Inhibition of Human Amylin Aggregation and Cellular Toxicity by Lipoic

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Inhibition of Human Amylin Aggregation and Cellular Toxicity by Lipoic Acid and Ascorbic Acid Sarah Kassem Azzam, Hyunwoo Jang, Myung Chul Choi, Habiba Alsafar, Suryani Lukman, and Sungmun Lee Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b01009 • Publication Date (Web): 30 Apr 2018 Downloaded from http://pubs.acs.org on May 2, 2018

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Molecular Pharmaceutics

Inhibition of Human Amylin Aggregation and Cellular Toxicity by Lipoic Acid and Ascorbic Acid

Sarah Kassem Azzam1, Hyunwoo Jang2, Myung Chul Choi2, Habiba Alsafar1,3, Suryani Lukman4, Sungmun Lee1* 1

Department of Biomedical Engineering, Khalifa University of Science and Technology, P.O. Box 127788, Abu Dhabi, United Arab Emirates

2

Department of Bio and Brain Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, South Korea

3

Khalifa University’s Center for Biotechnology, PO Box 127788, Abu Dhabi, United Arab Emirates

4

Department of Chemistry, College of Arts and Science, Khalifa University of Science and Technology, P.O. Box 127788, Abu Dhabi, United Arab Emirates

* Corresponding Author Sungmun Lee, Ph.D. Department of Biomedical Engineering Khalifa University of Science, Technology, and Research, Abu Dhabi Campus, PO Box 127788 Abu Dhabi, United Arab Emirates Tel: +971 2 401 8104 Fax: +971 2 447 2442 E-mail: [email protected]

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Abstract More than 30 human degenerative diseases result from protein aggregation such as Alzheimer’s disease (AD) and type 2 diabetes mellitus (T2DM). Islet amyloid deposits, a hallmark in T2DM, are found in pancreatic islets of more than 90 % of T2DM patients. An association between amylin aggregation and reduction in β-cell mass was also established by post-mortem studies. A strategy in preventing protein aggregationrelated disorders is to inhibit the protein aggregation and associated toxicity. In this study we demonstrated that two inhibitors, lipoic acid and ascorbic acid, significantly inhibited amylin aggregation. Compared to amylin (15 µM) as 100 %, lipoic acid and ascorbic acid reduced amylin fibril formation to 42.1 ± 17.2 % and 42.9 ± 12.8 % respectively, which is confirmed by fluorescence and TEM images. In cell viability tests, both inhibitors protected RIN-m5f β-cells from the toxicity of amylin aggregates. At 10:1 molar ratio of lipoic acid to amylin, lipoic acid with amylin increased the cell viability to 70.3 %, whereas only 42.8 % RIN-m5f β-cells survived in amylin aggregates.

For

ascorbic acid, an equimolar ratio achieved the highest cell viability of 63.3 % as compared to 42.8 % with amylin aggregates only. Docking results showed that lipoic acid and ascorbic acid physically interact with amylin amyloidogenic region (residues Ser20-Ser29) via hydrophobic interactions; hence reducing aggregation levels. Therefore, lipoic acid and ascorbic acid prevented amylin aggregation via hydrophobic interactions, which resulted in the prevention of cell toxicity in vitro. Keywords Amyloid inhibition, amylin, type 2 diabetes, ascorbic acid, lipoic acid


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Introduction Protein aggregation diseases, termed amyloidoses, are associated with protein misfolding and amyloid aggregation

1,2

. More than 30 human degenerative diseases

result from protein aggregation such as Alzheimer’s disease (AD), Parkinson’s disease (PD) and type 2 diabetes mellitus (T2DM)

3,4

. Islet amyloid deposits, a hallmark in

T2DM, are found in pancreatic islets of more than 90% of T2DM patients, which is confirmed by histopathological tests at autopsy

5,6

.

Post-mortem studies also

established an association between amylin aggregation and reduction in β-cell mass. Moreover, an association between islet amyloid depositions and reduced β-cell area and increased β-cell apoptosis was reported 7. Amylin, also known as Islet amyloid polypeptide (IAPP), is a 37-amino acid protein hormone that constitutes the major component of islet amyloid deposits in pancreatic islets of T2DM patients 8. Amylin is co-localized with insulin in secretory granules of pancreatic β-cells and amylin is co-secreted with insulin in response to β-cell stimulation 5

.

Physiological roles of amylin include regulating glucose metabolism, controlling

satiety, slowing-down gastric emptying, suppressing glucagon secretion from pancreatic α-cells, and inhibiting insulin secretion

3–5,9–11

. Secretion of mature amylin along with

insulin from β-cell occurs at approximately 1:100 molar ratio (amylin: insulin). In T2DM, the elevation of insulin levels results in the increase of amylin plasma levels 5.

In

addition to protein concentration, various factors can contribute to amyloidogenic amylin aggregation such as cellular environment, and genetic mutations. In terms of cellular environment, changes in pH affect amylin conformation and aggregation propensity, where a basic pH value of 8.8 is reported as a more favorable aggregation condition 3 ACS Paragon Plus Environment


compared to an acidic pH value of 4.0

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11

. In fact, this is in agreement with an elevation

in environmental pH value when human amylin is secreted from pancreatic secretory granules (pH 5.5) into the extracellular space (pH 7.4); the location of islet amyloid depositions 4. Amylin in humans, cats and non-human primates can form amyloid fibrils, whereas rodent amylin is found to be non-amyloidogenic

12

. The majority of differences in amylin

amino-acid sequence among species is found in the region of residues Ser20-Ser29, a critical amyloidogenic region in human amylin

11,12

. Three proline-residue substitutions

found in rodent amylin, and absent from human amylin, are reported to account for the lack of amyloidogenicity of rodent amylin, because proline is a well-known β-sheet breaker

3,9,12

. The S20G mutation is a natural missense mutation that is linked to early

onset of T2DM 3. This mutation has been reported in some T2DM Asian populations; suggesting the association of the S20G mutation with early onset of T2DM in Japanese 13,14

and Chinese 15 populations.

For the pathologic amylin amyloid formation implicated in T2DM, no inhibitors have yet been clinically approved; making the prevention of amylin amyloid formation a quite active field of research 4. In this study, we selected and characterized the potential inhibitory activities of six compounds, specifically benzoic acid, salicylic acid, acetylsalicylic acid, folic acid, lipoic acid, and ascorbic acid. All six compounds are acidic small-molecule drugs, with molecular weights below 500 g/mol. The isoelectric point (pI) of amylin is reported around 8.8 16, and amylin aggregation rate is slower at an acidic pH of 5.5 as compared to physiological pH of 7.4

17

. Hence, four aromatic acids

and two aliphatic acids were selected to investigate their inhibitory activity on amylin 4 ACS Paragon Plus Environment

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aggregation. Inhibitory activities of the selected drugs were tested via Thioflavin T (ThT) assay for amyloid fibrils staining, and importantly investigating drugs’ ability to inhibit amylin aggregates toxicity as affirmed by MTT assay. method for amyloid fibril detection

ThT assay is a well-known

18,19

. A schematic summary of amylin aggregation

inhibition mechanism is illustrated in figure 1. Possible interactions between monomeric amylin and inhibitors, were visualized via molecular docking studies, using ChimeraVina and LigPlot+ software. AutoDock Vina was utilized to allow amylin-inhibitor docking calculations followed by 3D-visualization of docking results in UCSF Chimera software. LigPlot+ tool was used to allow amylin-inhibitor 2D-visualization. The importance of this study is highlighted by the novel use of two well-known compounds, lipoic acid and ascorbic acid, in modulating amylin aggregation propensity and protecting T2DM patients’ pancreatic β-cells.

2. Experimental Section 2.1 Materials Human amylin (1-37), α-lipoic acid, ascorbic acid, folic acid, acetylsalicylic acid, thioflavin T (ThT) dye and 1, 1, 1, 3, 3, 3-hexafluoro-2-propanol (HFIP) were all purchased from Sigma-Aldrich (Saint Louis, MO, USA). Benzoic acid and salicylic acid were obtained from Merck (Darmstadt, Germany). 3-(4, 5-Dimethylthiazol-2-yl)-2, 5Diphenyltetrazolium Bromide (MTT) and cell culture medium, the Roswell Park Memorial Institute (RPMI) 1640 medium, were purchased from Thermo-Fisher Scientific 5 ACS Paragon Plus Environment


(Waltham, MA USA). Rat RIN-m5f pancreatic β-cells, fetal bovine serum (FBS) and penicillin-streptomycin were obtained from the American Type Culture Collection (ATCC; Manassas, VA USA). Stock solutions of the six inhibitors were prepared using 10 mM phosphate buffer (NaH2PO4) as a solvent at required concentrations. Fresh stock solutions were prepared just before each experiment. 2.2 Methods 2.2.1 Human amylin aggregation protocol Human amylin was dissolved in 100 % HFIP at 1 mM, aliquoted to yield required final protein concentration, and incubated for 8 hours. Next, aliquots were air-dried to ensure removal of solvent. Phosphate buffer (10 mM NaH2PO4, pH 7.4) was prepared using deionized water. Aliquots of human amylin (30 µM) were dissolved in phosphate buffer (pH 7.4). Human amylin samples (30 µM) were mixed with six selected inhibitors (30 µM) at 1:1 (v/v), which resulted in 15 µM human amylin and 15 µM inhibitors as a final concentration. Then, human amylin samples (15 µM) with or without inhibitors (15 µM) were incubated under the physiological conditions, namely pH 7.4 and at 37 °C; to allow the formation of amyloid fibrils and aggregation. Similarly, for the two most potent inhibitors, ascorbic acid and lipoic acid, human amylin (15 µM) was incubated with or without the inhibitors at five different mole concentration ratios (0.01:1, 0.1:1, 1:1, 10:1 and 100:1; inhibitor: human amylin). In the human amylin aggregation time study, human amylin samples were incubated for up to 24 hours. Remaining experiments were performed by incubating human amylin samples with or without the inhibitors, for up to 2 hours.


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2.2.2 Inhibitor screening assay using thioflavin-T (ThT) ThT stock solution (30 µM) was prepared in deionized water at pH 7.0, mixed thoroughly. In a 96-well plate, 80 µL of each sample (15 µM) was added, at each indicated time point, to 80 µL of ThT solution (30 µM). Fluorescence was measured by excitation at 440 nm and emission at 485 nm, using an Infinite® 200 Pro microplate reader (Tecan Trading AG, Switzerland). To eliminate background fluorescence from free dye, we subtracted fluorescence intensity value of ThT in phosphate buffer solution, from fluorescence intensity reading of each sample. Relative fluorescence intensity percentage was calculated by normalizing sample readings with respect to human amylin sample readings, as shown in the following equation: Relative ThT Fluorescence intensity % = (Sample fluorescence intensity) / (Amylin fluorescence intensity) ×100 2.2.3 Fluorescence microscopy Fluorescence Microscope (Zeiss ZEN, Germany) was used to obtain fluorescent images of amylin samples incubated in the absence or presence of ascorbic acid and lipoic acid. Dye used for sample staining and fluorescence detection is thioflavin-T. 5 µL of ThT dye (30 µM) was added to 5 µL of each test sample (15 µM), which was placed on a glass slide and covered using a cover slip. Samples were kept in the dark. 2.2.4 Transmission electron microscopy (TEM) For TEM imaging, amylin samples in the absence and presence of the two selected inhibitors, ascorbic acid and lipoic acid, were incubated at 37 °C for 2 hours. Then, 10 µL of each sample was deposited on carbon-coated copper grids (Ted-Pella Inc, Redding, CA, USA), blotted, and negatively stained with freshly prepared 1% uranyl 7 ACS Paragon Plus Environment


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formate solution. After sufficient air-dry, samples were observed under a 300-kV in-situ transmission electron microscope (JEM-3011 HR, JEOL, Tokyo, Japan). 2.2.5 Molecular docking studies To perform docking studies, a human amylin structure was initially obtained from Protein Data Bank (PDB), where a PDB entry of 2L86 resolved using NMR was selected. 2L86 PDB entry was selected as it presents amylin structure in its natively amidated form at physiological pH

20

; presenting a more physiologically relevant

structure to this research than amylin structure reported in another study

21

. PubChem

compound database was utilized to obtain inhibitors SMILES identifiers to allow building 3D-ligand molecules in UCSF Chimera v.1.11.2

22

. Amylin was prepared for docking

using UCSF Chimera, by adding hydrogens and protein charges using an AMBER force field. As for inhibitor molecules, hydrogens and Gasteiger charges were added using UCSF Chimera. AutoDock Vina

23

software was used to perform docking studies, and

docked structures were visualized in UCSF Chimera. Given that our purpose of using the selected drugs is to inhibit human amylin aggregation; a search volume was selected based on human amylin amyloidogenic region (residues S20NNFGAILSS29). 2D-representative structures of protein-inhibitor interactions were visualized using LigPlot+ v.1.4.5 24. 2.2.6 Cell culture RIN-m5f pancreatic β-cells were cultured in 75 cm2 flask using RPMI 1640 medium, as a base medium, supplemented with 10 % FBS, 100 IU/mL penicillin and 0.1 mg/mL streptomycin, to create complete growth medium. Cells were incubated at 37 °C under 5


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% CO2. Subsequent experiments involving drug treatments were performed after 24 hours of seeding cells in 96-well plates. 2.2.7 MTT [3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide] cell toxicity assay MTT assay was initially utilized to assess the effect of inhibitors only on pancreatic RINm5f cells. Next, MTT assay was used to assess the toxicity of human amylin samples with or without inhibitors on pancreatic RIN-m5f cell line; hence, allowing the evaluation of inhibitors’ capacity to rescue β-cells from toxic amyloid fibrils. RIN-m5f β-cells viability was determined using MTT assay as described by Tada et al.

25

. Cells were plated in

flat bottom 96-well plates at a density of 20,000 cells/well, and allowed to stabilize for 24 hours in a humidified incubator with 5 % CO2. Fresh stock solutions of inhibitors and aliquoted amylin were prepared using RPMI-1640 base medium (without FBS) as a solvent. Samples of human amylin in the absence or presence of inhibitors were incubated for 2 hours at pH 7.0 and 37 °C. After 24 hours of cells incubation, culture medium was discarded and cells were treated with human amylin samples in the absence or presence of inhibitors at desired concentrations, and incubated for 24 hours in 5 % CO2 humidified incubator. Following 24 hour incubation period, 10 µL of fresh MTT solution (5 mg/mL), prepared in fresh RPMI-1640 base medium and mixed thoroughly, was added per well. Plates were then re-incubated for 4 hours. 100 µL of 10 % SDS in 0.01 M HCl were added to each well, and 96-well plates were re-incubated overnight. SDS is used to completely solubilize formazan crystals, without the need to mix each well thoroughly

25

. Sample absorbance was recorded at 570 nm using an

Infinite® 200 Pro microplate reader (Tecan Trading AG, Switzerland). Cell viability 9 ACS Paragon Plus Environment


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percentages were calculated with respect to control wells (without sample treatment), according to the equation: Cell viability % = (Sample absorbance at 570 nm) / (Control absorbance at 570 nm) × 100 2.2.8 Statistical analysis Data were expressed as either mean values ± standard deviation (SD), or mean values ± standard error (SE). Comparisons between treated samples and control samples were performed using unpaired Student t test. Results were considered of statistical significance when the p-value was < 0.05.

3. Results 3.1 Evaluation of six candidate drugs’ inhibitory activity on amylin aggregation using ThT assay Thioflavin T (ThT) is one of the most widely used dyes for amyloid fibrils staining

18,19

.

This fluorometric assay is useful in elucidating amyloid fibril formation kinetics and in screening of drugs that can inhibit fibrils assembly

26

.

ThT assay was utilized in

screening six acidic small-molecule compounds in terms of their inhibitory activity on human amylin fibril formation after 2 hours of incubation, and results are illustrated in figure 2A. Compared to amylin (15 µM) as 100 %, two aliphatic acids, lipoic acid and ascorbic acid, showed the highest attenuation in ThT fluorescence intensity to 42.1 ± 17.2 % and 42.9 ± 12.8 % (respectively), when tested at an equimolar ratio to that of


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amylin. Significant attenuation in amylin fibril formation was also observed with the structurally-similar compounds: salicylic acid and benzoic acid, with ThT levels of 49.5 ± 12.7 % and 54.9 ± 10.6 %, respectively. Acetylsalicylic acid, commonly known as aspirin, showed insignificantly low attenuation in ThT fluorescence levels relative to amylin control sample. Folic acid failed to inhibit amylin amyloid formation with relative ThT fluorescence levels; appearing higher than those of control samples, yet did not reach significance levels; suggesting comparable levels to those of control samples only. Structures of the six selected inhibitory acidic compounds are shown in figure 2B. 3.2 Time study of amylin aggregation in the absence and presence of the two selected potent inhibitors (ascorbic acid and lipoic acid) Aliquots were withdrawn at the following time points (0, 1, 2, 4 and 24 hours), as indicated in figure 3A. Amylin fibril formation in the absence and presence of ascorbic acid or lipoic acid follows a nucleation-dependent polymerization model. For amylin only, an increase in ThT fluorescence emission was observed after a lag time of about less than 30 minutes, and reached saturation levels after 2 hours of incubation. ThT fluorescence intensity increased from 25.0 ± 3.06 a.u. (0 hour-time point) to 125 ± 17.7 a.u. (2 hour-time point; mean ± SE) for amylin sample. In comparison to amylin only, amylin incubated in the presence of ascorbic acid achieved lower ThT fluorescence levels, with a lag phase approximately less than 60 minutes, and a plateau reached after 2 hours. ThT fluorescence intensity increased from 23.7 ± 1.45 a.u. (0 hour-time point) to 52.7 ± 17.7 a.u. (2 hour-time point; mean ± SE) for amylin sample in presence of ascorbic acid. As for amylin incubated in the presence of lipoic acid, even further reduction in ThT fluorescence levels is observed as compared to ascorbic acid effect. 11 ACS Paragon Plus Environment


Amylin incubated with lipoic acid experiences a lag phase of about 2 hours, and reaches saturation levels after 4 hours. ThT fluorescence intensity increased from 13.3 ± 3.33 a.u. (0 hour-time point) to 35.7 ± 0.88 a.u. (2 hour-time point; mean ± SE) for amylin sample in presence of lipoic acid. 3.3 Effect of two selected amylin aggregation inhibitors on amylin morphology Fluorescence images do not provide a quantitative measure of fibrils formed or inhibited, yet they merely provide a visual comparison between amylin samples incubated in the absence or presence of ascorbic acid and lipoic acid. As can be seen from figure 3B, amylin incubated with ascorbic acid (figure 3B; b) as well as with lipoic acid (figure 3B; c) illustrates significantly less ThT fluorescence as compared to amylin only (figure 3B; a); suggesting inhibition of amylin fibril formation. As a means of confirming the inhibitory activity of ascorbic acid and lipoic acid, TEM was utilized. The morphology of amylin fibrils in the absence and presence of ascorbic acid or lipoic acid were observed under TEM at 1 hour and 2 hour-time points. Extensive and long amylin fibrils are clearly observed for amylin samples in the absence of inhibitory drugs (figure 3C; left). Lipoic acid at an equimolar ratio to amylin (15 µM), strongly decreases amylin fibrillar structure after 2 hour incubation, as observed by considerably less amount of amylin fibrils formed and their much shorter structures (figure 3C; right), as compared to amylin only. Ascorbic acid also at an equimolar ratio to amylin (15 µM), reduces fibrillar amylin structure as shown by fewer and noticeably shorter fibrils (figure 3C; middle) as compared to amylin only, but at a lower extent as compared to amylin in the presence of lipoic acid.


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3.4 Testing inhibitory activity of the two selected inhibitors at different concentrations Following two hours incubation of amylin with or without ascorbic acid at five different molar ratios (100:1, 10:1, 1:1, 0.1:1 and 0.01:1; inhibitor: amylin), ThT fluorescence measurements showed significant attenuation in ThT fluorescence intensity in the inhibitor-to-amylin ratio range 10:1 to 0.01:1, relative to amylin control samples; illustrating a relatively wide range for ascorbic acid inhibitory activity on amylin fibril formation. Significantly high attenuation in ThT levels is observed for ascorbic acid at 10:1 ratio (inhibitor: amylin), with ThT levels of 21.1 ± 4.94 %, relative to amylin control sample (100%). Relative ThT fluorescence intensity of aggregation inhibition using ascorbic acid is shown in figure 4A. Amylin protein samples (15 µM) were also incubated for two hours in the absence or presence of lipoic acid at different molar ratios including 100:1, 10:1, 1:1, 0.2:1, 0.1:1 and 0.01:1; (inhibitor: amylin). A significant reduction in ThT fluorescence intensity, relative to amylin control samples, is observed in the inhibitor-to-amylin ratio range 10:1 to 0.1:1 as can be seen in figure 4B; indicating that lipoic acid inhibitory activity on amylin aggregation is at a narrower concentration range as compared to ascorbic acid inhibitory activity. The highest attenuation in ThT fluorescence intensity using lipoic acid is observed at an equimolar ratio (1:1) with respect to amylin control sample (100%), with ThT levels of 7.22 ± 1.62 %. 3.5 Molecular docking studies



Molecular docking studies were performed to help visualize possible interactions between amylin and the two potent inhibitors, lipoic acid and ascorbic acid. Lipoic acid is predicted to form a hydrogen bond with Ser28 in amylin, has triple hydrophobic interactions with amylin at Gly24, Ala25, Leu27, and has a predicted docking score of 3.1 kcal/mol. As for ascorbic acid, it is expected that it forms 2 hydrogen bonds with amylin at residues Asn21, Ser28, and has hydrophobic interactions with amylin at Gly24, Leu27, and with a predicted docking score of -2.8 kcal/mol. Results of docked poses of lipoic acid and ascorbic acid with amylin are illustrated in figure 5. 3.6 MTT toxicity assay of human amylin in RIN-m5f Cells in the absence and presence of selected aggregation inhibitors The effect of inhibitors only was shown to be non-toxic on pancreatic RIN-m5f cells for all six inhibitors at 15 µM, and at different tested concentrations for lipoic acid and ascorbic acid (supplementary figures S-2 and S-3). An initial cell viability time study was performed, where amylin samples in the absence or presence of either ascorbic acid or lipoic acid were incubated at an equimolar ratio with respect to amylin for 2 hours, and aliquots were withdrawn at 0, 1 and 2 hour-time points, as observed in figure 6A. The initial MTT time study illustrated that the highest aggregates toxicity was produced with pre-formed fibrillar amylin with cell viability of 47.1 ± 5.67 % (2 hour-time point), as compared to freshly prepared monomeric amylin solution with cell viability of 93.0 ± 10.4 % (0 hour-time point) with p

Inhibition of Human Amylin Aggregation and Cellular Toxicity by Lipoic

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