ABSTRACT
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, USP30inh. Activity-based protein profiling mass spectrometry confirmed target engagement, high selectivity, and potency of USP30inh for USP30 against 49 other deubiquitylating enzymes in a neuroblastoma cell line. In vitro characterization of USP30inh 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 USP30inh, identifying structural rearrangements at the cleft of the USP30 thumb and palm subdomains. These studies suggest that USP30inh 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.
Subject(s)
Deubiquitinating Enzymes , Mitophagy , Deubiquitinating Enzymes/antagonists & inhibitors , Deubiquitinating Enzymes/metabolism , Mitochondrial Proteins/metabolism , Mitophagy/physiology , Ubiquitin-Protein Ligases/metabolism , Ubiquitination , Sulfonamides/pharmacologyABSTRACT
Residues in the histone substrate binding sites that differ between the KDM4 and KDM5 subfamilies were identified. Subsequently, a C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-one series was designed to rationally exploit these residue differences between the histone substrate binding sites in order to improve affinity for the KDM4-subfamily over KDM5-subfamily enzymes. In particular, residues E169 and V313 (KDM4A numbering) were targeted. Additionally, conformational restriction of the flexible pyridopyrimidinone C8-substituent was investigated. These approaches yielded potent and cell-penetrant dual KDM4/5-subfamily inhibitors including 19a (KDM4A and KDM5B Kiâ¯=â¯0.004 and 0.007⯵M, respectively). Compound cellular profiling in two orthogonal target engagement assays revealed a significant reduction from biochemical to cell-based activity across multiple analogues; this decrease was shown to be consistent with 2OG competition, and suggests that sub-nanomolar biochemical potency will be required with C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-one compounds to achieve sub-micromolar target inhibition in cells.
Subject(s)
Enzyme Inhibitors/pharmacology , Jumonji Domain-Containing Histone Demethylases/antagonists & inhibitors , Pyridines/pharmacology , Pyrimidinones/pharmacology , Cell Line, Tumor , Crystallography, X-Ray , Drug Screening Assays, Antitumor/methods , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Humans , Hydrophobic and Hydrophilic Interactions , Jumonji Domain-Containing Histone Demethylases/chemistry , Jumonji Domain-Containing Histone Demethylases/metabolism , Molecular Structure , Protein Binding , Pyridines/chemical synthesis , Pyridines/chemistry , Pyridines/metabolism , Pyrimidinones/chemical synthesis , Pyrimidinones/chemistry , Pyrimidinones/metabolism , Structure-Activity RelationshipABSTRACT
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 Fe2+-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 â¼50 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.
Subject(s)
Glycine/analogs & derivatives , Histones/metabolism , Niacinamide/analogs & derivatives , Retinoblastoma-Binding Protein 2/metabolism , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Crystallography, X-Ray , Glycine/chemistry , Glycine/metabolism , Glycine/pharmacology , HeLa Cells , Humans , Kaplan-Meier Estimate , Ketoglutaric Acids/chemistry , Ketoglutaric Acids/metabolism , Methylation , Multiple Myeloma/metabolism , Multiple Myeloma/mortality , Multiple Myeloma/pathology , Niacinamide/chemistry , Niacinamide/metabolism , Niacinamide/pharmacology , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/genetics , Protein Isoforms/metabolism , Pyridines/chemistry , Pyridines/metabolism , Pyridines/pharmacology , Retinoblastoma-Binding Protein 2/antagonists & inhibitors , Retinoblastoma-Binding Protein 2/genetics , Transcription Initiation SiteABSTRACT
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.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Heterocyclic Compounds, 2-Ring/pharmacology , Indazoles/pharmacology , Janus Kinases/antagonists & inhibitors , Lung Diseases/drug therapy , Protein Kinase Inhibitors/pharmacology , Skin Diseases/drug therapy , Administration, Cutaneous , Administration, Inhalation , Animals , Anti-Inflammatory Agents/administration & dosage , Anti-Inflammatory Agents/chemical synthesis , Anti-Inflammatory Agents/toxicity , Binding Sites , Crystallography, X-Ray , Dogs , Drug Design , Hepatocytes/metabolism , Heterocyclic Compounds, 2-Ring/administration & dosage , Heterocyclic Compounds, 2-Ring/chemical synthesis , Heterocyclic Compounds, 2-Ring/toxicity , Humans , Indazoles/administration & dosage , Indazoles/chemical synthesis , Indazoles/toxicity , Janus Kinase 1/antagonists & inhibitors , Janus Kinase 2/antagonists & inhibitors , Janus Kinase 3/antagonists & inhibitors , Mice, Inbred BALB C , Microsomes, Liver/metabolism , Phosphorylation , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/toxicity , Rats , SolubilityABSTRACT
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.
Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Jumonji Domain-Containing Histone Demethylases/antagonists & inhibitors , Nuclear Proteins/antagonists & inhibitors , Pyrimidinones/chemistry , Pyrimidinones/pharmacology , Repressor Proteins/antagonists & inhibitors , Caco-2 Cells , Cell Membrane Permeability , Enzyme Inhibitors/pharmacokinetics , Humans , Jumonji Domain-Containing Histone Demethylases/chemistry , Jumonji Domain-Containing Histone Demethylases/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Pyrimidinones/pharmacokinetics , Repressor Proteins/chemistry , Repressor Proteins/metabolismABSTRACT
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.
Subject(s)
Drug Discovery , Histone Demethylases/antagonists & inhibitors , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Amino Acid Sequence , Animals , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Histone Demethylases/chemistry , Histone Demethylases/metabolism , Humans , Models, Molecular , Molecular Sequence Data , Peptides/chemistry , Peptides/pharmacologyABSTRACT
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.
Subject(s)
Histone Demethylases/metabolism , Molecular Targeted Therapy/methods , Animals , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Enzyme Inhibitors/therapeutic use , Histone Demethylases/antagonists & inhibitors , Histone Demethylases/chemistry , Humans , Models, Molecular , Neoplasms/drug therapy , Neoplasms/metabolism , Protein BindingABSTRACT
A potent inhibitor of the JmjC histone lysine demethylase KDM2A (compound 35, pIC50 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.
ABSTRACT
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.
