An automated microliter-scale high-throughput screening system (MSHTS) for real-time monitoring of protein aggregation using quantum-dot nanoprobes

Protein aggregation is the principal component of numerous protein misfolding pathologies termed proteinopathies, such as Alzheimer’s disease, Parkinson’s disease, prion disease, and AA amyloidosis with unmet treatment needs. Protein aggregation inhibitors have great potential for the prevention and treatment of proteinopathies. Here we report the development of an automated real-time microliter-scale high throughput screening (MSHTS) system for amyloid aggregation inhibitors using quantum-dot nanoprobes. Screening 504 crude extracts and 134 low molecular weight aromatic compounds revealed the relationship of amyloid-β (Aβ) aggregation inhibitory activities of plant extracts using a plant-based classification. Within the eudicots, rosids, Geraniales and Myrtales showed higher activity. Screening low molecular weight aromatic compounds demonstrated that the structure of tropolone endows it with potential Aβ aggregation inhibitory activity. The activity of the most active tropolone derivative was higher than that of rosmarinic acid. MSHTS also identified three chaperone molecules as tau aggregation inhibitors. These results demonstrate that our automated MSHTS system is a novel and robust tool that can be adapted to a wide range of compounds and aggregation-prone polypeptides.

plate was incubated at 37 °C for 1 day, and observed under the same conditions as indicated above.
The SD value of the fluorescence intensity of each pixel was measured by the General Analysis program of NIS-Elements (Nikon) from ROI images (432 × 432 pixels). The half-maximal effective concentration (EC50) was estimated from the SD values by Prism software (GraphPad) using an EC50 shift by global fitting (Asymmetric sigmoidal, 5 parameter logistic) according to our previous method 1 .

Preparation of plant library
Five hundred and four plants growing in the wild or cultivated in Hokkaido were collected from June to November, 2004. Each was washed briefly, cut into small pieces using pruning shears and dried by hot-air at 50 °C for 24 h. Each dried material was powdered using a mixer before solvent extraction. Dried and powdered material (20 g for each sample) were extracted with 200 ml of 100% methanol at room temperature for 24 h. After filtration, the methanol solution was concentrated under reduced pressure to give a residue. Each crude extract was transferred to a 30 ml brown glass bottle and stored in a refrigerator at -20 °C. Average yields based on each plant's dry weight were about 25%. Each extract was dissolved in DMSO to a concentration of 100 mg/ml and the solution was stored in a refrigerator at -20 °C. A portion of each DMSO solution was used for several bioassays.
Preparation and management of drug solution of small molecule compound library Each compound was dissolved in DMSO as a stock solution at a concentration of 20 mM, identified by a code number, and registered in the searchable database created by ChemBioFinder Ultra 12.0. Both 20 mM stock solutions and the pure compounds were stored at -80 °C in an ultracold freezer and used for bioassays after thawing with warm (35 °C) water.

Construction of plasmids for the bacterial expression of tau MBD fragment
The tau cDNAs were cloned from a mouse cDNA library. DNA sequencing analysis revealed that the sequences were identical to tau (Accession No.: NM_010838). To construct expression plasmids for tau MBD fragments, a primer set for the tau gene (tau-forward; 5′-AGACATATGAGCAGCCCCGGCTC-3′, tau-reverse; 5′-CCCAAGCTTTCACAAACCCTGCTTGGCC-3′), both of which contained cleavage sites for NdeI and HindIII, was purchased from Hokkaido System Science. Thirty rounds of amplification were carried out using the following parameters: denaturation at 98 °C for 10 s, annealing at 55 °C for 15 s, and primer extension at 72 °C for 60 s, on a TaKaRa PCR Thermal Cycler SP (Takara Bio). The purified PCR products and pET-21a (+) vector were digested with NdeI and HindIII, and purified using NucleoTrap (Macherey-Nagel) after agarose gel electrophoresis. The vector and insert fragments were ligated using a DNA Ligation Kit (Takara Bio). The resultant plasmid expressed an N-terminal methionine residue, followed by a Pro-rich repeat, and tail regions of the genes for tau.

Preparation of tau MBD fragment
The bacterial expression and purification of tau MBD fragment was carried out as described previously 2 . Briefly, the expression plasmids were transformed into E. coli (Rosetta™ (DE3) pLys), and plasmid expression was induced by 1 mM isopropyl-1-thio-b-D-galactopyranoside. The heatstable fraction of each extract was subjected to successive column chromatographies using a Bio-Scale TM Mini UNOsphere TM S (Bio-rad) and a TOYOPEARL ® butyl column (Tosoh). Protein concentration was estimated using the method described by Lowry et al. (1951) 3 , using bovine serum albumin (BSA) as the standard. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to the method of Laemmli et al. (1970) 4 .
Sedimentation assay of tau protein 10 µM tau, 50 nM QDTau, and 10 µM heparin in PBS were incubated in the presence or absence of 10 mM DTT in microtubes for 24 h at 37 °C and centrifuged at 386,000 ×g for 15 min at 4 °C.
After removing the supernatant, the pellet was resuspended in the same volume of PBS.
Supernatants and resuspended pellets were electrophoresed on an SDS-polyacrylamide gel according to the method of Laemmli (1970) 4 .