Binding of the radioligand SIL23 to α-synuclein fibrils in Parkinson disease brain tissue establishes feasibility and screening approaches for developing a Parkinson disease imaging agent

Abstract

Accumulation of α-synuclein (α-syn) fibrils in Lewy bodies and Lewy neurites is the pathological hallmark of Parkinson disease (PD). Ligands that bind α-syn fibrils could be utilized as imaging agents to improve the diagnosis of PD and to monitor disease progression. However, ligands for α-syn fibrils in PD brain tissue have not been previously identified and the feasibility of quantifying α-syn fibrils in brain tissue is unknown. We report the identification of the (125)I-labeled α-syn radioligand SIL23. [(125)I]SIL23 binds α-syn fibrils in postmortem brain tissue from PD patients as well as an α-syn transgenic mouse model for PD. The density of SIL23 binding sites correlates with the level of fibrillar α-syn in PD brain tissue, and [(125)I]SIL23 binding site densities in brain tissue are sufficiently high to enable in vivo imaging with high affinity ligands. These results identify a SIL23 binding site on α-syn fibrils that is a feasible target for development of an α-syn imaging agent. The affinity of SIL23 for α-syn and its selectivity for α-syn versus Aβ and tau fibrils is not optimal for imaging fibrillar α-syn in vivo, but we show that SIL23 competitive binding assays can be used to screen additional ligands for suitable affinity and selectivity, which will accelerate the development of an α-syn imaging agent for PD.

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 K_d 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 K_d value was determined by fitting the data to the equation Y = B_max*X/(X+K_d). 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 Aβ_1–42 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 K_d 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.

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. K_d values for binding to PD-dementia brain samples range from 119.1 nM to 168.3 nM and B_max 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.

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 B_max 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 B_max 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.

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 K_d of 150.3 nM and a B_max of 65 pmol/mg insoluble protein. No significant [¹²⁵I]SIL23 binding was detected in M7 brain. The results were verified with two independent experiments.