作者: Nomura, Teiko Komori ; Endo, Satoshi ; Kuwano, Takuma ; Takashima, Shigeo ; Furuta, Kyoji ; Todo, Tomoki ; Hinoi, Eiichi ; Koyama, Hiroko ; Yamamoto, Takuhei ; Fukasawa, Kazuya ; Honda, Ryo

ARL-17477 is a selective neuronal nitric oxide synthase (NOS1) inhibitor that has been used in many preclinical studies since its initial discovery in the 1990s. In the present study, we demonstrate that ARL-17477 exhibits a NOS1-independent pharmacological activity that involves inhibition of the autophagy-lysosomal system and prevents cancer growth in vitro and in vivo. Initially, we screened a chemical compound library for potential anticancer agents, and identified ARL-17477 with micromolar anticancer activity against a wide spectrum of cancers, preferentially affecting cancer stem-like cells and KRAS-mutant cancer cells. Interestingly, ARL-17477 also affected NOS1-knockout cells, suggesting the existence of a NOS1-independent anticancer mechanism. Analysis of cell signals and death markers revealed that LC3B-II, p62, and GABARAP-II protein levels were significantly increased by ARL-17477. Furthermore, ARL-17477 had a chemical structure similar to that of chloroquine, suggesting the inhibition of autophagic flux at the level of lysosomal fusion as an underlying anticancer mechanism. Consistently, ARL-17477 induced lysosomal membrane permeabilization, impaired protein aggregate clearance, and activated transcription factor EB and lysosomal biogenesis. Furthermore, in vivo ARL-17477 inhibited the tumor growth of KRAS-mutant cancer. Thus, ARL-17477 is a dual inhibitor of NOS1 and the autophagy-lysosomal system that could potentially be used as a cancer therapeutic.

2020-02-01·American Journal of Physiology-Heart and Circulatory Physiology2区 · 医学

Nitric oxide synthase inhibitors negatively regulate respiration in isolated rodent cardiac and brain mitochondria

2区 · 医学

Article

作者: Katakam, Prasad V. G. ; Evans, Wesley R. ; Albuck, Aaron L. ; Sakamuri, Siva S. V. P. ; Satou, Ryousuke ; Mostany, Ricardo ; Sperling, Jared A. ; Dholakia, Monica H. ; Sure, Venkata N.

Nitric oxide (NO) is known to exert inhibitory control on mitochondrial respiration in the heart and brain. Evidence supports the presence of NO synthase (NOS) in the mitochondria (mtNOS) of cells; however, the functional role of mtNOS in the regulation of mitochondrial respiration is unclear. Our objective was to examine the effect of NOS inhibitors on mitochondrial respiration and protein S-nitrosylation. Freshly isolated cardiac and brain nonsynaptosomal mitochondria were incubated with selective inhibitors of neuronal (nNOS; ARL-17477, 1 µmol/L) or endothelial [eNOS; N5-(1-iminoethyl)-l-ornithine, NIO, 1 µmol/L] NOS isoforms. Mitochondrial respiratory parameters were calculated from the oxygen consumption rates measured using Agilent Seahorse XFe24 analyzer. Expression of NOS isoforms in the mitochondria was confirmed by immunoprecipitation and Western blot analysis. In addition, we determined the protein S-nitrosylation by biotin-switch method followed by immunoblotting. nNOS inhibitor decreased the state IIIu respiration in cardiac mitochondria and both state III and state IIIu respiration in brain mitochondria. In contrast, eNOS inhibitor had no effect on the respiration in the mitochondria from both heart and brain. Interestingly, NOS inhibitors reduced the levels of protein S-nitrosylation only in brain mitochondria, but nNOS and eNOS immunoreactivity was observed in the cardiac and brain mitochondrial lysates. Thus, the effects of NOS inhibitors on S-nitrosylation of mitochondrial proteins and mitochondrial respiration confirm the existence of functionally active NOS isoforms in the mitochondria. Notably, our study presents first evidence of the positive regulation of mitochondrial respiration by mitochondrial nNOS contrary to the current dogma representing the inhibitory role attributed to NOS isoforms. NEW & NOTEWORTHY Existence and the role of nitric oxide synthases in the mitochondria are controversial. We report for the first time that mitochondrial nNOS positively regulates respiration in isolated heart and brain mitochondria, thus challenging the existing dogma that NO is inhibitory to mitochondrial respiration. We have also demonstrated reduced protein S-nitrosylation by NOS inhibition in isolated mitochondria, supporting the presence of functional mitochondrial NOS.

2017-01-01·BioMed Research International3区 · 生物学

Calcium Ionophore, Calcimycin, KillsLeishmaniaPromastigotes by Activating Parasite Nitric Oxide Synthase

3区 · 生物学

ArticleOA

作者: Grekov, Igor ; Bartůněk, Petr ; Lipoldová, Marie ; Pombinho, António R. ; Kobets, Tatyana

Leishmaniasis is an infectious disease caused by protozoan parasites of the genusLeishmania. There is no vaccine against human leishmaniasis and the treatment of the disease would benefit from a broader spectrum and a higher efficacy of leishmanicidal compounds. We analyzed the leishmanicidal activity and the mechanism of action of the calcium ionophore, calcimycin.L. majorpromastigotes were coincubated with calcimycin and the viability of the cells was assessed using resazurin assay. Calcimycin displayed dose-dependent effect with IC50= 0.16 μM. Analysis of propidium iodide/LDS-751 stained promastigotes revealed that lower concentrations of calcimycin had cytostatic effect and higher concentrations had cytotoxic effect. To establish the mechanism of action of calcimycin, which is known to stimulate activity of mammalian constitutive nitric oxide synthase (NOS), we coincubatedL. majorpromastigotes with calcimycin and selective NOS inhibitors ARL-17477 or L-NNA. Addition of these inhibitors substantially decreased the toxicity of calcimycin toLeishmaniapromastigotes. In doing so, we demonstrated for the first time that calcimycin has a direct leishmanicidal effect onL. majorpromastigotes. Also, we showed thatLeishmaniaconstitutive Ca2+/calmodulin-dependent nitric oxide synthase is involved in the parasite cell death. These data suggest activation ofLeishmanianitric oxide synthase as a new therapeutic approach.

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适应症	最高研发状态	国家/地区	公司	日期
肌萎缩侧索硬化	临床2期	英国	Astra Arcus AB	-
肌萎缩侧索硬化	临床2期	美国	AstraZeneca R&D Charnwood	-
疼痛	临床2期	英国	Astra Arcus AB	-
疼痛	临床2期	美国	AstraZeneca R&D Charnwood	-
大脑中动脉梗塞	临床前	美国	Oakland University	2016-08-31
大脑中动脉梗塞	临床前	-	Henry Ford Hospital	2016-08-31
大脑中动脉梗塞	临床前	美国	Astra Arcus AB	2016-08-31