title: Apoptosis induced by NAD depletion is inhibited by KN-93 in a CaMKII-independent manner.
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.
authors.affiliation: Drug Discovery Research, Astellas Pharma Inc., Miyukigaoka 21, Tsukuba, Ibaraki 305-8585, Japan; Department of Microbiology and Molecular Genetics, Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chiba 260-8675, Japan. Electronic address: mikio.takeuchi@astellas.com.
authors.forename: Mikio
authors.initials: M
authors.lastname: Takeuchi
authors.affiliation: Department of Microbiology and Molecular Genetics, Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chiba 260-8675, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Yamamoto
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000096:biosynthesis; D000072224:Bcl-2-Like Protein 11; D001596:Benzylamines / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers; D015220:Calcium Channels / Q000378:metabolism*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000037:antagonists & inhibitors / Q000378:metabolism; D045744:Cell Line, Tumor; D016207:Cytokines / Q000037:antagonists & inhibitors; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects*; D008565:Membrane Proteins / Q000096:biosynthesis; D009243:NAD / Q000172:deficiency*; D054409:Nicotinamide Phosphoribosyltransferase / Q000037:antagonists & inhibitors; D009553:Nimodipine / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis; D013449:Sulfonamides / Q000494:pharmacology*; D014700:Verapamil / Q000494:pharmacology; D000096926:Benzenesulfonamides
chemical_list: D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; D000072224:Bcl-2-Like Protein 11; D001596:Benzylamines; D002118:Calcium; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D016207:Cytokines; D008565:Membrane Proteins; D009243:NAD; D054409:Nicotinamide Phosphoribosyltransferase; D009553:Nimodipine; D010455:Peptides; D011518:Proto-Oncogene Proteins; D013449:Sulfonamides; D014700:Verapamil; C000606775:BCL2L11 protein, human; C425780:KN 92; C085903:autocamtide-2; C072105:KN 93; D000096926:Benzenesulfonamides; C085455:nicotinamide phosphoribosyltransferase, human
keywords: AS1604498; Apoptosis; FK866; KN-92; KN-93; NAD; Nicotinamide phosphoribosyltransferase