Figure 1.
Phenothiazine structures and radiosynthesis of [125I]SIL23.
Structures of SIL23 analogues are shown in A. The radiosynthesis of [125I]SIL23 is shown in B.
Figure 2.
The radioligand [125I]SIL23 binds recombinant α-syn fibrils with a Kd of 148 nM.
Fibrils prepared from recombinant α-syn were incubated with increasing concentrations of [125I]SIL23. Nonspecific binding was determined in parallel reactions utilizing 50 µM ThioT as competitor. A representative plot of specific binding versus [125I]SIL23 concentration is shown in A. Data points represent mean +/− s.d. (n = 3). The data was analyzed by curve fitting to a one-site binding model using nonlinear regression. The Kd value was determined by fitting the data to the equation Y = Bmax*X/(X+Kd). Scatchard analysis of binding is shown in B. Similar results were obtained in more than three independent experiments.
Figure 3.
SIL23 competitive binding assays demonstrate higher affinity binding to recombinant α-syn fibrils for additional phenothiazine analogues, indicating that the structure of this compound class can be optimized to improve binding affinity using SIL23 assays.
α-Syn fibrils were incubated with 200 nM [125I]SIL23 and increasing concentrations of SIL22 (A), SIL26 (B), SIL3B (C), and SIL5 (D). The amount of bound radioligand is plotted as a function of the concentration of unlabeled competitor ligand in the incubation mixture. Data points represent mean +/− s.d. (n = 3). EC50 values were determined by fitting the data to the equation Y = bottom+(top-bottom)/(1+10(x-logEC50)). Similar results were obtained in two independent experiments.
Figure 4.
Radioligand binding studies demonstrate that [125I]SIL23 has selectivity for binding recombinant α-syn fibrils compared to synthetic Aβ1–42 fibrils or recombinant tau fibrils.
Binding affinities of [125I]SIL23 to Aβ (A) and tau (B) fibrils were determined in saturation binding studies. The determined Kd values of Aβ and tau fibrils were 635 nM and 230 nM, respectively. Data points represent mean +/− s.d. (n = 3). Similar results were obtained in more than three independent experiments for saturation binding studies.
Table 1.
Comparison of Ki values for SIL analogues in assays with α-syn, Aβ1–42, and tau fibrils illustrates relative selectivity for α-syn over Aβ1–42 and tau.
Figure 5.
[125I]SIL23 exhibits specific binding to insoluble protein from human PD brain samples but not control human brain samples.
Homogenized insoluble fractions from human brain samples (n = 8) were incubated with increasing concentrations of [125I]SIL23. Nonspecific binding was determined in parallel reactions utilizing 50 µM ThioT as competitor. Representative plots of specific binding versus [125I]SIL23 concentration are shown. A–D show four different PD cases and E–H show four control cases. The data was analyzed by curve fitting to a one-site binding model using nonlinear regression. Kd values for binding to PD-dementia brain samples range from 119.1 nM to 168.3 nM and Bmax values range from 13–25 pmol/mg insoluble protein. No significant binding of [125I]SIL23 to control samples was observed. Results were verified with at least two independent experiments.
Table 2.
Clinical and demographic information for autopsy cases utilized for binding studies.
Figure 6.
Binding of [125I]SIL23 is correlated with levels of insoluble α-syn present in PD brain.
Levels of α-syn were measured in insoluble fractions from human brain samples (n = 10) by sequential extraction and western blot. A representative syn1 western blot with SDS extracts from PD and control cases is shown in A. A quantitative syn303 western blot with SDS extracts from PD cases is shown in B, in which higher molecular weight α-syn species (asterisk) are observed in combination with monomeric α-syn (arrow). The correlation of Bmax values for [125I]SIL23 binding to levels of total insoluble α-syn quantified from the monomer band on western blot is shown in C. The correlation of Bmax values for [125I]SIL23 binding to levels of total insoluble α-syn quantified from monomer plus high molecular weight species on western blot is shown in D. Results were verified with more than three independent experiments.
Table 3.
Comparison of Ki values for SIL analogues in assays with recombinant α-syn fibrils versus human PD tissue.
Table 4.
Ki values of previously reported ligands for α-syn fibrils determined in [125I]SIL23 competitive binding assays with recombinant α-syn fibrils and PD tissue.
Table 5.
Bmax values determined in saturation binding studies with PD brain tissue samples.
Figure 7.
[125I]SIL23 binding sites are present at high density in a transgenic A53T α-syn mouse model for PD.
[125I]SIL23 binding was determined in mouse brain samples obtained from (A) M83 mice with transgenic expression of human A53T α-syn, and (B) M7 mice with transgenic expression of human WT α-syn. Representative plots of specific binding versus [125I]SIL23 concentration are shown. The data was analyzed by curve fitting to a one-site binding model using nonlinear regression. [125I]SIL23 binding was detected in M83 brain samples with a Kd of 150.3 nM and a Bmax of 65 pmol/mg insoluble protein. No significant [125I]SIL23 binding was detected in M7 brain. The results were verified with two independent experiments.