Identification and structural characterization of small molecule inhibitors of PINK1.

Mutations in PINK1 and Parkin cause early-onset Parkinson's Disease (PD). PINK1 is a kinase which functions as a mitochondrial damage sensor and initiates mitochondrial quality control by accumulating on the damaged organelle. There, it phosphorylates ubiquitin, which in turn recruits and activates Parkin, an E3 ubiquitin ligase. Ubiquitylation of mitochondrial proteins leads to the autophagic degradation of the damaged organelle. Pharmacological modulation of PINK1 constitutes an appealing avenue to study its physiological function and develop therapeutics. In this study, we used a thermal shift assay with insect PINK1 to identify small molecules that inhibit ATP hydrolysis and ubiquitin phosphorylation. PRT062607, an SYK inhibitor, is the most potent inhibitor in our screen and inhibits both insect and human PINK1, with an IC50 in the 0.5-3 µM range in HeLa cells and dopaminergic neurons. The crystal structures of insect PINK1 bound to PRT062607 or CYC116 reveal how the compounds interact with the ATP-binding pocket. PRT062607 notably engages with the catalytic aspartate and causes a destabilization of insert-2 at the autophosphorylation dimer interface. While PRT062607 is not selective for PINK1, it provides a scaffold for the development of more selective and potent inhibitors of PINK1 that could be used as chemical probes.

Structural and Functional Characterization of the Most Frequent Pathogenic PRKN Substitution p.R275W.

Mutations in the PINK1 and PRKN genes are the most frequent genetic cause of early-onset Parkinson disease. The pathogenic p.R275W substitution in PRKN is the most frequent substitution observed in patients, and thus far has been characterized mostly through overexpression models that suggest a possible gain of toxic misfunction. However, its effects under endogenous conditions are largely unknown. We used patient fibroblasts, isogenic neurons, and post-mortem human brain samples from carriers with and without PRKN p.R275W to assess functional impact. Immunoblot analysis and immunofluorescence were used to study mitophagy activation, and mitophagy execution was analyzed by flow cytometry of the reporter mitoKeima. The functional analysis was accompanied by structural investigation of PRKN p.R275W. We observed lower PRKN protein in fibroblasts with compound heterozygous p.R275W mutations. Isogenic neurons showed an allele-dose dependent decrease in PRKN protein. Lower PRKN protein levels were accompanied by diminished phosphorylated ubiquitin and decreased MFN2 modification. Mitochondrial degradation was also allele-dose dependently impaired. Consistently, PRKN protein levels were drastically reduced in human brain samples from p.R275W carriers. Finally, structural simulations showed significant changes in the closed form of PRKN p.R275W. Our data suggest that under endogenous conditions the p.R275W mutation results in a loss-of-function by destabilizing PRKN.

Basal activity of PINK1 and PRKN in cell models and rodent brain.

The ubiquitin kinase-ligase pair PINK1-PRKN recognizes and transiently labels damaged mitochondria with ubiquitin phosphorylated at Ser65 (p-S65-Ub) to mediate their selective degradation (mitophagy). Complete loss of PINK1 or PRKN function unequivocally leads to early-onset Parkinson disease, but it is debated whether impairments in mitophagy contribute to disease later in life. While the pathway has been extensively studied in cell culture upon acute and massive mitochondrial stress, basal levels of activation under endogenous conditions and especially in vivo in the brain remain undetermined. Using rodent samples, patient-derived cells, and isogenic neurons, we here identified age-dependent, brain region-, and cell type-specific effects and determined expression levels and extent of basal and maximal activation of PINK1 and PRKN. Our work highlights the importance of defining critical risk and therapeutically relevant levels of PINK1-PRKN signaling which will further improve diagnosis and prognosis and will lead to better stratification of patients for future clinical trials.