ABSTRACT
Fatty acid amide hydrolase (FAAH) is one of the main enzymes responsible for the degradation of the endocannabinoid anandamide (N-arachidonoylethanolamine, AEA). FAAH inhibitors may be useful in treating many disorders involving inflammation and pain. Although brain FAAH may be the relevant target for inhibition, rat studies show a correlation between blood and brain FAAH inhibition, allowing blood FAAH activity to be used as a target biomarker. Building on experience with a rat leukocyte FAAH activity assay using [³H]AEA, we have developed a human leukocyte assay using stably labeled [²H4]AEA as substrate. The deuterium-labeled ethanolamine reaction product ([²H4]EA) was analyzed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in the positive electrospray ionization (ESI) mode. The response for [²H4]EA was linear from 10 nM to 10 µM, and the analysis time was less than 6 min/sample. Results using the [²H4]AEA and HPLC-MS/MS method agreed well with those obtained using the [³H]AEA radiometric assay. In addition to using a nonradioactive substrate, the HPLC-MS/MS method had increased sensitivity with lower background. Importantly, the assay preserved partial FAAH inhibition resulting from ex vivo treatment with a time-dependent irreversible inhibitor, suggesting its utility with clinical samples. The assay has been used to profile the successful inhibition of FAAH in recent clinical trials.
Subject(s)
Amidohydrolases/blood , Chromatography, High Pressure Liquid/methods , Leukocytes/enzymology , Spectrometry, Mass, Electrospray Ionization/methods , Tandem Mass Spectrometry/methods , Amidohydrolases/antagonists & inhibitors , Biomarkers/blood , HumansABSTRACT
Installation of sites for metabolism in the lead compound PHA-767408 was the key focus of the IKK-2 inhaled program. This paper reports our efforts to identify a novel series of aminopyridinecarboxamide-based IKK-2 inhibitors, which display low nanomolar potency against IKK-2 with long duration of action (DOA), and metabolically labile to phase I and/or phase II metabolizing enzymes with potential capability for multiple routes of clearance. Several compounds have demonstrated their potential usefulness in the treatment of asthma and chronic obstructive pulmonary disease (COPD).
Subject(s)
Aminopyridines/chemical synthesis , Asthma/drug therapy , I-kappa B Kinase/antagonists & inhibitors , Niacinamide/analogs & derivatives , Protein Kinase Inhibitors/chemical synthesis , Pulmonary Disease, Chronic Obstructive/drug therapy , Pyrazoles/chemical synthesis , Administration, Inhalation , Aminopyridines/chemistry , Aminopyridines/pharmacology , Binding, Competitive , Drug Design , HEK293 Cells , Humans , Indazoles/chemistry , Indazoles/metabolism , Indazoles/pharmacology , Isonicotinic Acids/chemistry , Isonicotinic Acids/metabolism , Isonicotinic Acids/pharmacology , Microsomes, Liver/drug effects , Models, Molecular , Molecular Structure , Molecular Targeted Therapy , Niacinamide/chemical synthesis , Niacinamide/chemistry , Niacinamide/metabolism , Niacinamide/pharmacology , Phenethylamines/metabolism , Potassium Channel Blockers/metabolism , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Pyrazoles/chemistry , Pyrazoles/metabolism , Pyrazoles/pharmacology , Structure-Activity Relationship , Sulfonamides/metabolismABSTRACT
Nuclear factor (NF)-kappaB activation has been clearly linked to the pathogenesis of multiple inflammatory diseases including arthritis. The central role that IkappaB kinase-2 (IKK-2) plays in regulating NF-kappaB signaling in response to inflammatory stimuli has made this enzyme an attractive target for therapeutic intervention. Although diverse chemical classes of IKK-2 inhibitors have been identified, the binding kinetics of these inhibitors has limited the scope of their applications. In addition, safety assessments of IKK-2 inhibitors based on a comprehensive understanding of the pharmacokinetic/pharmacodynamic relationships have yet to be reported. Here, we describe a novel, potent, and highly selective IKK-2 inhibitor, PHA-408 [8-(5-chloro-2-(4-methylpiperazin-1-yl)isonicotinamido)-1-(4-fluorophenyl)-4,5-dihydro-1H-benzo[g]indazole-3-carboxamide]. PHA-408 is an ATP-competitive inhibitor, which binds IKK-2 tightly with a relatively slow off rate. In arthritis-relevant cells and animal models, PHA-408 suppresses inflammation-induced cellular events, including IkappaBalpha phosphorylation and degradation, p65 phosphorylation and DNA binding activity, the expression of inflammatory mediators, and joint pathology. PHA-408 was efficacious in a chronic model of arthritis with no adverse effects at maximally efficacious doses. Stemming from its ability to bind tightly to IKK-2, as a novelty, we demonstrated that PHA-408-mediated inhibition of IKK-2 activity correlated very well with its ability to modulate the fate of IKK-2 substrates and downstream transcriptional events. We ultimately directly linked IKK-2 activity ex vivo and in vivo to markers of inflammation with the inhibitor plasma concentrations. Thus, PHA-408 represents a powerful tool to further gain insight into the mechanisms by which IKK-2 regulates NF-kappaB signaling and validates IKK-2 as a therapeutic target.
Subject(s)
Arthritis/pathology , Enzyme Inhibitors/pharmacology , I-kappa B Kinase/antagonists & inhibitors , NF-kappa B/drug effects , Signal Transduction/drug effects , Animals , Arthritis, Experimental/chemically induced , Arthritis, Experimental/pathology , Blotting, Western , Cells, Cultured , Chromatography, High Pressure Liquid , Electrophoretic Mobility Shift Assay , Enzyme Inhibitors/metabolism , Enzyme-Linked Immunosorbent Assay , Female , Fibroblasts/drug effects , Fibroblasts/metabolism , I-kappa B Kinase/metabolism , Inflammation/chemically induced , Inflammation/pathology , Lipopolysaccharides/pharmacology , Rats , Rats, Inbred Lew , Recombinant Proteins/metabolism , Streptococcus/immunology , Synovial Fluid/cytology , Synovial Fluid/drug effects , Tandem Mass Spectrometry , Tomography, X-Ray Computed , Transcription Factor RelA/genetics , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitorsABSTRACT
The inhibition of PKC-zeta has been proposed to be a potential drug target for immune and inflammatory diseases. A series of 2-(6-phenyl-1H indazol-3-yl)-1H-benzo[d]imidazoles with initial high crossover to CDK-2 has been optimized to afford potent and selective inhibitors of protein kinase c-zeta (PKC-zeta). The determination of the crystal structures of key inhibitor:CDK-2 complexes informed the design and analysis of the series. The most selective and potent analog was identified by variation of the aryl substituent at the 6-position of the indazole template to give a 4-NH(2) derivative. The analog displays good selectivity over other PKC isoforms (alpha, betaII, gamma, delta, epsilon, mu, theta, eta and iota/lambda) and CDK-2, however it displays marginal selectivity against a panel of other kinases (37 profiled).
Subject(s)
Benzimidazoles/chemical synthesis , Chemistry, Pharmaceutical/methods , Enzyme Inhibitors/pharmacology , Imidazoles/chemical synthesis , Protein Kinase C/chemistry , Protein Kinase C/isolation & purification , Benzimidazoles/pharmacology , Crystallography, X-Ray , Cyclin A/chemistry , Cyclin-Dependent Kinase 2/metabolism , Drug Design , Humans , Imidazoles/pharmacology , Inhibitory Concentration 50 , Models, Chemical , Models, Molecular , Molecular Conformation , Protein IsoformsABSTRACT
A series of 21 novel 2-[(aminocarbonyl)amino]-5-acetylenyl-3-thiophenecarboxamides were synthesized and evaluated for the inhibition of IKK-2. In spite of their often modest activity on the enzyme, six selected analogs showed significant inhibition of the production of inflammatory cytokine IL-8 in IL-1beta stimulated rheumatoid arthritis-derived synovial fibroblasts, demonstrating their potential usefulness as NF-kappaB regulators.
