Introduction

The filamentous Gram-positive bacterial genus, Streptomyces, is well characterized as the prolific producer of secondary metabolites with a vast array of significant biological activities [1]. Bioactive molecules controlling physiological functions in various organisms have been discovered through extensive screening of the Streptomyces culture library [2,3,4,5,6,7,8]. Our group also discovered an 18-membered macrolide borrelidin with a necrotic activity against potato tubers [9] and a bipyrrole compound 4-methoxy-2,2′-bipyrrole-5-carbaldehyde as a strobilation inhibitor against moon jellyfish without inducing cytotoxicity [10].

Malaria is a major global infectious diseases caused by the mosquito-borne Plasmodium. Among the five species that infect humans, Plasmodium falciparum is responsible for most severe and fatal cases. In 2024, there were almost 282 million estimated malaria cases across 80 malaria-endemic countries. Furthermore, 610,000 deaths were associated with malaria in 2024. Unexpectedly, there is an increase of 12 million compared to 2023 [11]. Sustainable discovery and development of new antimalarial drugs are key element in combating malaria. However, the overuse or insufficient use of antimalarial drugs sometimes lead to the emergence of drug-resistant strains. To reduce the occurrence of malaria disease, structural modification of parent compounds and extensive exploration of bioresources are considered to be practical strategies. Chloroquine, a first-generation antimalarial agent, is associated with considerable side-effect and its overuse led to emerge drug-resistant strains. To improve its safety profile, hydroxychloroquine was developed by introducing a hydroxyl group, while largely retaining the antimalarial activity of chloroquine. However, hydroxychloroquine does not overcome chloroquine resistance. To address drug-resistant malaria, structurally distinct compounds such as mefloquine were subsequently developed. Thus, both the structural modification of parent compounds and the development of new chemical scaffolds are essential to improving biological activity and overcoming drug resistance in malaria treatment. Regarding extensive exploration of bioresources, we have previously discovered antimalarial compounds in traditional Kampo medicine [12] and in the leaves of Morinda morindoides [13, 14]. Streptomyces strains are potential antimalarial agents. For example, a phosphonate FR900098 was isolated from Streptomyces rubellomurius [8, 15] and an α-pyridone compound iromycin from Streptomyces sp. RBL-0292 [16]. Furthermore, some of our Streptomyces culture libraries showed promising antimalarial activity against both chloroquine/mefloquine-sensitive (3D7) and -resistant (Dd2) cell lines of P. falciparum [17]. Among the screened libraries, Streptomyces antibioticus HUT6035 (a synonym of strain NBRC3117) showed the highest activity and lowest cytotoxicity.

In this study, we isolated antimalarial compounds through the large-scale fermentation of the strain HUT6035 and subsequent purification using various chromatography techniques. Strain HUT6035 accumulated actinomycins X2 and X0ß, which are the 4-oxo and 4-hydroxy proline derivatives of actinomycin D, respectively. Their antimalarial activity, cytotoxicity, and selectivity index (SI) were investigated, and the results are described herein.

Materials and methods

Bacterial strain used in this study and preparation of the culture extract

Streptomyces antibioticus strain HUT6035 was cultured in YM medium (0.4% yeast extract, 1.0% malt extract, and 0.4% D-glucose, pH 7.3) at 28 °C with 120 rpm (revolutions per minute) for 5 days, according to our standard protocol [18].

Spectroscopic instruments

The active components of strain HUT6035 against P. falciparum strains 3D7 and Dd2 were analyzed using electrospray ionization-mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR). The ESI-MS spectra were obtained using an Orbitrap EclipseTM TribridTM mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). High-resolution ESI-MS was performed in positive ion mode. The NMR spectra (600 MHz for 1H NMR and 150 MHz for 13C NMR) were recorded using an ECZL600G spectrometer (JEOL, Tokyo, Japan) equipped with a field gradient accessory. The NMR chemical shifts were recorded as δ values in ppm. The coupling constants in 1H NMR were shown as J values in Hz. Deuteriochloroform was used as the solvent. Chemical shifts were recorded in δ value based on solvent signals (δC = 77.0 in CDCl3) or the internal standard tetramethylsilane (δH = 0).

Isolation of actinomycins from Streptomyces antibioticus strain HUT6035

The culture supernatant of HUT6035 cells (6 L) was extracted twice using an equal volume of ethyl acetate (EtOAc). The combined organic phases were dried (Na2SO4), filtered, and concentrated in vacuo. The crude extract was purified by gel filtration chromatography on Sephadex LH-20 (GE Healthcare, Chicago, IL, USA) in MeOH. All fractions (1 mL each; total 50 fractions) eluted with MeOH were subjected to a bioassay using P. falciparum 3D7 and Dd2 for antimalarial activity and primary Adult Mouse Brain (AMB) cells for cytotoxicity according to our previous report [17]. The fractions containing the active component(s) with antimalarial activity were combined, and the resulting reddish residue was further purified by silica gel chromatography in CHCl3–MeOH = 100:1–50:1 (v/v) to obtain two active components. These two components, HUT6035A and HUT6035B, appeared as reddish spots on TLC at Rf = 0.50 and 0.40 at CHCl3–MeOH = 15:1 (v/v), respectively. Compounds HUT6035A and HUT6035B were identified as actinomycin X2 and actinomycin X0ß, respectively, according to the reported data [19,20,21,22].

HUT6035A ( = actinomycin X2): The 1H and 13C NMR assignments are shown in Table S1. HRMS (positive ESI): m/z calculated for C62H84N12O17Na: 1291.5970 [M+Na]+; observed: 1291.5984.

HUT6035B ( = actinomycin X0ß): The 1H and 13C NMR assignments are shown in Table S2. HRMS (positive ESI): m/z calculated for C62H86N12O17Na: 1293.6126 [M+Na]+; observed: 1293.6140.

Bioassay for the antimalarial activity and the cytotoxicity

Malarial parasite cultures and AMB cells were previously described [17]. The detailed experimental procedure is described in the Supplementary data. Antimalarial activity and cytotoxicity were evaluated at 50% inhibitory concentration (IC50) and 50% cytotoxic concentration (CC50), respectively. We here analyzed these activities on purified actinomycin X2 (1), actinomycin X0ß (2), and commercially available actinomycin D (3). The SI is calculated as CC50/IC50.

Time-course production of actinomycins in strain HUT6035

The EtOAc extract from a 100-mL culture of strain HUT6035 (1-5 days of cultivation) was dissolved in MeOH (1 mL) and analyzed by HPLC and ESI-MS. For HPLC analysis, each aliquot (10 µL) was applied on a COSMOSIL CHOLESTER column (4.6 I.D. x 250 mm, Nacalai Tesque, Kyoto, Japan) and eluted with 70% aqueous MeOH at a flow rate of 1.0 mL min–1. The eluate was monitored at 450 nm using an MD-2010 multiwavelength photodiode array detector (JASCO Corporation, Tokyo, Japan). Owing to the overlapping peaks for 1 and 3 in the HPLC chromatogram, the production yields of 1 and 3 were evaluated by ESI-MS through the peak intensities of the monoisotopic signals. Purified natural 1 and 2, and the commercially available 3, were analyzed in the same manner.

DNA sequencing and assembly

Streptomyces total DNA was prepared according to a previously modified protocol [23]. Draft genome sequencing was performed using a paired-end sequencing strategy (2 × 300 bp) on an Illumina NextSeq 1000 platform (Illumina, Inc., San Diego, CA, USA). De novo assembly of the raw genome sequencing data was performed using SPAdes 4.2.0 [24]. The actinomycin biosynthetic gene cluster (BGC) was identified using antiSMASH ver. 8.0.3 [25], and its annotated sequence was deposited in the DDBJ/ENA/GenBank database under the accession number: LC912581.

Detailed experimental procedure for sample preparation of Streptomyces total DNA and next-generation sequencing is described in the Supplementary data.

Results and discussion

Isolation of actinomycins from Streptomyces antibioticus strain HUT6035

In our pilot screening of 28 actinomycete culture extracts, 17 samples showed parasite growth inhibition of more than 50% at a concentration of 50 µg/mL [17]. Among them, the culture extract of Streptomyces antibioticus strain HUT6035 showed the highest antimalarial activity with an IC50 value of 0.09 and 0.22 µg/mL against 3D7 and Dd2, and SI of 188 and 73.7, respectively [17]. Hence, we isolated antimalarial compound(s) with high SI values from strain HUT6035.

The culture extract (6-L culture) of HUT6035 was purified using Sephadex LH20 and silica gel chromatography, with a bioassay using P. falciparum strains 3D7 and Dd2 for antimalarial activity and AMB cells for cytotoxicity. Two reddish spots detected at Rf = 0.50 and 0.40 in CHCl3–MeOH = 15:1 (v/v) showed significant antimalarial activity. They were designated as HUT6035A and HUT6035B (average isolation yields of 25 mg and 3.3 mg per L, respectively), and further analyzed by ESI-MS and NMR analyses [19,20,21,22].

In high-resolution ESI-MS analysis, HUT6035A was established as C62H84N12O17. The 1H and 13C NMR assignments of HUT6035A are summarized in Table S1 (Supplementary Materials). In the 13C NMR spectrum of HUT6035A, 62 signals were detected and classified as 16 methyls, 7 methylenes, 16 methines, and 23 nonprotonated carbons. Their connectivity was further confirmed using 1D and 2D NMR techniques including HMQC and HMBC spectra (Figs. S1–S2), in good agreement with actinomycin X2 ( = actinomycin V) (Fig. 1; compound 1) [19,20,21,22]. In contrast, the molecular formula of HUT6035B was determined to be C62H86N12O17, with two protons larger than that of HUT6035A. The NMR assignment (Table S2) and spectra of HUT6035B (Figs. S3–S4) showed good agreement with the reported data for actinomycin X0ß (Fig. 1; compound 2) [21, 22].

Fig. 1
Fig. 1The alternative text for this image may have been generated using AI.
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Structures of actinomycin X2 (1) and actinomycin X0ß (2) isolated from Streptomyces antibioticus HUT6035. The representative actinomycin derivatives, actinomycin D, was also described as compound 3. Sar, sarcosine

Significant selectivity index of actinomycin derivatives for antimalarial activity

The two actinomycin derivatives together with commercially available actinomycin D (Fig. 1) were subjected to an antimalarial activity assay using P. falciparum strain 3D7. To determine the selectivity index (SI) of the actinomycins, their cytotoxicity was investigated using AMB cells (Table 1). Actinomycin X2 (1) showed an antimalarial activity (IC50 = 0.241 nM) and a cytotoxicity (CC50 = 6.71 nM), while actinomycin X0ß (2) showed IC50 = 7.69 nM and CC50 = 818 nM. Compared with commercially available actinomycin D (3) (IC50 = 0.469 nM and CC50 = 6.71 nM), both 1 and 2 had significant selectivity index (SI) values (CC50/IC50) of 27.9 and 106, respectively. The SI value is important for the safety of human cells, and the evaluation of drug candidates. These SI values were 2- and 7-fold higher than those of actinomycin D, respectively (SI = 14.3). Therefore, actinomycin derivatives are potential antimalarial agents. Alenazi et al. reported the antimalarial activity of actinomycin D, however, they did not mention the SI value [26]. In terms of the SI value, actinomycin D is considered to be a less promising candidate for an antimalarial drug. This tendency corresponds to our preliminary screening of Indonesian Streptomyces sp. AA018, an actinomycin D overproducer, was isolated from Karst in Sulawesi Island as part of our international collaborative project [27, 28]. The culture extract of strain AA018 was removed from the list of potential antimalarial drug candidates due to its low SI value. These findings indicate that the structural modification of the mother compound may lead to increased SI values, which are important for clinical antimalarial agents. Similarly, our group reported that structural modification of lankacidin through chemical modification led to improved antitumor activity by filling the binding pocket in microtubule [29, 30]. Hence, structural variants are worth studying for their biological activity.

Table 1 In vitro antimalarial activity and cytotoxicity of actinomycin derivativesa
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Time-course production of actinomycins in strain HUT6035

Culture extracts of strain HUT6035 (1–5 days of cultivation) were analyzed using HPLC and ESI-MS. Equal volumes of crude extracts (10 µL of 1 mL MeOH solution) at every period were injected for normalization, and their productivity was estimated by the peak area between peak curves and baseline. In our HPLC condition (Fig. 2a), actinomycins X0β (2) elutes at 15.5 min, and actinomycin X2 (1) and actinomycin D (3) elute at 24.5 min with overlapping. Hence, the production ratios of 1 and 3 were evaluated based on the peak intensities in their ESI-MS spectra monoisotopic signals at m/z 1291.60 for 1, m/z 1277.62 for 2, and m/z 1293.61 for 3. Compound 1 accumulated prior to 2 and 3 in strain HUT6035 (Fig. 2). The production ratios of 1–3 were 100:0:0 (2-days culture), 100:5:5 (3-days), 100:15:20 (4-days), and 100:20:20 (5-days), respectively, based on the peak intensities of their monoisotopic signals. Under our culture conditions for strain HUT6035, actinomycin derivatives rather than 1–3 could not be detected by either HPLC or ESI-MS analyses (Fig. 2). Compounds 1–3 were designated actinomycin X (a mixture of actinomycins) in S. antibioticus IMRU3720 [31]. Genome sequencing of strain HUT6035 using an Illumina NextSeq 1000 sequencer revealed the presence of an actinomycin BGC. Reported actinomycin BGCs are shown in Fig. 3a. The actinomycin BGC (actm cluster) in HUT6035 has an end-to-end similarity to that of Streptomyces antibioticus IMRU3720 (Fig. 3a, panel ii) [31]. They have a variety of BGC compared to Streptomyces chrysomallus ATCC11523 (Fig. 3a, panel iii) [31] and Streptomyces costaricanus SCSIO ZS0073 (Fig. 3a, panel iv) [32]. The substrate specificity of A-domain was predicted using a web-based NRPS A-domain predictors [PARAS (Predictive Algorithm for Resolving Adenylation domain Selectivity): https://paras.bioinformatics.nl/data_annotation] [33]. Substrate specificity determining residues in two NRPS enzymes ActmB and ActmC of strain HUT6035 were similar to those in S. antibiotics IMRU3720, rather than those in S. chrysomallus ATCC11523 (Fig. 3b). ActmM functions as a cytochrome P450 enzyme in coordination with ferredoxin ActmN, indicating the oxidation of ring-B in the biosynthesis of 1 and 2. ActmF presumably catalyzes the condensation of two pentapeptide chains into the phenoxazinone core during actinomycin biosynthesis (Fig. 3b) [32, 34, 35]. The following features are unique to actinomycin biosynthesis: (1) regiospecific oxidation of a proline residue occurs only in ring-B, not in ring-A, and (2) oxidized ring-B is preferentially coupled with ring-A through condensation to a phenoxazinone core in strain HUT6035. This observation was also reported by Wang et al. [22]. The preferential production of 1 and 2 over 3 indicates the rich building blocks of the oxidized pentapeptide chains, rather than the pentapeptide chain with non-oxidized proline, which is used for ring-A in 1–3 and ring-B in 3. The variable production of actinomycins with an oxidative degree strongly suggests relaxed substrate specificity for the condensation reaction between the two peptide chains and the phenoxazinone core.

Fig. 2
Fig. 2The alternative text for this image may have been generated using AI.
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Time-course production of actinomycins in strain HUT6035. Strain HUT6035 was cultivated and analyzed the production of actinomycins at 1–5 days periods using HPLC a and ESI-MS b analyses. (i) actinomycin X2 (1), (ii) actinomycin X0ß (2), (iii) actinomycin D (3), (iv) 1-day culture, (v) 2-day culture, (vi) 3-day culture, (vii) 4-day culture, and (viii) 5-day culture

Fig. 3
Fig. 3The alternative text for this image may have been generated using AI.
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Biosynthetic gene cluster for actinomycins in strain HUT6035. a Gene organization of actinomycin biosynthetic gene (actm) cluster in strain HUT6035 (panel i) and other producing strains, Streptomyces antibioticus IMRU3720 (panel ii), Streptomyces chrysomallus ATCC11523 (panel iii), and Streptomyces costaricanus SCSIO ZS0073 (panel iv). b Possible biosynthetic pathway of actinomycins

Conclusion

We have revealed that actinomycin derivatives, actinomycins X2 and X0ß, produced by strain HUT6035, showed higher SI values (27.9 and 106) compared with their mother compound, actinomycin D (14.3). Value-added derivatives are available through natural variants or artificial conversion (e.g., chemical synthesis and enzymatic modifications). Thus, structural variants are important resources for improving biological activity.