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1.
Exp Cell Res ; 335(1): 62-7, 2015 Jul 01.
Article in English | MEDLINE | ID: mdl-26024774

ABSTRACT

Nicotinamide phosphoribosyltransferase (NAMPT) is a key enzyme that catalyzes the synthesis of nicotinamide mononucleotide from nicotinamide (Nam) in the salvage pathway of mammalian NAD biosynthesis. Several potent NAMPT inhibitors have been identified and used to investigate the role of intracellular NAD and to develop therapeutics. NAD depletion induced by NAMPT inhibitors depolarizes mitochondrial membrane potential and causes apoptosis in a range of cell types. However, the mechanisms behind this depolarization have not been precisely elucidated. We observed that apoptosis of THP-1 cells in response to NAMPT inhibitors was reduced by the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN-93 via an unknown mechanism. The inactive analog of KN-93, KN-92, exhibited the same activity, but the CaMKII-inhibiting cell-permeable autocamtide-2-related inhibitory peptide II did not, indicating that the inhibition of THP-1 cell apoptosis was not dependent on CaMKII. In evaluating the mechanism of action, we confirmed that KN-93 did not inhibit decreases in NAD levels but did inhibit decreases in mitochondrial membrane potential, indicating that KN-93 exerts inhibition upstream of the mitochondrial pathway of apoptosis. Further, qPCR analysis of the Bcl-2 family of proteins showed that Bim is efficiently expressed following NAMPT inhibition and that KN-92 did not inhibit this expression. The L-type Ca(2+) channel blockers verapamil and nimodipine partially inhibited apoptosis, indicating that part of this effect is dependent on Ca(2+) channel inhibition, as both KN-93 and KN-92 are reported to inhibit L-type Ca(2+) channels. On the other hand, KN-93 and KN-92 did not markedly inhibit apoptosis induced by anti-cancer agents such as etoposide, actinomycin D, ABT-737, or TW-37, indicating that the mechanism of inhibition is specific to apoptosis induced by NAD depletion. These results demonstrate that NAD depletion induces a specific type of apoptosis that is effectively inhibited by the KN-93 series of compounds.


Subject(s)
Apoptosis/drug effects , Benzylamines/pharmacology , Calcium Channels/metabolism , Membrane Potential, Mitochondrial/drug effects , NAD/deficiency , Sulfonamides/pharmacology , Antineoplastic Agents/pharmacology , Apoptosis Regulatory Proteins/biosynthesis , Bcl-2-Like Protein 11 , Calcium/metabolism , Calcium Channel Blockers , Calcium-Calmodulin-Dependent Protein Kinase Type 2/antagonists & inhibitors , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Cell Line, Tumor , Cytokines/antagonists & inhibitors , Humans , Membrane Proteins/biosynthesis , Nicotinamide Phosphoribosyltransferase/antagonists & inhibitors , Nimodipine/pharmacology , Peptides/pharmacology , Proto-Oncogene Proteins/biosynthesis , Verapamil/pharmacology
2.
Biol Pharm Bull ; 37(1): 31-6, 2014.
Article in English | MEDLINE | ID: mdl-24389478

ABSTRACT

Nicotinamide phosphoribosyl transferase (NAMPT) is a key enzyme in the salvage pathway of mammalian nicotinamide adenine dinucleotide (NAD) biosynthesis, catalyzing the synthesis of nicotinamide mononucleotide from nicotinamide (Nam). The diverse functions of NAD suggest that NAMPT inhibitors are potential drug candidates as anticancer agents, immunomodulators, or other agents. However, difficulty in conducting high-throughput NAMPT assay with good sensitivity has hampered the discovery of novel anti-NAMPT drugs with improved profiles. We combined an in silico screening strategy with a radioisotope (RI)-based enzyme assay and rationally identified promising NAMPT inhibitors with novel structures. AS1604498 was the most potent inhibitor, with an IC50 of 44 nM, and inhibited THP-1 and K562 cell line growth with the IC50 of 198 nM and 673 nM, respectively. The mode of action was found to reduce intracellular NAD following apoptosis, suggesting that these compounds inhibit NAMPT in cell-based assay. This strategy can be used to discover new drug candidates with targets which are difficult to assess through high-throughput screening. Our hit compounds may be used as seed compounds for developing new therapeutics with NAMPT.


Subject(s)
Antineoplastic Agents/pharmacology , Computer Simulation , Drug Discovery/methods , Enzyme Inhibitors/pharmacology , Leukemia/enzymology , NAD/biosynthesis , Nicotinamide Phosphoribosyltransferase/antagonists & inhibitors , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Apoptosis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/therapeutic use , Humans , Inhibitory Concentration 50 , K562 Cells , Leukemia/drug therapy , Mice , Niacinamide/metabolism , Nicotinamide Mononucleotide/biosynthesis
3.
Naunyn Schmiedebergs Arch Pharmacol ; 374(4): 311-6, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17123065

ABSTRACT

Extracellular signal-regulated kinase (ERK), a serine/threonine protein kinase of the mitogen-activated protein kinase superfamily, is activated by various stimuli in inflammatory cells. We recently described FR180204 (5-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-1H-pyrazolo[3,4-c]pyridazin-3-amine), a novel selective ERK inhibitor. In this paper, we investigated the effect of FR180204 on collagen-induced arthritis (CIA) in DBA/1 mice, an animal model of rheumatoid arthritis (RA) mediated by type II collagen (CII)-reactive T cells and anti-CII antibodies. Preventive administration of FR180204 (100 mg/kg, i.p., b.i.d.) significantly ameliorated the clinical arthritis and body weight loss occurring in the CIA mice. Further, FR180204-treated mice showed a significant decrease in plasma anti-CII antibody levels (62%). FR180204 also attenuated delayed-type hypersensitivity in CII-immunized DBA/1 mice, an inflammatory response elicited by CII-reactive T cells, in a dose-dependent manner (52 and 62% inhibition at 32 and 100 mg/kg, respectively). Moreover, FR180204 inhibited in vitro CII-induced proliferation of lymph node cells prepared from CII-immunized mice, in which CII-specific T cells are known to undergo specific proliferation. In conclusion, our results suggest that ERK regulates both the cell-mediated and humoral immune responses in the development of CIA. ERK inhibitors may be useful as therapeutic reagents for the treatment of RA.


Subject(s)
Arthritis, Experimental/prevention & control , Collagen Type II/toxicity , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Pyrazoles/pharmacology , Pyridazines/pharmacology , Animals , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/therapeutic use , Arthritis, Experimental/chemically induced , Arthritis, Experimental/physiopathology , Collagen Type II/antagonists & inhibitors , Dose-Response Relationship, Drug , Glucocorticoids/pharmacology , Glucocorticoids/therapeutic use , Hypersensitivity, Delayed/prevention & control , Immunoglobulin Isotypes/blood , Injections, Intraperitoneal , Lymphocyte Activation/drug effects , Male , Methylprednisolone/pharmacology , Methylprednisolone/therapeutic use , Mice , Mice, Inbred DBA , Pyrazoles/administration & dosage , Pyrazoles/therapeutic use , Pyridazines/administration & dosage , Pyridazines/therapeutic use , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Thymidine/metabolism , Weight Loss/drug effects
4.
Anal Biochem ; 352(1): 15-23, 2006 May 01.
Article in English | MEDLINE | ID: mdl-16540075

ABSTRACT

The isolation of both specific and nonspecific binding proteins on affinity matrices bearing bioactive compounds hinders the identification of drug cellular targets. Although solid-phase elution and competition methods conventionally are used to distinguish between specific and nonspecific receptor-ligand interactions, these approaches often are severely restricted by low ligand solubility and/or slow kinetic dissociation. This article describes an alternative and versatile method, termed serial affinity chromatography, to identify ligand receptors using affinity resins.


Subject(s)
Carrier Proteins/analysis , Carrier Proteins/isolation & purification , Chromatography, Affinity/methods , Affinity Labels/chemistry , Affinity Labels/metabolism , Animals , Brain/cytology , Brain/metabolism , Carrier Proteins/chemistry , Cells, Cultured , Humans , Ligands , Models, Biological , Rats , Resins, Synthetic/chemical synthesis , Resins, Synthetic/chemistry , Resins, Synthetic/metabolism , Tacrolimus Binding Protein 1A/isolation & purification
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