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May 31, 2016 - Road, Jodhpur, Rajasthan, India 342011. •S Supporting Information. ABSTRACT: We have synthesized capsaicin-coated silver nanoparticle...
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Capsaicin-coated silver nanoparticles inhibit amyloid fibril formation of serum albumin Bibin G. Anand, Kriti Dubey, Dolat Singh Shekhawat, and Karunakar Kar Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00418 • Publication Date (Web): 31 May 2016 Downloaded from http://pubs.acs.org on June 5, 2016

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Capsaicin-coated silver nanoparticles inhibit amyloid fibril formation of serum albumin Bibin G. Anand, Kriti Dubey, Dolat Singh Shekhawat, Karunakar Kar* Center for Biologically Inspired Systems Science, Department of Biology, Indian Institute of Technology Jodhpur, Old Residency Road, Jodhpur, Rajasthan, India-342011. KEYWORDS. Capsaicin, Amyloid aggregation, Serum albumin, Silver nanoparticles, Thioflavin T Supporting Information ABSTRACT: Here, we have synthesized capsaicin-coated Cap

silver nanoparticles (AgNPs ) and have tested their antiamyloid activity, considering serum albumin (BSA) as a model protein. We found that amyloid formation of BSA was Cap strongly suppressed in the presence of AgNPs nanoparticles. However, isolated capsaicin and uncapped control nanoparticles did not show such inhibition effect. Bioinformatics analysis reveals CH- and H-bonding interactions between capsaicin and BSA in the formation of protein-ligand complex. These results suggest the significance of surface functionalization of nanoparticles with capsaicin which probably enables capsaicin to effectively interact with the key residues of the amyloidogenic core of BSA.

Over the past decade, much research has been developed to surface functionalization of nanoparticles with selected compounds to target several biological processes including amyloid formation of proteins. The process of amyloid formation of proteins is considered as one of the foundational 1,2 events for the onset of a number of health problems . So far ~35 different amyloidogenic proteins are known to be associ2,3 ated with the onset of several neurodegenerative diseases . One of the most effective strategies to target amyloid-linked diseases is to find potential inhibitors against the initiation of the process of amyloid formation. In the past decades, many studies have focused on targeting inhibition of amyloid formation through various compounds such as natural products 4-6 7-9 and selected peptides . Recent investigations have also looked at the effect of various nanoparticles on amyloid ag10-13 gregation of proteins . Some studies have suggested that nanoparticles coated with hydrophobic molecules are capable 11,14,15 of inhibiting the fibrillation process of proteins . Our group has recently synthesized tyrosine and tryptophan coated nanoparticles which not only suppress the amyloid formation of insulin but also trigger the disassembly of the ma12 ture amyloid fibrils. Though capsaicin has gained much attention for its versatile medicinal properties, the effect of capsaicin on amyloid formation of proteins is largely unknown. Capsaicin is a wellknown natural product that possesses multiple pharmacolog16 17,18 ical properties including its anti-inflammatory , analgesic 19,20 and antitumorigenic activities. Recent reports have also

revealed the ability of capsaicin to bind to different proteins 21-23 and to influence their functional properties . Binding affinity of any molecule for the native as well as aggregation-prone intermediate structures of proteins is believed to be a crucial factor for inhibition of protein aggregation. The capsaicin molecule appears to be an effective molecule to target aggregation-prone residues of an amyloidogenic protein due to its unique molecular structure comprising vanillyl, amide and 22 aliphatic chain components . To our knowledge, reports on the effect of capsaicin and capsaicin coated nanoparticles on amyloid aggregation of proteins are scarce in literature. Hence, in this study, considering BSA as a model protein we have tested the effect of capsaicin as well as capsaicin coated Cap silver nanoparticles (AgNPs ) on amyloid formation of BSA. We have also synthesized control silver nanoparticles without capsaicin to test the significance of surface functionalization. Finally, we have carried out molecular docking studies to understand possible molecular interactions between capsaicin and BSA. The results provide a new approach to target aggregation of proteins by stable nanoparticles coated with aromatic natural compounds such as capsaicin. Cap

Capsaicin coated silver nanoparticles AgNPs were syn24 thesized by following the established protocol (see materials and methods section in the supplementary materials). We first confirmed the formation of nanoparticles by recording UV-visible absorption profiles of the sample. UV visible specCap tra of the sample containing AgNPs nanoparticles showed 25 typical SPR band at ~421 nm (Figure 1B, red line) . We obtained hydrodynamic radius of the nanoparticles using DLS and the results, as shown in Figure 1D, reveal a diameter value that varies within ~10-40 nm. AFM data and transmission Cap electron micrographs of AgNPs samples are shown in Figure 1C and Figure 1E respectively. The obtained nanoparticles appeared spherical in shape displaying an average diameter value of ~50 nm. To understand the orientation of the surface functionalized capsaicin we performed FTIR measurements. cap Figure 1G shows FTIR signal of AgNPs nanoparticles where -1 we detected many characteristic bands (within 2700cm to -1 3000 cm ) that were observed to be similar to the reported 24 results on capsaicin coated silver nanoparticles . These data suggest that capsaicin molecules are functionalized to the surface of the nanoparticles through their C=O group as shown in the schematic diagram in Figure 1A. Further, the

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Figure 1. Characteristic properties of capsaicin coated silver nanoparticles. (A) Schematic representation of capsaCap icin coated silver nanoparticles (AgNPs ). (B) UV-visible spectra of the capsaicin coated silver nanoparticle samples, Cap AgNPs (▬) and control silver nanoparticles, AgNPs (▬); Cap (C) AFM topology image of AgNPs nanoparticles. (D) ParCap ticles size distribution histograms of AgNPs nanoparticles showing an average hydrodynamic radius of ~10-30nm. (E) Cap TEM image of AgNPs nanoparticles (F) TEM image of control AgNPs (G) FTIR data showing characteristic peaks for Cap AgNPs nanoparticles (▬) and capsaicin alone (▬). be -42 mV (Figure S3). These capsaicin capped nanoparticles were also observed to be very stable and the sample did not aggregate for a prolonged storage time at room temperature. These nanoparticles were also observed to be stable at 70°C at which amyloid reactions were performed (Supplementary Figure S10). In this study, we have also synthesized control silver nanoparticles (without capsaicin, as mentioned in the methods section), and the UV data of the control AgNPs (Figure 1B, blue curve) display a prominent peak at ~401 nm 25 . We also carried out size characterization of these nanoparticles using DLS (supplementary Figure S4) and visualized them using TEM (Figure 1F). Amyloid aggregation of BSA in PBS was studied at ~70°C 26 (close to the Tm of BSA) by monitoring the rise in the Thioflavin T signal at different time points. Monomers of BSA at ~5 M showed a characteristic aggregation curve (Figure 2,) comprising of a distinct growth phase and a saturation phase. The nature of mature amyloid fibrils of BSA, as evident from AFM (Figure 2B) and TEM (Figure 2C) data, showed typical 26 amyloid morphology as seen in the previous studies . The inhibition effect of capsaicin coated nanoparticles was examined at three different molar ratio values (of BSA:inhibitor) as mentioned in Figure 2A. We observed a gradual decrease in the ThT signals of the aggregating sample with increasing Cap concentrations of the AgNPs nanoparticles. This suggests a dose dependent inhibition of amyloid formation of BSA in the Cap presence of AgNPs nanoparticles (Figure 2A). To confirm this inhibition effect, as a next step, we performed two con-

trol experiments. First, we tested the effect of isolated capsaicin molecules (at similar concentrations) on amyloid aggregation of BSA and the results are shown in Figure 1A (), (▲) and (). Second, we studied the effect of control AgNPs (uncoated, without capsaicin) on the aggregation process of BSA (Figure 2A ▼). Obtained results from both these control exCap periments prove that only capsaicin coated AgNPs nanoparticles are capable of showing anti-amyloid property (Figure 2A: ,,►). However at higher concentrations isolated capsaicin molecules showed slight inhibition effect on amyloid aggregation of BSA (Figure 2, ). This result confirms that capsaicin molecules are highly effective in inhibiting the formation of amyloid fibrils only when they are attached to the surface of the nanoparticles. Similar observation on the importance of surface functionalization of nanoparticles to achieve inhibition of protein aggregation has been recently 12 Cap reported from our group . Notably, these AgNPs nanoparticles can be considered as effective inhibitors of BSA amyloid formation because strong suppression of the aggregation process was observed even at 1:1 molar ratio value of protein:inhibitor.

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Figure 2. Inhibition of amyloid formation of bovine serum albumin in the presence of capsaicin coated nanoCap particles. (A) Effect of capsaicin, AgNPs and AgNPs on aggregation of ~5 M BSA sample at different molar ratios: () 5M BSA only; () 5M BSA + 1M capsaicin; (▲) 5M BSA + 5M capsaicin; () 5M BSA + 10M capsaicin; (▼) 5M BSA + control AgNPs at equivalent concentration; () Cap Cap 5M BSA + 1M AgNPs ; () 5M BSA + 5M AgNPs ; Cap (►) 5M BSA + 10M AgNPs . (B) AFM image of mature BSA aggregates. Scale bar, 250nm (C) TEM image of mature BSA amyloid fibrils. Scale bar, 100 nm. (D) Native gelelectrophoresis of the BSA (~5 M) sample under aggregating conditions at 6 h time point. (1) Control soluble BSA; (2) BSA aggregates without inhibitors (~5 M); (3) BSA + capsaicin Cap (~5 M); (4) BSA + AgNPs nanoparticles (~1 M) (5) BSA + Cap Cap AgNPs nanoparticles (~5 M) (6) BSA + AgNPs nanoparticles (~10 M). (E) ATR-FTIR second derivative spectra of BSA amyloid fibers from an inhibited reaction in presence of Cap AgNPs nanoparticles.

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Figure 3. Molecular docking studies of a capsaicin molecule with BSA. The BSA-capsaicin complex shows fourteen possible interactions comprising of one hydrogen bond, two carbon bonds, one pi-sigma pi-donor, two alkyl and seven pi-alkyl bonds. Right panel of the figure lists all the interactions between capsaicin and BSA. Next, we examined the secondary structure contents of the mature amyloid fibrils of BSA taken from an inhibited reacCap tion in the presence of AgNPs . The second derivative FTIR data, as shown in Figure 2E, reveal several characteristic -1 -1 -1 -1 -1 peaks (1616 cm , 1635 cm , 1646 cm , 1667 cm and 1684 cm ), particularly suggesting the prevalence of intermolecular 27 sheets as reported for BSA . This result suggests that AgNPCap s nanoparticles may be delaying the formation amyloid fibril without altering the aggregation pathway. To further understand the observed inhibition effect of capsaicin, we carried out molecular docking studies on BSAcapsaicin interactions. Blind docking studies (see methods section) predicted a viable capsaicin-BSA interaction at a site within BSA’s A-chain which spans from Tyr 400 – Leu 582. The value of CDocker energy for the best pose (as shown in -1 Figure 3) was -31.2 kcal.mol and its corresponding interac-1 tion energy was observed to be -35.3 kcal.mol . Such energy values predict the formation of a stable protein-ligand complex. Capsaicin-BSA complex structure displays fourteen interactions as listed in the Figure 3. Notably, the docking studies have predicted five strong interactions (TYR400 : CapsaicinO2, ASN401 : CapsaicinH49, LYS524 : CapsaicinH34, PHE506:CapsaicinH27 and PHE550:CapsaicinH36) which mostly involve hydrophobic and electrostatic interactions. To identify the critical residues in the BSA sequence (PDB ID: 4F5S) which are predicted to promote amyloid aggregation, we used online tools (PASTA 2.0 and AGRESCAN3D) and the data are shown in the supplementary Figure S6-S9. The aggregation propensity and the degree of disorderness data (supplementary S6A and S6B) show that the region spanning from Tyr400 to Leu582 is the aggregation prone tract. This prediction is further supported by the information obtained from AGGRESCAN3D (Figure S7-S9). Interestingly, most of the amino acids that interact with capsaicin (from Figure 3) are from the predicted aggregation prone regions of BSA (supplementary Figure S8 and S9).

This work clearly reveals the potential of the capsaicin Cap coated silver nanoparticles (AgNPs ) to inhibit amyloid fibril formation of BSA. Our work confirms that surface functionalization of capsaicin molecule is vital for its inhibitory effect against amyloid formation because such inhibition effect was not observed in the presence of isolated capsaicin molecules as well as control uncapped nanoparticles. It has been suggested that structural constraints and specific aromatic interactions are two key factors to facilitate the interaction between the inhibitor and the amyloidogenic core of the 28 protein . Fundamentally, the degrees of freedom of a molecule would decrease if such compound is functionalized over nanoparticles. Hence, it is much likely that as a result of surCap face functionalization, the AgNPs nanoparticles would preferably allow the functional groups of the capsaicin molecules to participate in crucial interactions with the corresponding reactive groups of the protein molecule at the site of binding. Our FTIR data (Figure 1G) suggest the functionalization of capsaicin molecule on the nanoparticles though C=O group and such an orientation of the attached capsaicin would presumably allow its vanillyl group, amide group and the aliphatic tail region to enhance capsaicin’s binding affini22 ty for proteins . The stabilization of the native conformation of proteins due to ligand binding is believed to be an important factor for prevention of amyloid aggregation of pro29 teins . Our molecular docking studies and bioinformatics analysis predict capsaicin’s ability to participate in H-bond, CH- and electrostatic interactions with the key aggregationprone residues of BSA (Figure 3). Temperature induced amyloid formation process of BSA is believed to be mediated by intermolecular association between partially folded interme30 diate species with exposed hydrophobic residues . If the ligand molecule is capable of interfering with the intermolecular association of aggregation prone species, it may eventually lead to prevention of the process of protein aggregation. Additionally, stabilization of the native conformation would also reduce the population of aggregation prone intermediate

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species during the process of temperature induced amyloid fibril formation. Our results also indicate that the native confirmation of BSA is retained to some extent while aggregating Cap in the presence of AgNPs nanoparticles (Fig. 2D). Though Cap this data do not prove any BSA-AgNPs interaction, retention of native structures can be attributed to the suppression of protein aggregation. According to a recent study, the negatively charged hydrophobic nanoparticles can prevent Aamyloid formation by interfering with their hydrophobic 31 Cap and electrostatic interactions . Here, the AgNPs nanoparticles were found to possess a negative potential of -42mV (supplementary Figure S3) and with such a value of negative potential the nanoparticles are predicted to influence the electrostatic interaction between protein and the ligand, suppressing the inherent aggregation-propensity of the protein species. Hence, we believe that through surface functionalization of nanoparticles with capsaicin, the reactivity of capsaicin’s vanillyl and the aliphatic chain components towards the aggregation-prone hydrophobic residues of BSA is enhanced. In summary, we have revealed capsaicin’s anti-amyloid activity when it is surface functionalized with silver nanoparticles. Since strong inhibition of amyloid fibril formation was observed at 1:1 molar ratio of capsaicin:BSA, these amyloid inhibitors are highly relevant for future designing of therapeutics against amyloid-linked diseases.

ASSOCIATED CONTENT Supporting Information Figures S1, S2, S3, S4, S5, S6, S7, S8, S9, S10 and Methods. This material is available free of charge via internet at: http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Email: [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding This work was supported by BRNS grant (Grant no 37(1)/14/38/2014-BRNS) and Seed grant from IIT Jodhpur (KK).

ACKNOWLEDGMENT We thank IIT Jodhpur for research facilities. We thank IIT Bombay for use of the Cryo HR-TEM Central Facility.

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