Evaluation of Indolocarbazoles from Streptomyces sanyensis as a Novel Source of Therapeutic Agents against the Brain-Eating Amoeba Naegleria fowleri

Naegleria fowleri is an opportunistic pathogenic free-living amoeba which is able to rapidly colonize the central nervous system (CNS) and causes a lethal infection known as primary amoebic meningoencephalitis (PAM). Furthermore, more than 98% of the known cases of PAM are fatal and affect mainly children under 12 and young adults. Until now, no fully effective therapeutic agents against N. fowleri are available and hence the urgent need to find novel agents to treat PAM. At present, PAM therapy is based on the combination of amphotericin B, miltefosine, among others, with unwanted toxic effects. Recently, our team isolated various indolocarbazoles (ICZs) from the culture of a mangrove strain of Streptomyces sanyensis which showed activity against kinetoplastids and the Acanthamoeba genus. Hence, in this study, the activity of the previously isolated ICZs, staurosporine (STS), 7-oxostaurosporine (7OSTS), 4′-demethylamino-4′-oxostaurosporine, and streptocarbazole B, was evaluated against two type strains of N. fowleri. Furthermore, the performed activity assays revealed that STS was the most active ICZ presenting an inhibitory concentration 50 (IC50) of 0.08 ± 0.02 µM (SI 109.3). Moreover, STS induced programmed cell death (PCD) in the treated amoebae by triggering DNA condensation, mitochondrial disfunction, cell membrane disruption, and reactive oxygen species (ROS) generation. Therefore, STS could be a promising therapeutic agent against PAM.


Introduction
The free-living amoeboid species Naegleria fowleri is the causal agent of a fatal encephalitis known as primary amoebic meningoencephalitis (PAM) [1][2][3][4]. Furthermore, this species, also named brain-eating amoeba by the media, is able to colonize the brain after amoeboid-contaminated water Since 2000, the incidence of PAM has increased worldwide and it is often associated with aquatic risk 48 activities such as swimming and diving in warm-water bodies [10].

49
Regarding PAM clinical symptoms, they are non-specific, and hence the diagnosis is often 50 reached after the patients have died [11][12][13]. Apart from a late diagnosis, current therapeutic 51 approaches are based on a combination of compounds which include miltefosine and amphotericin 52 B as well as azithromycin and/or rifampin [14,15]. In the most recent reported cases, the addition of 53 miltefosine in combination with hypothermia has resulted in the survival of affected patients 54 [12,16,17]. Nevertheless, novel therapeutic molecules for PAM are needed since the current option 55 presents undesired toxic side effects [13].

63
Indolocarbazoles including STS have been previously reported to present protein kinase (PK) 64 and/or DNA topoisomerase I inhibition properties [22,[25][26][27][28][29][30]. Furthermore, focusing on PK inhibitors Our results revealed that STS statins caused induction of PCD-compatible effects in treated amoebae and hence, they could be a useful candidate for the development of novel therapeutic approaches for PAM.

Amoebic Strains and Cell Line Maintenance
The American Type Culture Collection strains ATCC ® 30808™ and ATCC ® 30215™ of Naegleria fowleri (LG Promochem, Barcelona, Spain) were used for the activity assays. Amoebae were grown at 37 • C in axenic conditions in 2% (w/v) bactocasitone medium (Thermo Fisher Scientific, Madrid, Spain) supplemented with 10% (v/v) foetal bovine serum (FBS), 0.3 µg/mL of penicillin G and 0,5 mg/mL of streptomycin sulphate (Sigma-Aldrich, Madrid, Spain). As required by the Spanish Government biosafety guidelines for this pathogen, amoebic strains were cultured in a biological security facility level 3 at the Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias, Universidad de La Laguna.

Activity Assays
The in vitro activity of the four ICZs included in this study was evaluated against trophozoites of two Naegleria fowleri type strains using a colorimetric assay [13]. Amoebae (2 × 10 5 cells/mL) were placed in a sterile 96-well microtiter plate. Next, 50 µL of a serial dilution of each of the ICZs (in N. fowleri culture medium) was added to the plate. In all the activity assays, 2% DMSO was used to dissolve the highest dose of the molecules to prevent generation of any effects in the amoebic cells. Negative controls were also included in the assays, consisting of the addition of medium alone to the seeded trophozoites as previously described [13]. Miltefosine and amphotericin B were used as positive controls whereas the addition of medium alone to the seeded trophozoites was considered as the negative control. Lastly, 10% of the alamarBlue ® reagent was placed in each well, and plates were incubated with slight agitation for 48 h at 37 • C. After that, activity plates were evaluated with an EnSpire ® Multimode Plate Reader (Perkin Elmer, Madrid, Spain) using a wavelength of 570 nm and a reference wavelength of 630 nm.
To calculate the percentages of growth inhibition and 50% inhibitory concentrations (IC 50 ), non-linear regression analyses were performed with a 95% confidence limit using the GraphPad Prism8.0.2 software program (GraphPad Software, San Diego, CA, USA). The IC 90 values of the compounds were calculated using a GraphPad calculator (GraphPad Software, San Diego, CA, USA) based on the calculated IC 50 and the Hill Slope (H). All experiments were performed in triplicate, and the mean values were also calculated. A paired two-tailed t-test was used for the analysis of the data, and P values < 0.05 were considered statistically significant as previously described [13,35].

Cytotoxicity Assays
Cytotoxicity of the compounds was evaluated using toxicity assays established in our laboratory based on the evaluation of the toxicity induced in a murine macrophages J774.A1 cell line (ATCC ® TIB-67™) [35,36]. Concisely, different concentrations of the ICZs were incubated with the cell line, and alamarBlue ® reagent (10% of medium volume) was placed in each well and incubated for 24 h, at 37 • C under 5% CO 2 conditions. Results were examined as described in Section 2.4.1.

Evaluation of Cellular Membrane Damage
The SYTOX Green assay (Life Technologies, Madrid, Spain) allows the detection of alterations in the permeability of the cell membrane. The SYTOX Green dye binds to DNA, showing intense green fluorescence, only if the plasmatic membrane is damaged and allows it to enter the cytoplasm [21]. Experiments were performed by incubation of 5 × 10 5 trophozoites incubated with the earlier calculated IC 90 of STS. After 24 h, 1 µM SYTOX Green (final concentration) was added to the cell culture and incubated for 15 min. Next, cells were examined in an EVOS FL Cell Imaging System AMF4300 (Life Technologies, Madrid, Spain).

Assessment of Mitochondrial Function Disruption in Treated Cells
The evaluation of mitochondrial function disruption in STS-treated amoebae was carried out using the Celltiter-Glo ® Luminescent Cell Viability Assay (Promega Biotech Ibérica, Madrid, Spain) and the JC-1 Mitochondrial Membrane Potential Detection Kit (Cayman Chemicals, Vitro SA, Madrid, Spain). The first kit detects the levels of ATP in STS-treated cells in comparison to non-treated cells.

ATP Detection Levels Using the Celltiter-Glo ® Luminescent Cell Viability Assay
Measurement of ATP levels involved incubation of amoebae at a concentration of 5 × 10 5 cells/mL for 24 h at 37 • C with the previously calculated IC 90 of STS. Finally, the data were obtained using an EnSpire ® Multimode Plate Reader (Perkin Elmer, Madrid, Spain) as described before [21].

JC-1 Assay for the Detection of Alteration in the Mitochondrial Membrane Potential
Mitochondrial Membrane Potential evaluation was started by using a stock solution of 5 × 10 5 cells/mL which were incubated in a 96-well plate with the IC 90 of STS for 24 h at 37 • C. Images were captured using an EVOS FL Cell Imaging System AMF4300 (Life Technologies, Madrid, Spain). The obtained staining pattern permitted the identification of two groups in a cellular population: live cells which would show only red fluorescence and cells presenting low mitochondrial membrane potential (undergoing PCD) which would show an intense level of green and red fluorescence [21].

Generation of Intracellular Reactive Oxygen Species (ROS)
The CellROX Deep Red fluorescent assay (Thermo Fisher Scientific, Madrid, Spain) was used to evaluate the generation of intracellular reactive oxygen species (ROS) in STS-treated amoebae. Experiments were carried out after incubation of 5 × 10 5 cells/mL with STS for 24 h at 37 • C. Next, cells were exposed to 5 µM CellROX Deep Red for 30 min at 37 • C in the dark and observed after using the EVOS FL Cell Imaging System AMF4300 (Life Technologies, Madrid, Spain). The presence of intracellular ROS was shown by the detection of high red fluorescence in the cells [21].

In Vitro Activity and Toxicity of the Tested Natural ICZs against Naegleria fowleri
Activity assays revealed that staurosporine (STS, 1) and 7-oxostaurosporine (7OSTS, 2) were the most active tested ICZs against both strains of N. fowleri as shown in Table 1. In contrast, 4 -demethylamino-4 -oxostaurosporine (4 D4 OSTS, 3) and streptocarbazole B (SCZ B, 4) were only active against the N. fowleri ATCC 30808 strain. Furthermore, STS (1) and 7OSTS (2) showed IC 50 values ranging from 0.08 ± 0.02 to 1.28 ± 0.35 µM for both tested strains. Therefore, ICZs 1 and 2 were more active than the currently used compounds miltefosine (from 38.74 ± 4.23 to 81.57 ± 7.23 µM depending on the strain, Table 1) and amphotericin B (from 0.12 ± 0.03 to 0.16 ± 0.02 µM, differing on the strain assayed, Table 1). Furthermore, STS (1) and 7OSTS (2) showed lower values of cytotoxicity than miltefosine after performance of toxicity assays (Table 1). In the case of amphotericin B, STS (1) showed similar levels of toxicity when compared to the reference drug. Hence, STS was selected as the candidate for the evaluation of PCD induction in treated cells due low IC 50 and toxicity.

Evaluation of PCD Induction in STS-Treated Amoebae
The chromatin condensation assay was positive after observation of IC 90 -treated N. fowleri cells (treatment was conducted for 24 h). Treated amoebae exhibited the characteristic intense blue fluorescent-stained nuclei as shown in Figures 2 and 3, which is caused by condensed chromatin. When cellular membrane damage was observed in STS-treated amoebae (SYTOX Green Assay), an intense green fluorescence was observed (Figures 4 and 5), and thus STS caused plasmatic membrane permeabilization in both strains of N. fowleri. Mitochondrial potential collapse was observed after treatment of cells with the IC 90 of STS which showed green fluorescence (JC-1 in monomeric form, Figures 6 and 7). The generation of intracellular ROS was shown in treated amoebae as an intense red fluorescence as shown in Figures 8 and 9.

Discussion
Indolocarbazoles (ICZs) have been intensely studied due to their inhibition properties and potential as chemotherapeutic agents and some of them have even reached clinical trial stages [38,39]. Moreover, these molecules are classified based on the targeted protein and hence are subdivided into (a) protein kinase and (b) DNA topoisomerase I inhibitors [40,41].
In this study, we aimed to evaluate the potential activity of four natural ICZs (1-4) previously isolated from the culture of the S. sanyensis PBLC04 strain which was collected from a mangrove in Ecuador [22][23][24]. All the mentioned compounds are reported PK inhibitors and have shown activity against other parasitic protozoa such as various species of Leishmania, Trypanosoma cruzi, and the Acanthamoeba genus [22,23,27,32]. Furthermore, activity assays revealed that STS (1) and 7OSTS (2) were the most active molecules, presenting IC 50 values ranging from 0.08 ± 0.02 to 1.28 ± 0.35 µM, against both strains of N. fowleri, and lower levels of cytotoxicity than miltefosine after performance of toxicity assays (Table 1). In addition, the two other evaluated ICZs were only active against the type strain ATCC 30808 which could be due to long-term culture of this strain in the laboratory (higher sensitivity to compounds) [42][43][44] and/or to differences in the PK profiles of both strains [45][46][47].
Previous studies have reported that N. fowleri presents several PKs related to important cell roles such as fibronectin interaction, differentiation, and survival [46,47], as well as protection of N. fowleri amoeba against complement-mediated lysis [46]. Moreover, around 250 PKs have been detected in the complete genome project of the species N. gruberi [48], and hence, their potential as druggable therapeutic targets against PAM has been previously discussed [49]. Therefore, inhibition of these kinases could lead to induction of cell death mechanisms which were evaluated in our study using STS, the most active compound among the tested ICZs.
Programmed Cell Death (PCD) involves multiple cellular changes such as chromatin condensation, cell shrinkage, and loss of mitochondrial potential, among other phenomena [21,22,[50][51][52]. In the present work, STS-treated amoeba presented cellular membrane damage, chromatin condensation, collapse of mitochondrial function as well as increased levels of intracellular ROS. Furthermore, effects at the cellular level were even observed after 30 min of treatment with STS. Based on the observed events, it is hypothesized that STS induces PCD through the intrinsic pathway due to the dramatic levels of mitochondrial function collapse which was observed. Moreover, no significant differences were observed in the activities and triggered mechanisms between the two strains of N. fowleri included in the study; hence the importance of STS as a novel therapeutic agent, since it could be effective against a wide range of N. fowleri strains/isolates.
Although blood brain barrier permeability could be an issue for PAM therapy, and the ability of ICZs to penetrate this barrier should be assayed, available data and modifications of ICZs to improve permeability should be the way forward [53]. Moreover, heterocyclic scaffolds, such as those based on the indole, carbazole, and indolocarbazole ones, have been highlighted as promising anticancer agents against tumours of the CNS. In addition, no clinical trials are available for PAM due to the rare frequency of these infections. As a consequence, most of the available data regarding drug efficacy are based on in vitro studies and/or clinical case reports [14]. Undoubtedly, future in vivo studies using STS in a PAM model should be performed in the near future.
Previously reported interaction studies of ICZs and PKs [39,41] have established a critical hydrogen bond interaction between the heteroatoms of the lactam moiety of STS with a conserved glutamic residue at the protein active site [22]. In addition, in closely STS-related compounds, the methylamino group at C-4 is involved in the formation of two hydrogen bonds with amino acids (Glu, Asp) involved in the catalytic pocket. Furthermore, as previously reported [22], these interactions fix a boat-type conformation of the sugar moiety, which is perpendicularly located to the planar sp2 ICZ fragment and is related to their inhibitory activity of PKs. Hence, those ICZs exhibiting functionalization at the carbon C-4 present an increased activity in the function of the number of hydrogen bonds between the nitrogen at the methyl amino moiety of neighboring protein residues [22]. Therefore, and based on this model, STS (1) and 7OSTS (2) (assuming that STS is the leader compound) presented similar activities even though STS was much more active against both N. fowleri strains. These compounds present the lactam group and the methyl amine at the C-4 position with similar orientation and conformation ( Figure 1). Thus, both compounds interact with the conserved aminoacidic residues in amoebic PKs and hence show similar activities. These observations are also supported by the fact that similar results have been reported in other parasitic protozoa [22,23]. In addition, the two others tested ICZs which differ from STS (1) and 7OSTS (2) in the sugar moiety were less active and only affected the viability of one of the strains (Table 1).
methylamino group at C-4′ is involved in the formation of two hydrogen bonds with amino acids 309 (Glu, Asp) involved in the catalytic pocket. Furthermore, as previously reported [22], these 310 interactions fix a boat-type conformation of the sugar moiety, which is perpendicularly located to the 311 planar sp2 ICZ fragment and is related to their inhibitory activity of PKs. Hence, those ICZs exhibiting 312 functionalization at the carbon C-4′ present an increased activity in the function of the number of 313 hydrogen bonds between the nitrogen at the methyl amino moiety of neighboring protein residues 314 [22].

315
Therefore, and based on this model, STS (1) and 7OSTS (2) (assuming that STS is the leader 316 compound) presented similar activities even though STS was much more active against both N. 317 fowleri strains. These compounds present the lactam group and the methyl amine at the C-4′ position 318 with similar orientation and conformation ( Figure 1). Thus, both compounds interact with the 319 conserved aminoacidic residues in amoebic PKs and hence show similar activities. These 320 observations are also supported by the fact that similar results have been reported in other parasitic 321 protozoa [22,23]. In addition, the two other tested ICZs which differ from STS (1) and 7OSTS (2) in 322 the sugar moiety were less active and only affected the viability of one of the strains (Table 1).

323
In order to provide an initial structure-activity relationship (SAR), the commercial ICZs 324 rebeccamycin (5), K252a (6), K252b (7), K252c (8), and arcyriaflavin A (9) were also evaluated ( Figure   325 10 and Table 2).    Among these compounds, only K252a (6) showed activity ranging from 0.118 ± 0.026 (ATCC 30215) to 0.338 ± 0.090 (ATCC 30808). K252a (6) is a known cell-permeable PKA and PKC inhibitor, and consequently, differences in the activities against the tested strains could be due to variation among their profiles of PKA and PKC. Thus, natural and commercial ICZs with log P values between 3.60 and 3.90 are those that show the highest of activity. Finally, rebeccamycin (5), a known DNA topoisomerase-I inhibitor, was not active against the pathogenic amoebae, supporting that the tested natural ICZs affect the viability of Naegleria fowleri via PKs. In the case of the respective aglycones of STS and 7OSTS, K252c (8), and arcyriaflavin A (9), no activity was observed ( Table 2), confirming that the sugar moiety is highly relevant for the inhibition process of the amoebic PKs as previously reported [22,23].

Conclusions
The evaluation of the activity of STS (1) revealed high activity against the trophozoite stage of Naegleria fowleri. Moreover, a minor ICZ isolated from the S. sanyensis strain used as a source in this study, 7OSTS (2), also showed high activity values but was excluded from further assays due to higher toxicity. Nevertheless, both compounds presented high activities against N. fowleri which is due to the similar structure and conformation features of these compounds which interact in a similar orientation with the aminoacidic residues of the amoebic PKs. Moreover, STS induced PCD in treated amoebae via the mitochondrial (intrinsic) pathway. Therefore, STS is presented as a highly active PCD inducer and low-toxic anti-Naegleria fowleri compound which could be considered for future PAM therapy.