Mechanistic Studies of the Inhibition of Insulin Fibril Formation by

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Mechanistic Studies of the Inhibition of Insulin Fibril Formation by Rosmarinic Acid Qiuchen Zheng, and Noel D Lazo J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b00689 • Publication Date (Web): 05 Feb 2018 Downloaded from http://pubs.acs.org on February 6, 2018

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The Journal of Physical Chemistry

Mechanistic Studies of the Inhibition of Insulin Fibril Formation by Rosmarinic Acid Qiuchen Zheng and Noel D. Lazo* Carlson School of Chemistry and Biochemistry, Clark University, 950 Main Street, Worcester, MA 01610

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ABSTRACT The self-assembly of insulin to form amyloid fibrils has been widely studied because it is a significant problem in the medical management of diabetes and is an important model system for the investigation of amyloid formation and its inhibition. A few inhibitors of insulin fibrillation have been identified with potencies that could be higher. Knowledge of how these work at the molecular level is not known but important for the development of more potent inhibitors. Here we show that rosmarinic acid completely inhibits amyloid formation by dimeric insulin at pH 2 and 60 ˚C. In contrast to other polyphenols, rosmarinic acid is soluble in water and does not degrade at elevated temperatures, and thus we were able to decipher the mechanism of inhibition by a combination of solution-state 1H NMR spectroscopy and molecular docking. Based on 1H chemical shift perturbations, intermolecular nuclear Overhauser effect enhancements between rosmarinic acid and specific residues of insulin, and slowed dynamics of rosmarinic acid in the presence of insulin, we show that rosmarinic acid binds to a pocket found on the surface of each insulin monomer. This results in the formation of a mixed tetramolecular aromatic network on the surface of insulin dimer resulting in increased resistance of the amyloidogenic protein to thermal unfolding. This finding opens new avenues for the design of potent inhibitors of amyloid formation and provides strong experimental evidence for the role of surface aromatic clusters in increasing the thermal stability of proteins.


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INTRODUCTION Insulin is a small hormone that is essential for glucose metabolism. Since its discovery in 1922,1 it has been the focus of many biochemical, biophysical and clinical studies. These investigations have led to the development of new forms of insulin for type 1 and type 2 diabetes mellitus2,3 and have made insulin one of the most well characterized proteins to date. Insulin is composed of two peptide chains, a chain A composed of 21 amino acid residues and a chain B composed of 30 amino acid residues, linked by two interchain disulfide bonds (Figure 1A). It stimulates the uptake of glucose by muscle and adipose cells by binding to specific transmembrane receptors as a monomer.4 While the functional form of insulin is monomeric, the protein has a strong propensity to form dimers, tetramers and hexamers depending on solution conditions.5,6 In the secretory granules of pancreatic β cells, insulin is stored as a hexamer (Figure 1B) presumably for efficient packing and protection from unwanted physical and chemical transformations.7 At highly acidic pH, it forms native-like dimers (Figure 1C). The A chain contains an N-terminal αhelix (A2-A8) (helix 1), a non-canonical turn (A9-A12) and a C-terminal α-helix (A13-A19) (helix 2). The B-chain contains an unstructured N-terminal region (B1-B7), central α-helix (B8B19) (helix 3), β-turn (B20-B23), C-terminal β-strand (B24-B27) and an unstructured C-terminal region (B28-B30). An unwanted physical transformation in solutions of insulin is the self-assembly of the protein to form insoluble fibrils. These fibrils possess characteristics of amyloid fibrils including an elongated and unbranched ultrastructure,8 an X-ray diffraction pattern consistent with the presence of cross β-sheet,9 binding of thioflavin T (ThT) resulting in enhanced ThT fluorescence,10-12 binding of Congo red resulting in birefringence,13 and nucleation-dependent kinetics.14 Insulin fibrillation is accelerated by agitation, acidification and heating.15 It is a major


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concern in the administration of insulin because it can significantly decrease insulin potency and trigger a deleterious immune response in diabetic patients.16 Insulin fibrillation at the site of injection may cause poor penetration of the injected insulin.17-19 Fibrillation in devices for insulin delivery may cause occlusion and thus insufficient rates of delivery.16,20 Pharmaceutical formulations of insulin therefore contain zinc and phenolic excipients to increase the stability of hexamers16,21-23 over monomers which are more susceptible to fibrillation.24 Increasing the stability of hexamers, however, delays the execution of insulin function. To circumvent this, analogues of insulin that keep the protein in the monomeric state have been developed,2 but these fast-acting analogues have an increased propensity to self-assemble to form fibrils.16 A potential solution to this problem is the addition of inhibitors of insulin fibrillation to formulations of fastacting insulin analogues.25,26 Apart from complicating the medical management of diabetes, the self-assembly of insulin is an important model for mechanistic studies of amyloid formation. Relative to other amyloidogenic proteins and peptides, insulin is highly soluble in aqueous solutions, an important factor that limits the experimental investigation of other amyloidogenic systems. Indeed, insulin has been used widely to decipher key generic features of fibril formation, including the intermediacy of partially unfolded amyloid precursors and the hierarchical nature of protein selfassembly to fibrils. In comparison to amyloidogenic peptides and proteins including amyloid-β in Alzheimer’s disease, islet amyloid polypeptide in type 2 diabetes and α-synuclein in Parkinson’s disease, known inhibitors of insulin fibrillation are relatively few. Peptide-based inhibitors have been identified26-28 but suboptimal pharmacokinetics of peptide-based drugs due in part to susceptibility to proteolytic degradation is a major drawback. Small-molecule inhibitors have


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been identified29-32 with varying potencies. Knowledge of how these inhibitors work at the molecular level is not known but is important in the development of more potent inhibitors. Here we show that rosmarinic acid (RA) (Figure 2), a polyphenol found in common herbal plants inhibits insulin fibrillation completely, as indicated by ThT fluorescence and supplemented by circular dichroism and solution-state NMR. In contrast to other small-molecule inhibitors, RA is highly soluble in water and does not degrade under conditions commonly used to accelerate insulin fibrillation and thus, we were able to decipher using solution-state 1H NMR the underlying mechanism for inhibition. EXPERIMENTAL SECTION Materials and Preparation of Stock Solutions. Recombinant human insulin was purchased from Sigma-Aldrich (St. Louis, MO) and used without additional purification. Rosmarinic acid, thioflavin T (ThT) and deuterium oxide were also purchased from Sigma-Aldrich. Aspirin was purchased from Bayer AG (Germany). Deuterated acetic acid was obtained from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA). Stock solutions of insulin, rosmarinic acid, and aspirin were prepared in 10 mM sodium phosphate and 10 mM NaCl at pH 2 and their concentrations were determined by UV absorbance using molar extinction coefficients of 6190 M-1cm-1 (at 275 nm),33

19000 M-1cm-1 (at 328 nm),34

and 1100 M-1cm-1 (at 276 nm),35

respectively. Samples containing insulin and rosmarinic acid or aspirin were prepared by combining aliquots from their stock solutions that result in the desired insulin to small-molecule mole to mole ratio. Circular Dichroism (CD). All far-UV CD spectra were acquired at 25°C using a JASCO J815 spectropolarimeter. Stoppered quartz cuvettes with a path length of 1 mm were used in all acquisitions. All CD spectra reported here are the averages of 8 repeats, with each spectrum


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recorded every 1 nm over a wavelength range of 260-195 nm using an averaging time of 1s and a bandwidth of 1 nm. Observed ellipticity (θobs) in units of mdeg was converted to mean residue ellipticity ([θ]) in units of deg cm2 dmol-1 by using the equation: [θ]=θobs(M/10dcn) where M is the molar mass of the protein in g mol-1, c is the concentration of the protein in g L-1, d is the path length of the cuvette in cm and n is the number of amino acid residues of the protein. Thioflavin T (ThT) Fluorescence. To determine the extent of fibril formation as a function of time, ThT fluorescence was used. Samples were prepared in a manner that the concentrations of insulin and ThT were both set at 30 µM, i.e., the insulin:ThT ratio in all samples is 1:1 (mole:mole). Fluorescence spectra were recorded using a Cary Eclipse fluorescence spectrophotometer. Following excitation at 440 nm, emission spectra were recorded from 450 nm to 600 nm. The slit width for both excitation and emission was set at 5 nm. Transmission Electron Microscopy (TEM). Aliquots taken periodically from samples used for CD were spotted on carbon-coat copper grids and stained with 1% uranyl acetate. TEM was performed at the Core Electron Microscopy Facility of University of Massachusetts Medical School using a Phillips CM10 microscope. NMR Spectroscopy. All one-dimensional (1D) and two-dimensional (2D) 1H NMR spectra were recorded at 25 or 60 °C using a Varian INOVA spectrometer operating at 600 MHz. For referencing of chemical shifts, the methyl peak of 2, 2-dimethyl-2-silapentane-5-sulfonate (DSS) was set at 0 ppm. Nuclear Overhauser effect spectroscopy (NOESY) spectra were obtained in phase sensitive mode using mixing times of 100, 200, 300 and 400 ms. The peak due to water was suppressed using WATERGATE.36


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Docking Studies. Two molecules of RA, both in the extended conformation, were docked onto the two binding pockets of insulin dimer that were identified by the intermolecular NOEs between RA and insulin, using the docking routine found in the Molecular Operating Environment (MOE, 2015.10 version) (Chemical Computing Group, Montreal, Canada). The triangle matcher algorithm was used in placement of each RA. The force field used in the docking was Amber10: EHT, which is parameterized for large molecules using Amber and for small molecules using extended Hückel theory (EHT). Each bound structure was scored according to London dG defined as the free energy of binding with units of kcal mol-1 and refined using the induced fit algorithm. RESULTS AND DISCUSSION Insulin fibril formation by circular dichroism, ThT fluorescence and transmission electron microscopy. The self-assembly of insulin to form fibrils was first observed in the mid1940’s by Waugh.37,38 Waugh introduced the classical method to produce insulin fibrils in vitro, i.e., heating insulin solutions at pH 2 at elevated temperatures.38 We and others have used a temperature of 60 °C and shown that the time required for fibrillation is inversely proportional to the concentration of insulin.10,11,14,24 Figure 3A presents far UV CD spectra of insulin (30 µM) at pH 2 before and after heating at 60 °C for 7 days. Before heating, the dichroic spectrum of the protein shows two minima at 208 and 222 nm, consistent with the predominant presence of αhelix. The ratio of the mean-residue ellipticity at 222 nm to the mean-residue ellipticity at 208 nm, i.e., [θ]222/[θ]208, has been shown to be sensitive to the assembly state of insulin.39,40 This ratio ranges from 0.739 to 0.840 for dimeric insulin and is ∼0.5 for monomeric insulin.40 In all helical dichroic spectra of insulin, [θ]222/[θ]208 is 0.75 ± 0.005, indicating the presence of dimers. After heating at 60 °C for 7 days, the spectrum of the protein changed to show a single minimum


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at 215 nm consistent with the predominant presence of β-sheet. To unambiguously detect the presence of insulin fibrils, we used TEM and ThT fluorescence spectroscopy. TEM of samples that produced the β-sheet dichroic spectrum in Figure 3A showed the presence of indeterminately long fibrils with a uniform diameter of approximately 10 nm (Figure S1A). ThT binds specifically to cross β-sheet present in amyloid fibrils.10,41,42 In the absence of amyloid fibrils, ThT does not fluoresce but when amyloid fibrils are present, it fluoresces intensely, with maximum emission at 482 nm following excitation at 440 nm.42 Addition of ThT to aliquots of samples that produced the β-sheet dichroic spectrum in Figure 3A yielded the typical emission spectrum of fibril-bound ThT showing dramatic enhancement of fluorescence and maximum emission at 482 nm (Figure 3B). Together, our CD, TEM and ThT fluorescence results show that α-helical insulin at pH 2.0 when heated at 60 °C undergoes conformational rearrangement to form cross-β-sheet-containing fibrillar assemblies that bind ThT, consistent with previous reports by us and others.10,11,15,43,44 In sharp contrast, insulin (30 µM, pH 2) incubated in the presence of RA present in a 1:1 ratio (mol RA:mol insulin), remained α-helical after heating at 60 °C for 7 days (Figure 3C), indicating that RA prevents the α-helix to β-sheet conformational rearrangement associated with fibril formation. Additionally, the ratio [θ]222/[θ]208, in all spectra recorded after heating is 0.68 ± 0.02, indicating that insulin remains a dimer. TEM of samples heated for 7 days showed the absence of fibrillar assemblies (Figure S1B). ThT in these samples did not fluoresce (Figure 3D), indicating the absence of assemblies that contain cross β-sheet. We also investigated the effect of aspirin (Figure S2A) on fibril formation by insulin. Aspirin has been used by others as negative control in studies of potential inhibitors of amyloid formation.45,46 Samples containing insulin (30 µM) and aspirin present at 1:1 ratio (mol aspirin:mol insulin) at pH 2.0, after heating at 60 °C for 7 days, produced dichroic spectra


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indicating the presence of β-sheet (Figure S2B). TEM (Figure S2C) and ThT fluorescence (Figure S2D) unambiguously confirm the presence of fibrils. In summary, taking together the results of our studies of insulin alone, insulin + RA, and insulin + aspirin, we conclude that RA inhibits fibril formation by insulin. We compared RA with other phenolic compounds that have been reported to inhibit insulin fibrillation, including epigallocatechin-3-gallate (EGCG),31 curcumin,32 quercetin,29 ferulic acid,47 and gallic acid48 (Table 1). Using fluorescence of ThT at 482 nm as an indicator of the ability of the compound to inhibit the formation of fibrillar assemblies, RA and quercetin work best. ThT fluorescence at 482 nm in the presence of RA (Figure 3D) or quercetin29 is 0, indicating complete inhibition of insulin fibril formation. Quercetin, however, is insoluble in water and requires dimethyl sulfoxide for solubilization.29 Insulin fibril formation by NMR. To determine the mechanism of inhibition by RA, we also studied the fibrillation of insulin by 1D and 2D solution-state 1H NMR. Figure 4A presents the fingerprint regions of 2D [1H↔1H] NOESY49 spectra of insulin at pH 2 before and after heating at 60 °C for 2 days. The spectrum recorded before heating contains numerous cross peaks, each due to the nuclear Overhauser effect (NOE) resulting from cross relaxation between two protons within 5 Å of each other. We noted that the spectrum is similar to published NOESY spectra of insulin under similar conditions.24 Based on published 1H chemical shifts of insulin,24,50 we identified several intermonomer cross peaks indicating the dominant presence of insulin dimers (vide infra). After heating the sample at 60 °C for 2 days, all peaks are gone (Figure 4A). This must be due to the formation of NMR-invisible assemblies whose large size and thus, long rotational correlation times, broadens peaks beyond detection.51-53 Indeed, inspection of the NMR tube with the naked eye showed a dispersion of white thread-like assemblies (Figure S3A).


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On the other hand, heating insulin + RA (1:1 ratio, mol:mol) produced NOESY spectra that were essentially identical to spectra recorded before heating (Figure 4B), indicating that RA prevents the formation of NMR-invisible assemblies. No assemblies in the NMR tube were detected with the naked eye (Figure S3B). Thermal stability of rosmarinic acid. Next, we investigated the thermal stability of RA by 1

H NMR. Our motivation for doing so lies in the observation that some polyphenolic inhibitors

of insulin fibrillation including EGCG decompose at elevated temperatures.54 Figure 5 presents 1D 1H NMR spectra of RA before and after heating at 60 °C for 7 days. Our assignments of the peaks in Figure 5 are presented in Table S1 and are consistent with published 1H chemical shifts of RA.55,56 Notably, the spectra recorded before and after heating are essentially identical (Figure 5, Table S1), indicating that RA does not degrade with prolonged heating at 60 °C. This result is important because it shows that it is full-length, monomeric RA that is responsible for the inhibition of insulin fibril formation. Intermonomer NOE enhancements in NOESY spectra of insulin at pH 2. Although strongly acidic conditions inhibit the formation of insulin hexamers, presumably because of electrostatic repulsion between protonated histidine residues and Zn2+, insulin at 25 °C and pH 2 is a dimer, as indicated by X-ray crystallography,57 solution-state NMR24,58,59 and circular dichroism as discussed above. Insulin at pH 2 crystallizes to form a dimer whose structure is similar to the dimer found in insulin hexamers (Figure 1B).57 Insulin dimers are retained in solution as shown by 1H NMR.24,58,59 Several intermonomer nuclear Overhauser effect (NOE) enhancements in insulin dimer have been identified including between TyrB16-CβH (3.05 ppm) and TyrB26- CδH (7.01 ppm), between SerB9-CαH (4.00 ppm) and TyrB16- CεH (6.83 ppm), between SerB9-CβH (3.83 ppm) and TyrB16- CεH (6.83 ppm), between GlyB8-CαH (3.93 ppm) and


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TyrB16-CεH (6.83 ppm), between SerB9-CαH (4.01 ppm) and TyrB16-CδH (7.10 ppm), between GlyB23-CαH (3.83 ppm) and TyrB26-CδH (7.01 ppm), between ValB12-CγH (0.92 ppm) and TyrB16CεH (6.83 ppm), between TyrB16-CβH (2.93 ppm) and ValB12-CαH (3.39 ppm), between ThrB27CβH (4.02 ppm) and PheB24-NH (7.76 ppm), between ProB28-CβH (1.89 ppm) and PheB24-CεH (7.12 ppm) and between ProB28-CγH (1.91 ppm) and TyrB16-CεH (6.83 ppm).24,59 The NOESY spectra of unheated samples of insulin and insulin + RA show cross peaks for these intermonomer NOE enhancements (Table S2), indicating that insulin in these samples is dimeric. We noted that eight of the eleven NOE enhancements involve TyrB16, a residue essential for dimer formation.60 For negative control, we recorded NOESY spectra of insulin in 20% acetic acid (Figure S4), a condition in which insulin exists as a monomer.57 No cross peaks for intermonomer NOEs were detected in these spectra (Table S2). After heating at 60 °C for 2 days, only insulin + RA (1:1 ratio, mol:mol) samples produced spectra that contain cross peaks for intermonomer NOEs (Table S2). We also recorded NOESY spectra of insulin + RA at 60 °C. At a mixing time of 400 ms, we detected seven of the eleven intermonomer NOEs (Table S3). Together, our results indicate that RA stabilizes insulin dimer, presumably by binding to it in a manner that prevents the dissociation of the dimer to monomers, which are susceptible to fibrillation at 60 °C.24 Binding of rosmarinic acid to insulin dimer but not to insulin monomer. NMR is widely used to detect the binding of ligands to their protein receptors. The basis for the detection of binding by NMR is simple. First, binding results in changes in the chemical environment of the nuclear spins on the ligand. The change in chemical environment generally results in chemical shift perturbations and intermolecular NOEs between the protein and ligand. We noted that the chemical shifts of the eight of the nine resolved resonances of RA are perturbed in the presence


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of insulin dimer (Table S4). Additionally, we identified several intermolecular NOEs resulting from cross relaxation between some protons in RA and some protons in nine residues of insulin including IleA2, ValA3, TyrA19, CysA20, LeuB15, GlyB23, PheB25, ThrB27 and LysB29 (Table S5). Interestingly, these nine residues together with TyrB26 form a pocket on the surface of each insulin monomer (vide infra). Second, binding results in slowed dynamics of the ligand. We compared the signs of cross and diagonal peaks of RA in NOESY spectra of RA and of insulin + RA at 25 °C (Figure 6A) and at 60 °C (Figure 6B).

A small molecule in solution tumbles rapidly and thus its rotational

correlation time is short. The molecule will yield weak, positive NOEs, i.e., the sign of the cross peaks in the NOESY spectrum of the molecule is opposite to the sign of the diagonal peaks. When the small molecule binds to a large biopolymer, it tumbles more slowly and its correlation time becomes similar to that of the biopolymer. The bound molecule will yield strong, negative NOEs, i.e., the sign of cross peaks due to cross relaxation between protons in the small molecule is the same as the sign of the diagonal peaks.10,61,62 Figures 6A and 6B show that the sign of the cross peak due to cross relaxation between proton 5 (6.38 ppm) and proton 3 (7.19 ppm) in the NOESY spectra of RA at 25 and 60 °C is opposite to the sign of the diagonal peak at 7.19 ppm. In contrast, the NOESY spectra of insulin + RA show that the sign of the cross peak due to cross relaxation between protons 5 and 3 is the same as the diagonal peak at 7.14 ppm. The other cross peaks in NOESY spectra of RA also show the same sign change in NOESY spectra of the molecule in the presence of insulin (Figure S5). Taking together the perturbations of the 1H chemical shifts of RA (Table S4), intermolecular NOEs between RA and insulin (Table S5), and the change in sign of the NOEs within RA (Figures 6 and S5), we conclude that RA binds to insulin dimer.


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Interestingly, NOESY spectra of insulin + RA show cross peaks for both short- and long-range NOEs in RA (Table S6), in contrast to NOESY spectra of RA which show the short-range NOEs only (Table S7). The long-range NOEs include NOEs between aromatic proton 2 and aromatic proton 11, between olefinic proton 4 and aromatic proton 9, between olefinic proton 4 and proton 11, between olefinic proton 5 and proton 9, and between olefinic proton 5 and proton 11. These results suggest that RA undergoes a conformational change from an extended to a bent conformation in binding to insulin dimer. Analysis of 1D and NOESY spectra of insulin + RA in 20% acetic acid, on the other hand, show no perturbations of the chemical shifts of RA (Table S4) and the absence of cross peaks that could be assigned to intermolecular NOEs between RA and insulin (Table S5) and to longrange NOEs within RA (Table S6). Together, these results indicate that RA does not bind to insulin monomer. Computational docking of rosmarinic acid. If our hypothesis that RA binds in the pocket noted above is correct, we should have observed additional intermolecular NOEs including NOEs between RA and the side chains of TyrB26. However, we noted that potential cross peaks for intermolecular NOEs between protons of RA and other protons in the pocket cannot be unambiguously assigned because of severe signal overlap. To further probe the binding of RA to insulin dimer, we utilized computational docking, a powerful approach that is widely used in rational drug design. We used the docking routine in the Molecular Operating Environment (MOE) which has been used by others to elucidate structures of protein-ligand complexes.63,64 We docked RA in the extended conformation to the binding pocket localized by intermolecular NOEs between RA and insulin dimer (Figure 7A). In silico docking generated bound RA in a bent conformation that is consistent with the long-range NOEs detected in NOESY spectra of the


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molecule in the presence of insulin (Table S6). The bound structure of RA in the bent conformation can be classified into four families (Families I-IV, Figure S6). Family I gave the lowest average London dG (free energy of binding of the ligand).

Figure 7B shows the

conformation from Family I that has the lowest London dG. One of the dihydroxyphenyl rings of RA mediates aromatic interactions between TyrA19 and PheB25 to form a TyrA19-RA-PheB25 aromatic cluster. Because there are 2 molecules of RA bound per insulin dimer, two TyrA19-RAPheB25 aromatic clusters form on the surface of the dimer. We noted that the arrangement of pairwise aromatic-aromatic interactions in each cluster, i.e., TyrA19-RA and RA-PheB25, is Tshaped edge-to-face structure (Figure 7C) which is the predominant arrangement of aromatic residues in proteins involved in stabilizing π-π interactions.65,66 Resulting mechanism of inhibition. The mechanism of the fibrillation of insulin at acidic pH and elevated temperatures has been studied well.14,24,43,57 There is now general agreement that the key initial event is the dissociation of the dimer to form two partially folded monomers which then self-assemble to form an unknown nucleus en route to fibrillation (Figure 8A). Insights into the structure of the monomer prior to detectable self-assembly have been obtained by 1H NMR.24 Several nonpolar side chains that are buried in the native monomer become exposed to the solvent, providing potential sites for non-native self-assembly. The N-terminal segments of the A and B chains including helix 1 and helix 2 in the A chain are mostly disordered resulting in the complete exposure of the hydrophobic core of the native monomer consisting of TyrA19, LeuB15, and PheB24 to the solvent. The C-terminus of the B chain which participates in β-sheet formation in the native dimer is also exposed to the solvent completely, making the aromatic side chains of PheB24, PheB25 and TyrB26 available for aberrant protein-protein interactions.


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Figure 8B presents a schematic diagram of the mechanism for the inhibition of insulin fibrillation by RA. The binding of RA results in a conformational change in the small molecule from a planar to a bent conformation. A similar conformational change in RA occurs upon binding of the small molecule on the surface of Aβ42 oligomers.56 EGCG also undergoes a conformational change upon binding to Aβ40 oligomers.67 The bent conformation of RA allows for the intercalation of the molecule into the binding pocket localized by intermolecular NOEs between RA and insulin (Figure 7A). Importantly, two molecules of RA on the two binding pockets result in the formation of two TyrA19-RA-PheB25 aromatic clusters. These two clusters form an enlarged aromatic network (i.e., TyrA19-RA-PheB25-PheB25-RA-TyrA19) on the surface of insulin dimer (Figure 8B). Aromatic clusters found on the surface of thermophilic proteins have been hypothesized to provide the stabilization needed for thermal stability.68-70 Studies applying protein engineering methods have shown that the introduction of aromatic clusters on the surface of proteins increases their thermal stability.65,71 Taking together our results and these published work on the stabilization of thermophilic proteins by aromatic networks, we conclude that RA inhibits fibril formation of insulin at acidic pH and elevated temperatures by preventing the dissociation of the dimer to aggregation-prone monomers. The thermal denaturation of proteins begins with the unfolding of secondary structures found on the surface resulting in the exposure of the hydrophobic core.72 A plausible explanation therefore for the increased thermal stability of insulin dimer is stabilization of helix 1 and helix 2 by RA. These two helices limit the exposure of the hydrophobic core of insulin24 including LeuB15 and PheB24 to the solvent. Many experimental studies have shown that helix capping interactions stabilize α-helices.73 Our docking studies show that the N-cap residue of helix 1 (IleA2) forms a π-hydrogen bond66 with the 3,4-dihydroxyphenyllactic acid moiety of RA.


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Additionally, the C-cap residue of helix 2 (TyrA19) forms an aromatic edge-to-face interaction66 with the caffeic acid moiety of RA (Figure 7B). We postulate that these interactions prevent the heat-induced unwinding of helix 1 and helix 2 and thus the characteristic α-helical dichroic spectrum of insulin persists at elevated temperatures (Figure 3C). In conclusion, RA is a potent inhibitor of the fibrillation of insulin with a mode of inhibition that has not been reported until now. RA inhibits the fibrillation of insulin by preventing the dissociation of insulin dimer to aggregation-prone partially folded monomers. In binding on the surface of insulin dimer, an extended aromatic network is formed which increases the resistance of the dimer to thermal unfolding. Finally, polyphenols have been shown to modulate the selfassembly of Aβ,74 islet amyloid polypeptide75 and α-synuclein76 with potencies that could be better. Our work suggests that a folded polyphenol that has the ability to form mixed aromatic clusters following its binding to aromatic-rich regions of the amyloid precursor may inform the design of polyphenol analogues with enhanced inhibitory potencies.


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AUTHOR INFORMATION Corresponding Author *E-mail, [email protected] ORCID Noel D. Lazo: 0000-000301769-7572 Notes The authors declare no competing financial interest. ACKNOWLEDGMENT Parts of this work were funded by the Lise Anne and Leo E. Beavers II endowment to Clark University. The authors thank Dr. Guoxing Lin for maintaining the NMR spectrometer used in this work. SUPPORTING INFORMATION AVAILABLE Additional data including Figures S1-S6 and tables of NMR data (Tables S1-S7) including tables of chemical shifts, chemical shift perturbations, and proton pairs involved in intermonomer NOE enhancements in insulin dimer, intermolecular NOE enhancements between insulin and RA, and intramolecular NOE enhancements in RA. This information is available free of charge via the Internet at http://pubs.acs.org.


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Table 1. Phenolic compounds that inhibit the fibrillation of insulin at pH 2 and elevated temperatures. Solubility in water (25° C)

Inhibitor to insulin ratio (mol:mol)

Inhibition of fibril formation by ThT fluorescence

Reference

Rosmarinic acid

Y

1:1

FI* at 482 nm ∼0 Complete inhibition

This work

Epigallocatechin3-gallate (EGCG)

Y

1:1

Not performed

31

Quercetin

N

1:1

FI at 482 nm ∼0 Complete inhibition

29

Ferulic acid

Y

5:1

FI at 482 nm > 0 Incomplete inhibition

47

Curcumin

N

1:22


32

Gallic acid

Y

5:1


48

Inhibitor

Structure

*FI, fluorescence intensity


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Figure 1. Structure of human insulin. (A) Chains A and B of insulin are composed of 21 and 30 amino acid residues, respectively, and are linked by two interchain disulfide bonds. (B) In its hexameric form (1MSO), the insulin molecule is predominantly α-helical and forms three identical dimers symmetrically arranged around Zn2+ which coordinate to the HisB10 side chains of the three dimers. (C) At pH 2, insulin forms native-like dimers (1GUJ).


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Figure 2. Structure of rosmarinic acid. RA is an ester of caffeic acid and 3,4dihydroxyphenyllactic acid. Nonexchangeable protons are numbered (RA-1 to RA-11) to facilitate the assignment and discussion of 1H NMR spectra herein.


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Figure 3. Insulin at pH 2 in the absence and presence of rosmarinic acid was studied by circular dichroism and ThT fluorescence spectroscopy before and after heating at 60 °C. (A) Far-UV CD spectra of insulin in the absence of RA prior to heating (solid line) and after heating for 7 days (broken line). After heating, insulin underwent conformational rearrangement from an α-helix to β-sheet. (B) Fluorescence spectra of ThT in the samples that yielded the CD spectra in (A). Dramatic enhancement of ThT fluorescence at 482 nm was observed in the CD sample that showed the presence of β-sheet. (C) Far-UV CD spectra of insulin in the presence of RA before (solid line) and after heating for 7 days (broken line). After heating, insulin remained α-helical. (D) Fluorescence spectra of ThT in the samples that yielded the CD spectra in (C), show that the fluorescence of ThT at 482 nm is essentially 0, consistent with the absence of insulin fibrils. The RA to insulin ratio in RA-containing samples is 1:1 (mol:mol).


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Figure 4. Human insulin at pH 2 was investigated by 2D 1H solution-state NMR before and after heating at 60 °C. NOESY spectra of insulin in the (A) absence and (B) presence of RA before and after heating at 60 °C for 2 days. In the absence of RA, no cross peaks were observed after heating. In the presence of RA, however, the cross peaks due to insulin remained after heating. NOESY spectra recorded before and after The RA to insulin ratio in the samples that produced the spectra in (B) is 1:1 (mol:mol). The concentration of insulin in both samples is approximately 1 mM. All spectra shown here were acquired using a mixing time of 400 ms.


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Figure 5. Rosmarinic acid at pH 2 was investigated by 1D 1H solution-state NMR before and after heating at 60 °C. Spectra recorded before (A) and after heating for 7 days (B) are essentially identical. The peaks are assigned according to the labels shown in Figure 2.


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Figure 6. The binding of RA on insulin dimer was detected by NOESY. Spectra acquired at (A) 25 °C and at (B) 60 °C show that the sign of cross peaks resulting from cross relaxation of protons in RA within 5 Å of each other, including protons 5 and 3, is opposite to the sign of diagonal peaks in NOESY spectra of RA only but the same as the sign of diagonal peaks in NOESY spectra of RA in the presence of insulin dimer.


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Figure 7. Docking of rosmarinic acid to insulin dimer. (A) RA in the extended conformation was docked to the two binding pockets in insulin dimer (1GUJ) localized by intermolecular NOEs. The docking generated bound structures of RA in the bent conformation resulting in the interaction of the two aromatic moieties of RA with residues in the binding pocket. (B) The caffeic acid moiety mediates aromatic interactions with TyrA19 and PheB25 through the formation of a TyrA19-RA-PheB25 aromatic cluster. The 3,4-dihyroxyphenyllactic acid moiety interacts with the N-H of IleA2. (C) The geometry of the PheB25-RA and TyrA19-RA interactions is T-shaped, i.e., aromatic edge-to-face interactions. The N-H/π contact between IleA2 and the 3,4dihydroxyphenyllactic acid moiety is also T-shaped.


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Figure 8. Schematic representations of the mechanism of insulin fibrillation and its inhibition by RA. (A) Dissociation of insulin dimer to partly folded insulin monomers leads to the formation of a nucleus en route to insulin fibrils. (B) The binding of two molecules of RA on two binding pockets leads to the formation of a mixed tetramolecular aromatic cluster, an extended aromatic network (TyrA19-RA-PheB25-PheB25-RA-TyrA19), which increases the resistance of insulin dimer to thermal unfolding. The dissociation of the dimer to aggregation-prone monomers is thus prevented.


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TOC GRAPHIC


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Mechanistic Studies of the Inhibition of Insulin Fibril Formation by

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