Fig 1.
Chemical structures of YI compounds.
A chemical library of 26 fluorescent indolizine derivatives.
Fig 2.
In vitro assays to evaluate anti-amyloidogenic properties of indolizine-derived YI compounds.
(A) Aβ aggregation inhibition test by ThT assay. Aβ42 (50 μM) was incubated with or without YI compounds (0.5, 5, 50 μM) for three days. (B)Aβ aggregates dissociation test by ThT assay. Aβ42 aggregates (50 μM, 3-day pre-aggregation) were incubated with or without YI compounds (0.5, 5, 50 μM) for additional three days. (C) SDS-PAGE with PICUP and silver staining for disaggregated Aβ (50 μM) with the incubation of YI compounds (250 μM). Sizes of Aβ species according to the protein size markers are monomers (5 kDa), dimers (10 kDa), oligomers (15 to 75 kDa), and larger aggregates or fibrils (embedded at the top of the gels). Fluorescent intensities of all samples were normalized to 3-day Aβ aggregates data (100%). Denormalized data of ThT assay (S1 Fig) and whole gel images are shown in the (S2 Fig). Abbreviations: 3d = 3-day incubation of Aβ,– = Aβ monomer, + = 3-day incubation of Aβ, ++ = 3-day pre-incubation of Aβ and additional 3-day incubation of Aβ and/or compounds. Data represents the mean of triplicated experiments ± SEMs and one-way anova was applied followed by Bonferroni’s post-hoc comparison test (*P < 0.033, **P < 0.002, ***P < 0.001).
Fig 3.
Fluorescence spectra of YI compounds in the presence of Aβ42.
Selected 15 YI compounds (250 μM) were added to 25 μM Aβ42 monomers or aggregates obtained by 3-day incubation. Emission spectra of YI compounds with Aβ42 in 3:1 ratio were recorded with the individual excitation wavelength as following: YI-01, 362 nm; YI-02, 472 nm; YI-03, 330 nm; YI-04, 412 nm; YI-05, 320 nm; YI-07, 332 nm; YI-08, 310 nm; YI-12, 410 nm; YI-13, 394 nm; YI-14, 440 nm; YI-15, 475 nm; YI-16, 330 nm; YI-17, 486 nm; YI-22, 480 nm; YI-26, 415 nm. Fluorescent intensity of ThT (5 μM) in the presence of Aβ42 monomers or aggregates (25 μM) was measured as a control. Spectra of all samples were acquired using an Infinite 200 PRO plate reader. Abbreviations: Cpd. = compound, FI = fluorescence intensity, AU = arbitrary unit.
Fig 4.
Ex vivo analyses of fluorescent YI-13 to confirm it as an imaging agent targeting insoluble and soluble Aβ.
(A) A scheme of YI-13 ex vivo analyses. A brain hemisphere of aged mice was used for histochemistry (TG and WT, 12-month old, n = 2 and 2). Dissected regions of young mouse brain hemisphere (TG and WT, 5-month old, n = 3 and 3) were lysed to obtain soluble Aβ and verified by fluorescence scans and dot blots. The pellet fraction was further lysed in guanidine-hydrochloride buffer (GdnHCl) to obtain insoluble Aβ and verified by fluorescence scans. The illustration was drawn using Adobe Photoshop software. (B) Brain slides of TG co-stained by 6E10 and YI-13 (500 μM). Arrowheads indicate Aβ plaques (scale bars = 100 μm). (C-D) Soluble Aβ obtained by brain lysis of TG or WT mouse was analyzed. (C) Fluorescence spectral scan of YI-13 (250 μM) analyses (394/582 nm, ex/em). (D) Dot blot analyses. The levels of soluble Aβ were determined by using anti-Aβ antibody, 6E10. Original images of blotting analysis are shown in S5 Fig. (E) Insoluble Aβ obtained from pellet lysates was analyzed by fluorescence spectral scan of YI-13 (250 μM) (394/582 nm, ex/em). Blank in each scanning graph indicates YI compound only without any Aβ sample. The data collected from WT littermates is shown in the (S6 Fig). Abbreviations: HIP = hippocampus, CTX = cortex, WT = wild-type, TG = transgenic, AU = arbitrary unit.
Fig 5.
In vitro analysis of fluorescent YI-13 to detect its interaction with Aβ oligomers and dimers.
(A) A scheme of isolating oligomers from heterogeneous mixture of Aβ aggregates (50 μM). Through 100 kDa membrane filter, high molecular weight Aβ oligomers over 100 kDa were removed from the mixture of Aβ aggregates. The filtrate sample was applied to the 30 kDa filter to exclude low molecular weight Aβ aggregates. Only Aβ oligomers sized from 30 to 100 kDa were used for further experiment. The illustration was drawn using Adobe Photoshop software. (B-C) Fluorescence spectral scan of YI-13 (250 μM) when applied to Aβ. Blank indicates fluorescent intensities of YI-13 without Aβ. (B) Fluorescent analyses of YI-13 with the isolated Aβ oligomers. (C) Fluorescent analyses of YI-13 with the parallel Aβ dimer (25 μM). (D) Structure of synthesized parallel Aβ dimer with a lysine linker and spacer sequences, (GGGS)2. Abbreviations: FI = fluorescence intensity, AU = arbitrary unit.
Table 1.
Fluorescent properties of selected YI-13 with and without Aβ aggregates.
Fig 6.
A scheme of synthesizing new indolizines.
Fig 7.
A scheme of synthesizing YI-09.
Fig 8.
A scheme of synthesizing YI-10.
Fig 9.
A scheme of synthesizing YI-23.