Toyocamycin

Toyocamycin is an inhibitor of phosphatidylinositol kinase. It is known as an antifungal antibiotic. [1]

Phosphatidylinositol kinase is one of the important enzymes that take part in the regulation of the pathway of phosphatidylinositol turnover. Phosphatidylinositol turnover is studied to be involved in the cellular response to mitogens and transformation.[1]

Toyocamycin can suppress thapsigargin-, tunicamycin- and 2-deoxyglucose-induced XBP1 mRNA splicing in HeLa cells. This suppression doesn’t affect the activating of transcription factor 6 (ATF6) and PKR-like ER kinase (PERK)’s activation. Toyocamycin prevents IRE1a-induced XBP1 mRNA cleavage in vitro. [2]

In mammalian cells, toyocamycin inhibits RNA synthesis. Toyocamycin induces apoptosis of MM cells including bortezomib-resistant cells at nanomolar levels in a dose-dependent manner. It also inhibited growth of xenografts in an in vivo model of human multiple myeloma. It is also a lead compound for developing anti-MM therapy and XBP1 as an appropriate molecular target for anti-multiple myeloma therapy.[2]

References:
[1]Nishioka H, Sawa T, etal. , Inhibition of phosphatidylinositol kinase by toyocamycin. J Antibiot (Tokyo). 1990 Dec;43(12):1586-9.
[2]Ri M, Tashiro E, Oikawa D, etal.  , Identification of Toyocamycin,an agent cytotoxic for multiple myeloma cells, as a potent inhibitor of ER stress-induced XBP1 mRNA splicing. Blood Cancer J. 2012 Jul;2(7):e79.

Characterization of representative rpoB gene mutations leading to a significant change in toyocamycin production of Streptomyces diastatochromogenes 1628.[Pubmed:26790416]

J Ind Microbiol Biotechnol. 2016 Apr;43(4):463-71.

Modification of enzymes involved in transcription- or translation-processes is an interesting way to increase secondary metabolite production in Streptomycetes. However, application of such methods has not been widely described for strains which produce nucleoside antibiotics. The nucleoside antibiotic Toyocamycin (TM) is produced by Streptomyces diastatochromogenes 1628. For improving TM production in S. diastatochromogenes 1628, the strain was spread on rifamycin-resistant (Rif(r)) medium. Several spontaneous mutants were obtained with mutations in the rpoB gene which encodes a RNA polymerase beta-subunit. The mutants which showed increased TM production were detected at a frequency of 7.5 % among the total Rif(r) mutants. Mutant 1628-T15 harboring amino acid substitution His437Arg was the best TM producer with a 4.5-fold increase in comparison to that of the wild-type strain. The worst producer was mutant 1628-T62 which also showed a poor sporulation behavior. RT-PCR was performed to study the transcription levels of the TM biosynthetic gene toyG in the parental strain as well as in mutants 1628-T15 and 1628-T62. The transcriptional level of toyG was higher in mutant 1628-T15 than that in parental strain 1628, while much lower in mutant 1628-T62. In mutant strain 1628-T62 the expression of adpA sd gene, which is required for morphological differentiation, was also much lower. Our studies also indicate that the introduction of mutations into rpoB is an effective strategy to improve the production of TM which is an important nucleoside antibiotic.

Toyocamycin induces apoptosis via the crosstalk between reactive oxygen species and p38/ERK MAPKs signaling pathway in human prostate cancer PC-3 cells.[Pubmed:27912102]

Pharmacol Rep. 2017 Feb;69(1):90-96.

BACKGROUND: Toyocamycin, an antibiotic agent isolated from Streptomyces species, has been shown to have anticancer and chemopreventive effects on various cancer cells. Until now, Toyocamycin-induced apoptosis has not been reported to be involved in the regulation between mitogen-activated protein kinases (MAPKs) and reactive oxygen species (ROS) production. METHODS: Cell viability assay, western blot, cell-cycle arrest, annexin V/propidium iodide assay, reactive oxygen species (ROS) production, mitochondrial membrane potential and intracellular Ca(2+) flux were assayed. RESULTS: We investigated the apoptotic effect of Toyocamycin and the underlying molecular mechanism in prostate cancer PC-3 cells. Toyocamycin treatment resulted in reduced cell viability of PC-3 cells, but not of non-malignant RWPE-1 cells. Toyocamycin enhanced apoptosis, mitochondrial dysfunction, and ROS production in PC-3 cells. In addition, MAPK proteins were activated upon Toyocamycin treatment. The p38 and extracellular signal-regulated kinases (ERK) activities were regulated by ROS-mediated signaling pathway underlying the Toyocamycin-induced apoptosis. Pretreatment with N-acetyl-l-cysteine (NAC) recovered the Toyocamycin-induced mitochondrial dysfunction, ROS, and apoptosis. Additionally, p38 stimulated ROS production and inhibitory effects on ERK activation, while ERK inhibited the ROS production and had no effect on p38 activation. CONCLUSION: ROS-mediated activation of p38/ERK partially contributes to Toyocamycin-induced apoptosis, and p38/ERK MAPKs regulate the ROS production in PC-3 cells.

Toyocamycin attenuates free fatty acid-induced hepatic steatosis and apoptosis in cultured hepatocytes and ameliorates nonalcoholic fatty liver disease in mice.[Pubmed:28278289]

PLoS One. 2017 Mar 9;12(3):e0170591.

BACKGROUND AND AIMS: A high serum level of saturated free fatty acids (FFAs) is associated with the development of nonalcoholic fatty liver disease (NAFLD). X-box binding protein-1 (XBP-1) is activated by FFA treatment upon splicing. XBP-1 is a transcription factor induced by the endoplasmic reticulum (ER) stress sensor endoribonuclease inositol-requiring enzyme 1 alpha (IRE1alpha). However, the role of XBP-1 in NAFLD remains relatively unexplored. Toyocamycin was recently reported to attenuate the activation of XBP-1, possibly by inducing a conformational change in IRE1alpha. In this study, we examined the effect of Toyocamycin on hepatocyte lipoapoptosis and steatosis. We also explored the effects of Toyocamycin in a mouse model of NAFLD. METHODS: Huh-7 cells and isolated rat primary hepatocytes were treated with palmitic acid (PA), which is a saturated FFA, in the presence or absence of Toyocamycin. In addition, male C57BL/6J mice were fed a diet rich in saturated fat, fructose, and cholesterol (FFC) for 4 months, after which the effect of Toyocamycin was assessed. RESULTS: Toyocamycin attenuated FFA-induced steatosis. It also significantly reduced PA-induced hepatocyte lipoapoptosis. In addition, Toyocamycin reduced the expression of cytosine-cytosine-adenosine-adenosine-thymidine enhancer-binding protein homologous protein (CHOP), which is a key player in ER stress-mediated apoptosis, as well as its downstream cell death modulator, death receptor 5. In the in vivo study, Toyocamycin ameliorated the liver injury caused by FFC-induced NAFLD. It also reduced hepatic steatosis and the expression of lipogenic genes. CONCLUSIONS: The data we obtained suggest that Toyocamycin attenuates hepatocyte lipogenesis and ameliorates NAFLD in vivo and may therefore be beneficial in the treatment of NAFLD in humans.

Selection of an efficient promoter and its application in toyocamycin production improvement in Streptomyces diastatochromogenes 1628.[Pubmed:28058639]

World J Microbiol Biotechnol. 2017 Feb;33(2):30.

The selection of efficient promoter is usually very crucial for gene expression and metabolic engineering in Streptomycetes. In this study, the synthetic promoters SPL-57and SPL-21, and the engineered promoter kasOp(*)were selected and their activities were examined by using a reporter gene assay based on GUS. All selected promoters which have been reported to be stronger than promoter permE(*), which was used as control promoter. As host we were choosing S. diastatochromogenes 1628, the producer of Toyocamycin (TM). Our results indicate that all tested promoters can be used to express genes in S. diastatochromogenes 1628. Interesting, promoter SPL-21 showed the strongest transcriptional and expression level and gave rise to a 5.2-fold increase in GUS activity compared with control. In order to improve TM production, the promoters were used to control expression of toyF. This gene encodes an adenylosuccinate lyase involved in TM biosynthesis. Among all different recombinant strains, the strain 1628-21F, in which over-expression of toyF gene was driven by SPL-21, exhibited the largest increase in TOYF activity and TM production. In a 5-l fermenter this strain produced more than two times more TM compared with the wild-type strain.

Identification of Toyocamycin, an agent cytotoxic for multiple myeloma cells, as a potent inhibitor of ER stress-induced XBP1 mRNA splicing.[Pubmed:22852048]

Blood Cancer J. 2012 Jul;2(7):e79.

The IRE1alpha-XBP1 pathway, a key component of the endoplasmic reticulum (ER) stress response, is considered to be a critical regulator for survival of multiple myeloma (MM) cells. Therefore, the availability of small-molecule inhibitors targeting this pathway would offer a new chemotherapeutic strategy for MM. Here, we screened small-molecule inhibitors of ER stress-induced XBP1 activation, and identified Toyocamycin from a culture broth of an Actinomycete strain. Toyocamycin was shown to suppress thapsigargin-, tunicamycin- and 2-deoxyglucose-induced XBP1 mRNA splicing in HeLa cells without affecting activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) activation. Furthermore, although Toyocamycin was unable to inhibit IRE1alpha phosphorylation, it prevented IRE1alpha-induced XBP1 mRNA cleavage in vitro. Thus, Toyocamycin is an inhibitor of IRE1alpha-induced XBP1 mRNA cleavage. Toyocamycin inhibited not only ER stress-induced but also constitutive activation of XBP1 expression in MM lines as well as primary samples from patients. It showed synergistic effects with bortezomib, and induced apoptosis of MM cells including bortezomib-resistant cells at nanomolar levels in a dose-dependent manner. It also inhibited growth of xenografts in an in vivo model of human MM. Taken together, our results suggest Toyocamycin as a lead compound for developing anti-MM therapy and XBP1 as an appropriate molecular target for anti-MM therapy.

Identification of inhibitors of ribozyme self-cleavage in mammalian cells via high-throughput screening of chemical libraries.[Pubmed:16556935]

RNA. 2006 May;12(5):797-806.

We have recently described an RNA-only gene regulation system for mammalian cells in which inhibition of self-cleavage of an mRNA carrying ribozyme sequences provides the basis for control of gene expression. An important proof of principle for that system was provided by demonstrating the ability of one specific small molecule inhibitor of RNA self-cleavage, Toyocamycin, to control gene expression in vitro and vivo. Here, we describe the development of the high-throughput screening (HTS) assay that led to the identification of Toyocamycin and other molecules capable of inhibiting RNA self-cleavage in mammalian cells. To identify small molecules that can serve as inhibitors of ribozyme self-cleavage, we established a cell-based assay in which expression of a luciferase (luc) reporter is controlled by ribozyme sequences, and screened 58,076 compounds for their ability to induce luciferase expression. Fifteen compounds able to inhibit ribozyme self-cleavage in cells were identified through this screen. The most potent of the inhibitors identified were Toyocamycin and 5-fluorouridine (FUR), nucleoside analogs carrying modifications of the 7-position and 5-position of the purine or pyrimidine bases. Individually, these two compounds were able to induce gene expression of the ribozyme-controlled reporter approximately 365-fold and 110-fold, respectively. Studies of the mechanism of action of the ribozyme inhibitors indicate that the compounds must be incorporated into RNA in order to inhibit RNA self-cleavage.

Inhibition of phosphatidylinositol kinase by toyocamycin.[Pubmed:2177464]

J Antibiot (Tokyo). 1990 Dec;43(12):1586-9.

We have screened Toyocamycin as an inhibitor of phosphatidylinositol kinase. It inhibited the enzyme of A431 cell membrane with an IC50 of 3.3 micrograms/ml. Adenosine and formycin A also inhibited the enzyme, but other 6 related nucleosides did not. Although orobol and 2,3-dihydroxybenzaldehyde that inhibit phosphatidylinositol kinase inhibited in situ phosphatidylinositol turnover, Toyocamycin did not.

Toyocamycin (Vengicide) is an adenosine analog produced by Actinomycete, acts as an XBP1 inhibitor, inhibits IRE1α-induced ATP-dependent XBP1 mRNA cleavage, with an IC50 of 80 nM. Toyocamycin (Vengicide) induces apoptosis. Toyocamycin (Vengicide) shows no effect on IRE1α auto-phosphorylation.

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