Structure-Activity Relationships of Novel Tau Ligands: Passive Fibril Binders and Active Aggregation Inhibitors

Abstract

Intrinsically disordered proteins (IDPs) are core components of many biological processes and are central players in several pathologies. Despite being important drug targets, attempts to design small-molecule ligands that would help understand and attenuate their behavior are frustrated by the structural diversity exhibited by these flexible proteins. To accommodate the dynamic nature of IDPs, we developed a procedure that efficiently identifies active small-molecule ligands for disordered proteins. By exploring the chemical space around these ligands, we refined their effect on aggregation and identified molecular features critical for activity and affinity. Notably, the discovery of this new family of disordered protein ligands was achieved more quickly and with less expense than conventional high-throughput screening (HTS) or docking alone would have allowed. The resulting ligands include tau aggregation inhibitors as well as at least one compound that binds fibrils potently but does not appear to perturb the extent of kinetics of aggregation.

Figure 1.

Structures of Compounds 1 and 6, as well as examined analogs. Compound 1’s rings are labeled with red letters to facilitate discussion on chemical analogs.

Figure 2:

Effects of Compound 1 and analogs on tau4RD aggregation. A) ThT fluorescence-based aggregation assay showing compound activities at 10μM. Dose response of compounds 1.4 (panel B), 1.5 (C), and 1.7 (D) was assayed at concentrations of 1, 3, and 10μM, compared to vehicle control (black) and 10μM Compound 1 (blue). Data shows three technical replicates ± S.E.M. and is representative of three independent experiments.

Figure 3:

A) Compound 6 increases the ThT fluorescence signal associated with amyloid fibrils only when the protein aggregates. B) After overnight aggregation, the absorbance of compound 6 is notably decreased, consistent with binding to fibrillar aggregates.

Figure 4:

Estimation of Compound 6 affinity for Tau4RD fibrils. The points show the fraction of compound that co-precipitates with fibrils, plotted against the concentration of precipitated fibrils, for two different concentrations of Tau4RD and a range of heparin concentrations. The solid lines show fits to the model defined by equations 3–6. The recovered value of K_f (affinity of the compound towards Tau4RD fibril), along with uncertainties estimated by resampling (Supplementary Figure 4), are shown at the top of the graph.

Figure 5:

Top) ThT traces of Tau4RD with 10μM of selected compounds shows that compounds 6.1, 6.2, and 6.3 increase ThT fluorescence in a similar manner as compound 6. ThT data shows mean + SEM of 3 technical repeats and is representative of 3 independent experiments. Bottom) Gel densitometry of end-point samples illustrate that the amount of aggregation is not significantly different from vehicle samples, as would be suggested by ThT data.