M., Teplow D. the rational identification of a range of candidate drugs directed against neurodegenerative disorders. model of A-mediated toxicity. RESULTS Chemical kinetics-based therapeutic strategies allow to combat A aggregation The drug discovery strategy that we describe in this work to target the aggregation of A42 consists of four main actions (Fig. 1). First, a fragment-based approach is applied to identify small molecules that could interfere with A aggregation (Fig. 1, step 1 1). Fragment-based drug design approaches are based on the screening of a limited number of small molecules to identify fragments with low binding affinities, typically with represents in each case either models We further evaluated the effects of bexarotene on the formation of toxic A42 species in a model of A42-mediated dysfunction, denoted GMC101 (termed the A worm model) (models of A42-mediated toxicity by preventing A42 aggregation.(A) Experimental procedure for the measurement of the effects of bexarotene around the frequency of body bends and on the quantity of aggregates in GMC101 (that is, the A worm model) and CL2122 (that is, the control worm model) models. Bexarotene was given to the worms at larval stages L1 and L4. (B) Measurements of the effect of increasing concentrations of bexarotene ranging from 5 to 10 M around the frequency of body bends in the A worm model. Normalized values with respect to day 0 are shown. The experimental data are shown for a single experiment but are representative in each case of three impartial experiments. Complete recovery of the motility of the A worm model can be observed at 10 M bexarotene; the inset shows the dose dependence of the effects of bexarotene on A worms at day 3 of adulthood. (C) L-165,041 In vivo imaging of aggregates stained using the amyloid-specific dye NIAD-4 in the absence and in the presence of 10 M bexarotene; images from days 6 and 9 only are shown for clarity. (D) Time course of the reaction of amyloid aggregates formed in the A worms in the absence and in the presence of 1 M bexarotene. Quantification of fluorescence intensity was performed using ImageJ software (see Materials and Methods). In all panels, error bars represent the SEM. (E) Insoluble fraction of the protein extracts from in the presence and in the absence of bexarotene with immunodetection of A and -tubulin (see Materials and Methods). We then explored the effect of increasing concentrations of bexarotene added at the larval stages of the life cycle (Fig. 6A) and observed a concentration-dependent maintenance of the motility of the A worm model. Indeed, the motility of the A worms Fst was maintained completely upon addition of 10 M bexarotene in two bursts at the L1 and L4 larval stages, that is, reached the level observed in the control worms (Fig. 4, A and B). In addition, imaging studies (see Materials and Methods) showed that this addition of 10 M bexarotene to the A worms suppressed the formation of A42 fibrils for 9 days of adulthood (Fig. 6D). The level of A42 expression in the A worms in the absence and in the L-165,041 presence of bexarotene was found to be closely comparable (Fig. 6E). This result indicates that maintaining the level of motility in the A worms could be achieved by preventing the aggregation of A42 by bexarotene. In accord with this conclusion, the addition of bexarotene did not show any effects in the control worm model (fig. S5A). The normal motility observed for the A worms in the presence of bexarotene could be consistent with two distinct scenarios. One possibility could be a strong delay in primary nucleation, which would substantially delay the aggregation of A42 and hence maintain the motility of the A worms to values similar to those of the control worms. Alternatively, these results could arise from a combination of effects on primary and secondary nucleation. This latter possibility is particularly relevant because although bexarotene preferentially inhibits primary nucleation, it could also affect secondary nucleation when present in excess. Indeed, our experiments using 5% seeds show that bexarotene is usually capable of acting on surface-catalyzed secondary nucleation as well as on primary nucleation (Fig. 4, F to H). Furthermore, we also observed that adding bexarotene only at day 2 did not show any detectable effect on the motility of the A worms (fig. S5, B and C), suggesting that this finding from the in vitro studies that bexarotene specifically affects nucleation events in A42 aggregation is also the L-165,041 case in vivo. DISCUSSION We have described a drug discovery strategy that aims at targeting A42 aggregation with small molecules. This strategy involves the identification of compounds from an A-specific fragment-based library and the subsequent application of chemical kinetics.