The mitochondrial deubiquitylase USP30 negatively regulates the selective autophagy of damaged mitochondria. We present the characterisation of an N-cyano pyrrolidine compound, FT3967385, with high selectivity for USP30. We demonstrate that ubiquitylation of TOM20, a component of the outer mitochondrial membrane import machinery, represents a robust biomarker for both USP30 loss and inhibition. A proteomics analysis, on a SHSY5Y neuroblastoma cell line model, directly compares the effects of genetic loss of USP30 with chemical inhibition. We have thereby identified a subset of ubiquitylation events consequent to mitochondrial depolarisation that are USP30 sensitive. Within responsive elements of the ubiquitylome, several components of the outer mitochondrial membrane transport (TOM) complex are prominent. Thus, our data support a model whereby USP30 can regulate the availability of ubiquitin at the specific site of mitochondrial PINK1 accumulation following membrane depolarisation. USP30 deubiquitylation of TOM complex components dampens the trigger for the Parkin-dependent amplification of mitochondrial ubiquitylation leading to mitophagy. Accordingly, PINK1 generation of phospho-Ser65 ubiquitin proceeds more rapidly in cells either lacking USP30 or subject to USP30 inhibition.

Despite the ubiquitous use of phosphoramidite chemistry in the synthesis of biophosphates, catalytic asymmetric phosphoramidite transfer remains largely unexplored for phosphate ester synthesis. We have discovered that a tetrazole-functionalized peptide, in the presence of 10-Å molecular sieves, functions as an enantioselective catalyst for phosphite transfer. This chemistry in turn has been used as the key step in a streamlined synthesis of myo-inositol-6-phosphate. Mechanistic insights implicate phosphate as a directing group for a highly selective kinetic resolution of a protected inositol monophosphate. This work represents a distinct and efficient method for the selective catalytic phosphorylation of natural products.kinetic resolution | organocatalysis T he inositol phosphates and their phosphoinositide relatives are hubs for an extraordinary amount of biochemistry, and there appears to be no wane in the pace of discovery in this field (1-3). Many early efforts have focused on the biochemistry of the inositol lipids, whereas the last decade has seen a revival of interest in the myriad of inositol phosphates and polyphosphates not associated with the lipid bilayer (1). Total syntheses of these compounds and their analogs have played a critical role in enabling high-precision studies of their biochemistry (4). Historically, the strategy for synthesis in this area has been dominated by protecting group manipulations and classical resolution (5). Our laboratory endeavored to take a step forward in 2001, introducing a class of catalysts that rely on catalytic enantioselective transfer of P(V) reagents ( Fig. 1A) (6, 7). This work enabled access to a wide variety of myo-inositol phosphates (8), phosphatidyl-myo-inositol phosphates (9-11), and related analogs, as well as facilitating studies of their biochemistry (12-15).Although catalytic enantioselective P(V) chemistry continues to prove useful, we have observed limitations. First, the nearly exclusive requirement of phenyl substituents on the chlorophosphate P(V) reagent necessitates additional protecting group manipulations in many syntheses (8-11) due to reagent stability and the incompatibility of phenyl phosphate hydrolysis conditions with synthetic schemes. Despite their utility in concert with peptide-based catalysts, chlorophosphates are generally regarded as less reactive and less versatile reagents compared to their phosphorous(III) analogues. In fact, these realities were an impetus for the development of P(III) reagents for complex phosphate and polynucleotide synthesis (16). Second, from the standpoint of asymmetric, catalytic syntheses of phosphoinositides, the P(V) methodology has thus far failed to effectively differentiate the respective 4-or 6-positions of myo-inositol (Fig. 1B) (17), even though it is highly selective for differentiation of the enantiotopic 1-and 3-positions of the myo-inositol ring.We speculate that this discrepancy in 4∕6 vs 1∕3 desymmetrization is due to local structure of the reacting hydroxyl groups (Fig. 2). Whereas the...

Peptide-based catalysts have been applied to the enantioselective syntheses of the title compounds, with this being the first report of the synthesis of an ent-PI5P analogue. The key steps in the synthesis involve asymmetric phosphorylation catalysis. Additional maneuvers were developed with a protecting groups scheme that enabled efficient, streamlined syntheses of these important mediators of biochemical events.

The mitochondrial deubiquitylase USP30 negatively regulates the selective autophagy of damaged mitochondria. It has been proposed as an actionable target to alleviate the loss of function of the mitophagy pathway governed by the Parkinson's Disease associated genes PINK1 and PRKN. We present the characterisation of a N-cyano pyrrolidine derived compound, FT3967385, with high selectivity for USP30. The compound is well tolerated with no loss of total mitochondrial mass. We demonstrate that ubiquitylation of TOM20, a component of the outer mitochondrial membrane import machinery that directly interacts with USP30, represents a robust biomarker for both USP30 loss and inhibition. We have conducted proteomics analyses on a SHSY5Y neuroblastoma cell line model to directly compare the effects of genetic loss of USP30 with selective inhibition in an unbiased fashion. We have thereby identified a subset of ubiquitylation events consequent to mitochondrial depolarisation that are USP30 sensitive. Within responsive elements of the ubiquitylome, several components of the outer mitochondrial membrane transport (TOM) complex are most prominent. Thus, our data support a model whereby USP30 can regulate the availability of ubiquitin at the specific site of mitochondrial PINK1 accumulation following membrane depolarisation. In this model, USP30 deubiquitylation of TOM complex components dampens the trigger for the Parkin-dependent amplification of mitochondrial ubiquitylation leading to mitophagy. Accordingly, PINK1 generation of phospho-Ser65 Ubiquitin proceeds more rapidly and to a greater extent in cells either lacking USP30 or subject to USP30 inhibition.

When a ribosome stalls during translation, it runs the risk of collision with a trailing ribosome. Such an encounter leads to the formation of a stable di-ribosome complex, which needs to be resolved by a dedicated machinery. The initial stalling and the subsequent resolution of di-ribosomal complexes requires activity of Makorin and ZNF598 ubiquitin E3 ligases, respectively, through ubiquitylation of the eS10 and uS10 subunits of the ribosome. We have developed a specific small-molecule inhibitor of the deubiquitylase USP9X. Proteomics analysis, following inhibitor treatment of HCT116 cells, confirms previous reports linking USP9X with centrosome-associated protein stability but also reveals a loss of Makorin 2 and ZNF598. We show that USP9X interacts with both these ubiquitin E3 ligases, regulating their abundance through the control of protein stability. In the absence of USP9X or following chemical inhibition of its catalytic activity, levels of Makorins and ZNF598 are diminished, and the ribosomal quality control pathway is impaired.