Biology subjects

Murphy, E. J.

Publications and source records attributed to Murphy, E. J..

4 recordsLinked to original sources

bioRxiv2025

USP30 inhibition improves mitochondrial health through both PINK1-dependent and independent mechanisms

Mitochondrial dysfunction is a key feature of many pathologies, including Parkinsons disease. The selective vulnerability of dopaminergic neurons is thought to be influenced by mitochondrial dysfunction and mutations in the mitophagy regulating proteins PINK1 and Parkin that are known to cause early-onset Parkinsonism in an autosomal recessive manner. Augmentation of mitophagy through inhibition of USP30 may be a viable therapeutic strategy for a number of diseases including Parkinsons. USP30 inhibition has been demonstrated to augment PINK1/PRKN mitophagy but also potentiate basal mitophagy to support the removal of dysfunctional mitochondria. Therefore, long-term de-regulation of mitophagy has been proposed to lead to mitochondrial depletion. We have used an integrated approach across cell lines, primary neurons and iPSC-derived dopaminergic neuronal cultures to assess the short and long-term effects of USP30 inhibition on mitochondrial health and neuronal activity. We investigated the dependence of USP30 inhibition phenotypes on the PINK1/Parkin pathway using genetic ablation and in iPSC-derived neurons from Parkinsons patients with PINK1 or PRKN mutations. Loss of USP30 through CRISPR/Cas9-mediated knockout resulted in increased basal and depolarisation-induced mitophagy in SH-SY5Y cells. Loss of USP30 or pharmacological inhibition altered mitochondrial morphology and led to increases in membrane potential and ATP levels with decreased oxygen consumption, suggesting that USP30 loss results in a more efficient mitochondrial network. These changes in morphology were found to be independent of PINK1 or Parkin. Chronic pharmacological inhibition of USP30 or CRISPRi-mediated knockdown of USP30 did not impact dopaminergic neuronal activity, as assessed by electrophysiological profiling, but did potentiate depolarisation-induced mitophagy in primary and iPSC-derived neuronal cultures. We observed minimal changes in mitophagy levels in iPSC-derived dopaminergic neurons from Parkinsons patients with PINK1 or PRKN mutations that were independent of the ability to produce p65Ub. Importantly, within this experimental paradigm, pharmacological USP30 inhibition increased depolarisation-induced mitophagy in both PINK1 and PRKN patients to the same extent as control neurons. These results support a role for USP30 in modulating the trigger threshold for mitophagy and suggest that USP30 inhibitors may be beneficial in patients with impairments in PINK1/Parkin-mediated mitophagy.

neuroscience↗

bioRxiv2024

Structural Dynamics of the Ubiquitin Specific Protease USP30 in Complex with a Cyanopyrrolidine-Containing Covalent Inhibitor

Inhibition of the mitochondrial deubiquitinating enzyme USP30 is neuroprotective and presents therapeutic opportunities for the treatment of idiopathic Parkinsons Disease and mitophagy-related disorders. We have integrated structural and quantitative proteomics with biochemical assays to decipher the mode of action of covalent USP30 inhibition by a small molecule containing a cyanopyrrolidine reactive group, USP30-I-1. The inhibitor demonstrated high potency and selectivity for endogenous USP30 in neuroblastoma cells. Enzyme kinetics and Hydrogen Deuterium eXchange mass spectrometry (HDX-MS) infers that the inhibitor binds tightly to regions surrounding the USP30 catalytic cysteine and positions itself to form a binding pocket along the thumb and palm domains of the protein, thereby interfering its interaction with ubiquitin substrates. A comparison to a non-covalent USP30 inhibitor containing a benzosulfonamide scaffold revealed a slightly different binding mode closer to the active site Cys77, which may provide the molecular basis for improved selectivity towards USP30 against other members of the DUB enzyme family. Our results highlight advantages in developing covalent inhibitors, such as USP30-I-1, for targeting USP30 as treatment of disorders with impaired mitophagy.

biochemistry↗

bioRxiv2023

Regulation of inositol 5-phosphatase activity by the C2 domain of SHIP1 and SHIP2

SHIP1, an inositol 5-phosphatase, plays a central role in cellular signalling. As such, it has been implicated in many conditions. Exploiting SHIP1 as a drug target will require structural knowledge and the design of selective small molecules. We have determined apo, and magnesium and phosphate-bound structures of the phosphatase and C2 domains of SHIP1. The C2 domains of SHIP1 and the related SHIP2 modulate the activity of the phosphatase domain. To understand the mechanism, we performed activity assays, hydrogen-deuterium-exchange mass spectrometry and molecular dynamics on SHIP1 and SHIP2. Our findings demonstrate that the influence of the C2 domain is more pronounced for SHIP2 than SHIP1. We determined 91 structures of SHIP1 with fragments bound, with some near the interface between the two domains. We performed a mass spectrometry screen and determined four structures with covalent fragments. These structures could act as starting points for the development of potent, selective probes.

biochemistry↗

bioRxiv2022

Structural Premise of Selective Deubiquitinase USP30 Inhibition by Small-Molecule Benzosulfonamides

Dampening functional levels of the mitochondrial deubiquitylating enzyme USP30 has been suggested as an effective therapeutic strategy against neurodegenerative disorders such as Parkinsons Disease. USP30 inhibition may counteract the deleterious effects of impaired turnover of damaged mitochondria which is inherent to both familial and sporadic forms of the disease. Small-molecule inhibitors targeting USP30 are currently in development, but little is known about their precise nature of binding to the protein. We have integrated biochemical and structural approaches to gain novel mechanistic insights into USP30 inhibition by a small-molecule benzosulfonamide containing compound, 39. Activity-based protein profiling (ABPP) mass spectrometry confirmed target engagement, the high selectivity, and potency of 39 for USP30 against 49 other deubiquitylating enzymes in a neuroblastoma cell line. In vitro characterization of 39 enzyme kinetics infers slow and tight binding behavior, which is comparable with features of covalent modification of USP30. Finally, we blended hydrogen-deuterium exchange mass spectrometry and computational docking to elucidate the molecular architecture and geometry of USP30 complex formation with 39, identifying structural rearrangements at the cleft of the USP30 thumb and palm subdomains. These studies suggest that 39 binds to the thumb-palm cleft that guides the ubiquitin C-terminus into the active site, thereby preventing ubiquitin binding and isopeptide bond cleavage, and confirming its importance in the inhibitory process. Our data will pave the way for the design and development of next-generation inhibitors targeting USP30 and associated deubiquitinylases.

biochemistry↗