) and 1 mM (e) of SBE7β-CD. (B) Fluorescence decay traces of ThT in lysozyme (140 µM) solution after incubation (~13 hrs, 60 °C) in the absence (a) and presence (b) of 140 µM SBE7β-CD. Decay trace of ThT in mature lysozyme fibrils post treated with 10 µM of SBE7β-CD (c). λex = 374 nm and λmon =480 nm, L represents the instrument response function.

Alternatively, fluorescence microscopy (FM), atomic force microscopy (AFM) and/or dynamic light scattering (DLS) measurements are commonly used to obtain direct information on the morphology or dimension of the fibril structures. Aliquots of appropriately diluted samples of Ins and Lyz, incubated in the absence and presence of SBE7β-CD, were spotted and dried on cover slip/mica sheet and were subjected to FM/AFM measurements. The FM images are presented in Fig. 3. While abundant mature fibrils were visible as in Fig. 3 (i) and (iv) for Ins and Lyz, respectively, no such strands were visible in the Ins/Lyz system treated along with SBE7β-CD (Fig. 3 (ii) and (v). Similarly, the AFM measurements shown in Fig. S3 (A, B) clearly established the formation of fibrillar strands in both the cases. Correspondingly, increase in particle sizes were also observed from the DLS measurements during the incubation process as displayed in Fig. S3 (C). However, the formation of such larger particles were not seen when the Ins/Lyz protein systems were treated with SBE7β-CD before the onset of incubation (Fig. S3 (C)), substantiating the contended inhibition process in presence of SBE7β-CD. These findings


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are further supported by the zeta potential measurements, which displayed changes in the surface charges in commensurate with that expected for fibrillation or inhibition (Fig. S3 (D)). The formation/inhibition process has also been looked from the changes in the alpha to beta conversion of these protein strands. In the FTIR spectra obtained for the Ins/Lyz proteins and their respective fibrils, the amide bond for proteins shows peak at around 1658 cm-1 (corresponds to the α-helix structure),7 and the fibril shows the peak at ~1636 cm-1 (corresponds to the β-sheet of fibrils) (Fig. S4).7 Whereas the proteins in presence of SBE7β-CD host after the incubation (6 hrs for insulin and 26 hrs for the lysozyme at 60 oC) shows the peak at 1658 cm-1 which clearly indicates that the α-helix form of the protein is stabilized by the host molecules, proving the inhibition of beta strand formation/fibrillation. All these results are in excellent agreement with the inhibition of fibrillation contended from the steady state and time resolved fluorescence measurements discussed above. To recognize the role of the cavitand macrocycle, we have obtained the lysozyme fibrillation in the presence of p-hydroxybenzenesulphonic acid (pHBSA), a noncyclic small molecule. Similar to the case of Ins reported earlier,24 for both these proteins we did not observe any major effect on the fibrillation process (Fig. S5), underlining the importance of the macrocyclic cavity in inhibition. To further authenticate the inhibition of fibril formation by SBE7β-CD, control measurements were carried out in the presence of βcyclodextrin (β-CD) macrocycle having the same hydrophobic core cavity, the portal sulfobutylether arms of SBE7β-CD.31 Intriguingly, normal fibril formation were observed and the fibrillation plots generated (Fig. 1A (f), 1B (e)), well matched with that obtained from the respective control solutions of insulin or lysozyme alone. In other words, the inability of β-CD to inhibit the fibrillation process would point to the role of the extended


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Figure 3. FM pictures of Ins after incubation (pH 2, 60 °C, 6 hrs) without SBE7β-CD (i) and with SBE7β-CD (ii). FM images of Lyz after incubation (pH 2, 60 °C, 50 hrs) without SBE7β-CD (iv) with SBE7β-CD (v). FM images recorded after treating the mature fibrils of Ins and Lyz with SBE7β-CD (iii), (vi), respectively.

sulfobutylether arms of SBE7β-CD in effecting the fibril inhibition. Supporting this contention, it has been reported that β-CD displays a binding constant of the order of ~102 M−1,44 whereas the SBE7β-CD displays much higher association constant (~103 M−1)45 with amino acid residues. In the present study, also we have independently evaluated the binding constant values of SBE7βCD with Ins and Lyz proteins through competitive binding experiments (see Fig. S6, SI, discussed later). This hints that the encapsulation of amino acid units by the SBE7β-CD is the most probable reason for the inhibition of fibril formation. Mechanistically, the early stages of fibril formation pass through spherical oligomers or protofibrils, which eventually mature in to fibrils.38,46,47 These pre-fibrillar structures are gaining increased attention in recent years as they are being recognized as the toxic intermediate in fibril associated issues. Any interference in the initial structural folding/rearrangement of the native


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protein, thus would be crucial in influencing the fibrillation process.48 In this context, it is more likely that the interactions of the Ins/Lyz amino acid residues with the SBE7β-CD cavity and/or its extended portal arms sufficiently stable to resist the structural changes/unfolding of the protein required for fibril formation.48 It has been reported that SBE7β-CD shows reasonably good binding interaction (K ~103 M−1)45 with aromatic aminoacids such as tryptophan (Trp) and their derivatives at pH ~3.45 In a competitive binding approach and using DAPI as the emissive probe, we evaluated the interaction of SBE7β-CD with Ins/Lyz.31 The data generated from the competitive binding method (Fig. S6, SI) provided the overall binding constant for the interaction of SBE7β-CD with Ins to be ~4.1x1011 M−3 and with Lyz to be ~9.8x107 M−2. Since the effective binding interaction of the macrocyclic host may depend on the specific amino acid residues and also on the particulars of the binding site provided by the folded structure, it is quite likely that the presence of compatible amino acid guests and its approach by the large macrocyclic cavity is more practical in case of Ins revealing larger binding constants, in agreement to that reported earlier for Ins with another cationic receptor macrocycle, i.e. cucurbit[7]uril.23 Our efforts to supplement the binding constants obtained for SBE7βCD:Ins/Lyz systems from ITC measurements became futile as the titrated solutions turned turbid at the planned concentrations of protein and SBE7β-CD and need adequate optimization of the experimental parameters and hence demand detailed investigation. During fibrillation Ins/Lyz undergoes structural changes from predominantly α-helical state to a β-sheet rich conformer. As discussed above, the inhibition may arise due to the conformational changes in Ins/Lyz brought out by the complexation with the SBE7β-CD and can be probed by circular dichroism (CD) experiments.45 The CD spectroscopic changes during fibrillation recorded for Ins and Lyz are presented in Fig. S7 and Fig. 4, respectively. As


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presented in Figure 4A (Fig. S7 (A) for Ins), initially, the CD spectra of Ins/Lyz showed negative band at ~212/209 nm with a shoulder at ~222 nm, attributable to the α-helical strands.49

Figure 4. CD data showing formation (A), inhibition (B) and disintegration (C) of Lyz fibrils. (A); Lyz (140 µM, pH 2) 0 hrs (1), 13 hrs (2), 21 hrs (3) and 26 hrs (4) of incubation at 60 °C. (B); CD spectra of Lyz (140 µM, pH 2) along with SBE7β-CD (140 µM), 0 hrs (1) 26 hrs (2) at 60 °C. (C); CD spectra of Lyz mature fibrils before the addition of SBE7β-CD (1) and after treating with 1 mM (2), 4 mM (3) and 10 mM of SBE7β-CD (4).

As the incubation time advanced, the 222 nm band progressively developed in to a distinctive trough, indicating β-sheet conformation.36,

50

At the same time, the Ins/Lyz samples having

SBE7β-CD (1:1 ratio), provided a blue shifted spectrum with trough maximum ~206 nm for Lyz and 210 nm for Ins (Fig. 4B and Fig. S7(A), SI). These spectral features, which remained unchanged, document the retention of α-helical conformations and no transformation into the βsheet, supporting inhibition. The above spectroscopic evidences establish that the presence of appropriate cavitands inhibit the structural transformation of native proteins in to amyloid fibrils. At the same time, it


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becomes imperative that a therapeutic approach to cleanse the amyloid deposits, for a therapeutic action, demands the dissolution/disintegration of such chunks in a facile chemical treatment. In our earlier study using p-sulfonatocalix[4/6]arenes, we have shown that the negatively charged sulfonate groups of p-sulfonatocalixarene are mainly responsible for the disintegration of insulin fibrils.22 Experimentally, with gradual addition of SBE7β-CD the fluorescence changes in the ThT probe have been recorded for both Ins and Lyz fibrils. As displayed in Fig. 5, the fluorescence intensity of ThT decreased significantly with the addition of 10 µM and 250 µM of SBE7β-CD into Lyz and Ins fibril, respectively. Consequently, the decay time of ThT decreased considerably from 1.9 ns / 1.4 ns (without SBE7β-CD)29 to 0.82 ns / 0.8 ns (in its presence) in Ins and Lyz fibrils, respectively (see Fig. 2). This can be explained by two ways; first one is the dislocation of the dye from the fibrillar hollows to the aqueous medium or in to SBE7β-CD host and alternatively, SBE7β-CD prompts degeneration fibrils, thus placing probe dye in more flexible surrounding having no support for emission enhancement.

Figure 5. The fluorescence spectra of ThT in lysozyme (A) and insulin (B) fibril at different concentrations of SBE7β-CD. (A) [SBE7β-CD]/µM (1) 0.0, (2) 2.5 (3) 5.0 & (4) 10.0. (B) [SBE7βCD]/mM (1) 0.0, (2) 0.25 (3) 1.0 & (4) 1.8. Respective insets show the decrease in the fluorescence intensity of ThT with increasing concentration of SBE7β-CD.


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In order to understand the disintegration process of fibrils on post addition of SBE7β-CD, we have carried out the CD measurements and the details are presented in Fig. 4C for Lyz fibrils (Fig. S7(B), SI for Ins). In both the cases of Lyz/Ins fibrils, the CD bands having a spectral minimum at ~222 nm,36 turned into a feature less spectrum on addition of SBE7β-CD, documenting degeneration of the mature fibrils. The FM scan of the Ins/Lyz fibril samples posttreated with SBE7β-CD displayed small particles of degenerated fibrils (Fig. 3 (iii) and (vi), which in the absence of SBE7β-CD looked very (A)

(B)

Size distribution(s)

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Size distribution(s)

%in class

%in class

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(C)

(D) 40

10

20

5

10

50 100 Diameter (nm)

500 1000

5000

5

10

50 100 Diameter (nm)

500 1000

5000

Figure 6. AFM images after treating the mature fibrils of Ins (A) and Lys (B) with SBE7β-CD. DLS data obtained after treating the mature fibrils of Ins (C) and Lyz (D) with SBE7β-CD.

prominent as in Fig. 3(i)/(iv), substantiating partial dissolution/disintegration in to fine oligomeric particles. This is further corroborated by the AFM and DLS measurements displayed Fig. 6. While the AFM images from both the proteins post treated with SBE7β-CD were clear


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with scattered small particles (Fig. 6 (A, B) the DLS measurements from these samples revealed the presence of dispersed fine particles of ~500-600 nm size (Fig. 6 (C, D).

Figure 7. Zeta potential curves of mature lysozyme fibril with and without SBE7β-CD. [SBE7β-CD]/µM: 0 (1), 1 (2), 10 (3), 50 (4) and 250 (5).

Interpreting the cause of such beneficial interaction of the SBE7β-CD host, we understand that both the fibrillar strands do carry fairly positive charge of ~31 mV (at pH 2) on its surface as estimated by zeta potential experiments.51 However, with the addition of SBE7βCD, the potential decreased and turned negative to ~-22 mV with 100 µM SBE7β-CD for Lyz fibril and ~-29 mV with 1.75 mM SBE7β-CD for Ins fibril (Fig. 7, Table S1, SI). Thus the above results categorically put forward that the collapse of the fibrillar strands is the end result of interaction of sulfonate portals of SBE7β-CD with the positively charged fibril surface. Experimentally, the addition of small concentration (~20 µM) of SBE7β-CD to the fibril solution, the solution became more transparent with the presence of small dispersed oligomeric particles. However, the solution becomes turbid on further addition of SBE7β-CD and the particles settles down after few hrs. In other words, this indicates that the larger chunks are


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disintegrated into smaller fine particles. The above demonstrated fibril inhibition and disintegration pathways in Lyz and Ins proteins with SBE7β-CD are schematically shown in Scheme 1B. Towards its practical implication, recent reports suggest that the toxic pre-fibrillar structures are mainly blamable advancement of the neurodegenerative diseases.47 To project SBE7β-CD to counter amyloidosis, we assessed whether SBE7β-CD introduces any added toxicity. The fibril samples, inhibited or disintegrated by SBE7β-CD were administered to CHO cells and carried out the MTT assay. As shown in Fig.8, the cell viability data for fibrils (Fig. 8A(ii)) in comparison with that from the respective control medium (Fig. 8A(i)), revealed ~25% toxicity of the Ins fibrils. In the same way, Ins/Lyz proteins pre-treated with SBE7β-CD (for inhibition, Fig. 8A(iii)), the cell viability data is seen to be comparable with the reference system (i). However, studies with fibrils post-treated with SBE7β-CD exhibited the cell viability slightly higher than that of earlier two cases, eventually exhibiting mitigation of toxicity by SBE7β-CD. On the other hand, similar procedure with lysozyme fibril shows negligible toxicity (4%) and addition of SBE7β-CD before and after fibrillation does not show any toxicity to the CHO cell lines (Fig. 8B).


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Figure 8. CHO cell viability data with insulin (A) and lysozyme (B) systems, inhibited or disintegrated on adding SBE7β-CD. Control medium (i), with Ins/Lys fibrils (~12 µM) (ii), Ins/Lys solution containing SBE7β-CD during incubation (inhibited) (iii) and fibrils post-treated with SBE7β-CD (disintegrated) (iv).

Conclusions In summary, we report a supramolecular strategy to accomplish the inhibition and/or disintegration of amyloid fibrils produced from human insulin and lysozyme proteins by the treatment of SBE7β-CD macrocycle. The methodology has been established from the results of absorption, emission, circular dichroism, fluorescence microscopy, zeta potential and the cytotoxicity measurements. Host-guest interaction of the SBE7β-CD macrocycle with the aminoacid residues of the proteins deter the transformation of the native α-helics into the β-sheet conformers, effectively inhibiting the fibril formation. In the disintegration mechanism, the negatively charged sulfonato groups of SBE7β-CD plays a major role. The surface charges on the fibrils being positive, the interaction of the SO3- groups of SBE7β-CD apparently destabilizes the extended fibrillar structure into soluble or fine particles. Beneficially, the measurements with CHO cell lines asserted that the addition of SBE7β-CD did not introduce any additional toxicity in the system and is highly promising as therapeutics for amyloidosis. Further studies are being


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carried out with different protein systems/clinical samples to evaluate the effectiveness of SBE7β-CD in cellular medium.

ASSOCIATED CONTENT Supporting Information Additional figures (S1-S7) and table S1 for the control experiments, FTIR and zeta potential measurements of both proteins in the absence and presence of SBE7β-CD during incubation are given in the supporting information. The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org AUTHOR INFORMATION $

Authors MNS and RK share equal contribution

Corresponding Author *Email: [email protected] (A.C.B.) *Email: [email protected] (J.M.) Notes The authors declare no competing financial interest. ACKNOWLEDGMENT The authors sincerely acknowledge the support from Bhabha Atomic Research Centre (BARC). We thank Dr. V. Sudarsan and K. G. Girija, ChD, BARC for AFM data and Dr. K. C. Barick, ChD, BARC for DLS/zeta potential/fluorescence microscopic data and Dr. A. Kunwar, RPCD,


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BARC for MTT assay. M.N.S. thanks Dr. (Mrs) A. Kumbhar, SPPU, Pune for support and BARC-SPPU PhD program for financial assistance. R. K. acknowledges BARC for providing him a research fellowship. REFERENCES (1)

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TOC Graphic

Sulfobutylether-β-Cyclodextrin for Inhibition and Rupture of Amyloid Fibrils M. N. Shinde, R. Khurana, N. Barooah, A. C. Bhasikuttan, and J. Mohanty


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Sulfobutylether-Î²-Cyclodextrin for Inhibition and Rupture of Amyloid

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