Streptomyces sp. BV410 isolate from chamomile rhizosphere soil efficiently produces staurosporine with antifungal and antiangiogenic properties

Abstract Applying a bioactivity‐guided isolation approach, staurosporine was separated and identified as the active principle in the culture extract of the new isolate Streptomyces sp. BV410 collected from the chamomile rhizosphere. The biotechnological production of staurosporine by strain BV410 was optimized to yield 56 mg/L after 14 days of incubation in soy flour–glucose–starch–mannitol‐based fermentation medium (JS). The addition of FeSO4 significantly improved the staurosporine yield by 30%, while the addition of ZnSO4 significantly reduced staurosporine yield by 62% in comparison with the starting conditions. Although staurosporine was first isolated in 1977 from Lentzea albida (now Streptomyces staurosporeus) and its potent kinase inhibitory effect has been established, here, the biological activity of this natural product was assessed in depth in vivo using a selection of transgenic zebrafish (Danio rerio) models, including Tg(fli1:EGFP) with green fluorescent protein‐labeled endothelial cells allowing visualization and monitoring of blood vessels. This confirmed a remarkable antiangiogenic activity of the compound at doses of 1 ng/ml (2.14 nmol/L) which is below doses inducing toxic effects (45 ng/ml; 75 nmol/L). A new, efficient producing strain of commercially significant staurosporine has been described along with optimized fermentation conditions, which may lead to optimization of the staurosporine scaffold and its wider applicability.


| INTRODUC TI ON
It has been widely recognized that Actinomycetes, especially members of the genus Streptomyces, are one of the most prolific sources of bioactive natural products, including the most important antimicrobial drug classes such as β-lactams, tetracyclines, rifamycins, polyenes, indocarbazoles, and others (Genilloud, 2017). They also produce various secondary metabolites that are cytotoxic, cytostatic, anti-inflammatory, antiparasitic, antimalaria, antiviral, antioxidant, and antiangiogenic (Liu, Deng, & Liu, 2018). Frequent rediscovery of the same compounds from the soil isolates has made them less attractive for screening programs in the recent years. Nevertheless, classical screening strategies based on whole cell assays are still successful in discovery of novel bioactive molecules or new biological activities of known compounds from microbial extracts (Donadio, Maffioli, Monciardini, Sosio, & Jabes, 2010;Riahi, Hosni, Raies, & Oliveira, 2019) providing appropriate prioritization approaches (Crüsemann et al., 2017;Xie et al., 2014). For example, the natural product rapamycin (generic name sirolimus), produced by a strain of S. hygroscopicus, has been isolated originally as an antifungal agent with excellent activity against Candida spp. (Sehgal, 2003); subsequently, its impressive antitumor and immunosuppressive activities have been revealed (Li, Kim, & Blenis, 2014).
Having transitioned from the rare incidence to a serious problem and a leading cause of morbidity in immunocompromised patients, members of the genus Candida have recently been added to the list of priority pathogens (McCarthy & Walsh, 2017;Perfect, 2017).
Candidiasis is now one of the most frequent hospital-acquired infections with around 60 000 cases recorded annually (McCarty & Pappas, 2016;Rodloff, Koch, & Schaumann, 2011). Polyene natural products, such as nystatin and amphotericin B, isolated from Streptomyces nursei and S. nodosus, respectively, represent the oldest family of antifungal drugs and are still useful in the treatment of invasive fungal infections (Chandrasekar, 2011). However, the need for new antifungal drugs is evident, as resistance against polyenes have been on the rise (Dalhoff, 2018). As a part of our effort to identify new antifungal compounds, we have introduced other species of Candida (C. krusei, C. parapsilosis and C. glabrata) to complement the standard C. albicans in the functional antifungal screen of Streptomyces culture extracts (Mojićević et al., 2019), reasoning that following this approach will increase the chances to discover novel lead structures. Herein, we investigated Streptomyces sp. BV410, a soil isolate associated with the rhizosphere of chamomile, which was selected for further chemical characterization and investigation due to the antifungal properties of the crude culture extract (minimal inhibitory concentration (MIC) against C. albicans of 8 µg/ml).

| Microbial strain isolation and identification
Streptomyces sp. BV410 was isolated from a soil sample associated with rhizosphere of Matricaria chamomilla collected in Serbia in 2016 using conditions favouring the growth of sporulating Actinomyces (Mojićević et al., 2019). The strain has been cultivated in tryptone soy broth (Difco) at 30°C for 4 days shaking at 180 rpm for DNA isolation (Nikodinovic, Barrow, & Chuck, 2003). Strain has been identified by 16S rDNA sequence analysis using universal bacterial primer set: 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) and the sequence has been deposited under GenBank accession number: MH128156 (Mojićević et al., 2019). The strain has been deposited at the Institute of Soil Science culture collection ISS WDCM375 under accession number ISS625. The phylogenetic tree was constructed by the maximum likelihood algorithm using Jukes-Cantor distance correction and bootstrap resampling method, all included in the MEGA7 package (Kumar, Stecher, & Tamura, 2016). The tree was rooted using the 16S rRNA gene sequence of Bacillus subtilis MF993342.1 as an outgroup.
Sequences of the nearest type strains, as well as the outgroup strain, were obtained from the GenBank database.
The strain was grown on mannitol-soy flour (MSF; mannitol 20 g/L, soybean flour 20 g/L, agar 20 g/L) agar that was found to promote good growth and sporulation of a majority of Streptomyces spp. from laboratory culture collection at 30°C for 4-14 days. Spore suspension of Streptomyces sp. BV410 was stored in glycerol (20%, v/v), maintained at −80°C, and used for the inoculation of cultures for further experiments.

| Scanning electron microscopy (SEM)
In order to assess the surface morphological characteristics of the hyphae and spores of BV410 isolate, scanning electron microscope was used. Scanning electron micrographs of BV410 grown on MSF agar were obtained by a high-resolution field emission Zeiss Ultra Plus-SEM (Carl Zesis AG) using InLens detector with an accelerating voltage of 5 kV at working distance of 5 mm. Prior to imaging, BV410 were fixed on to the SEM stubs using carbon tape and sputtered with gold/palladium (80/20 ratio) for 10 s.
wide range of small molecules of differing polarity (from nonpolar to polar), was performed by vigorous mixing at 30°C for 12 hr. The EtOAc extract was separated from the cell debris and the aqueous phase by centrifugation (4,000 g for 20 min at 4°C; Eppendorf 5804R benchtop centrifuge). The mycelium residue was afterward extracted with methanol (MeOH) (1/10 of the original culture volume) by vigorous mixing at 30°C for 30 min. The MeOH extract was separated from the cell debris by centrifugation (4,000 g for 20 min at 4°C; Eppendorf 5804R benchtop centrifuge). Both extracts were then separately dried with anhydrous MgSO 4 , followed by drying under vacuum (BUCHI Rotavapor ® R-300, Germany), and the dry mass of extracts was determined.

| Optimization of culture conditions
The effect of substituting soy flour from the JS medium with yeast extract (3%, w/v; JSYE) and the addition of KH 2 PO 4 (1%, w/v; JSYEP) as well as two additional media TSB (20 g/L) and R2YE (Kieser, Bibb, Buttner, Chater, & Hopwood, 2000) were assessed as production media. The initial pH of JS medium was adjusted to pH of 6.5, 7.5 and 8.5 using 0.1 mol/L HCl or NaOH. The addition of methyl oleate (Sigma-Aldrich, 2 ml/L), ZnSO 4 ·7H 2 O (0.5 g/L) and FeSO 4 ·7H 2 O (0.5 g/L) to JS medium (with adjusting initial pH to 8.5) was also examined.

| Isolation, purification, and characterization of the active compound
The 2 g of ethyl acetate extract was completely resuspended in H 2 O (40 ml). This H 2 O (re)suspension was re-extracted using an equal volume of heptane (excluded to remove nonpolar compounds) for further purification, followed be re-extraction with EtOAc (using two times equal volume of solvent). The EtOAc and aqueous phases were dried under vacuum and then dissolved in 5 ml of MeOH, followed by separation by size-exclusion chromatography on Sephadex LH20 (Sigma) with methanol as the mobile phase. Fractions found to contain our molecule of interest (based on activity screening) were pooled and concentrated under vacuum prior to final purification by preparative HPLC. The isolated molecule was used for downstream structure elucidation by NMR and for biological activity assays.
Commercially available staurosporine (Fisher BioReagents, Fisher Scientific) was used as an analytical standard ( Figure A1 in Appendix 1).

| Liquid chromatography-mass spectrometry, high-resolution ESI mass spectrometry and NMR analysis
Samples were analyzed by LCMS on an UltiMate 3,000 LC System coupled to a LCQ Fleet Ion Trap Mass Spectrometer (Thermo Scientific). Chromatographic separation was performed on a Hypersil Gold aQ C18 column (150 × 2.1 mm, 3 µm particle size).
Water (A) and acetonitrile (B) were used as the eluents, both supplemented with 0.1% formic acid. The separation method was performed at 0.7 ml/min using a gradient as follows: 5% B at 0 min to 95% B by 8 min followed by washing the column at 100% B for 2 min and re-equilibration of the column at 5% B for 2 min prior to the next injection. HR-ESI-MS spectra were recorded with a Thermo LTQ-FT Ultra coupled with a Dionex UltiMate 3,000 HPLC system. NMR spectra were recorded on Bruker AVHD300, Bruker AVHD400, Bruker AVHD500 (only 1 H NMR spectra), or Bruker AV500-cryo spectrometers. The chemical shifts are listed as parts per million (ppm) and refer to (TMS; Tetramethylsilane) = 0. The spectra were calibrated using residual undeuterated solvent as an internal reference (CDCl 3 = 7.26 ppm, (CD 3 ) 2 CO = 2.05 ppm, (CD 3 ) 2 SO = 2.50 ppm, CD 3 OD = 3.31 for 1H NMR; CDCl3 = 77.0 ppm, (CD 3 ) 2 CO = 29.8 ppm, (CD 3 ) 2 SO = 39.5 ppm, CD 3 OD = 49.0 for 13 C NMR).

| Anti-Candida assays
Antifungal activity was tested by standard disc diffusion assays against type strains: C. albicans ATCC 10231, C. krusei ATCC 6258, C. parapsilosis ATCC 22019, C. glabrata ATCC 2001. These strains are among top five species most commonly associated with candidiasis (Turner & Butler, 2014). MICs were determined for crude BV410 culture extracts and for purified staurosporine according to CLSI broth microdilution guidelines (CLSI M27-A4, 2012 and CLSI M27-A3, 2008). MICs were determined in RPMI -1640 medium (Sigma, Aldrich) as specified in the standards. The MIC value corresponds to the lowest concentration that inhibited the growth of the respective test organism after 24 hr at 37°C. The highest concentration of the tested compounds used in these assays was 250 µg/ml.
Isolated staurosporine was dissolved in DMSO and filter sterilized (0.2 µm, EMD Millipore) to prepare a stock solution (50 mg/ml) and added to the cells at a concentration of 0.78-50 ng/ml for a treatment that lasted for 48 hr. Results were presented as percentage of the DMSO-treated control that was set to 100%. The percentage viability values were plotted against the log of concentration, and a sigmoidal dose-response curve was calculated by nonlinear regression analysis using the GraphPad Prism software, version 5.0 for Windows (GraphPad Software). Cytotoxicity was expressed as the concentration of the compound inhibiting growth by 50% (IC 50 ).

| In vivo embryotoxicity assay
The in vivo toxicity assessment of isolated staurosporine was car- The effect of staurosporine on the zebrafish embryos survival and development was examined according to the OECD 2013 guidelines for the testing of chemicals (OECD, 2013) and following previously described protocol (Warżajtis et al., 2017) with some modifications.
Briefly, zebrafish embryos were produced by pair-wise mating of wildtype adults, collected and distributed into 24-well plates containing 10 embryos per well and 1 ml embryos water (0.2 g/L of Instant Ocean ® Salt in distilled water), and raised at 28°C. For assessing lethal and developmental toxicity, embryos at the 6 hr postfertilization (hpf) stage were treated with eight concentrations of staurosporine (1,10,20,30,35,40,50, and 60 ng/ml). DMSO (0.125%, v v −1 ) was used as a negative control. Experiments were performed in triplicate using 30 embryos per concentration. Apical endpoints used for the toxicity evaluation (Table A1; Appendix 1) were recorded at 24, 48, 72, 96, and 120 hpf using an inverted microscope (CKX41; Olympus).
Dead embryos were counted and discarded every 24 hr. At 120 hpf, embryos were inspected for heartbeat rate, anesthetized by addition of 0.1% (w v −1 ) tricaine solution (Sigma-Aldrich), photographed and killed by freezing at −20°C for ≥24 hr.
The LC 50 value (the concentration upon which 50% embryos were dead) and the EC 50 value (the concentration affecting 50% of embryos) were determined by the program ToxRatPro (ToxRat ® , Software for the Statistical Analysis of Biotests, ToxRat Solution GmbH, Version 2.10.05) using the probit analysis with linear maximum likelihood regression.

| Hepatotoxicity and myelotoxicity evaluation in the zebrafish model
In order to examine the isolated staurosporine for a possible hepatotoxic effect in vivo, the transgenic Tg(fabp10:EGFP) zebrafish embryos with the fluorescently labeled liver were treated at the 72 hpf stage (when the liver is fully functional) with five doses (30,35,40,45, and 50 ng/ml) of the tested compound. DMSO (0.125%, v v −1 ) was used as a negative control. The hepatotoxicity was determined according to the change of liver area index compared to the control group, calculated as the ratio between liver area and embryonic lateral area × 100% (Zhang et al., 2017).
To address the possible myelotoxicity of the isolated staurosporine, transgenic zebrafish embryos Tg(mpx:GFP) expressing enhanced green fluorescent protein (EGFP) in neutrophils were used. The assay was performed according to the previously described protocol  with slight modifications. Briefly, transgenic embryos were generated by natural spawning of Tg(mpx:GFP) and wild-type adults and reared in the fish embryo water at 28°C. At the 6 hpf stage, embryos were exposed to three doses (25, 30, and 35 ng/ml) of staurosporine upon which no embryonic malformations were observed. DMSO (0.125%, v/v) was used as a negative control. The transgenic embryos were inspected at 72 hpf stage under a fluorescence microscope (Olympus BX51, Applied Imaging Corp.) for the neutrophils presence and fluorescence intensity. Neutrophils occurrence (fluorescence) was determined by ImageJ program (NIH public domain software).

| Antiangiogenic potential evaluation in the zebrafish model
The antiangiogenic activity of the isolated staurosporine was evaluated using transgenic zebrafish Tg(fli1:EGFP) embryos with EGFPlabeled endothelial cells, as was previously described . Briefly, transgenic embryos were generated by natural spawning of wild-type and Tg(fli1:EGFP) adults and reared in embryo water at 28°C. At 6 hpf, embryos were exposed to the range of nontoxic staurosporine concentrations and incubated at 28°C.
After the treatments, embryos were anesthetized with 0.02% tricaine and subsequently photographed. The development of intersegmental blood vessels (ISVs), dorsal longitudinal anastomotic vessels (DLAVs), and of subintestinal vessel (SIV) plexus was inspected and imaged in embryos at 48 hpf and 72 hpf, respectively, under a fluorescence microscope (Olympus BX51, Applied Imaging Corp.). Sunitinib malate (Suten Pfizer), an antiangiogenic drug of clinical relevance, was used as the positive control (Chimote et al., 2014).

| Statistical analysis
The results were expressed as mean values ± standard deviation (SD) and analyzed using Student's t test at a threshold level of p = .05. This analysis was carried out using SPSS 20 (SPSS Inc.) software.

| Streptomyces sp. BV410 isolate from the rhizosphere of chamomile
Soil isolate BV410 was associated with rhizosphere of Matricaria chamomilla, with considerable antifungal activity observed when ethyl acetate extracts of the whole culture grown in JS medium were tested against C. albicans, C. parapsilosis, and C. glabrata (Mojićević et al., 2019). The extract of BV410 showed a MIC against C. albicans of 8 µg/ml and the ability to inhibit formation of biofilm at 125 µg/ml (Mojićević et al., 2019). Therefore, strain BV410 was selected for further characterization and chemical investigation.
BV410 grows well on a variety of standard solid media utilized for Streptomycetes, including ISP-2, TSB, and oatmeal agar (Kieser et al., 2000). We have chosen MSF for general propagation of this strain Using 16S rDNA sequence analysis, strain BV410 was confirmed to belong to the genus Streptomyces (the sequence has been deposited under GenBank Accession number MH128156).

| Isolation, purification, and characterization of the active compound
Ethyl acetate extracts of BV410 whole culture were analyzed using analytical HPLC and further fractionated by semi-preparative HPLC. Antifungal bioactivity assays of each fraction revealed the active component as a major compound within fraction 6 (retention time from 14.5 to 15.5 min) of the extract (Figure 2). For preparative compound isolation, the BV410 crude extract was further extracted with solvents ranging in polarity (heptane/ethyl acetate/aqueous). The active compound was predominantly found in the ethyl acetate and aqueous extracts, which was further separated by size-exclusion chromatography using Sephadex LH-20 followed by purification using preparative HPLC. The isolation using an acidic buffer system. This lead to a major conformational change in this molecular portion with the observed changes in NMR chemical shifts, as previously described in the literature (Link et al., 1996). We furthermore corroborated our NMR structure elucidation results by comparison of our material to an authentic commercial standard by HPLC-UV-MS ( Figure A3 in Appendix 3). Both compounds indeed perfectly matched (HPLC retention time, UV spectrum, MS spectrum), thus validating the above assignment.

| Optimization of staurosporine production by BV410
Having determined that the active secondary metabolite produced  The crude extract was collected as multiple fractions (red bars) which were individually tested for antifungal activity. Fraction 6 (*) with a retention time of 14.5-15.5 min showed potent antifungal activity. Fraction 6 was further purified, and a UV absorbance spectrum was obtained (inlet) Having established that JS was optimal for the staurosporine production over 14 days, the initial pH values were adjusted to 6.5, 7.5, and 8.5 with the unadjusted pH value of the JS medium being 7.8 (Figure 3c). Lowering the initial pH value of the production medium turned out to be suboptimal for the final staurosporine yield (reduction of 13% and 26% has been observed), while the increase to pH 8.5 resulted in the slight increase in the staurosporine yield (2%-5%). The addition of methyl oleate, ZnSO 4, and FeSO 4 to JS medium with the initial pH adjusted to 8.5 on the staurosporine yield was also assessed (Figure 3c). The presence of methyl oleate and ZnSO 4 resulted in the 35% and 62% reduction, respectively, while the addition of FeSO 4 caused 30% increase in staurosporine amount.
Alternatively, methyl oleate and especially ZnSO 4 had beneficial effect on the biomass yield (Figure 3c inlet). Overall, this initial optimization of the staurosporine production resulted in the defining of the stable fermentation medium and protocol yielding 56.25 mg/L of staurosporine.

| Biological activity evaluation of the isolated staurosporine
We next comprehensively evaluated the anti-Candida activity of isolated staurosporine, in vitro cytotoxicity against two cell lines (healthy MRC5 and cancer cell line A549) as well as the in vivo embryotoxicity and antiangiogenic properties in zebrafish (Table 1).

Isolated staurosporine inhibited all four Candida strains (MIC values between 24 and 390 ng/ml) although the crude extract of BV410
was not active against C. krusei. The most sensitive test strain was

C. glabrata with MIC values between 4-and 16-fold lower in com-
parison with the other three Candida spp. In a comparison, under the same conditions MIC values for standard antifungal nystatin were between 0.125 and 2 µg/ml (Table A2).  Zebrafish embryos were exposed to 12 different concentrations (between 1 and 100 ng/ml) of isolated staurosporine up to 120 hpf, and its overall toxicity as well as hepatotoxicity were evaluated ( Figure 4). Staurosporine did not cause any observable embryos malformations up to a concentration of 35 ng/ml, while at a dose of 40 ng/ml 50% of the embryos were dead, 23% appeared to be teratogenic (nonresorbed yolk and scoliosis), while 27% of the embryos were without visible deformations (Figure 4a).

Potential cardiotoxicity and liver toxicity of staurosporine
were assessed daily from 72 to 120 hpf as these present common F I G U R E 4 Toxicity assessment of staurosporine in the zebrafish model. (a) The dose-dependent survival/teratogenicity, (b, c) cardio-and hepatotoxicity, and (d, e) myelotoxicity are shown. At a staurosporine dose of 50 ng/ml, embryos were seriously affected, showing large pericardial edema (solid arrow), reduced and damaged liver (white arrow) and whole-body edema (b). The changes in the liver area index assessed in 120-hr old zebrafish embryos were not observed between DMSO-treated (Control) and staurosporine-treated embryos (35 ng/ ml), contrary to the group upon a dose of 50 ng/ml (c). Staurosporine was not myelotoxic at doses up to 35 ng/ml, while at 35 ng/ml, it caused weak neutropenia in the treated zebrafish embryos, as detected at 72 hpf. *p < .05, **p < .01, ***p < .0001 drawbacks of drugs approved for human use. The results showed that no embryos exhibited signs of cardiotoxicity at doses ≤35 ng/ ml, such as an appearance of pericardial edema, changed heart morphology (Figure 4b), nor disturbed heartbeat rates (data not shown). At doses ≥35 ng/ml, staurosporine induced teratogenic malformations, such as scoliosis (at ≥40 ng/ml), pericardial and whole-body edema (at ≥45 ng/ml). The transgenic Tg(fabp10:EGFP) zebrafish embryos with fluorescently labeled liver was used to evaluate the potential hepatotoxicity. The hepatotoxicity was evaluated at 120 hpf old embryos, according to the liver area index (the ratio between liver area and lateral body area) which was shown to be an adequate measure to assess liver damage (Zhang et al., 2017). As shown in Figure 4b Therefore, strain BV410 was selected for further characterization and chemical investigation. Bioactive compound was identified undoubtedly as staurosporine through comprehensive purification and chemical analysis steps (Figure 2, Figure A2, and Figure   A3).

The closest similarity of Streptomyces sp. BV410 was to
Streptomyces europaeiscabiei, isolated from potato scab lesions in Korea (Figure 1c) (Song et al., 2004). These two grouped with S. flavogriseus and S. longisporoflavus, supported by 83% of bootstrap replicates. Interestingly, Streptomyces sp. BV410 was not found within the same branch with staurosporine producer S. staurosporininus isolated from hay meadow soil from the Cockle Park (Northumberland, United Kingdom) (Kim, Zucchi, Fiedler, & Goodfellow, 2012).
For all bioactive compounds, there is a general interest to obtain high productivities and high titers, as it ensures a cost-effective production. With Streptomycetes, this has been achieved using classical strain improvement, optimization of process parameters as well as genetic engineering (Kieser et al., 2000;Olmos et al., 2013). The influence of the nutritional parameters on the production of antibiotics is undisputable (Bundale, Begde, Nashikkar, Kadam, & Upadhyay, 2015;Görke & Stülke, 2008). Through the initial optimization of staurosporine production, isolate BV410 was shown to produce 56.25 mg/L of staurosporine ( Figure 3). As a comparison, the staurosporine yield of 23 mg/L from a 400-l fermentation of Streptomyces sp. AM-2282 (the originally described staurosporine producer, now Lentzea albida AM-2282) has been reported after 72 hr cultivation in a medium containing glucose 3%, soybean meal 1.5%, and CaCO 3 0.4% for 72 hr (Omura et al., 1977). Several other species of Streptomyces have been reported to produce staurosporine and its derivatives with comparable or lower titers (Cheng et al., 2016;Onaka, Taniguchi, Igarashi, & Furumai, 2002;Park et al., 2006;Zhou, Q in, Ding, & Ma, 2018). Staurosporine has also been isolated from the marine ascidian Cystodytes solitus (Reyes et al., 2008). Because 56.25 mg/L (0.9 mg/g mycelia) of staurosporine from 400 ml culture in 2 L flask was obtained within this study, future optimization of staurosporine production in BV410 should result in further yield increase.
Notably, the addition of exogenous methyl oleate that was previously proposed to cause the alteration in Streptomyces spp. membrane permeability and stimulate the accumulation of branched amino acids (Huang, Xia, Li, Wen, & Jia, 2013;Mouslim, David, Pétel, & Gendraud, 1993), negatively affected staurosporine production. Surprisingly, the addition of ZnSO 4 even more drastically reduced the production of staurosporine in this strain (Figure 3c).

This was not in line with the previous findings that both ZnSO 4
and FeSO 4 stimulated neomycin production in Streptomyces fradiae (Vastrad & Neelagund, 2014).
Staurosporine showed anti-proliferative activity against healthy and cancer cell lines (MRC5 and A549) at a comparable level (2 and 3 ng/ml), which is in line with previous reports on staurosporine nonselective cytotoxicity via potent inhibition of protein kinases, especially tyrosine kinase (Manns et al., 2011;Nakano & Omura, 2009;Tamaoki et al., 1986). On the other hand, in vivo toxicity assessment in zebrafish embryos revealed no signs of toxicity at 35 ng/ml (LC 50 value of 45 ng/ml) (Table 1, Figure 4). Taken together, our results indicated only limited potential of staurosporine as an antifungal agent, but encouraged further evaluation of the compound in the zebrafish platform. Indeed, using transgenic zebrafish line suitable to monitor angiogenesis, its potent antiangiogenic activity was shown ( Figure 5). The antiangiogenic effect of staurosporine was previously studied in a different in vivo assay involving chorioallantoic membranes of growing chick embryos, with the efficient concentrations of 71 pmol per egg (Oikawa et al., 1992). This was followed up with synthesis of a number of staurosporine derivatives that also showed antiangiogenic potential by inhibiting endothelial cell proliferation even in mice (Li et al., 2000;Monnerat et al., 2004). Nowadays, staurosporine is the control agent of choice to induce apoptosis in zebrafish (Eimon & Ashkenazi, 2010).
Our results demonstrate that staurosporine still have high therapeutic potential (therapeutic window, Table A3) especially in the angiogenesis-related pathologies such as cancer, inflammation, retinopathy, and others. Only tivozanib, vascular endothelial growth factor receptor (VEGFR) inhibitor, has been shown to have better antiangiogenic properties in the zebrafish model (Chimote et al., 2014) compared to staurosporine. Tivozanib was associated with the complete regression in SIVs at 5 nM with acute toxicity in zebrafish at 45 nM and is currently in clinical trials (Chimote et al., 2014).
In this study, although initially aiming at the discovery of novel antifungal compounds against C. albicans and non-albicans strains,

CO N FLI C T O F I NTE R E S T
None declared.

DATA AVA I L A B I L I T Y S TAT E M E N T
The strain has been deposited at the Institute of Soil Science b Malformation of eyes was recorded for the retardation in eye development and abnormality in shape and size. c Presence of no, one or more than two otoliths per sacculus, as well as reduction and enlargement of otoliths and/or sacculi (otic vesicles). d Tail malformation was recorded when the tail was bent, twisted or shorter than to control embryos as assessed by optical comparison. e Growth retardation was recorded by comparing with the control embryos in development or size (before hatching, at 24 and 48 hpf) or in a body length (after hatching, at and onwards 72 hpf) using by optical comparison using a inverted microscope (CKX41; Olympus).  The values given for staurosporine and sunitinib are in ng/mL and µg/mL, respectively, while those in brackets are in nM and µM, respectively.

F I G U R E
LC 50 -the concentration inducing the lethal effect of 50% embryos. ECx 50 -the concentration affecting 50% embryos (survival and developmental defects). IC 50 values -the concentration upon which 50% of embryos displayed anti-angiogenic phenotype. EC 50 (SIVs and ISVs) -the effective concentration resulting in 50% decrease of SIVs basket or ISVs length compared to the DMF-treated control group. Tw-the ratio between LC 50 and IC 50 values. a The data for sunitinib-malate have previously been reported by our group (Pavic et al. Jour Inorg Biochem, 174 (2017), 156-168).