ABSTRACT
Cyclic GMP-AMP synthase (cGAS) is the primary sensor for aberrant intracellular dsDNA producing the cyclic dinucleotide cGAMP, a second messenger initiating cytokine production in subsets of myeloid lineage cell types. Therefore, inhibition of the enzyme cGAS may act anti-inflammatory. Here we report the discovery of human-cGAS-specific small-molecule inhibitors by high-throughput screening and the targeted medicinal chemistry optimization for two molecular scaffolds. Lead compounds from one scaffold co-crystallize with human cGAS and occupy the ATP- and GTP-binding active site. The specificity and potency of these drug candidates is further documented in human myeloid cells including primary macrophages. These novel cGAS inhibitors with cell-based activity will serve as probes into cGAS-dependent innate immune pathways and warrant future pharmacological studies for treatment of cGAS-dependent inflammatory diseases.
Subject(s)
Drug Discovery/methods , Enzyme Inhibitors/pharmacology , Nucleotidyltransferases/antagonists & inhibitors , Autoimmune Diseases/drug therapy , Autoimmune Diseases/immunology , Autoimmune Diseases/pathology , Cells, Cultured , Crystallography, X-Ray , DNA/immunology , DNA/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/therapeutic use , High-Throughput Screening Assays/methods , Humans , Immunity, Innate/drug effects , Interferons/immunology , Interferons/metabolism , Macrophages , Models, Molecular , Nucleotides, Cyclic/immunology , Nucleotides, Cyclic/metabolism , Nucleotidyltransferases/immunology , Nucleotidyltransferases/isolation & purification , Nucleotidyltransferases/metabolism , Primary Cell Culture , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolismABSTRACT
Catechol O-methyl transferase belongs to the diverse family of S-adenosyl-l-methionine transferases. It is a target involved in the treatment of Parkinson's disease. Here we present a fragment-based screening approach to discover noncatechol derived COMT inhibitors which bind at the SAM binding pocket. We describe the identification and characterization of a series of highly ligand efficient SAM competitive bisaryl fragments (LE = 0.33-0.58). We also present the first SAM-competitive small-molecule COMT co-complex crystal structure.
Subject(s)
Catechol O-Methyltransferase Inhibitors , S-Adenosylmethionine/metabolism , Animals , Binding Sites , Catechol O-Methyltransferase/chemistry , Humans , Kinetics , Mice , Models, Molecular , Protein Conformation , Pyrazoles/chemistry , Rats , S-Adenosylmethionine/chemistry , Structure-Activity Relationship , Thiazoles/chemistry , Triazoles/chemistryABSTRACT
A series of N-hydroxy-3-[3-(1-substituted-1H-benzoimidazol-2-yl)-phenyl]-acrylamides (5a-5ab) and N-hydroxy-3-[3-(1,4,5-trisubstituted-1H-imidazol-2-yl)-phenyl]-acrylamides (12a-s) were designed, synthesized, and found to be nanomolar inhibitors of human histone deacetylases. Multiple compounds bearing an N1-piperidine demonstrate EC(50)s of 20-100 nM in human A549, HL60, and PC3 cells, in vitro and in vivo hyperacetylation of histones H3 and H4, and induction of p21(waf). Compound 5x displays efficacy in human tumor xenograft models.
Subject(s)
Benzimidazoles/chemistry , Histone Deacetylase Inhibitors/chemistry , Histone Deacetylases/chemistry , Imidazoles/chemistry , Acetylation , Animals , Benzimidazoles/chemical synthesis , Benzimidazoles/pharmacology , Cell Line, Tumor , HL-60 Cells , Histone Deacetylase Inhibitors/chemical synthesis , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylases/metabolism , Humans , Imidazoles/chemical synthesis , Imidazoles/pharmacology , Mice , Mice, Nude , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
A series of N-(2-amino-5-substituted phenyl)benzamides (3-21) were designed, synthesized and evaluated for their inhibition of HDAC2 and their cytotoxicity in HCT116 cancer cells. Multiple compounds from this series demonstrated time-dependent binding kinetics that is rationalized using a co-complex crystal structure of HDAC2 and N-(4-aminobiphenyl-3-yl)benzamide (6).
Subject(s)
Benzamides/chemistry , Histone Deacetylase 2/antagonists & inhibitors , Histone Deacetylase Inhibitors/chemical synthesis , Benzamides/chemical synthesis , Benzamides/toxicity , Binding Sites , Catalytic Domain , Crystallography, X-Ray , HCT116 Cells , Histone Deacetylase 2/metabolism , Histone Deacetylase Inhibitors/chemistry , Histone Deacetylase Inhibitors/toxicity , Humans , Kinetics , Structure-Activity RelationshipABSTRACT
Modulation of the acetylation state of histones plays a pivotal role in the regulation of gene expression. Histone deacetylases (HDACs) catalyze the removal of acetyl groups from lysines near the N termini of histones. This reaction promotes the condensation of chromatin, leading to repression of transcription. HDAC deregulation has been linked to several types of cancer, suggesting a potential use for HDAC inhibitors in oncology. Here we describe the first crystal structures of a human HDAC: the structures of human HDAC8 complexed with four structurally diverse hydroxamate inhibitors. This work sheds light on the catalytic mechanism of the HDACs, and on differences in substrate specificity across the HDAC family. The structure also suggests how phosphorylation of Ser39 affects HDAC8 activity.
