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Inhibition of the mitochondrial deubiquitinating (DUB) enzyme USP30 is neuroprotective and presents therapeutic opportunities for the treatment of idiopathic Parkinson's disease and mitophagy-related disorders. We 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 indicated 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 noncovalent 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 toward 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.

Dampening functional levels of the mitochondrial deubiquitylating enzyme Ubiquitin-specific protease 30 (USP30) has been suggested as an effective therapeutic strategy against neurodegenerative disorders such as Parkinson's 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, USP30 inh . Activity-based protein profiling mass spectrometry confirmed target engagement, high selectivity, and potency of USP30 inh for USP30 against 49 other deubiquitylating enzymes in a neuroblastoma cell line. In vitro characterization of USP30 inh enzyme kinetics inferred 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 USP30 inh , identifying structural rearrangements at the cleft of the USP30 thumb and palm subdomains. These studies suggest that USP30 inh binds to this thumb-palm cleft, which 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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Methylation of lysine residues on histone tail is a dynamic epigenetic modification that plays a key role in chromatin structure and gene regulation. Members of the KDM5 (also known as JARID1) sub-family are 2-oxoglutarate (2-OG) and Fe 2+ -dependent oxygenases acting as histone 3 lysine 4 trimethyl (H3K4me3) demethylases, regulating proliferation, stem cell self-renewal, and differentiation. Here we present the characterization of KDOAM-25, an inhibitor of KDM5 enzymes. KDOAM-25 shows biochemical half maximal inhibitory concentration values of <100 nM for KDM5A-D in vitro, high selectivity toward other 2-OG oxygenases sub-families, and no off-target activity on a panel of 55 receptors and enzymes. In human cell assay systems, KDOAM-25 has a half maximal effective concentration of ∼50 μM and good selectivity toward other demethylases. KDM5B is overexpressed in multiple myeloma and negatively correlated with the overall survival. Multiple myeloma MM1S cells treated with KDOAM-25 show increased global H3K4 methylation at transcriptional start sites and impaired proliferation., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

By use of a structure-based computational method for identification of structurally novel Janus kinase (JAK) inhibitors predicted to bind beyond the ATP binding site, a potent series of indazoles was identified as selective pan-JAK inhibitors with a type 1.5 binding mode. Optimization of the series for potency and increased duration of action commensurate with inhaled or topical delivery resulted in potent pan-JAK inhibitor 2 (PF-06263276), which was advanced into clinical studies.

There is increasing interest in targeting histone N-methyl-lysine demethylases (KDMs) with small molecules both for the generation of probes for target exploration and for therapeutic purposes. Here we update on previous reviews on the inhibition of the lysine-specific demethylases (LSDs or KDM1s) and JmjC families of N-methyl-lysine demethylases (JmjC KDMs, KDM2-7), focusing on the academic and patent literature from 2014 to date. We also highlight recent biochemical, biological, and structural studies which are relevant to KDM inhibitor development.

We report the discovery of N-substituted 4-(pyridin-2-yl)thiazole-2-amine derivatives and their subsequent optimization, guided by structure-based design, to give 8-(1H-pyrazol-3-yl)pyrido[3,4-d]pyrimidin-4(3H)-ones, a series of potent JmjC histone N-methyl lysine demethylase (KDM) inhibitors which bind to Fe(II) in the active site. Substitution from C4 of the pyrazole moiety allows access to the histone peptide substrate binding site; incorporation of a conformationally constrained 4-phenylpiperidine linker gives derivatives such as 54j and 54k which demonstrate equipotent activity versus the KDM4 (JMJD2) and KDM5 (JARID1) subfamily demethylases, selectivity over representative exemplars of the KDM2, KDM3, and KDM6 subfamilies, cellular permeability in the Caco-2 assay, and, for 54k, inhibition of H3K9Me3 and H3K4Me3 demethylation in a cell-based assay.

N-Methylation of lysine and arginine residues has emerged as a major mechanism of transcriptional regulation in eukaryotes. In humans, N(ε)-methyllysine residue demethylation is catalysed by two distinct subfamilies of demethylases (KDMs), the flavin-dependent KDM1 subfamily and the 2-oxoglutarate- (2OG) dependent JmjC subfamily, which both employ oxidative mechanisms. Modulation of histone methylation status is proposed to be important in epigenetic regulation and has substantial medicinal potential for the treatment of diseases including cancer and genetic disorders. This article provides an introduction to the enzymology of the KDMs and the therapeutic possibilities and challenges associated with targeting them, followed by a review of reported KDM inhibitors and their mechanisms of action from kinetic and structural perspectives., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A potent inhibitor of the JmjC histone lysine demethylase KDM2A (compound 35 , pIC 50 7.2) with excellent selectivity over representatives from other KDM subfamilies has been developed; the discovery that a triazolopyridine compound binds to the active site of JmjC KDMs was followed by optimisation of the triazole substituent for KDM2A inhibition and selectivity.

A series of acidic triazoles with activity as soluble guanylate cyclase stimulators is described. Incorporation of the CF(3) triazole improved the overall physicochemical and drug-like properties of the molecule and is exemplified by compound 25., (Copyright © 2011 Elsevier Ltd. All rights reserved.)