Systematic identification of antiprion drugs by high-throughput screening based on scanning for intensely fluorescent targets

Abstract

Conformational changes and aggregation of specific proteins are hallmarks of a number of diseases, like Alzheimer's disease, Parkinson's disease, and prion diseases. In the case of prion diseases, the prion protein (PrP), a neuronal glycoprotein, undergoes a conformational change from the normal, mainly alpha-helical conformation to a disease-associated, mainly beta-sheeted scrapie isoform (PrP(Sc)), which forms amyloid aggregates. This conversion, which is crucial for disease progression, depends on direct PrP(C)/PrP(Sc) interaction. We developed a high-throughput assay based on scanning for intensely fluorescent targets (SIFT) for the identification of drugs which interfere with this interaction at the molecular level. Screening of a library of 10,000 drug-like compounds yielded 256 primary hits, 80 of which were confirmed by dose response curves with half-maximal inhibitory effects ranging from 0.3 to 60 microM. Among these, six compounds displayed an inhibitory effect on PrP(Sc) propagation in scrapie-infected N2a cells. Four of these candidate drugs share an N'-benzylidene-benzohydrazide core structure. Thus, the combination of high-throughput in vitro assay with the established cell culture system provides a rapid and efficient method to identify new antiprion drugs, which corroborates that interaction of PrP(C) and PrP(Sc) is a crucial molecular step in the propagation of prions. Moreover, SIFT-based screening may facilitate the search for drugs against other diseases linked to protein aggregation.

FIG. 1.

Schematic representations of the assay components without and with therapeutic compound, respectively. (A and B) Antibodies against human PrP are depicted as red Ys. Mouse rPrP is symbolized by green cubes. Human PrP^Sc aggregates are drawn as chains of pink cubes. The white circle indicates the laser focus that is used to scan the assay mixture. In B, therapeutic compounds are symbolized by blue spheres. (C and D) Two-dimensional fluorescence intensity distribution histograms without and with 17 μM DOSPA, respectively. Red fluorescence intensity is given on the vertical axis, and the green fluorescence intensity is given on the horizontal axis as photons/bin. The frequency of bins with identical red and green intensities is color coded, ranging from yellow for single events through blue and green up to white for increasing bin numbers. The presence of the inhibitory compound DOSPA shifts the high-intensity events away from the “green” sectors at the bottom to the “red” sectors at the left, as less green-labeled rPrP probe is bound to the PrP^Sc aggregates.

FIG. 2.

Primary SIFT screening. (A) Fluorescence intensity distribution of a typical primary screening experiment for 80 compounds and eight control reactions. The number of high-intensity bins found in each of the 18 sectors (cf. Fig. 1) is plotted for each compound and control reaction. Intensity distributions of screened compounds are shown in grey, control samples containing no antiprion compounds are shown in green, and controls containing 17 μM DOSPA are shown in red. Controls containing only rPrP and MAb mix are shown in blue. (B) SIFT primary activity values for the DIVERSet1 library compounds (solid), negative controls (dotted), and positive DOSPA controls (dashed). The activities of negative and positive controls are narrowly distributed around 0 and 1, respectively, and are clearly separated. The threshold value to define an active compound was set to 0.5.

FIG. 3.

Inhibition of PrP^Sc formation in scrapie-infected N2a cells. (A, B) ScN2a cells were treated for 2 days with compounds at 15 μM (A) or 10 μM (B). PrP present in the detergent-soluble (S) and detergent-insoluble (P) fractions, respectively, was analyzed by Western blotting. The relative amount of the cytosolic chaperone Hsp70 present in the detergent-soluble fraction was analyzed in parallel (B, a-Hsp70). Incubation with DOSPA served as a positive control for antiprion activity.

FIG. 4.

Dose response analysis of new antiprion drugs. (A) Selected compounds were tested at different concentrations as described in the legend of Fig. 3. The detergent-insoluble fraction was treated with PK prior to Western blot analysis. (B) Quantitative analysis of the experiments shown in A. To quantify the reduction of PrP^Sc by the different compounds, the relative amount of PrP^Sc present in control cells was set at 100%. Reduction was calculated from three independent experiments.

FIG. 5.

Compounds with cell culture activity. The molecular structures of these substances and their activities in the three steps of the screening are shown. The first column shows the activities in the primary SIFT screening (SIFT prim. activ.). All these substances were validated in SIFT dilution series, and, where possible, EC₅₀ values were determined. The last column combines the results of several steps in the cell culture system. Four of these compounds share an N′-benzylidene-benzohydrazide core.

FIG. 6.

Structure-activity relationships for N′-benzylidene-benzohydrazide derivatives. SIFT primary activities of eight substance classes are shown. The substance classes are characterized in that they contain the depicted structure motifs. The boxes and large vertical bars within the strongly occupied activity distributions mark the median and the quartiles; that statistic has been omitted for classes containing only a few members.