Antikinetoplastid Activity of Indolocarbazoles from Streptomyces sanyensis.

Chagas disease and leishmaniasis are neglected tropical diseases caused by kinetoplastid parasites of Trypanosoma and Leishmania genera that affect poor and remote populations in developing countries. These parasites share similar complex life cycles and modes of infection. It has been demonstrated that the particular group of phosphorylating enzymes, protein kinases (PKs), are essential for the infective mechanisms and for parasite survival. The natural indolocarbazole staurosporine (STS, 1) has been extensively used as a PKC inhibitor and its antiparasitic effects described. In this research, we analyze the antikinetoplastid activities of three indolocarbazole (ICZs) alkaloids of the family of staurosporine STS, 2–4, and the commercial ICZs rebeccamycin (5), K252a (6), K252b (7), K252c (8), and arcyriaflavin A (9) in order to establish a plausive approach to the mode of action and to provide a preliminary qualitative structure–activity analysis. The most active compound was 7-oxostaurosporine (7OSTS, 2) that showed IC50 values of 3.58 ± 1.10; 0.56 ± 0.06 and 1.58 ± 0.52 µM against L. amazonensis; L. donovani and T. cruzi, and a Selectivity Index (CC50/IC50) of 52 against amastigotes of L. amazonensis compared to the J774A.1 cell line of mouse macrophages.

A short group of NTDs caused by the so-called kinetoplastid parasites of increasing research interest include HAT, Chagas disease (American trypanosomiasis) and leishmaniasis. Kinetoplastids cell death of the most promising molecules compared to the commercial analogues rebeccamycin (5), K252a (6), K252b (7), and their respective aglycones K252c (8) and arcyriaflavin A (9) (Figure 1) against Leishmania spp. and T. cruzi by confirming the different characteristic events that occur in these protozoa. The antiparasitic drugs in current use have several limitations [4,[6][7][8], and therefore new candidate drugs are required.
Biomolecules 2020, 10, x 3 of 14 The aim of this research is to analyze the antikinetoplastid activity of the natural ICZs 2-4 isolated from the S. sanyensis PBLC04 strain collected in Ecuador, and to elucidate the mechanism of induced cell death of the most promising molecules compared to the commercial analogues rebeccamycin (5), K252a (6), K252b (7), and their respective aglycones K252c (8) and arcyriaflavin A (9) (Figure 1) against Leishmania spp. and T. cruzi by confirming the different characteristic events that occur in these protozoa. The antiparasitic drugs in current use have several limitations [4,[6][7][8], and therefore new candidate drugs are required.

General Methods
NMR spectra were acquired on a Bruker AVANCE 500 MHz or 600 MHz (Bruker Biospin, Falländen, Switzerland) instrument spectrometer at 300 K) when required. Bruker AVANCE 600 MHz spectrometer is equipped with a 5 mm TCI inverse detection cryoprobe (Bruker Biospin, Falländen, Switzerland). Standard Bruker NMR pulse sequences were utilized. NMR spectra were obtained by dissolving samples in CDCl3 (99.9%). EnSpire ® Multimode Reader (Perkin Elmer, Waltham, MA, USA) to analyze plates using absorbance values of AlamarBlue ® reagent (Bio-Rad Laboratories, Oxford, UK). Thin-layer chromatography (TLC) silica gel plates were used to monitor column chromatography, visualized by UV light (254 nm) and developed with cobalt chloride (2%) as spraying reagent. All reagents and solvents were commercially available and used as received.
S. sanyensis PBLC04 was cultured in modified seawater-based medium (A1) (10 g starch, 4 g yeast extract, 2 g proteose peptone, 1 g calcium carbonate, supplemented with 5 mL/L of a solution of potassium bromide (67 mM) and ferric sulfate (20 mM), in 75% seawater) and extracted as previously described [24]. The extract (12.6) g was fractionated by gel filtration on Sephadex LH-20 column (MeOH) to afford four main fractions (SF1-SF4), grouped according to their similar chemical content by TLC. The bioassay analysis of the obtained fractions led us to select the active fractions

General Methods
NMR spectra were acquired on a Bruker AVANCE 500 MHz or 600 MHz (Bruker Biospin, Falländen, Switzerland) instrument spectrometer at 300 K) when required. Bruker AVANCE 600 MHz spectrometer is equipped with a 5 mm TCI inverse detection cryoprobe (Bruker Biospin, Falländen, Switzerland). Standard Bruker NMR pulse sequences were utilized. NMR spectra were obtained by dissolving samples in CDCl 3 (99.9%). EnSpire ® Multimode Reader (Perkin Elmer, Waltham, MA, USA) to analyze plates using absorbance values of AlamarBlue ® reagent (Bio-Rad Laboratories, Oxford, UK). Thin-layer chromatography (TLC) silica gel plates were used to monitor column chromatography, visualized by UV light (254 nm) and developed with cobalt chloride (2%) as spraying reagent. All reagents and solvents were commercially available and used as received.

Parasite Strain
The activity of compounds 1-9 was evaluated against the promastigotes and amastigote stage of L. amazonensis (MHOM/BR/77/LTB0016), promastigotes of L. donovani (MHOM/IN/90/GE1F8R) and epimastigote of T. cruzi (Y strain). Cytotoxicity assays of molecules 1-9 were performed against the macrophage J774A.1 cell line, cultured in an RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) at 37 • C and 5% CO 2 atmosphere. Promastigotes of both strains of Leishmania were cultured in Schneider's medium (Sigma-Aldrich, Madrid, Spain) supplemented with 10% FBS at 26 • C and were grown to the log phase before performing all the experiments. To carry out the assays, the parasites were cultured in RPMI 1640 medium (Gibco), with or without phenol red. Epimastigotes were cultured in Liver Infusion Tryptose (LIT) medium supplemented with 10% FBS at 26 • C and were grown to the log phase for use in further experiments.

Leishmanicidal Capacity Assay
The leishmanicidal assay was performed against the promastigote stage of L. amazonensis and L. donovani. In a sterile 96-well plate, a serial dilution of compounds 1-9 was done in RPMI-1640 supplemented with 10% FBS with a final volume of 100µL. Parasites were added to wells to reach a concentration of 10 6 /well. AlamarBlue ® at 10% was added into each well and the plate was incubated for 72 h at 26 • C [25]. Subsequently, the most active molecules were tested against the intra-macrophages stage of L. amazonensis. The anti-amastigote activity was measured according to Jain et al. [26]. Macrophages of the J774A.1 cell line were seeded in a 96-well flat bottom plate at a concentration of 2 × 10 5 /mL in RPMI-1640 supplemented with 10% FBS and was incubated at 37 • C in a 5% CO 2 environment to allow the almost complete differentiation of the cells. After one hour of incubation, 100 µL of stationary phase promastigotes of 7-day-old culture was added in a 10:1 ratio (2 × 10 6 /mL) and the plates were re-incubated at 37 • C for 24 h to achieve a maximum infection. After the incubation, the wells were washed at least three times to remove the remaining promastigotes and 50 µL of the culture medium (RPMI-1640 with 10% FBS) were added into each well. Separately, and in a 96-deep well plate, a serial dilution of the selected compounds was made with the same medium and then 50 µL of each dilution was added to each well. The plates were incubated at 37 • C, 5% CO 2 for 24 h. After this incubation, we removed the medium from each well and 30 µL of Schneider medium containing 0.05% SDS was added to each well. The plate was shacked for 30 s and 170 µL of Schneider medium were added to each well. AlamarBlue ® at 10% was added into each well and incubated at 26 • C for 72 h. The fluorescence in each well was measured using a spectrofluorimeter at 544 nm excitation, 590 nm emission. Miltefosine (Cayman Chemicals, Vitro SA, Madrid, Spain) was used as reference drug.

Trypanocidal Capacity Assay
The assay was carried out in vitro against epimastigote stage of T. cruzi. In a 96-well plate, a serial dilution of compounds 1-9 was incubated for 72 h with the parasite at a concentration of 10 5 parasite/well. A total of 10% of AlamarBlue ® was added to each well and the IC 50 was calculated. Benznidazole (Sigma-Aldrich, Madrid, Spain) was used as a reference drug.

Cytotoxicity Assay
The cytotoxicity of active compounds was evaluated in J774A.1 macrophage cell line. Serial dilutions of compounds 1-9 were plated and incubated with the appropriate cell concentration of macrophages. After 24 h, cell viability was determined using AlamarBlue ® method [24]. Miltefosine (Cayman Chemicals, Vitro SA, Madrid, Spain) and benznidazole (Sigma-Aldrich, Madrid, Spain) were used as reference drugs.

Plasma Membrane Permeability
The SYTOX ® Green assay was performed to detect the membrane permeability alterations in parasites. Briefly, 1 × 10 7 parasites/mL were incubated with the previously calculated IC 90 for 24 h. SYTOX ® Green was added at a final concentration of 1 µM (Molecular Probes). After 15 min of incubation, the increase in fluorescence due to the binding of the dye to the parasitic DNA was observed in an EVOS FL Cell Imaging System AMF4300, Life Technologies, Bothell, WA, USA.

Analysis of Mitochondrial Membrane Potential
The decrease in the mitochondrial membrane potential was detected using a JC-1 Mitochondrial Membrane, Potential Assay Kit, Cayman Chemical. After 24 h of incubation, the previously calculated IC 90 of the tested molecules, the cells were centrifuged at 1500 rpm for 10min. The pellet was resuspended in JC-1 buffer. After that, 100 µL of each treated culture was added to a black 96-well plate (PerkinElmer) and 10 µL of JC-1 was added, and the plate was incubated for half an hour at 26 • C. Green and red fluorescence intensity was measured using an Enspire microplate reader (PerkinElmer, Massachusetts, USA) for 30 min. In addition, the depolarization of the mitochondrial membrane potential was confirmed by microscopic observation using EVOS FL Cell Imaging System AMF4300, Life Technologies, USA.

Measurement of ATP
ATP level was measured using a Cell Titer-Glo ® Luminescent Cell Viability Assay (Promega). The effect of the drug on the ATP production was evaluated by incubating (10 7 cells/mL) with the previously calculated IC 90 of the tested molecules for 24 hours. The luminescence was measured using an Enspire microplate reader (PerkinElmer, Waltham, MA, USA).

Statistical Analysis
The half maximal inhibitory concentration (IC 50 ) and the cytotoxicity concentration (CC 50% ) were determined by nonlinear regression analysis with 95% confidence limits. All experiments were performed three times, in duplicates for each concentration tested, and the mean values were also calculated. A Tukey test was used for analysis of the data.

Antikinetoplastid Activities
Leishmanicidal and trypanocidal activities of natural ICZ compounds 1-4 and the structurally related commercial analogues 5-9 were determined based on a dose-dependent application against promastigotes of both L. amazonensis and L. donovani and epimastigotes of T. cruzi. The obtained values of concentrations inhibiting 50% (IC 50 ) of parasites are summarized in Table 1 and expressed in µM. Compounds 3 and 4 did not show activity against L. donovani at concentrations below 40 µM. Rebeccamycin 5 and the aglycones 8 and 9 were completely inactive against all tested parasites. The natural ICZ metabolites 1 and 2 showed the lowest IC 50 values, comparable to the reference drug for leishmanicidal (miltefosine IC 50 = 6.48 ± 0.24 µM) or trypanocidal (benznidazole IC 50 = 6.94 ± 1.94 µM) treatments.
On the other hand, the toxicity of all compounds was evaluated against the J774A.1 cell line of mouse macrophages as cytotoxic concentration 50 (CC 50 ), a concentration in which the population of cells is reduced to 50%. The results are summarized in Table 2 to show the low toxicity of ICZs 3, 4 and 7 and the aglycones 8-9. The most toxic compounds were rebeccamycin (5) and K252a (6) with CC 50 values of 1.42 ± 0.19 µM and 1.07 ± 0.21 µM, respectively. The effect of the natural ICZs 1-4 on amastigotes of L. amazonensis is shown in Table 3. All tested compounds are active with similar IC 50 values compared to miltefosine, with the exception of 7OSTS (2), which is the most potent compound tested among minor metabolites with an IC 50 of 0.10 ± 0.00 µM. Furthermore, the calculated selectivity index (SI) of 2 is over 2-fold the value obtained for the reference drug to treat leishmaniasis.

Mechanisms of Cell Death
Programmed cell death (PCD) pathways are critical for parasite development and infection, and, consequently, the ability of a molecule to target those mechanisms are considered of relevance in terms of therapeutic potential [31]. The promising results showed by 7OSTS (2) prompted us to continue the experimental analysis of its mechanisms of action.

Mitochondrial Damage in Leishmania amazonensis Induced by 7-oxostaurosporine (2)
The effect of 7OSTS (2) on the mitochondrial membrane potential was measured in promastigotes of L. amazonensis and L. donovani, and T. cruzi epimastigotes. We could observe an intense effect of the mitochondrial membrane potential (∆Ψm), when L. amazonensis promastigotes were treated with 2 at the IC 90 concentration (8.36 µM) (Figures 2 and 3). The IC 90 value was used to increase the population of affected parasites and to reduce the experimental time. The presence of JC-1 dye in the cytoplasm in its monomeric form (green fluorescence) confirms the depolarization of L. amazonensis mitochondrial membrane (Figure 3). In contrast, we did not observe any change in L. donovani promastigotes or T. cruzi epimastigotes treated with the IC 90 of the 7OSTS (2) (Figure 2).

Cytoplasmic Membrane Permeability in Leishmania donovani and Trypanosoma cruzi Induced by 7-oxostaurosporine (2)
The cytoplasmic membrane permeability of L. amazonensis, L. donovani and T. cruzi after 24 h treatment with the IC90 of 7OSTS (2) by the SYTOX Green assay, reveals a remarkable membrane alteration in cultures of L. donovani and T. cruzi, as shown in Figure 4. Similarly, the same effect is

Cytoplasmic Membrane Permeability in Leishmania donovani and Trypanosoma cruzi Induced by 7-oxostaurosporine (2)
The cytoplasmic membrane permeability of L. amazonensis, L. donovani and T. cruzi after 24 h treatment with the IC 90 of 7OSTS (2) by the SYTOX Green assay, reveals a remarkable membrane alteration in cultures of L. donovani and T. cruzi, as shown in Figure 4. Similarly, the same effect is also observed in death cells by propidium iodide staining. Interestingly, the cytoplasmic membrane of L. amazonensis does not seem to be permeable under the experimental conditions. Biomolecules 2020, 10, x 9 of 14 also observed in death cells by propidium iodide staining. Interestingly, the cytoplasmic membrane of L. amazonensis does not seem to be permeable under the experimental conditions.

Discussion
The family of ICZs have been the focus of intense research as chemotherapeutics, and some of them have advanced into clinical trials [32,33]. According to the proteins they target, they have been subdivided into two broad groups. One is represented by STS (1) and includes the ICZ compounds that are potent inhibitors of protein kinases (PKC, PKA, CDK2, etc.), whereas the second group, modified in the sugar moiety such as rebeccamycin (5), are potent stabilizers of DNA topoisomerase-I [33][34][35][36].
The initials studies on the STS-PK complex used PKA and CDK2 kinase models [34]. These studies and those completed later revealed a critical hydrogen bond interaction between the heteroatoms of the lactam moiety of STS with a conserved glutamic residue at the protein active site ( Figure 5A). Moreover, in closely STS-related compounds, the methyl amine group at C-4′ is involved in the formation of two hydrogen bonds with amino acids involved in the catalytic pocket, such as Glu and Asp ( Figure 5B). These interactions fix a boat-type conformation of the sugar moiety, which is perpendicularly located to the planar sp 2 ICZ fragment. This specific conformation has been related with the inhibitory activity of protein kinases. Thus, ICZs functionalized at the carbon C-4′ show an increased activity in the function of the number of hydrogen bonds between the nitrogen at the methyl amino moiety of neighboring protein residues ( Figure 5B) [34,35].

Discussion
The family of ICZs have been the focus of intense research as chemotherapeutics, and some of them have advanced into clinical trials [32,33]. According to the proteins they target, they have been subdivided into two broad groups. One is represented by STS (1) and includes the ICZ compounds that are potent inhibitors of protein kinases (PKC, PKA, CDK2, etc.), whereas the second group, modified in the sugar moiety such as rebeccamycin (5), are potent stabilizers of DNA topoisomerase-I [33][34][35][36].
The initials studies on the STS-PK complex used PKA and CDK2 kinase models [34]. These studies and those completed later revealed a critical hydrogen bond interaction between the heteroatoms of the lactam moiety of STS with a conserved glutamic residue at the protein active site ( Figure 5A). Moreover, in closely STS-related compounds, the methyl amine group at C-4 is involved in the formation of two hydrogen bonds with amino acids involved in the catalytic pocket, such as Glu and Asp ( Figure 5B). These interactions fix a boat-type conformation of the sugar moiety, which is perpendicularly located to the planar sp 2 ICZ fragment. This specific conformation has been related with the inhibitory activity of protein kinases. Thus, ICZs functionalized at the carbon C-4 show an increased activity in the function of the number of hydrogen bonds between the nitrogen at the methyl amino moiety of neighboring protein residues ( Figure 5B) [34,35]. Biomolecules 2020, 10, x 10 of 14 Some protein kinases form an additional hydrogen bond with the oxygen atom present at carbon C-7 in oxidized ICZs compounds ( Figure 5C). In these cases, water molecules are involved in the coordination. Therefore, it appears to be a differentiating element that may cause a reinforcement in the interaction of oxidized derivatives at C-7 with PKs and, consequently, increased activity. One example is UCN-01 (7-hydroxystaurosporine), which shows similar inhibition profiles to STS with eleven kinases. Five of those kinases have a residue equivalent to Thr222 in PDK1; and PKB and PKC have Thr at the Val143 position of PDK1, thus all may have formed an additional hydrogen bond to the 7-hydroxy group [33].
Based on this interaction model, in the present study the antiprotozoal activities of the natural compounds 1-4 beside the commercial ICZs rebeccamyccin (5), K252a (6), K252b (7), K252c (8), and arcyriaflavin A (9) have been analyzed in order to establish a plausive approach to the mode of action and to provide a preliminary structure-activity relationship (SAR). Thus, the DNA topoisomerase-I inhibitor rebeccamycin (5) showed no activity (IC50 > 40 µM) against all tested parasites, suggesting that the most probable inhibition mechanism for natural compounds 1-4 affects parasite PKs. Similarly, the aglycones of STS (1) and 7OSTS (2), K252c (8) and arcyriaflavin A (9), respectively, were inactive at concentrations below 40 µM, and confirm the relevance of the sugar moiety in the inhibition of parasite PKs.
The most active STS-related compound was 7OSTS (2), which showed IC50 values of 3.58 ± 1.10; 0.56 ± 0.06 and 1.58 ± 0.52 µM against L. amazonensis; L. donovani and T. cruzi, respectively (Table 1), which are slightly improved with respect to those of STS (1)-treated L. donovani and T. cruzi, and could be due to the presence of a carbonyl group at C-7 in 2. Of note is the Selectivity Index (CC50/IC50) of 7OSTS 2 against amastigotes of L. amazonensis (Table 3) when compared with murine macrophages J774A.1, which improves that of miltefosine. Thus, when L. donovani is treated with 2 at 10 µM, parasites suffer morphological changes compared to control cells, with differences in the size and appearance of the flagellar pocket (increased with treatment), and invagination of the plasma membrane in the place where the flagellar system is assembled to the body of the parasite (Figure 6), characteristic damages for PKs inhibition [9,19,37,38]. Some protein kinases form an additional hydrogen bond with the oxygen atom present at carbon C-7 in oxidized ICZs compounds ( Figure 5C). In these cases, water molecules are involved in the coordination. Therefore, it appears to be a differentiating element that may cause a reinforcement in the interaction of oxidized derivatives at C-7 with PKs and, consequently, increased activity. One example is UCN-01 (7-hydroxystaurosporine), which shows similar inhibition profiles to STS with eleven kinases. Five of those kinases have a residue equivalent to Thr222 in PDK1; and PKB and PKC have Thr at the Val143 position of PDK1, thus all may have formed an additional hydrogen bond to the 7-hydroxy group [33].
Based on this interaction model, in the present study the antiprotozoal activities of the natural compounds 1-4 beside the commercial ICZs rebeccamyccin (5), K252a (6), K252b (7), K252c (8), and arcyriaflavin A (9) have been analyzed in order to establish a plausive approach to the mode of action and to provide a preliminary structure-activity relationship (SAR). Thus, the DNA topoisomerase-I inhibitor rebeccamycin (5) showed no activity (IC 50 > 40 µM) against all tested parasites, suggesting that the most probable inhibition mechanism for natural compounds 1-4 affects parasite PKs. Similarly, the aglycones of STS (1) and 7OSTS (2), K252c (8) and arcyriaflavin A (9), respectively, were inactive at concentrations below 40 µM, and confirm the relevance of the sugar moiety in the inhibition of parasite PKs.
The most active STS-related compound was 7OSTS (2), which showed IC 50 values of 3.58 ± 1.10; 0.56 ± 0.06 and 1.58 ± 0.52 µM against L. amazonensis; L. donovani and T. cruzi, respectively (Table 1), which are slightly improved with respect to those of STS (1)-treated L. donovani and T. cruzi, and could be due to the presence of a carbonyl group at C-7 in 2. Of note is the Selectivity Index (CC 50 /IC 50 ) of 7OSTS 2 against amastigotes of L. amazonensis (Table 3) when compared with murine macrophages J774A.1, which improves that of miltefosine. Thus, when L. donovani is treated with 2 at 10 µM, parasites suffer morphological changes compared to control cells, with differences in the size and appearance of the flagellar pocket (increased with treatment), and invagination of the plasma membrane in the place where the flagellar system is assembled to the body of the parasite (Figure 6), characteristic damages for PKs inhibition [9,19,37,38]. Furthermore, ICZ analogues, K252a (6) and K252b (7), first isolated from the actinomycete Nocardiopsis [39] differ from STS (1) in the sugar moiety (Figures 1 and 5). Whereas 6 is a reversible cell-permeable inhibitor of phosphorylase kinase (IC50 = 1.7 nM), protein kinase A (PKA) (IC50 = 140 nM), and protein kinase C (PKC) (IC50 = 470 nM) [40,41], 7 is used as a non-permeable PKC inhibitor [42][43][44]. The antikinetoplastid screening show a similar behavior of L. amazonensis when treated with K252a (6) and 7OSTS (2), whereas the effectiveness of K252b (7) is lower (IC50 = 20.62 ± 4.50 µM), indicating that the most probable mechanism of action of 2 affects intracellular PKs of L. amazonensis. On the contrary, L. donovani and T. cruzi responded in a similar way when treated with 6 and 7.
In summary, 7OSTS (2) possesses potent activity against all three tested species, similar to that showed by STS (1). These results could be explained based on the fact that, structurally, both compounds possess the lactam group and the methyl amine at the C-4' position, with similar orientation and conformation, and thus, 1 and 2 interact with the conserved aminoacidic residues in parasite PKs. In addition, the differences found between STS (1) and 7OSTS (2) could be justified based on the positive or negative interaction with the active core of the target parasite PKs, due to the additional functionalization at C-7 position ( Figure 5C). For the rest of tested substances, 3-9, the crucial interactions between the N-Me moiety at the C-4′ position with the active center of the PK cannot be produced, and therefore their activity is lower than those compounds that contain the methyl amino fragment.

Conclusions
We have found a clear correlation between the antikinetoplastid activities observed and the structural elements of the studied ICZs. Both STS (1) and 7OSTS (2) possess potent activities against all three tested species. Their similar structural features, orientation and conformation assure the interaction with conserved aminoacidic residues of the PKs of parasites. Among all tested compounds, 7OSTS (2) was also revealed to be particularly selective against the amastigote stage of L. amazonensis, and new studies should be oriented to explore the therapeutic potential and mode of action of this molecule in order to develop new antileishmanial compounds.