ABSTRACT
A ((1S,2R)-2-hydroxy-2,3-dihydro-1H-inden-1-yl) succinamide derivative (here referred to as Compound 12) shows significant activity toward many matrix metalloproteinases (MMPs), including MMP-2, MMP-8, MMP-9, and MMP-13. Modeling studies had predicted that this compound would not bind to ADAMTS-5 (a disintegrin and metalloproteinase with thrombospondin motifs-5) due to its shallow S1' pocket. However, inhibition analysis revealed it to be a nanomolar inhibitor of both ADAMTS-4 and -5. The observed inconsistency was explained by analysis of crystallographic structures, which showed that Compound 12 in complex with the catalytic domain of ADAMTS-5 (cataTS5) exhibits an unusual conformation in the S1' pocket of the protein. This first demonstration that cataTS5 can undergo an induced conformational change in its active site pocket by a molecule like Compound 12 should enable the design of new aggrecanase inhibitors with better potency and selectivity profiles.
Subject(s)
ADAM Proteins/chemistry , Amides/chemistry , Protein Conformation , ADAMTS5 Protein , Animals , Catalytic Domain , Cattle , Drug Design , Humans , Matrix Metalloproteinase Inhibitors , Matrix Metalloproteinases/metabolism , Models, Molecular , Molecular Sequence Data , Molecular Structure , SuccinatesABSTRACT
Combinatorial cyclizations of imidates and hydrazides with methylene linked R groups, generated from the corresponding nitriles and carboxylic acids, respectively, provided a large library of 3,5-dimethylene substituted 1,2,4-trizoles. [formula: see text].
Subject(s)
Combinatorial Chemistry Techniques/methods , Methane/analogs & derivatives , Small Molecule Libraries/chemistry , Triazoles/chemistry , Carboxylic Acids/chemical synthesis , Carboxylic Acids/chemistry , Cyclization , Hydrazines/chemical synthesis , Hydrazines/chemistry , Imidoesters/chemical synthesis , Imidoesters/chemistry , Molecular Structure , Nitriles/chemical synthesis , Nitriles/chemistry , Small Molecule Libraries/chemical synthesis , Triazoles/chemical synthesisABSTRACT
A new 1,4-dihydropyridine 5a, containing a cyano group at the C3 position, was recently reported to possess excellent mineralocorticoid receptor (MR) antagonist in vitro potency and no calcium channel-blocker (CCB) activity. In the present study, we report the structure-activity relationships of this novel series of cyano ester dihydropyridines that resulted in R6 substituted analogues with improved metabolic stability while maintaining excellent MR antagonist activity and selectivity against other nuclear receptors. Further structure optimization with the introduction of five-membered ring heterocycles at R6 resulted in compounds with excellent MR antagonist potency and a suitable pharmacokinetic profile. In vivo studies of a promising tool compound in the Dahl salt-sensitive rat model of hypertension showed similar blood pressure (BP) reduction as the steroidal MR antagonist eplerenone, providing proof-of-concept (POC) for a nonsteroidal, orally efficacious MR antagonist.
Subject(s)
Antihypertensive Agents/chemical synthesis , Mineralocorticoid Receptor Antagonists , Nitriles/chemical synthesis , Pyridines/chemical synthesis , Animals , Antihypertensive Agents/pharmacokinetics , Antihypertensive Agents/pharmacology , Blood Pressure/drug effects , Male , Models, Molecular , Nitriles/pharmacokinetics , Nitriles/pharmacology , Pyridines/pharmacokinetics , Pyridines/pharmacology , Rats , Rats, Inbred Dahl , Rats, Sprague-Dawley , Stereoisomerism , Structure-Activity RelationshipABSTRACT
A number of known 1,4-dihydropyridine CCBs were identified as having comparable potency to the steroidal MR antagonist eplerenone. Chiral resolution of mebudipine revealed that MR and CCB activity reside in opposite enantiomers. Small molecule X-ray crystal structures showed that the C4 stereochemistry of optimized selective MR analogues, e.g. 5, is consistent with MR-active mebudipine. Molecular modeling supports a binding pose consistent with that previously proposed for DHP diesters.
Subject(s)
Dihydropyridines/chemistry , Mineralocorticoid Receptor Antagonists , Animals , Calcium Channel Blockers/chemistry , Calcium Channel Blockers/pharmacology , Calcium Channels, L-Type/metabolism , Cell Line , Crystallography, X-Ray , Dihydropyridines/pharmacology , Eplerenone , Genes, Reporter , Humans , Luciferases/genetics , Models, Molecular , Protein Binding , Rats , Receptors, Mineralocorticoid/chemistry , Receptors, Mineralocorticoid/genetics , Spironolactone/analogs & derivatives , Spironolactone/chemistry , Spironolactone/pharmacology , Stereoisomerism , Structure-Activity RelationshipABSTRACT
The classical nuclear factor kappaB (NF-kappaB) signaling pathway is under the control of the IkappaB kinase (IKK) complex, which consists of IKK-1, IKK-2, and NF-kappaB essential modulator (NEMO). This complex is responsible for the regulation of cell proliferation, survival, and differentiation. Dysregulation of this pathway is associated with several human diseases, and as such, its inhibition offers an exciting opportunity for therapeutic intervention. NEMO binding domain (NBD) peptides inhibit the binding of recombinant NEMO to IKK-2 in vitro. However, direct evidence of disruption of this binding by NBD peptides in biological systems has not been provided. Using a cell system, we expanded on previous observations to show that NBD peptides inhibit inflammation-induced but not basal cytokine production. We report that these peptides cause the release of IKK-2 from an IKK complex and disrupt NEMO-IKK-2 interactions in cells. We demonstrate that by interfering with NEMO-IKK-2 interactions, NBD peptides inhibit IKK-2 phosphorylation, without affecting signaling intermediates upstream of the IKK complex of the NF-kappaB pathway. Furthermore, in a cell-free system of IKK complex activation by TRAF6 (TNF receptor-associated factor 6), we show that these peptides inhibit the ability of this complex to phosphorylate downstream substrates, such as p65 and inhibitor of kappaB alpha (IkappaB alpha). Thus, consistent with the notion that NEMO regulates IKK-2 catalytic activity by serving as a scaffold, appropriately positioning IKK-2 for activation by upstream kinase(s), our findings provide novel insights into the molecular mechanisms by which NBD peptides exert their anti-inflammatory effects in cells.
Subject(s)
Anti-Inflammatory Agents/pharmacology , I-kappa B Kinase/metabolism , I-kappa B Kinase/pharmacology , Multiprotein Complexes/metabolism , Peptides/pharmacology , Transcription Factor RelA/metabolism , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/metabolism , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Cytokines/metabolism , Humans , I-kappa B Kinase/antagonists & inhibitors , I-kappa B Kinase/chemistry , Multiprotein Complexes/antagonists & inhibitors , Peptides/chemistry , Peptides/metabolism , Phosphorylation , Protein Binding/drug effects , Protein Structure, Tertiary , TNF Receptor-Associated Factor 6/metabolism , Transcription Factor RelA/antagonists & inhibitorsABSTRACT
Several inhibitors of a series of cis-1(S)2(R)-amino-2-indanol-based compounds were reported to be selective for the aggrecanases, ADAMTS-4 and -5 over other metalloproteases. To understand the nature of this selectivity for aggrecanases, the inhibitors, along with the broad spectrum metalloprotease inhibitor marimastat, were independently bound to the catalytic domain of ADAMTS-5, and the corresponding crystal structures were determined. By comparing the structures, it was determined that the specificity of the relative inhibitors for ADAMTS-5 was not driven by a specific interaction, such as zinc chelation, hydrogen bonding, or charge interactions, but rather by subtle and indirect factors, such as water bridging, ring rigidity, pocket size, and shape, as well as protein conformation flexibility.
Subject(s)
Endopeptidases/chemistry , Enzyme Inhibitors/chemistry , ADAM Proteins/chemistry , ADAMTS4 Protein , ADAMTS5 Protein , Animals , Cattle , Humans , Hydrogen Bonding , Procollagen N-Endopeptidase/chemistry , Protein Structure, Tertiary , Structural Homology, Protein , Zinc/chemistryABSTRACT
The intracellular distribution of fluorescent-labeled polyamides was examined in live cells. We showed that BODIPY-labeled polyamides accumulate in acidic vesicles, mainly lysosomes, in the cytoplasm of HCT116 colon cancer cells and human rheumatoid synovial fibroblasts (RSF). Verapamil blocked vesicular accumulation and led to nuclear accumulation of the BODIPY-labeled polyamide in RSFs. We infer that the basic amine group commonly found at the end of synthetic polyamide chains is responsible for their accumulation in cytoplasmic vesicles in mammalian cells. Modifying the charge on a polyamide by replacing the BODIPY moiety with a fluorescein moiety on the amine tail allowed the polyamide to localize in the nucleus of the cell and bypass the cytoplasmic vesicles in HCT116 cells.