Structure-activity relationship of cyanine tau aggregation inhibitors

Abstract

A structure-activity relationship for symmetrical cyanine inhibitors of human tau aggregation was elaborated using a filter trap assay. Antagonist activity depended on cyanine heterocycle, polymethine bridge length, and the nature of meso- and N-substituents. One potent member of the series, 3,3'-diethyl-9-methylthiacarbocyanine iodide (compound 11), retained submicromolar potency and had calculated physical properties consistent with blood-brain barrier and cell membrane penetration. Exposure of organotypic slices prepared from JNPL3 transgenic mice (which express human tau harboring the aggregation prone P301L tauopathy mutation) to compound 11 for one week revealed a biphasic dose response relationship. Low nanomolar concentrations decreased insoluble tau aggregates to half those observed in slices treated with vehicle alone. In contrast, high concentrations (> or =300 nM) augmented tau aggregation and produced abnormalities in tissue tubulin levels. These data suggest that certain symmetrical carbocyanine dyes can modulate tau aggregation in the slice biological model at concentrations well below those associated with toxicity.

Figure 1. Polymethine bridge length modulates inhibitory activity

Tau protein (4 μM) was incubated with ODS (50 μM) without agitation (24 h at 37°C) in the presence of either thiacarbocyanines 1 - 5 or DMSO vehicle alone, then assayed for aggregation by filter trap assay. (a) Concentration dependence of inhibition, where each point represents aggregation expressed as a normalized percentage of aggregation measured in the presence of DMSO vehicle alone (triplicate determination ± SD), and each solid line represents best fit of the data points to eq 4. All tested compounds were active, but less so than lead compound 1. (b) Replot of IC₅₀ values determined in Panel (a) versus bridge length for compounds 2 - 5. Inhibitory potency was maximal when the polymethine bridge consisted of three carbon atoms. *, p < 0.05; **, p < 0.01 when compared with compound 3 by Student’s t test.

Figure 2. Multiple heterocycles support inhibitory activity

Tau protein (4 μM) was incubated with ODS (50 μM) without agitation (24 h at 37°C) in the presence of either 3, 6, 7, 8 (composed of benzothiazole, benzoxazole, quinoline, and dimethylindole heterocycles, respectively) or DMSO vehicle alone, then assayed for aggregation by filter trap assay. Each point represents normalized aggregation relative to the DMSO vehicle control (mean of triplicate determination ± SD), whereas each solid line represents best fit of the data points to eq 4. All heterocycles except benzoxazole supported inhibitory activity under these conditions.

Figure 3. Meso- and N-substituents influence antagonist potency

Tau protein (4 μM) was incubated with ODS (50 μM) without agitation (24 h at 37°C) in the presence of either thiacarbocyanines 9 - 13 or DMSO vehicle alone, then assayed for aggregation by filter trap assay. Each point represents normalized aggregation relative to the DMSO vehicle control (mean of triplicate determination ± SD), whereas each solid line represents best fit of the data points to eq 4. Data for compounds 1 and 3 are replotted from Figure 1 for comparison. Extension of the N-substituent beyond two carbons decreased potency, whereas introduction of a methyl meso substituent increased potency. The combination of benzothiazole nucleus, three carbon bridge, methyl meso substituent, and ethyl N-substituents yielded an inhibitor (compound 11) with the efficacy and potency of the starting compound 1, but with physicochemical properties more appropriate for biological experimentation.

Figure 4. Thiacarbocyanine 11 is a stronger aggregation antagonist than Methylene blue

Tau protein (4 μM) was incubated with ODS (50 μM) without agitation (24 h at 37°C) in the presence of either 11, Methylene blue, or DMSO vehicle alone, then assayed for aggregation. Each point represents normalized aggregation relative to the DMSO vehicle control (mean of triplicate determination ± SD), whereas each line represents best fit of the data points to eq 4. Compound 11 was equally efficacious when assayed by either filter trap assay (○) or direct measurement of total filament length by quantitative electron microscopy assays (●), confirming that inhibitory activity did not depend on assay modality. However, 11 was significantly more potent and efficacious than Methylene blue (■) (electron microscopy assay format).

Figure 5. Inhibitory potency does not correlate with cyanine aggregation propensity

When dissolved in methanol at 5 μM concentration, compounds 11 (a), 12 (b), and 8 (c) yield simple spectra (dotted lines) consistent with monomeric structure (M). In assembly buffer (solid lines), increasing concentrations of compounds 11 and 12, but not 8, revealed the presence of both monomeric (M) and aggregated (D) species, with the latter more pronounced at high concentrations. These data suggest that 11 and 12, but not 8, underwent aggregation in aqueous solution at the concentrations tested.

Figure 6. The principal cyanine aggregate formed in solution is a dimer

Concentrations of monomeric (C_m) and aggregate (C_t-C_m) forms of 11, 12, and 5 were calculated from absorbance spectra and replotted on double logarithmic scales. Each point represents a spectrum collected at a different bulk compound concentration, whereas each line represents best fit of the data points to a linear regression. Regression slopes for 11, 12, and 5 were 2.3 ± 0.1, 1.8 ± 0.1, and 1.7 ± 0.1, respectively, consistent with dimerization being mostly responsible for the spectral shifts of all three compounds.

Figure 7. Compound 11 inhibits tau aggregation in organotypic slices

Slice cultures prepared from JNPL3 transgenic mice were incubated with either DMSO vehicle control or varying concentrations of 11 for seven days, then extracted and analyzed for levels of sarkosyl-insoluble tau (a measure of tau aggregation). (a) Two representative immunoblots collected after incubation with DMSO vehicle control (C) or thiacarbocyanine 11 (T) are shown. The presence of 1 nM 11 reduced the amount of tau aggregation in the slices relative to DMSO vehicle control, whereas 1 μM 11 exacerbated aggregation. (b) Sarkosyl-insoluble tau immunoreactivity was quantified by densitometry (n = three observations) and plotted as a normalized percentage of aggregation measured in the presence of DMSO vehicle alone. Tau aggregation was suppressed at 11 concentrations below 100 nM, but was aggravated at higher concentrations (≥300 nm). *, p < 0.05; **, p < 0.01 when compared to slices treated with vehicle alone by Student’s t test.

Figure 8. Compound 11 does not induce apoptosis in organotypic slices

Slices prepared from JNPL3 transgenic mice were incubated with either DMSO vehicle control or varying concentrations of 11 for seven days, then extracted and analyzed for levels of α-tubulin and cleaved PARP1. (a) Representative immunoblots collected after incubation with DMSO vehicle control (C) or 11 (T) are shown. (b) Levels of cleaved PARP1 (hollow bar) and α-tubulin (solid bar) immunoreactivity were quantified by densitometry (n = three observations) and plotted as a normalized percentage relative to treatment with vehicle alone. Cleaved PARP1 (CP) levels were unaffected after exposure to 11, suggesting that executioner caspase activity was not activated even at the highest concentration tested. Similarly, efficacious concentrations of 11 (1 nM) did not modulate levels of α-tubulin immunoreactivity in tissue extracts. However, high concentrations of 11 associated with induction of tau aggregation (1 μM) led to marked decreases in α-tubulin immunoreactivity. *, p < 0.05; **, p < 0.01 when compared to slices treated with vehicle alone by Student’s t test.