Staurosporine from Streptomyces sanyensis activates Programmed Cell Death in Acanthamoeba via the mitochondrial pathway and presents low in vitro cytotoxicity levels in a macrophage cell line.

Recently, the search for novel therapeutic agents against Acanthamoeba species has been focused on the evaluation of natural resources. Among them, marine microorganisms have risen as a source of bioactive compounds with the advantage of the ability to obtain unlimited and constant amounts of the compounds in contrast to other natural sources such as plants. Furthermore, marine actinomycetes have recently been reported as highly rich in bioactive agents including salinosporamides, xiamycines, indolocarbazoles, naphtyridines, phenols, dilactones such as antimycines and macrolides among others. In this study, staurosporine (STS) was isolated from a strain of Streptomyces sanyensis and tested against Acanthamoeba to characterize the therapeutic potential of STS against this protozoan parasite. We have established that STS is active against both stages of the Acanthamoeba life cycle, by the activation of Programmed Cell Death via the mitochondrial pathway of the trophozoite. We have also established that STS has relatively low toxicity towards a macrophage cell line. However, previous studies have highlighted higher toxicity levels induced on other vertebrate cell lines and future research to lower these toxicity issues should be developed.

Bioactive staurosporine derivatives from the Streptomyces sp. NB-A13.

Six new (1-6) and nine known (7-15) staurosporine derivatives were isolated from the rice solid fermentation of the marine-derived Streptomyces sp. NB-A13. The structures of the new staurosporine derivatives were established by extensive spectroscopic data interpretation. The absolute configurations of 1 and 2 were assigned by quantum chemical calculations of the electronic circular dichroism (ECD) spectra. All of these compounds were screened for their cytotoxic activities against PC-3 and SW-620 cell lines. Compound 7 exhibited stronger inhibitory activity against SW-620 cell lines than the positive control staurosporine (25.10 nM), with IC50 values of 9.99 nM. Moreover, compounds 1-5, 8-13 and 15 also showed significant cytotoxicities with IC50 values ranging from 0.02 to 16.60 µM, while 6 exhibited no cytotoxic potency. Additionally, compounds 1-7 were also tested for enzyme inhibition activities of Protein kinase C theta (PKC-θ), and showed activity with IC50 values ranging from 0.06 to 9.43 µM except for compound 6, which has no inhibition activity.

Suppressing activity of staurosporine from Streptomyces sp. MJM4426 against rice bacterial blight disease.

AIM: To identify the active compounds from the Streptomyces sp. MJM4426 that can protect rice from bacterial blight disease (BB), and to evaluate the potential of this Streptomyces strains and the compound for biocontrol of rice bacterial blight disease. METHODS AND RESULTS: The ethyl acetate extract of Streptomyces sp. MJM4426 can significantly protect rice leaf explants from the infection of Xanthomonas oryzae pv. oryzaeKACC 10331 (Xoo), the pathogen which cause BB. To identify the active compounds, the ethyl acetate extract of Streptomyces sp. MJM4426 was fractionated through a Sephadex LH-20 column chromatography, and further purified by preparative HPLC guided by the inhibitory activity against BB in rice leaf explants. UPLC-Q-TOF/MS analysis showed the active compound displayed its m/z values at [M+H](+) 467·2086 and [M+FA-H](-) 511·1963, and the molecular formula was estimated as C28 H26 N4 O3 which is identical to commercial standard staurosporine. In this study, the isolated staurosporine dramatically suppressed bacterial blight in rice leaf explants with the lowest concentration at 12·5 µmol l(-1) , however, it exhibited low inhibitory activity against Xoo with the MIC value at 256 µg ml(-1) . In addition, greenhouse study showed both crude extract and purified staurosporine can suppress the bacterial blight at the concentration of 5000 and 200 µg ml(-1) respectively. CONCLUSION: Streptomyces sp. MJM4426 can protect rice leaf explants from the infection of Xoo by producing staurosporine, but not by direct inhibitory activity against Xoo. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report that staurosporine can protect rice leaf against bacterial blight disease and showed the potential of Streptomyces sp. MJM4426 as an alternative to chemical bactericide for bacterial blight disease in rice.

Melleolides induce rapid cell death in human primary monocytes and cancer cells.

The melleolides are structurally unique and bioactive natural products of the basidiomycete genus Armillaria. Here, we report on cytotoxic effects of melleolides from Armillaria mellea towards non-transformed human primary monocytes and human cancer cell lines, respectively. In contrast to staurosporine or pretubulysin that are less cytotoxic for monocytes, the cytotoxic potency of the active melleolides in primary monocytes is comparable to that in cancer cells. The onset of the cytotoxic effects of melleolides was rapid (within <1 h), as compared to the apoptosis inducer staurosporine, the protein biosynthesis inhibitor cycloheximide, and the DNA transcription inhibitor actinomycin D (>5 h, each). Side-by-side comparison with the detergent triton X-100 and staurosporine in microscopic and flow cytometric analysis studies as well as analysis of the viability of mitochondria exclude cell lysis and apoptosis as relevant or primary mechanisms. Our results rather point to necrotic features of cell death mediated by an as yet elusive but rapid mechanism.

Staurosporine from the endophytic Streptomyces sp. strain CNS-42 acts as a potential biocontrol agent and growth elicitor in cucumber.

Chinese medicinal plants and their surrounding rhizospheric soil serve as promising sources of actinobacteria. A total of 180 actinobacteria strains were isolated from the rhizosphere soil, leaves, stems, and roots of nine selected plants and have been identified as potential biocontrol agents against Fusarium oxysporum f. sp. cucumerinum. An endophytic strain CNS-42 isolated from Alisma orientale showed the largest zone of inhibition demonstrating a potent effect against F. oxysporum f. sp. cucumerinum and a broad antimicrobial activity against bacteria, yeasts, and other pathogenic fungi. The in vivo biocontrol assays showed that the disease severity index was significantly reduced (P < 0.05), and plant shoot fresh weight and height increased greatly (P < 0.05) in plantlets treated with strain CNS-42 compared to the negative control. This isolate was identified as Streptomyces sp. based on cultural, physiological, morphological characteristics, and 16S rRNA gene analysis. Further bioassay-guided isolation and purification revealed that staurosporine was responsible for its antifungal and plant growth promoting activities and the latter property of staurosporine is reported for the first time. The in vivo assay was further performed and indicated that staurosporine showed good growth promoting effect on the plant shoot biomass of cucumber. This is the first critical evidence identifying CNS-42 as a biocontrol agent for the soil borne pathogen, F. oxysporum f. sp. cucumerinum.

New indolocarbazoles from a mutant strain of the marine-derived actinomycete Streptomyces fradiae 007M135.

Fradcarbazoles A-C (1-3), three new indolocarbazoles, were isolated from a mutant strain of the marine-derived actinomycete Streptomyces fradiae 007M135. Their structures were established by spectroscopic analysis, quantum chemical calculation, CD spectra, and chemical transformation. Fradcarbazole A (1) possessed a unique skeleton consisting of a staurosporine core, a thiazole ring, and an indole fragment. Compounds 1-3 displayed significant cytotoxicity against HL-60, K562, A-549, and BEL-7402 cell lines and inhibitory effects on the kinase PKC-α with IC(50) values of 0.001-4.58 µM.

Structure elucidation and anticancer activity of 7-oxostaurosporine derivatives from the Brazilian endemic tunicate Eudistoma vannamei.

The present study reports the identification of two new staurosporine derivatives, 2-hydroxy-7-oxostaurosporine (1) and 3-hydroxy-7-oxostaurosporine (2), obtained from mid-polar fractions of an aqueous methanol extract of the tunicate Eudistoma vannamei, endemic to the northeast coast of Brazil. The mixture of 1 and 2 displayed IC50 values in the nM range and was up to 14 times more cytotoxic than staurosporine across a panel of tumor cell lines, as evaluated using the MTT assay.

A new staurosporine analog from Actinomycetes Streptomyces sp. (172614).

A new staurosporine analog, 10'-{5″-[(methoxycarbonyl)amino]-2″-methyl}-phenylaminocarbonylstaurosporine (1), together with staurosporine (2), was obtained from the culture broth of Actinomycetes Streptomyces sp. (172614). Their structures were elucidated by comprehensive spectroscopic analysis including UV, MS, NMR, and CD spectra. Pharmacological experiments revealed that 1 and 2 showed significant cytotoxicity against human colon tumor cell HCT-116.

Identification, in vitro activity and mode of action of phosphoinositide-dependent-1 kinase inhibitors as antifungal molecules.

Although protein kinases have recently emerged as important drug targets, the anti-infective potential of protein kinase inhibitors has not been developed extensively. We identified the mammalian PDK1 inhibitor KP-372-1 as a potent antifungal molecule with activity against yeast and fungal biofilms using a screening strategy for protein kinase inhibitors that block the cell wall stress response in yeast. Genetic and biochemical studies indicate that KP-372-1 inhibits fungal PDK1 orthologs (Pkh kinases) as part of its mode of action and support a role for Pkh kinases in eisosome assembly. Two other structurally distinct molecules that inhibit PDK1, OSU-03012 and UCN-01, also have antifungal activity. Taken together, these data indicate that fungal PDK1 orthologs are promising targets for new antifungal drug development.

Anti-parasitic compounds from Streptomyces sp. strains isolated from Mediterranean sponges.

Actinomycetes are prolific producers of pharmacologically important compounds accounting for about 70% of the naturally derived antibiotics that are currently in clinical use. In this study, we report on the isolation of Streptomyces sp. strains from Mediterranean sponges, on their secondary metabolite production and on their screening for anti-infective activities. Bioassay-guided isolation and purification yielded three previously known compounds namely, cyclic depsipeptide valinomycin, indolocarbazole alkaloid staurosporine and butenolide. This is the first report of the isolation of valinomycin from a marine source. These compounds exhibited novel anti-parasitic activities specifically against Leishmania major (valinomycin IC(50) < 0.11 microM; staurosporine IC(50) 5.30 microM) and Trypanosoma brucei brucei (valinomycin IC(50) 0.0032 microM; staurosporine IC(50) 0.022 microM; butenolide IC(50) 31.77 microM). These results underscore the potential of marine actinomycetes to produce bioactive compounds as well as the re-evaluation of previously known compounds for novel anti-infective activities.

Protein kinase inhibition of clinically important staurosporine analogues.

The isolation in 1977 of the microbial alkaloid staurosporine inaugurated research into several distinct series of related natural and synthetic compounds. This has especially included research into applications as anticancer drugs, beginning with the observation of low nanomolar inhibition of protein kinases. At present, several staurosporine cognates are in advanced clinical trials as anticancer agents, with the potential to join the 10 other protein kinase inhibitors now approved for clinical use. Staurosporine is a broadly selective and potent protein kinase inhibitor, with submicromolar binding to the vast majority of the protein kinases tested, and binding most of them more tightly than 100 nM. Crystal structures have shown the extended buried surface area interactions between the protein kinase adenine binding site and the extended aromatic plane of the inhibitor, together with protein-saccharide interactions in the ribose binding site. Together with structures of closely related analogues, there are now some 70 X-ray crystal structures in the Protein Data Bank that enable analysis of target binding properties of the clinical compounds. In this manuscript we review the discovery of these compounds, revisit crystal structures and review the observed interactions. These support the interpretation of kinase selectivity profiles of staurosporine and its analogues, including midostaurin (PKC412), for which a co-crystal structure is not yet available. Further, the mix of purely natural, biosynthetically and chemically modified compounds described here offer insights into prospects and strategies for drug discovery via bioprospecting.

Cytotoxic staurosporines from the marine ascidian Cystodytes solitus.

Two new indolocarbazole alkaloids, 7-oxo-3,8,9-trihydroxystaurosporine (1) and 7-oxo-8,9-dihydroxy-4'-N-demethylstaurosporine (2), were isolated from samples of the marine ascidian Cystodytes solitus. Their structures were determined by a combination of spectroscopic techniques, including (+)-HRMALDITOFMS and 1D and 2D NMR spectroscopy, and comparison with published data for related structures. Both compounds displayed strong cytotoxicity against three human tumor cell lines.

N-carboxamido-staurosporine and selina-4(14),7(11)-diene-8,9-diol, new metabolites from a marine Streptomyces sp.

In our screening of micro-organisms for novel bioactive natural products, a new staurosporinone, N-carboxamido-staurosporine (1c), and a new sesquiterpene, (5S,8S,9R, 10S)-selina-4(14),7(11)-diene-8,9-diol (2a), were isolated from the culture broth of the marine-derived Streptomyces sp. QD518. Their structures were determined by spectroscopic methods and by comparison of the NMR data with those of structurally related known natural products, which were isolated from the same strain.

Isolation and antifungal and antioomycete activities of staurosporine from Streptomyces roseoflavus strain LS-A24.

The actinomycete strain LS-A24 active against some plant fungal and oomycete pathogens was isolated from a soil sample of the Sunghwan Lake in Korea. The cell wall composition and spore shape of strain LS-A24 were LL-diaminopimelic acid and spiral type, respectively. On the basis of the physiological and biochemical characteristics and 16S ribosomal DNA sequence analysis, strain LS-A24 was identical to Streptomyces roseoflavus. An antifungal and antioomycete antibiotic was isolated from LS-A24 using various chromatographic procedures. The molecular formular of the antibiotic was determined to be C(28)H(26)N(4)O(3), and on the basis of the NMR data, the antibiotic was confirmed to be staurosporine, 2,3,10,11,12,13-hexahydro-10R-methoxy-9S-methyl-11R-methylamino-9S,13R-epoxy-1H,9H-diindolo[1,2,3-gh:3',2',1'-lm]pyrrolo[3,4-j][1,7]benzodiazonin-1-one. Staurosporine completely inhibited the mycelial growth of Colletotrichum orbiculare, Phytophthora capsici, Rhizoctonia solani, Botrytis cinerea, and Cladosporium cucumerinum with minimum inhibitory concentration (MIC) values of 1-50 microg/mL for MICs. Staurosporine also was active against Saccharomyces cerevisiae, Bacillus subtilis ssp. subtilis, and Xanthomonas vesicatoria. Staurosporine and the commercial fungicide metalaxyl inhibited the development of Phytophthora blight on pepper plants. However, the control efficacy of staurosporine against the Phytophthora disease was somewhat less than that of metalaxyl. This is the first study to isolate staurosporine from S. roseoflavus and demonstrate its in vitro and in vivo antioomycete activity against P. capsici.

Deciphering the late steps in the biosynthesis of the anti-tumour indolocarbazole staurosporine: sugar donor substrate flexibility of the StaG glycosyltransferase.

The indolocarbazole staurosporine is a potent inhibitor of a variety of protein kinases. It contains a sugar moiety attached through C-N linkages to both indole nitrogen atoms of the indolocarbazole core. Staurosporine biosynthesis was reconstituted in vivo in a heterologous host Streptomyces albus by using two different plasmids: the 'aglycone vector' expressing a set of genes involved in indolocarbazole biosynthesis together with staG (encoding a glycosyltransferase) and/or staN (coding for a P450 oxygenase), and the 'sugar vector' expressing a set of genes responsible for the biosynthesis of the sugar moiety. Attachment of the sugar to the two indole nitrogens of the indolocarbazole core was dependent on the combined action of StaG and StaN. When StaN was absent, the sugar was attached only to one of the nitrogen atoms, through an N-glycosidic linkage, as in the indolocarbazole rebeccamycin. The StaG glycosyltransferase showed flexibility with respect to the sugar donor. When the 'sugar vector' was substituted by constructs directing the biosynthesis of l-rhamnose, L-digitoxose, L-olivose and D-olivose, respectively, StaG and StaN were able to transfer and attach all of these sugars to the indolocarbazole aglycone.

Further new staurosporine derivatives from the ascidian Eudistoma toealensis and its predatory flatworm Pseudoceros sp.

Three new indolocarbazole alkaloids, 3-hydroxy-4'-N-methylstaurosporine (3), 3-hydroxy-4'-N-demethylstaurosporine (4), and 3'-demethoxy-3'-hydroxy-4'-N-demethylstaurosporine (5), were isolated from the marine ascidian Eudistoma toealensis and its predatory flatworm Pseudoceros sp. in addition to two known staurosporines. The structures were determined by 1D and 2D homonuclear and 1H-detected heteronuclear NMR spectroscopy and from comparisons with published data. CD measurements for these five staurosporine derivatives, as well as the previously described seven staurosporines, are reported, confirming that all derivatives possess the 2'S,3'R,4'R,6'R configuration.

Anti-proliferative effects of new staurosporine derivatives isolated from a marine ascidian and its predatory flatworm.

Nine indolocarbazole alkaloids of the staurosporine type, including three new derivatives, were evaluated for their potential as inhibitors of cell proliferation and macromolecule synthesis. Four derivatives were tested as inhibitors of cell proliferation with twelve human leukemia cell lines and demonstrated powerful antiproliferative activities, with 3-hydroxystaurosporine being the most potent. IC(50) values were determined using the cell line MONO-MAC-6 and with an IC(50) of 13 ng/ml, 3-hydroxystaurosporine turned out to be one of the most active staurosporine-type inhibitors described so far. All derivatives, except 3-hydroxy-3'-demethoxy-3'-hydroxystaurosporine and 4'-N-methylstaurosporine very strongly reduced RNA and DNA synthesis with 3-hydroxystaurosporine again being the strongest inhibitor. Analysis of structure-activity relationships demonstrated that hydroxylation of staurosporine at position 3 of the indolocarbazole moiety caused an increase in anti-proliferative activity, while hydroxylation at carbon 11 resulted in a decrease in activity. Our results suggest that not only the presence or absence of hydrophilic substitutions, but also the position of the alteration within the molecule, is important in the antiproliferative properties of the various staurosporine analogues.