Discovery of small-molecule positive allosteric modulators of Parkin E3 ligase

Abstract

Pharmacological activation of the E3 ligase Parkin represents a rational therapeutic intervention for the treatment of Parkinson's disease. Here we identify several compounds that enhance the activity of wildtype Parkin in the presence of phospho-ubiquitin and act as positive allosteric modulators (PAMs). While these compounds activate Parkin in a series of biochemical assays, they do not act by thermally destabilizing Parkin and fail to enhance the Parkin translocation rate to mitochondria or to enact mitophagy in cell-based assays. We conclude that in the context of the cellular milieu the therapeutic window to pharmacologically activate Parkin is very narrow.

Keywords: Biochemistry; Biochemistry Applications; Chemistry.

Graphical abstract

Figure 1

Primary assay development (A) Schematic of the biochemical assay including detection by an FK2 antibody, which recognizes polyubiquitin and provides an FRET signal to the FITC-labeled ubiquitin. The components of the reaction are depicted with circles. (B) Comparison of the FRET ratio of the WT-, W403A- and C431S-Parkin after 1 h incubation. (C) Silver stain indicating that W403A-Parkin was more active than WT-Parkin in the presence of 1 μM pUb, as indicated by the disappearance of the Parkin band and the appearance of a high-molecular weight smear. (D) Dependency of WT- (blue) and W403A-Parkin (red) activity on pUb concentration. The C431S-Parkin (yellow) is inactive at all pUb concentrations. (E) Titration of pUb in TR-FRET assay with WT-Parkin (blue) at a 3 h reaction time and calculation of Ec₅₀. Each data point represents 2 technical replicates. (F) At a given time (i.e., 180 min), we can enable a large window between WT-Parkin (blue) and the W403A-Parkin positive control (red). Each data point represents 2 technical replicates. Statistical test performed: ANOVA with Dunnett’s MCT versus WT ∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. For panel B, error bars represent standard deviation with n = 3 technical replicates. See also Figure S1.

Figure 2

High-throughput screen for Parkin activators (A) Structural similarity map based on Unity fingerprints demonstrating that there are more than 6 clusters of hits, with a singleton, BIO-1741376 (THPP series). Compounds with an E_max> 75% are designated as green circles and compounds with an E_max 45–75% are designated as orange circles. The lead THPP compound BIO-1741376 is identified as a singleton. (B) Dose-response curve for cluster A representative compound, BIO-0030130. Each point on the curve represents n = 2 technical replicates. BIO-0030130 is a racemic mixture whose active enantiomer BIO-1953559 is detailed in Figure S2C. (C) Dose-response curve for cluster B representative compound BIO-0550701; each point on the curve represents n = 2 technical replicates. (D) Dose-response curve for THPP singleton, BIO-1741376; each point on the curve represents n = 2 technical replicates. Concentration response curves in n = 3 biological replicates for these compounds are shown in Figure S2C. (E) Confirmation of FRET assay using Western blot with increasing concentrations of cluster A compound. Smears indicate polyubiquitination of Parkin, and were measured after 5 min reaction times, while TR-FRET reactions were carried out over a 2 h time course at 37°C. (F) Same as E for cluster B representative compound BIO-0550701. (G) Western blot demonstrating that cluster A and THPP compound BIO-1967174 not only autoubiquitinate Parkin, but can enhance the rate of monoubiquitination of Miro1 substrate to a level comparable to that of W403A-Parkin. Note that PRK8 antibody does not recognize W403A-Parkin as previously noted (Zhu et al., 2018). Western blots represent n = 2 biological replicates. See also Figure S2 and Table S1.

Figure 3

THPP series activity in biochemical assays (A) BIO-2007817 structure and TR-FRET concentration response curve. (B and C) (B) Parkin autoubiquitination and (C) Miro1 ubiquitination induced by increasing concentrations of BIO-2007817. W403A-Parkin is shown as a positive control. (D) BIO-2007818 structure and concentration response curve. (E and F)(E) Parkin autoubiquitination and (F) Miro1 ubiquitination induced by increasing concentrations of BIO-2007818. (G) BIO-1984542 structure and concentration response curve. (H and I) (H) Parkin autoubiquitination and (I) Miro1 ubiquitination induced by increasing concentrations of BIO-1984542. Concentration response curves represent n = 2 technical replicates. Western blots represent n = 1 biological replicates. See also Table 1.

Figure 4

Activator compounds directly engage Parkin in the presence of pUb (A) Gel shift upon formation of Ub-VS linked Parkin demonstrating that THPP compounds retain the same activity ranking in the Ub-VS charging assay as in the biochemical assay. Reactions were run for 2 h (WT-Parkin). (B) Quantitation of the % charged with respect to compound concentration in (A). This panel represents n = 1 technical replicates, but n = 3 biological replicates is shown in Figure S3A and quantitated in Figure S3C. Activating mutant W403A-Parkin is also activated by compounds in the presence of pUb and with the same activity ranking as in the biochemical assay. Reactions were run for 1 h (W403A-Parkin). (C and D) (C) Gel shift upon formation of Ub-VS linked W403A-Parkin after 1 hr incubation. (D) Quantitation of the % Parkin charged with respect to compound concentrations in (C). This panel represents n = 1 technical replicates, but n = 3 biological replicates is shown in Figure S3B and quantitated in Figure S3D. Compound activation hierarchy is preserved in both cases. (E) C431S-Parkin is not responsive to activating compound BIO-2007817 over a range of pUb concentrations. (F) At a fixed concentration of 200 μM BIO-2007817, WT-Parkin charging is steeply dependent on pUb concentrations with EC₅₀ = 1.8 ± 0.4 μM. (G) At a fixed concentration of 200 μM BIO-2007817, W403A-Parkin charging is dependent on pUb concentrations, and activation occurs at lower pUb concentrations in the presence of the compound, as evidenced by a reduction in EC₅₀ for pUb (EC₅₀^-‘817 = 5.4 ± 0.3 μM vs EC₅₀^+‘817 = 1.6 ± 0.2 μM). (H) Quantitation of % charged with respect to compound concentrations in (E). (I) Quantitation of % charged with respect to pUb concentrations in (F). (J) Quantitation of % charged with respect to pUb concentrations in (G). Each experiment in (E), (F) and (G) represents n = 1 technical and biological replicates. See also Figures S3, S4, and S6.

Figure 5

Compounds do not affect the rate of Parkin translocation to mitochondria nor the number of mitochondria within lysosomes (A) The half-max time (T₅₀) for W403A-Parkin translocation to mitochondria is within 40 min, the half-max time for WT- Parkin is 90 min, and the half-max time for C431A Parkin is never reached (inactive). (B) The translocation time for W403A-Parkin is 56% faster than WT-Parkin. (C) Calculated fold change in T₅₀ relative to compound concentration for active (green), partially active (orange) and inactive (gray) THPP series compounds. (D–F)(D) BIO-2007817, (E) BIO-2007818 and (F) BIO-1984542 (10 μM) translocation curves relative to control (DMSO with CCCP treatment). (G) Baseline imaging for MitoQC in SH-SY5Y cells. MitoQC is a pH-sensitive mitochondrial fluorescent probe. 24 h of 5 μM Antimycin +10μM Oligomycin stimulates an increased level of mCherry puncta signal indicating mitolysosomes, whose pH is neutralized with BafA1. (H) Quantitation of number of mitochondria in lysosomes under different conditions described in (G). (I) At a fixed timepoint (8 h) there is no dose-dependent increase in mitolysosomes for the active compound (green) relative to the reduced activity compound (orange) or inactive compound (gray). In A, n = 2 technical replicates. In C, n = 3 biological replicates. In (D)–(F) n = 2 technical replicates and n = 3 biological replicates. See also Figure S5.