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.