K 252a

Polyploidization of 2nH1 (ES) cells by K-252a and staurosporine.[Pubmed:17949348]

Hum Cell. 2007 Nov;20(4):91-9.

Mouse 2nH1 (ES) cells were examined for polyploidization using K-252a and staurosporine. Though 2nH1 cells were polyploidized by both K-252a and staurosporine, tetraploid cells, 4nH1K cells, were obtained only from cell populations exposed to K-252a. The probability of successful establishment of tetraploid cells was 2/9, suggesting that the highly polyploidized-tetraploid transition might occur infrequently. Cell cycle parameters of 4nH1K cells were almost the same as those of 2nH1 cells, suggesting that the rate of DNA synthesis was about twice that of the diploid cells. The cell volume of 4nH1K cells was about twice of that of diploid cells, indicating that 4nH1K cells contained about twice as much total intracellular material as 2nH1 cells. The morphology of the 4nH1K cells was flagstone-like, thus differing from that of the spindle-shaped 2nH1 cells, suggesting that morphological transformation occurred during the diploid-tetraploid transition. 4nH1K cells exhibited alkaline phosphatase activity and formed teratocarcinomas, implying that they were pluripotent. These characteristics of 4nH1K cells were similar to those of tetraploid 4nH1 cells that have been established through polyploidization by demecolcine, suggesting that 4nH1K and 4nH1 cells are similar irrespective of the different mechanisms of polyploidization.

Molecular cloning, sequence analysis and functional characterization of the gene cluster for biosynthesis of K-252a and its analogs.[Pubmed:19756308]

Mol Biosyst. 2009 Oct;5(10):1180-91.

Among the indolocarbazole alkaloids of antitumor antibiotics, K-252a represents a structurally unique indolocarbazole glycoside and exhibits potent neuroprotective and broad anticancer activities. K-252a consists of K-252c and the unusual dihydrostreptose moiety, linked together with oxidative and glycosidic C-N bonds. Herein, we reported a complete sequence of an approximately 45 kb genomic fragment harboring the gene cluster for the biosynthesis of indolocarbazole alkaloids in Nocardiopsis sp. K-252 (NRRL15532). The sequence of 35 open reading frames discovered several new, critical genes, hence shedding new light on biosynthesis, resistance and regulation of K-252a and its analogs. To functionally characterize the gene cluster in vitro and in enzyme level, a multigene expression cassette containing the K-252c biosynthetic genes was constructed and successfully overexpressed in Escherichia coli to yield soluble proteins for cell-free tandem enzymatic assays. Consequently, the heterologous expression with soluble NokA and NokB led to in vitro production of chromopyrrolic acid (CPA), thereby providing functional evidence for K-252c biosynthesis. Moreover, a facile production of CPA in culture broth was successfully accomplished by using an in vivo biotransformation of L-tryptophan with E. coli harboring the gene cassette. Importantly, by sequence analysis and the functional characterization here and in the companion paper, biosynthetic pathways leading to formation of K-252a and its analogs were hence proposed. Together, the results provide critical information and materials useful for combinatorial biosynthesis of K-252a and its analogs for therapeutic applications.

Biochemical characterization and substrate specificity of the gene cluster for biosyntheses of K-252a and its analogs by in vitro heterologous expression system of Escherichia coli.[Pubmed:19756309]

Mol Biosyst. 2009 Oct;5(10):1192-203.

The indolocarbazole family of natural products has attracted great attention because of their unique structural features and potential therapeutic applications. Structurally distinct in the family, K-252a is characterized by an unusual dihydrostreptose moiety cross-bridged to K-252c aglycone with two C-N linkages. K-252a has served as a valuable lead for treatments of various cancers and neurodegenerative disorders. Recent cloning of the nok gene cluster for biosyntheses of K-252a and its analogs from Nocardiopsis sp. K-252 (NRRL15532) has revealed the nokABCD genes indispensible for K-252c biosynthesis and the key gene (nokL) coding for N-glycosylation. Herein, we report the first, successful demonstration of in vitro sugar transferase activity of indolocarbazole N-glycosyltransferase (NokL) by use of soluble protein expressed from Escherichia coli. Notably, NokL was found to exhibit peculiar mode of substrate promiscuity. Moreover, NokA and NokB reactions were biochemically characterized thoroughly by natural and alternative (e.g. fluoro-) substrates and by ammonium hydroxide (NH(4)OH). Interestingly, the in vitro expression of NokA revealed high substrate stereoselectivity, giving several indole-3-pyruvic acid-derived compounds, including indol-3-carboxaldehyde (ICA) and indole-3-acetic acid. The use of NH(4)OH successfully dissected the in vitro NokA/NokB coupled reaction, revealing mechanistic insight into the enzymes and their cross-talking relationship. Also, a simple, useful method to synthesize K-252d, ICA and chromopyrrolic acid (the NokB product) was developed by the E. coli expression systems of NokL, NokA and NokA/NokB, respectively. Together with NokA and NokB, NokL may serve as a useful tool for combinatorial engineering of K-252a and its analogs for improved therapeutic values.

Synthesis of new aza-analogs of staurosporine, K-252a and rebeccamycin by nucleophilic opening of C2-symmetric bis-aziridines.[Pubmed:19194586]

Org Biomol Chem. 2009 Feb 21;7(4):706-16.

Stable, water-soluble aminosugar staurosporine, K-252a and rebeccamycin analogs have been prepared by nucleophilic opening of C(2)-symmetric N-activated bis-aziridines by bis-indolylmaleimides. This divergent strategy allows the synthesis of unsymmetrical substituted derivatives and provides an easy access to the piperidine and pyrrolidine analogs.

K252a inhibits the oncogenic properties of Met, the HGF receptor.[Pubmed:12118367]

Oncogene. 2002 Jul 25;21(32):4885-93.

The ATP analog K252a is a potent inhibitor for receptor tyrosine kinases of the Trk family. Here we show that nanomolar concentrations of K252a prevent HGF-mediated scattering in MLP-29 cells (30 nM), reduce Met-driven proliferation in GTL-16 gastric carcinoma cells (100 nM), and cause reversion in NIH3T3 fibroblasts transformed by the oncogenic form of the receptor, Tpr-Met (75 nM). K252a inhibits Met autophosphorylation in cultured cells and in immunoprecipitates and prevents activation of its downstream effectors MAPKinase and Akt. Interestingly, K252a seems to be more effective at inhibiting the mutated form of Met (M1268T) found in papillary carcinoma of the kidney than the wild type receptor. Pretreatment of both Tpr-Met-transformed NIH3T3 fibroblasts and of GTL-16 gastric carcinoma cells with K252a results in loss of their ability to form lung metastases in nude mice upon injection into the caudal vein. These observations suggest that K252a derivatives, which are active in vivo as anti-cancer drugs in models of Trk-driven malignancies, should also be effective for treatment of Met-mediated tumors.

K-252 compounds: modulators of neurotrophin signal transduction.[Pubmed:1431889]

J Neurochem. 1992 Dec;59(6):1987-96.

K-252 compounds, which share a common polyaromatic aglycon structure, are rather general and potent inhibitors of various protein kinases, including protein kinase C and tyrosine-specific protein kinases, and possibly act by interfering at or near the ATP binding site. However, chemical modifications in their sugar moiety can result in high specificity of the inhibitory action and, furthermore, can induce other stimulatory and inhibitory effects on nerve cells. These compounds are of particular interest because, in intact cells, they inhibit the actions of NGF and other neurotrophins without diminishing comparable actions of other growth factors. This effect seems to reflect a direct inhibitory action on trk neurotrophin receptor proteins. At concentrations lower than those necessary to inhibit neurotrophin actions, K-252a and K-252b have been shown to potentiate the stimulatory effects of NT-3 on different neurons in culture and on PC12 cells. The structural requirements for this effect seem to be different from those for the inhibition of neurotrophin actions. These findings raise the possibility of development of compounds of high selectivity, able to inhibit or potentiate the transduction mechanisms of individual neurotrophins, and identify K-252a and K-252b as lead compounds for the development of such selective molecules. Specific inhibitors and stimulators of neurotrophins would be valuable tools to investigate biological functions of the neurotrophins in vitro and in vivo. Furthermore, it is possible that, in the future, highly selective drugs with agonistic or antagonistic actions on neurotrophin mechanisms could become therapeutically useful in the treatment of neurological disease and injury.

K-252a, a potent inhibitor of protein kinase C from microbial origin.[Pubmed:3759657]

J Antibiot (Tokyo). 1986 Aug;39(8):1059-65.

K-252a, a metabolite isolated from the culture broth of Nocardiopsis sp. K-252a, was found to exhibit an extremely potent inhibitory activity on protein kinase C. The IC50 value was 32.9 nM.

K-252a, a staurosporine analog isolated from Nocardiopsis sp. soil fungi, inhibits protein kinase, with IC50 values of 470 nM, 140 nM, 270 nM, and 1.7 nM for PKC, PKA, Ca2+/calmodulin-dependent kinase type II, and phosphorylase kinase, respectively. K-252a is a potent inhibitor (IC50 of 3 nM) of the tyrosine protein kinase (TRK) activity of the NGF receptor gp140trk, the product of the trk protooncogene.

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