A dual druggable genome-wide siRNA and compound library screening approach identifies modulators of parkin recruitment to mitochondria

Abstract

Genetic and biochemical evidence points to an association between mitochondrial dysfunction and Parkinson's disease (PD). PD-associated mutations in several genes have been identified and include those encoding PTEN-induced putative kinase 1 (PINK1) and parkin. To identify genes, pathways, and pharmacological targets that modulate the clearance of damaged or old mitochondria (mitophagy), here we developed a high-content imaging-based assay of parkin recruitment to mitochondria and screened both a druggable genome-wide siRNA library and a small neuroactive compound library. We used a multiparameter principal component analysis and an unbiased parameter-agnostic machine-learning approach to analyze the siRNA-based screening data. The hits identified in this analysis included specific genes of the ubiquitin proteasome system, and inhibition of ubiquitin-conjugating enzyme 2 N (UBE2N) with a specific antagonist, Bay 11-7082, indicated that UBE2N modulates parkin recruitment and downstream events in the mitophagy pathway. Screening of the compound library identified kenpaullone, an inhibitor of cyclin-dependent kinases and glycogen synthase kinase 3, as a modulator of parkin recruitment. Validation studies revealed that kenpaullone augments the mitochondrial network and protects against the complex I inhibitor MPP+. Finally, we used a microfluidics platform to assess the timing of parkin recruitment to depolarized mitochondria and its modulation by kenpaullone in real time and with single-cell resolution. We demonstrate that the high-content imaging-based assay presented here is suitable for both genetic and pharmacological screening approaches, and we also provide evidence that pharmacological compounds modulate PINK1-dependent parkin recruitment.

Urolithin A improves muscle strength, exercise performance, and biomarkers of mitochondrial health in a randomized trial in middle-aged adults

“…Enzymes such as UBE2N and UBE2R2 were previously shown to be essential for PINK1/Parkin-mediated mitophagy. 41 , 42 A trend toward the increase in these mitophagy-related proteins was observed also at the 1,000 mg dose ( Figure S5 D). Protein levels of the PINK1/Parkin-independent mitophagy regulator BNIP3 decreased in the 1,000 mg cohort ( Figure S5 E; Table S2 ), indicating a specific effect of UA on the PINK1/Parkin-mediated mitophagy axis.…”

Structural Landscape of nsp Coding Genomic Regions of SARS-CoV-2-ssRNA Genome: A Structural Genomics Approach Toward Identification of Druggable Genome, Ligand-Binding Pockets, and Structure-Based Druggability

“…Druggable genome-wide therapeutic siRNA discovery is one of the effective approaches for developing antisense oligonucleotide therapeutics [ 41 – 43 ]. Cao et al have studied the SARS-CoV-2 RNA genome architecture and siRNA-based therapeutic design.…”

Cracking the Monoubiquitin Code of Genetic Diseases

“…This indicates that there is a need to develop novel strategies for targeted therapies of genetic diseases [125]. Several screens identified compounds activating PARKIN ubiquitin ligase activity [126] and enhancing mitophagy [127], such as the compound described in patent WO2018023029. While no in vivo validation is available for this compound yet, this demonstrates the feasibility of identification of E3 ligase activators, opening novel therapeutic options for patients with genetic disorders.…”