ABSTRACT
EZH2 (enhancer of zeste homologue 2) is the catalytic subunit of the polycomb repressive complex 2 (PRC2) that catalyzes the methylation of lysine 27 of histone H3 (H3K27). Dysregulation of EZH2 activity is associated with several human cancers and therefore EZH2 inhibition has emerged as a promising therapeutic target. Several small molecule EZH2 inhibitors with different chemotypes have been reported in the literature, many of which use a bicyclic heteroaryl core. Herein, we report the design and synthesis of EZH2 inhibitors containing an indoline core. Partial saturation of an indole to an indoline provided lead compounds with nanomolar activity against EZH2, while also improving solubility and oxidative metabolic stability.
Subject(s)
Enhancer of Zeste Homolog 2 Protein/antagonists & inhibitors , Enzyme Inhibitors/chemical synthesis , Indoles/chemical synthesis , Animals , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Indoles/chemistry , Indoles/pharmacology , Mice , Microsomes, Liver/metabolism , Polycomb Repressive Complex 2/antagonists & inhibitors , Stereoisomerism , Structure-Activity RelationshipABSTRACT
There are three isoforms of dimeric nitric oxide synthases (NOS) that convert arginine to citrulline and nitric oxide. Inducible NOS is implicated in numerous inflammatory diseases and, more recently, in neuropathic pain states. The majority of existing NOS inhibitors are either based on the structure of arginine or are substrate competitive. We describe the identification from an ultra high-throughput screen of a novel series of quinolinone small molecule, nonarginine iNOS dimerization inhibitors. SAR studies on the screening hit, coupled with an in vivo lipopolysaccharide (LPS) challenge assay measuring plasma nitrates and drug levels, rapidly led to the identification of compounds 12 and 42--potent inhibitors of the human and mouse iNOS enzyme that were highly selective over endothelial NOS (eNOS). Following oral dosing, compounds 12 and 42 gave a statistical reduction in pain behaviors in the mouse formalin model, while 12 also statistically reduced neuropathic pain behaviors in the chronic constriction injury (Bennett) model.
Subject(s)
Drug Discovery , Fluoroquinolones/administration & dosage , Fluoroquinolones/pharmacology , Nitric Oxide Synthase Type II/antagonists & inhibitors , Pain/drug therapy , Protein Multimerization/drug effects , Pyrazines/administration & dosage , Pyrazines/pharmacology , Quinolones/administration & dosage , Quinolones/pharmacology , Administration, Oral , Animals , Cell Line , Constriction, Pathologic/chemically induced , Constriction, Pathologic/drug therapy , Disease Models, Animal , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Fluoroquinolones/chemistry , Fluoroquinolones/therapeutic use , Formaldehyde/toxicity , Humans , Inhibitory Concentration 50 , Lipopolysaccharides/toxicity , Mice , Nitric Oxide Synthase Type II/chemistry , Nitric Oxide Synthase Type II/metabolism , Protein Structure, Quaternary , Pyrazines/chemistry , Pyrazines/therapeutic use , Quinolones/chemistry , Quinolones/therapeutic use , Structure-Activity Relationship , Substrate SpecificityABSTRACT
We have identified and synthesized a series of thiophene containing inhibitors of kinesin spindle protein. SAR studies led to the synthesis of 33, which was co-crystallized with KSP and determined to bind to an allosteric pocket previously described for other known KSP inhibitors.
Subject(s)
Chemistry, Pharmaceutical/methods , Kinesins/antagonists & inhibitors , Thiophenes/chemistry , Allosteric Site , Amides/chemistry , Antimitotic Agents/pharmacology , Cell Line, Tumor , Crystallization , Crystallography, X-Ray , Drug Design , Humans , Inhibitory Concentration 50 , Kinesins/chemistry , Models, Chemical , Models, Molecular , Molecular ConformationABSTRACT
Anaphylatoxin C5a is a potent inflammatory mediator associated with pathogenesis and progression of several inflammation-associated disorders. Small molecule C5a receptor (C5aR) antagonist development is hampered by species-specific receptor biology and the associated inability to use standard rat and mouse in vivo models. Gerbil is one rodent species reportedly responsive to small molecule C5aR antagonists with human C5aR affinity. We report the identification of the gerbil C5aR cDNA using a degenerate primer PCR cloning strategy. The nucleotide sequence revealed an open reading frame encoding a 347-amino acid protein. The cloned receptor (expressed in Sf9 cells) bound recombinant human C5a with nanomolar affinity. Alignment of the gerbil C5aR sequence with those from other species showed that a Trp residue in transmembrane domain V is the only transmembrane domain amino acid unique to small molecule C5aR antagonist-responsive species (i.e. gerbil, human, and non-human primate). Site-directed mutagenesis was used to generate human and mouse C5aRs with a residue exchange of this Trp residue. Mutation of Trp to Leu in human C5aR completely eliminated small molecule antagonist-receptor interaction. In contrast, mutation of Leu to Trp in mouse C5aR enabled small molecule antagonist-receptor interaction. This crucial Trp residue is located deeper within transmembrane domain V than residues reportedly involved in C5a- and cyclic peptide C5a antagonist-receptor interaction, suggesting a novel interaction site(s) for small molecule antagonists. These data provide insight into the basis for small molecule antagonist species selectivity and further define sites critical for C5aR activation and function.
Subject(s)
Cell Membrane/chemistry , Gerbillinae , Receptor, Anaphylatoxin C5a/antagonists & inhibitors , Receptor, Anaphylatoxin C5a/chemistry , Tryptophan , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Cloning, Molecular , Gene Expression , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Humans , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , Rats , Receptor, Anaphylatoxin C5a/genetics , Sequence Alignment , Species Specificity , Structure-Activity RelationshipABSTRACT
On the basis of screening hits (1a,b), a series of selective, high affinity prostacyclin receptor antagonists was developed. The optimized lead compound 25d [(4,5-dihydro-1H-imidazol-2-yl)-[4-(4-isopropoxybenzyl)phenyl]amine] had analgesic activity in the rat.
Subject(s)
Aniline Compounds/chemical synthesis , Aniline Compounds/pharmacology , Imidazoles/chemical synthesis , Imidazoles/pharmacology , Receptors, Epoprostenol/antagonists & inhibitors , Administration, Oral , Animals , Binding, Competitive , Biological Assay , Biological Availability , Carrageenan/antagonists & inhibitors , Carrageenan/pharmacology , Humans , Hyperalgesia/chemically induced , Hyperalgesia/drug therapy , Mice , Rats , Structure-Activity RelationshipABSTRACT
A functional cell-based screen identified 3-(4-chlorophenyl)-3-(2-(dimethylamino)ethyl)isochroman-1-one hydrochloride (AC-7954, 1) as a nonpeptidic agonist of the urotensin-II receptor. Racemic 1 had an EC50 of 300 nM at the human UII receptor and was highly selective. Testing of the enantiopure (+)- and (-)- 1 revealed that the UII receptor activity of racemic 1 resides primarily in (+)-1. Being a selective nonpeptidic druglike UII receptor agonist, (+)-1 will be useful as a pharmacological research tool and a potential drug lead.
