An Improved Screening Model To Identify Inhibitors Targeting Zinc

Anal. Chem. 2009, 81, 6944–6951

An Improved Screening Model To Identify Inhibitors Targeting Zinc-Enhanced Amyloid Aggregation Pei-Teh Chang,† Fan-Lu Kung,† Rahul Subhash Talekar,† Chien-Shu Chen,†,‡ Shin-Yu Lai,† Hsueh-Yun Lee,† and Ji-Wang Chern*,†,§ School of Pharmacy and Department of Life Science, College of Life Science, National Taiwan University, No. 1, Section 1, Ren-Ai Road, Taipei, 100, Taiwan, and School of Pharmacy, China Medical University, No. 91, Hsueh-Shih Road, Taichung, 404, Taiwan Zinc, which is abundant in senile plaques consisting mainly of fibrillar β-amyloid (Aβ), plays a critical role in the pathogenesis of Alzheimer’s disease. Treatment with zinc chelators such as clioquinol has been used to prevent Aβ aggregation in Alzheimer’s patients; however, clioquinol produces severe side effects. A simple, easy, inexpensive, and versatile screen to identify zinc chelators for inhibition of Aβ aggregation is currently unavailable. We thus developed a high-throughput screen that identifies zinc chelators with anti-Aβ aggregation activity. The recombinant Aβ peptides, aggregated on solid-phase microplates, formed Aβ-immunopositive β-sheet-containing structures in the presence of zinc. Formation of these Aβ fibrils was specifically blocked by metal ion chelators. This screening model improves identification of zinc-enhanced Aβ fibrils and anti-Aβ aggregation mediated by zinc chelating. The convenient system could qualitatively and quantitatively assay a large sample pool for Aβ aggregation inhibition and dissolution of Aβ aggregates. This screen is practical, reliable, and versatile for comprehensive detection of amyloid fibrillation and identification of inhibitors of Aβ aggregation. One of the major neuropathologic abnormalities observed in the brains of Alzheimer’s disease (AD) patients is the aggregation of β-amyloid (Aβ). Aβ aggregates are believed to play a causative role in the dysregulation of synaptic function and the loss of neurons in AD patients.1,2 Aβ assembles into a variety of higherorder structures including dimers, oligomers, protofibrils, and fibrils.3-5 The mechanisms by which Aβ monomers are converted to high molecular mass species are still under debate. The factors * To whom correspondence should be addressed. Tel: +886-2-2393-9462. Fax: +886-2-2393-4221. E-mail: [email protected]. † School of Pharmacy, National Taiwan University. ‡ China Medical University. § Department of Life Science, National Taiwan University. (1) Hamos, J. E.; DeGennaro, L. J.; Drachman, D. A. Neurology 1989, 39, 355– 361. (2) Tanzi, R. E. Nat. Neurosci. 2005, 8, 977–979. (3) Cleary, J. P.; Walsh, D. M.; Hofmeister, J. J.; Shankar, G. M.; Kuskowski, M. A.; Selkoe, D. J.; Ashe, K. H. Nat. Neurosci. 2005, 8, 79–84. (4) Walsh, D. M.; Klyubin, I.; Fadeeva, J. V.; Cullen, W. K.; Anwyl, R.; Wolfe, M. S.; Rowan, M. J.; Selkoe, D. J. Nature 2002, 416, 535–539. (5) Kayed, R.; Head, E.; Thompson, J. L.; McIntire, T. M.; Milton, S. C.; Cotman, C. W.; Glabe, C. G. Science 2003, 300, 486–489.

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that influence the aggregation state of Aβ include pH, Aβ concentration, incubation time, temperature, Aβ-specific binding proteins,6-8 and the presence of certain metal ions, especially zinc.9 Human and transgenic mouse Aβ plaques show significant accumulation of zinc in vivo.10,11 The accumulation of zinc within neuronal lesions correlates with the presence of Aβ, which contains binding sites for zinc at His6, His13, and His14.9 Though still under debate, zinc binding to Aβ has been suggested to accelerate the aggregation process, which produces reactive oxygen species and leads to neuronal damage.12-15 Increasing evidence suggests that zinc plays a crucial role in Aβ aggregation and influences the neurotoxicity of Aβ, providing a potential strategy to prevent and treat AD by targeting the interaction between zinc and Aβ. Currently, there are only a few zinc chelators that successfully prevent Aβ aggregation in vivo. Clioquinol, which specifically chelates zinc ion, has had some positive effects on cognition in a phase II clinical trial, and it was also reported to significantly lower plasma Aβ levels in AD patients.16 However, clioquinol was later withdrawn from the trial due to its association (6) Carrotta, R.; Manno, M.; Bulone, D.; Martorana, V.; San Biagio, P. L. J. Biol. Chem. 2005, 280, 30001–30008. (7) Kim, Y. S.; Randolph, T. W.; Stevens, F. J.; Carpenter, J. F. J. Biol. Chem. 2002, 277, 27240–27246. (8) Jarrett, J. T.; Berger, E. P.; Lansbury, P. T., Jr. Biochemistry 1993, 32, 4693–4697. (9) Bush, A. I.; Pettingell, W. H.; Multhaup, G.; d Paradis, M.; Vonsattel, J. P.; Gusella, J. F.; Beyreuther, K.; Masters, C. L.; Tanzi, R. E. Science 1994, 265, 1464–1467. (10) Howell, G. A.; Welch, M. G.; Frederickson, C. J. Nature 1984, 308, 736– 738. (11) Suh, S. W.; Jensen, K. B.; Jensen, M. S.; Silva, D. S.; Kesslak, P. J.; Danscher, G.; Frederickson, C. J. Brain Res. 2000, 852, 274–278. (12) Huang, X.; Atwood, C. S.; Hartshorn, M. A.; Multhaup, G.; Goldstein, L. E.; Scarpa, R. C.; Cuajungco, M. P.; Gray, D. N.; Lim, J.; Moir, R. D.; Tanzi, R. E.; Bush, A. I. Biochemistry 1999, 38, 7609–7616. (13) Mantyh, P. W.; Ghilardi, J. R.; Rogers, S.; DeMaster, E.; Allen, C. J.; Stimson, E. R.; Maggio, J. E. J. Neurochem. 1993, 61, 1171–1174. (14) Yoshiike, Y.; Tanemura, K.; Murayama, O.; Akagi, T.; Murayama, M.; Sato, S.; Sun, X.; Tanaka, N.; Takashima, A. J. Biol. Chem. 2001, 276, 32293– 32299. (15) Garai, K.; Sahoo, B.; Kaushalya, S. K.; Desai, R.; Maiti, S. Biochemistry 2007, 46, 10655–10663. (16) Ritchie, C. W.; Bush, A. I.; Mackinnon, A.; Macfarlane, S.; Mastwyk, M.; MacGregor, L.; Kiers, L.; Cherny, R.; Li, Q. X.; Tammer, A.; Carrington, D.; Mavros, C.; Volitakis, I.; Xilinas, M.; Ames, D.; Davis, S.; Beyreuther, K.; Tanzi, R. E.; Masters, C. L. Arch. Neurol. 2003, 60, 1685–1691. 10.1021/ac901011e CCC: $40.75  2009 American Chemical Society Published on Web 07/21/2009

with subacute myelo-optic neuropathy.17 Re-evaluation of the clinical impact and function of zinc chelators has led to an attractively dual therapeutic approach that targets Aβ through metal chelation; this approach inhibits Aβ aggregation and also reduces the levels of aggregated Aβ by promoting solubilization of Aβ deposits. It is therefore desirable to identify more potent, but safer, zinc chelators that block AD progression. A variety of methodologies have been developed to dissect the stage phenomena and/or kinetics of Aβ aggregation. Turbidity18 or binding assays using thioflavin T (ThT)19 or Congo red (CR),20 which specifically bind β-sheet structure, and surface plasmon resonance (SPR)21,22 are being used to elucidate the kinetics of Aβ aggregation. Other methodsssuch as a centrifugation assay with fluoro-/or radio-labeled amyloid;23,24 enzyme-linked immunosorbent assay25 with a specific antibody to Aβ; separation techniques including gel electrophoresis,24,26 capillary electrophoresis,27 liquid chromatography-mass spectrometry,28,29 and gel electrophoresis for DNA-associated Aβ fibrils;30 and fluorescence correlation spectroscopys31 are currently available end point analyses for quantification of higher-order Aβ aggregates. In addition to quantification of Aβ aggregates in a high-throughput setting, it is also desirable to be able to study their morphologies. Differential interference contrast microscopy,32 circular dichroism spectroscopy,33 electron microscopy,34 X-ray fiber diffraction,35 and atomic force microscopy36 are commonly used for this purpose. These methods provide powerful image analysis capacity but lack the practicality for use in high-throughput screening (HTS). Each of the above methods has its own limitations, such as high cost, long assay time, relatively poor signal-to-noise ratio, and high (17) Cherny, R. A.; Atwood, C. S.; Xilinas, M. E.; Gray, D. N.; Jones, W. D.; McLean, C. A.; Barnham, K. J.; Volitakis, I.; Fraser, F. W.; Kim, Y.; Huang, X.; Goldstein, L. E.; Moir, R. D.; Lim, J. T.; Beyreuther, K.; Zheng, H.; Tanzi, R. E.; Masters, C. L.; Bush, A. I. Neuron 2001, 30, 665–676. (18) Jarrett, J. T.; Lansbury, P. T., Jr. Biochemistry 1992, 31, 12345–12352. (19) Wood, S. J.; Maleeff, B.; Hart, T.; Wetzel, R. J. Mol. Biol. 1996, 256, 870– 877. (20) Klunk, W. E.; Pettegrew, J. W.; Abraham, D. J. J. Histochem. Cytochem. 1989, 37, 1273–1281. (21) Myszka, D. G.; Wood, S. J.; Biere, A. L. Methods Enzymol. 1999, 309, 386– 402. (22) Cannon, M. J.; Williams, A. D.; Wetzel, R.; Myszka, D. G. Anal. Biochem. 2004, 328, 67–75. (23) Su, Y.; Chang, P. T. Brain Res. 2001, 893, 287–291. (24) Burdick, D.; Soreghan, B.; Kwon, M.; Kosmoski, J.; Knauer, M.; Henschen, A.; Yates, J.; Cotman, C.; Glabe, C. J. Biol. Chem. 1992, 267, 546–554. (25) Brown, A. M.; Tummolo, D. M.; Rhodes, K. J.; Hofmann, J. R.; Jacobsen, J. S.; Sonnenberg-Reines, J. J. Neurochem. 1997, 69, 1204–1212. (26) Yang, A. J.; Knauer, M.; Burdick, D. A.; Glabe, C. J. Biol. Chem. 1995, 270, 14786–14792. (27) Sabella, S.; Quaglia, M.; Lanni, C.; Racchi, M.; Govoni, S.; Caccialanza, G.; Calligaro, A.; Bellotti, V.; De Lorenzi, E. Electrophoresis 2004, 25, 3186– 3194. (28) Cheng, X.; van Breemen, R. B. Anal. Chem. 2005, 77, 7012–7015. (29) Nettleton, E. J.; Tito, P.; Sunde, M.; Bouchard, M.; Dobson, C. M.; Robinson, C. V. Biophys. J. 2000, 79, 1053–1065. (30) Ahn, B. W.; Song, D. U.; Jung, Y. D.; Chay, K. O.; Chung, M. A.; Yang, S. Y.; Shin, B. A. Anal. Biochem. 2000, 284, 401–405. (31) Pitschke, M.; Prior, R.; Haupt, M.; Riesner, D. Nat. Med. 1998, 4, 832– 834. (32) Schuler, B.; Rachel, R.; Seckler, R. J. Biol. Chem. 1999, 274, 18589–18596. (33) Tomski, S. J.; Murphy, R. M. Arch. Biochem. Biophys. 1992, 294, 630– 638. (34) Fraser, P. E.; Duffy, L. K.; O’Malley, M. B.; Nguyen, J.; Inouye, H.; Kirschner, D. A. J. Neurosci. Res. 1991, 28, 474–485. (35) Serpell, L. C.; Fraser, P. E.; Sunde, M. Methods Enzymol. 1999, 309, 526– 536. (36) Parbhu, A.; Lin, H.; Thimm, J.; Lal, R. Peptides 2002, 23, 1265–1270.

propensity for false positives, making them unsuitable for HTS. The optical or antibody-based approaches37-41 have been used as an independent means of HTS to identify aggregation inhibitors or Aβ imaging agents. Another model combining capillary electrophoresis and ThT is also capable of identifying Aβ aggregation inhibitors.42 The detection, however, is limited to oligomers containing ThT reactivity. To date, there is no single HTS model that quantitatively and qualitatively addresses the conformational aspects or folding dynamics throughout the stages of Aβ aggregation in the presence of inhibitors. Here we report the development of a simple and robust screen that could evaluate the extent of Aβ aggregation as well as dynamic changes in Aβ fibril formation with the following advantages: (a) The entire process is run in a 96-well (or 384well) format and minimizes sample handling. (b) The aggregation reaction would mimic different pathophysiological conditions (such as variations in incubation time, pH, or the addition of certain metals) in vitro. (c) Morphological analyses of the distributions and structures of Aβ aggregates as well as synchronous quantification of the level of aggregation would be carried out in the same module rapidly and easily. (d) Various exchangeable detection approaches could be used (e.g., simple chemicals or antibodies against either Aβ or protein tags). (e) The method could be used to study the anti-Aβ effects at different stages of Aβ aggregation. (f) Related events, such as dissolution of Aβ fibrils or free radicals generation in aggregates, could also be analyzed. (g) The method would be easy to perform and provide rapid results, and most importantly, it would rapidly measure the concentration-dependent effects of each compound. (h) The method would be highly cost-effective due to use of recombinant Aβ instead of costly synthetic Aβ in a 96-well plate model. The rapid and simple solid-phase-based HTS approach is reproducible and compatible with semi-high-throughput microscopy and provides quantitative information on the aggregation process to identify novel Aβ inhibitors by targeting Aβ or zinc-Aβ interactions. EXPERIMENTAL SECTION Stock Preparation. Lyophilized recombinant His-tagged Aβ1-42 (His-Aβ) (its preparation is described in Supporting Information), synthetic Aβ1-42, Aβ1-40, or reverse Aβ (Aβ42-1) peptides from Bachem (Switzerland) were first dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, Sigma) at a concentration of 5 mg/mL,43,44 shaken at 4 °C for 2 h, and then stored at -80 °C. Before use, HFIP was removed by evaporation under a gentle stream of nitrogen, and the peptide was dissolved in DMSO to make a 200 µM stock solution. Zinc chelators clioquinol and EDTA and the Aβ-aggregation inhibitors rifampicin and nicotinic acid (the latter did not have any zinc chelation activity) used in (37) Dolado, I.; Nieto, J.; Saraiva, M. J.; Arsequell, G.; Valencia, G.; Planas, A. J. Comb. Chem. 2005, 7, 246–252. (38) Szekeres, P. G.; Leong, K.; Day, T. A.; Kingston, A. E.; Karran, E. H. J. Biomol. Screen. 2008, 13, 101–111. (39) Haugabook, S. J.; Yager, D. M.; Eckman, E. A.; Golde, T. E.; Younkin, S. G.; Eckman, C. B. J. Neurosci. Methods 2001, 108, 171–179. (40) Inbar, P.; Yang, J. Bioorg. Med. Chem. Lett. 2006, 16, 1076–1079. (41) Inbar, P.; Bautista, M. R.; Takayama, S. A.; Yang, J. Anal. Chem. 2008, 80, 3502–3506. (42) Kato, M.; Kinoshita, H.; Enokita, M.; Hori, Y.; Hashimoto, T.; Iwatsubo, T.; Toyo’oka, T. Anal. Chem. 2007, 79, 4887–4891. (43) Wei, G.; Shea, J. E. Biophys. J. 2006, 91, 1638–1647. (44) Abedini, A.; Singh, G.; Raleigh, D. P. Anal. Biochem. 2006, 351, 181–186.


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this study were purchased from Sigma. Clioquinol analogs 1-4 were synthesized by condensation of substituted 8-hydroxy-2methylquinoline with the corresponding benzaldehydes in pyridine.45 Compound 5 was obtained through amide coupling of substituted quinoloine-2-carboxylic acid with anisidine.46 All synthetic compounds were dissolved in DMSO to form 20 mM stock solutions. Preparation of His-Aβ Peptide-Immobilized Plate. The 96well plates (NUNC, flat-bottom polystyrene) were coated with purified His-Aβ peptide as described in Cantarero et al.47 and Kaur et al.48 with several modifications. Briefly, the acid [47% (v/v) HNO3 in concentrated H2SO4] was freshly prepared, and 200 µL of the acid was loaded into each well of the microtiter plate and incubated at room temperature for 30 min with gentle shaking. After washing the plate twice with distilled H2O, 200 µL of 5% aminopropyltriethoxysilane solution (pH 6.9) was added into each well and incubated at 62 °C for 2 h. Meanwhile, His-Aβ peptides (10 mg, recombinant or synthetic) were mixed with N-hydroxysuccinimide (1.7 mg) and N,N′-dicyclohexyl carbodiimide (6.2 mg) in 1.3 mL of dimethylformamide, and the mixture was incubated at room temperature for 4 h prior to the coating reaction. After centrifuging the soultion at 10000g for 10 min at room temperature to remove precipitate, the Aβ solution was reconstituted in carbonate buffer [0.15% (w/v) Na2CO3, 0.1% (w/v) MgCl2 · 6H2O, 0.3% (w/v) NaHCO3, and 0.1% (w/v) sodium azide, pH 9.6] to a final concentration of 3.1 µg/mL. This Aβ solution (100 µL) was then added to either unmodified or chemically modified wells and incubated at 4 °C overnight. After aspiration of the coating buffer, the coated modules were washed twice with ice-cold phosphate-buffered saline (PBS, 137 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl, 2 mM KH2PO4, pH 7.4), air-dried, and then blocked with formaldehyde (0.2 M in PBS) by incubating the plate at 37 °C for 2 h. The formaldehyde solution was then removed, and the plates were washed three times with cold distilled H2O. Before the aggregation step, the efficiency of His-Aβ immobilization was determined by the micro BCA protein assay (Pierce, Rockford, IL). After incubating with 100 µL of BCA reagent at 37 °C for 2 h, the optical density at 562 nm was measured and the coating efficiency (%) was determined by taking the value of 0/0.31 µg of His-Aβ as 0/100%. The structural transitions of these immobilized His-Aβs were identified by CR and ThT analysis. After adding 100 µL of 30 µM ThT (in 50 mM sodium phosphate, pH 6.0), the fluorescence intensity was immediately obtained with a microplate fluorometer (Molecular Devices, Sunnyvale, CA; excitation ) 450 nm, emission ) 482 nm). The ThT response (%) was defined by taking the aggregation level of 0.31 µg of His-Aβ (after incubating at 37 °C for 24 h) as 100% and the value for vehicle only (without Aβ) as 0%. These immobilized His-Aβs were photographed with a bright-field microscope (T100, Nikon) at 40× for CR-reacted specimens or a fluorescence microscope (BX50, Olympus) at 40× for ThT(45) Chen, C. S.; Lai, S. Y.; Hsu, P. S.; Tsai, C. Y.; Fang, C. W.; Su, M. J.; Cheng, F. C.; Kao, C. L.; Chern, J. W. Chin. Pharm. J. 2002, 54, 353–374. (46) Talekar, R. S.; Chen, G. S.; Lai, S. Y.; Chern, J. W. J. Org. Chem. 2005, 70, 8590–8593. (47) Cantarero, L. A.; Butler, J. E.; Osborne, J. W. Anal. Biochem. 1980, 105, 375–382. (48) Kaur, J.; Suri, C. R. http://www.natureprotocols.com/2007/12/06/ direct_hapten_coated_elisa_for.php.

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reactive specimens. For CR or ThT staining, one hundred µL of the CR (200 µM in 150 mM NaCl, 5 mM KH2PO4, pH 7.4) or ThT (30 µM in 50 mM sodium phosphate, pH 6.0) were incubated at room temperature for 1 h or 30 min, respectively. Optimizing in-Well Aggregation. The aggregations were performed on His-Aβ-immobilized plates and incubated under various conditions (different incubation temperature, pH, time, the amounts of His-Aβ, and the presence of metal ions, etc.) to optimize Aβ aggregation on the solid-phase. The aggregations were detected as previously described, but with minor modification in CR analysis. Here CR (100 µL of 5 µM in 150 mM NaCl, 5 mM KH2PO4, pH 7.4) was added into each well and incubated at room temperature for 30 min. The absorbance (At) at 477 and 540 nm was measured using a microplate reader. The amount of CR bound to aggregated Aβ was calculated according to the following equation:19 CR binding (µM) ) (540At/ 25295) - (477At/46306). For immunoassays, the wells were treated with anti-Aβ1-16 (100 µL, 1:250 dilution in PBS) (Biosource, Camarillo, CA) for 2 h followed by another incubation with a TRITC-conjugated secondary antibody (100 µL, 1:250 dilution in PBS) (Jackson, West Grove, PA) for 1 h at 37 °C and fluorescence was measured (excitation ) 547 nm, emission ) 572 nm). All experiments were performed in triplicate. Readings obtained from each assay were normalized by taking the aggregation level at 37 °C and pH 5.8 in the absence of zinc in vehicle control as 100% and readings obtained in the absence of Aβ as 0%. In Vitro HTS Model for in-Well Solid-Phase Aggregation. To monitor the progression of toxic Aβ aggregate formation within a 96-well plate, the extension of His-Aβ fibrils is executed by polymerization between the immobilized His-Aβ and exogenous His-Aβ on the bottom of the microplate wells (Figure 1), as described in the following. Solid-phase aggregation was performed by adding excess (typically 5 µg) His-tagged Aβ in pH 5.8, 50 mM MES buffer into the His-Aβ-immobilized 96-well plate in the presence or absence of 10 µM zinc chloride at pH 5.8 in MES buffer and incubating at 37 °C for 4 h (or 24 h) followed by ThT staining or other measurements. To determine the inhibitory effects of compounds on Aβ aggregation, each compound (0, 1, 5, 10, or 25 µM) was added simultaneously with exogenous HisAβ. After incubation, the supernatant was aspirated, the pellet was washed with 50 µL of PBS and air-dried, and then the elongated Aβ fibrils extending from the solid phase were examined by the above-mentioned four types (CR, ThT, immunoassay, and BCA) of quantitative analysis or three kinds (CR, ThT, and immunoassay) of microscopy as described in the following section. HTS Model: Quantitative Evaluation and Microscopic Detection. Quantitative analyses (CR, ThT, immunoassay, and BCA) were performed as described in the preparation and optimization sections. Readings obtained from each assay were normalized by taking the aggregation level in the presence of zinc in vehicle control as 100% and readings obtained in the presence of zinc but in the absence of Aβ as 0%. All experiments were performed in triplicate, and IC50 values for zinc chelators on Aβ aggregation were calculated. The signal-to-noise ratio, coefficient of variation, and Z′ factors (a characteristic parameter of the quality assay conditions, without intervention of testing compounds) were evaluated according to Zhang et al.49

Figure 1. Schematic diagram of the solid-phase aggregation system for identifying the behaviors of Aβ aggregation. The immobilized His-Aβs provided the aggregation-initiation sites on solid-phase. The elongations of Aβ aggregates on solid-phase were carried in various environment conditions. The zinc chelators entered the solid-phase aggregation either at the initiation of aggregation or after elongation was completed and the floating Aβs were removed. The reactions on microplates were conducted with different methods of detection.

Antiaggregation activity of clioquinol was also examined and used as a positive control (Supporting Information, Figure S-3). Microscopic analyses (CR, ThT, and immunoassay) were performed as previously described. After chemical staining or immunostaining, the solution was aspirated, and the specimens were examined and photographed under bright-field/fluorescence microscopes at 40× for CR- or ThT-positive signals or Aβimmunoreactivities. HTS To Identify Metal Chelators That Dissolve Deposited Aβ Aggregates. To quantify the effect of zinc chelators on existed amyloidogenic deposits, the aforementioned HTS model was slightly modified. Five micrograms of His-Aβ in pH 5.8 MES buffer was added into the His-Aβ-immobilized microplate well in the absence of zinc ion and incubated at 37 °C for 24 h. The Aβ fibrils were retained on the solid-phase after aspirating the supernatant and washed once with PBS (50 µL). MES buffer (80 µL) containing 10 µM zinc ion and the chelator of interest at a specified concentration was then added into the wells containing preformed His-Aβ fibrils and incubated for another 24 h at 37 °C to disassemble His-Aβ fibrils. After washing, the fibrils remaining on the solid surface were reacted with ThT, CR, anti-Aβ, or BCA reagents for quantitative analyses and microscopic analyses. The lost amount of aggregates was calculated and normalized by taking the total amount of preformed Aβ aggregates as 100%. All

Figure 2. Characterization of the quantity and structural transition of immobilized His-Aβ on solid-phases. (A) Quantification of His-Aβ immobilized on unmodified or chemically modified surfaces as determined by BCA protein assay, and R-helix to β-sheet structural transitions of these Aβ were measured by ThT fluorometry. Both measurements (mean ( SD) were normalized to the quantity or ThT response of aggregated His-Aβ (0.31 µg, 100%). (B) Photographs of three types of immobilized His-Aβ under a bright- or dark-field microscope at 40× after staining with 200 µM CR or 30 µM ThT are shown.

experiments were performed in triplicate, and 50% effective concentration (EC50) values for zinc chelators on Aβ aggregates were calculated.

RESULTS Characterization and Optimization of the Solid-PhaseBased Aggregation. The utility of our solid-phase-based aggregation system, in which the readout would be used to quantitatively measure changes in Aβ aggregation to identify potential aggregation inhibitors, was evaluated prior to initiating the screen. As shown in Figure 2A, the coating efficiency increased from 35 to 90% when covalent binding was introduced by chemical modification. These immobilized His-Aβs had no response to CR or ThT and yielded much less β-sheet signals as compared with aggregated His-Aβ, suggesting that the chemically modified surfaces improved the immobilization efficiency of His-Aβ and maintained His-Aβ in the R-helix monomeric form. The His-Aβs used here have similar amyloidogenic activity as the native Aβs, as suggested by the results shown in Figure S-2 (Supporting Information). We then prescreened a number of His-Aβ immobilized plates under diverse conditions for optimal solid-phase aggregation (Figure 3). Aggregation reactions were first carried out with various amounts Analytical Chemistry, Vol. 81, No. 16, August 15, 2009

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Figure 3. The optimization of His-Aβ aggregation on a solid phase. The aggregation reactions were carried out on His-Aβ immobilized solidphase at different temperatures with increasing His-Aβ (A, B at pH 5.8 for 24 h), increasing incubation time (C, D at 37 °C and pH 5.8), or at different pH values (E, for 4 h at 37 °C; F, for 24 h at 37 °C) and then subject to ThT (A, C and E) or BCA (B, D and F) analyses. Five micrograms of His-Aβ (G) or synthetic Aβ1-40 (H) was incubated for 24 h at 37 °C/pH 5.8 in the presence of zinc or copper ion with/without clioquinol.

of His-Aβ at 25 or 37 °C (Figure 3A,B). Five micrograms of HisAβ or Aβ42-1 was then incubated at 37 °C for 1, 4, 12, 24, and

36 h (Figure 3C,D). The effects of pH value and the presence of metal ions were examined using different buffers [MES, pH 5.8;

(49) Zhang, J. H.; Chung, T. D.; Oldenburg, K. R. J. Biomol. Screen. 1999, 4, 67–73.

(50) Bush, A. I.; Pettingell, W. H., Jr.; Paradis, M. D.; Tanzi, R. E. J. Biol. Chem. 1994, 269, 12152–12158.

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Table 1. Concentration-Dependent His-Aβ Aggregation Is Enhanced by Zinc Iona aggregation (%) zinc (µM) thioflavin T Congo red immunoassay BCA

a

0 µg of His-Aβ

0 1 10 0 1 10 0 1 10 0 1 10

0.0 0.5 0.0 0.0 1.0 3.2 0.0 0.0 0.0 0.0 0.4 0.2

(0.2) (0.4) (0.2) (0.9) (0.2) (1.2) (0.1) (0.1) (0.1) (0.3) (0.4) (0.1)

1.25 µg of His-Aβ 1.6 7.9 18.2 6.0 27.1 46.5 1.7 4.5 14.9 15.4 24.2 40.8

(0.5) (0.6) (0.5) (1.8) (0.3) (1.6) (0.4) (0.2) (0.5) (1.2) (0.8) (0.6)

2.5 µg of His-Aβ 18.7 20.2 35.3 21.4 27.8 63.9 20.2 40.5 52.5 22.1 34.5 73.8

5 µg of His-Aβ

(0.5) (2.2) (1.3) (1.7) (0.2) (0.4) (1.8) (4.5) (3.5) (0.7) (0.2) (0.5)

47.4 60.1 100.0 57.5 73.7 100.0 53.4 73.6 100.0 51.5 64.6 100.0

(5.5) (3.5) (3.3) (2.5) (3.9) (11.0) (2.0) (2.2) (3.0) (2.7) (2.8) (1.0)

Data are mean percent aggregation with standard deviations in parentheses from three independent measurements.

Table 2. Inhibitory Effects of Metal Chelators on His-Tagged Aβ Solid-Phase Aggregationa

antiaggregation (IC50, µM) compound clioquinol 1 2 3 4 1 EDTA rifampicin nicotinic acid

R1

R2

Ar

H Cl Cl Cl Cl

H H Cl Cl I

3,5-diBr-4-OH-Ph 3,5-diBr-4-OH-Ph Ph 2,3,4-tri-OMe-Ph 4-OMe-Ph

thioflavin T

Congo red

immunoassay

BCA

6.8 9.9 >25 7.2 6.1 9.3 8.4 >25 >25

7.1 21.3 >25 7.6 4.5 14.9 8.9 >25 >25

8.3 >25 15.0 7.5 3.3 21.0 8.2 >25 >25

9.1 10.0 21.8 15.0 9.0 22.3 9.1 >25 >25

a Compounds 1-5 all contain an 8-OH quinoline structure. Data are obtained from three independent measurements, and the inhibitory capacities of each test compound were expressed as average IC50 values.

PBS, pH 7.4; or artificial cerebrospinal fluid (aCSF, 126 mM NaCl, 2.5 mM KCl, 1.24 mM NaH2PO4, 1.3 mM MgSO4, 2.4 mM CaCl2, 26 mM NaHCO3, 10 mM D-glucose, pH 7.6)] (Figure 3E,F) and different divalent ions (zinc or copper ion) (Figure 3G,H). In the latter case, clioquinol was used to deplete the ions. Aβ peptides were found to rapidly form fibrils under low pH (pH 5.8) and high temperature (37 °C) in the absence of zinc ion, as previously reported.6,7 The rate and degree of β-sheet transition or aggregation were dependent on the amount of His-Aβ as well as the incubation time, but started to plateau at 5 µg of His-Aβ (Figure 3A) after 4 or 24 h of incubation (Figure 3C,D), respectively. The higher level of zinc-induced Aβ aggregation is reversed more dramatically by clioquinol (Figure 3G,H). These findings, which suggest that zinc has a greater aggregation-promotion ability and lower affinity with Aβ, are consistent with reported by others.50-53 In a further study, the degree of His-Aβ aggregation was found to be greatly enhanced dose-dependently by zinc ion (Table 1). HTS Quantification of Aggregation Inhibition. An initial screening for potential aggregation inhibitors was then conducted using our solid-phase system. Level changes of His-Aβ aggregations in wells were determined by direct protein measurements or indirect measurements based on β-sheet binding or immunoresponse. The inhibitory capacities of each test compound were

expressed as average IC50 values. As shown in Table 2, clioquinol, EDTA, and compounds 3-5 were moderately potent inhibitors of Aβ aggregation with IC50 values in the low micromolar range, whereas the others were not as effective. As it is strongly desirable to have a relatively higher resolution and better reproducibility in a large-scale screening model, several quality factors were also examined for our system. The signal-to-noise ratio for the ThT assay, CR assay, immunoassay, and BCA assay was, respectively, 81.1, 12.4, 37.4, and 19.4; the coefficient of variation of the signal was 6.6, 5.3, 7.2, and 2.4; and the Z′ factor, which is indicative of the separation of the signal and background populations, was 0.39, 0.63, 0.75, and 0.62. These results indicated that the immunoassay was the best analytical method, with the protein assay being the second. ThT and CR detection are, however, still reliable quantitative methods. The solid-phase-based aggregation system equipped with these detection approaches satisfied the criteria for an HTS assay, (51) Clements, A.; Allsop, D.; Walsh, D. M.; Williams, C. H. J. Neurochem. 1996, 66, 740–747. (52) Atwood, C. S.; Scarpa, R. C.; Huang, X.; Moir, R. D.; Jones, W. D.; Fairlie, D. P.; Tanzi, R. E.; Bush, A. I. J. Neurochem. 2000, 75, 1219–1233. (53) Hu, W. P.; Chang, G. L.; Chen, S. J.; Kuo, Y. M. J. Neurosci. Methods 2006, 154, 190–197.


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Table 3. Potencies of Compounds 4 and 5 on the Depolymerization of His-Aβ Fibrilsa dissolution effect (EC50, µM) compound

thioflavin T

Congo red

immunoassay

BCA

clioquniol 4 5

18.0 9.6 19.8

6.9 5.6 7.9

7.8 5.9 12.6

6.9 5.9 14.1

a The preformed His-Aβ fibrils on the solid-phase were incubated with clioquinol and compounds 4 and 5 in the absence of exogenous His-Aβ. Data are obtained from three independent measurements and expressed as EC50 values.

and none of the chelators tested influenced the HTS system (unpublished data). Morphometry-Based HTS To Assess Inhibition of Aβ Aggregation. Zinc-induced His-Aβ fibrillar deposits were examined by microscopy (Figure S-4, Supporting Information). The large number of ultrastructures containing ThT green fluorescence indicated the presence of zinc-induced His-Aβ fibrils having an extended deformed polygonal pattern. Similar patterns were observed when zinc-enhanced His-Aβ fibrils were analyzed by CR staining or immunoassays. Formation of these ultrastructures, which are characteristic of Aβ fibrils, correlated with the zinc ion concentration. We then examined whether metal chelators (clioquinol, EDTA, and compounds 4 and 5) reduced the zincenhanced His-Aβ aggregation. The largest inhibitory effect on HisAβ aggregation was via treatment with EDTA or compound 4. The inhibitory effect of compound 4, another synthetic zinc chelator,45 on Aβ aggregation was superior to that of clioquinol, but neither inhibitor completely eliminated the polygonal structures. Interestingly, there were fewer Aβ polygonal aggregates in the presence of zinc chelators as compared with zinc-free Aβ aggregation, suggesting that these compounds may also influence aggregation through alternate pathways. HTS To Identify Metal Chelators That Dissolve Accumulated Aβ Aggregates. Blockade of Aβ fibrillation by zinc chelators such as clioquinol and compound 4 may be due to other factors in addition to zinc chelation. Thus, we proposed that zinc chelators may be able to interact directly with Aβ, i.e., in the absence of zinc. Consistent with other reports,22,23 our result indicated that preformed fibrils self-depolymerized spontaneously and slowly in the absence of monomeric His-Aβ (Figure S-5, Supporting Information). Indeed, treating His-Aβ deposits with chelators accelerated depolymerization. Zinc chelators disassembled the majority of Aβ deposits and also reduced the proportion of observable β-sheet structure in the remaining undissolved deposits (Table 3 and Figure S-5, Supporting Information). While Clioquinol and compounds 4 and 5 were all capable of enhancing the dissolution of preformed Aβ aggregates, compound 4 had the greatest effect among them all (Table 3). DISCUSSION The experimental challenge in designing an HTS system for Aβ is the apparent incompatibility between the rapid acquisition of large amounts of aggregation data and the need to obtain image information under varying conditions within a facile aggregation model. To fulfill both these requirements, we designed a solidphase-based aggregation system that conveniently characterizes 6950


Aβ-plaque-forming processes as well as the antiaggregation effects of various compounds and is compatible with image-based measurements of multiple aggregation features (Figure S-4, Supporting Information). As the model detects surface-linked Aβ polymers in microplate wells, it is critical to prevent spontaneous His-Aβ aggregation prior to the assay. To achieve it, His-Aβ was maintained as an R-helix monomer in the preparation (Figure S-1, Supporting Information) and immobilization processes (Figure 2) before aggregation began. The chemically modified surfaces improve the immobilization efficiency of His-Aβ, giving more numerous aggregation initiation sites. In-well aggregation gives a linear accumulation of His-Aβ deposits on the surfaces. It also provides a versatile platform for Aβ aggregation, on which the effects of reaction times, temperatures, pHs, and various additives (including metals, chemicals, Aβ binding proteins) could be investigated. Zinc then triggers higher levels of solid-phase aggregation by way of increasing β-sheet transition and aggregation tendency of Aβ. Though an opposite role of zinc on aggregation/toxicity of Aβ has been proposed by some researchers,14,15 this discrepancy may result from variations in the experimental conditions (e.g., in vivo vs in vitro, different Aβ species, and different pH setting). These zinc-enhanced Aβ aggregations are dramatically blocked merely by zinc chelators. Preliminary screening data from our system revealed that the inhibitory effect of 8-hydroxyquinoline derivatives (3-5) on Aβ aggregation was comparable to that of clioquinol. While compound 4 with an electron donor on styrylquinoline showed better antiaggregation activity than clioquinol, the anti-Aβ aggregation activities of compounds 1 and 2 with a more electron-withdrawing group on 8-hydroxyquinoline were lower. It was proposed that an electron-donating/-withdrawing group on 8-hydroxyquinoline will modulate its metal-chelating ability. Along with interest in antiaggregation effects comes a greater awareness of dissolution in Aβ aggregates. Our system can also be used to characterize the dissolution effects of zinc chelators on Aβ fibrils, an aspect that might be therapeutically useful for AD patients. The disassociation of zinc-dependent aggregates is potentially enhanced by the presence of chelators. This reaction is metal chelation-independent (Figure S-5, Supporting Information) and leads to the stabilization of Aβ in β-sheet-containing lower molecular weight structures (Table 3). Aβ interaction with certain zinc chelators may differ in the absence or presence of zinc ions, resulting in different changes in Aβ conformation. Novel therapeutics for Aβ plaque formation may be identified via screening for chelators that prevent further aggregation or that destabilize preformed fibrils. The use of a solid-phase aggregation system to screen for anti-Aβ inhibitors has several advantages. The optical measurements of Aβ fibrils can be enhanced by fluorophores or chromophores, which diminish the background noise caused by turbidity analysis.18 Further, imagebased screens offer a quick and easy end-point analysis for intact Aβ fibrils in the micromolar range, even though controversy remains regarding detailed Aβ structures observed by electron microscopy (EM) or atomic force microscopy (AFM).34,36 Our system could identify all of the aggregated Aβ species linked to the surfaces, not merely certain sizes of aggregates.42 Antibody detection utilizes different capture techniques to detect both the motif on Aβ1-42 and the tag on Aβ. Nonetheless, there are a

few limitations to be considered for the HTS model. The system is unable to distinguish various aggregated Aβ species as efficiently as by Western blotting, although it provides information on the size of aggregates on the Aβ-immobilized surface. This image-based screen expresses faster than EM and AFM but cannot compare with high content analysis carried out by CCD imaging microplate readers. However, our semi-HTS imaging system has potential to be applied in CCD imaging HTS setting. Another issue of concern with an HTS system is its potential for false positives; molecules designed to target Aβ aggregation might occupy binding sites for antibodies, β-sheet-binding agents, or metals on Aβ,23,40,41 resulting in the loss of signal due to steric hindrance. To overcome these obstacles and obtain more reliable and realistic screening results, our system monitors the aggregation process using two kinds of β-sheetbinding agents, an Aβ specific antibody and a BCA protein assay. This strategy may result in the slight difference of aggregation estimation between the four detection techniques, as well as the IC50 values of each inhibitor. The solid-phase-based HTS system provides a platform to conduct multiple studies in parallel and eliminate false positives originating from any individual assay system. Our system yields reproducible and reliable outcomes for antiaggregation activities; moreover, the sensitivity of the end-point Aβ aggregation kinetic analysis is in the micromolar range for large chemical libraries. The entire process, from aggregation to detection, occurs and is monitored in microwells, which are easy to handle and require fewer reagents than larger vessels. The use of His-Aβ, which has similar aggregation profile as native Aβ, reduces the cost of this system dramatically, while efficiency and versatility remain. As an illustration of multiple utilities of this platform, the technique could be slightly modified to make it applicable to the estimation of neuronal and cognitive impairments after therapy by detecting the level of Aβ1-42, a marker for early diagnosis of

AD, in cerebrospinal fluid or serum samples from AD patients. Likewise, modifications of this technique may also effect its application in evaluating the therapeutic effects of drugs, such as β-secretase inhibitors, that lower the levels of plasma Aβ. An additional appliance of this system is to evaluate behaviors and inhibitors of other amyloidogenic proteins, which could be used as immobilized ligands and analytes instead of HisAβ. This system can also be modified to measure free radical generation during Aβ aggregation and used for the screening of free redial scavengers/inhibitors (Table S-1, Supporting Information). CONCLUSION Our solid-phase aggregation system proves to be a valued platform by processing and identifying aggregation in well with higher sensitivity and reliability. The platform could be used for a complete and comprehensive study of Aβ aggregation processes under varying conditions with semicontinuous and near-real-time measurements, offering consistent and compatible antiaggregation determinations. ACKNOWLEDGMENT This work was supported by research grant to Prof. J.-W. Chern from National Science Council of Taiwan (NSC96-2320-B-002-017 and NSC97-2323-B-002-010). SUPPORTING INFORMATION AVAILABLE Method for the preparation of lyophilized recombinant Histagged Aβ1-42, Figures S-1-S-5, and Table S-1. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review May 10, 2009. Accepted July 8, 2009. AC901011E


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An Improved Screening Model To Identify Inhibitors Targeting Zinc

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