Antischistosomal Activity of Oxindolimine-Metal Complexes

Abstract

In recent years, a class of oxindole-copper and -zinc complex derivatives have been reported as compounds with efficient proapoptotic activity toward different tumor cells (e.g., neuroblastomas, melanomas, monocytes). Here we assessed the efficacy of synthesized oxindole-copper(II), -zinc(II), and -vanadyl (VO²⁺) complexes against adult Schistosoma mansoni worms. The copper(II) complexes (50% inhibitory concentrations of 30 to 45 μM) demonstrated greater antischistosomal properties than the analogous zinc and vanadyl complexes regarding lethality, reduction of motor activity, and oviposition.

TEXT

Schistosomiasis (also known as bilharzia) is a major neglected tropical disease caused by blood-dwelling flatworms of the genus Schistosoma (1). It is the second most important human parasitic disease after malaria, with more than 200 million people infected and over 250,000 deaths annually (2, 3). Despite considerable efforts over the past decades, only one efficient and safe drug is currently available, namely, praziquantel {RS-2-(cyclohexylcarbonyl)-1,2,3,6,7,11b-hexahydro-4H-pyrazino[2,1-a]isoquinolin-4-one}. The development of resistance raises concerns about the urgent need for alternative drugs to control this neglected disease in the long term (4). Therefore, new efficient antischistosomal compounds should be developed.

During recent years, despite a lack of interest from the pharmaceutical industry, considerable efforts have been made to develop novel schistosomicidal agents. As a result, many compounds with promising antischistosomal properties have been identified by academia (5). Among these, piplartine (piperlongumine) and curcumin have also been described as promising anticancer agents (6, 7). Additionally, artemisinin and its derivatives, some of the most interesting antischistosomal compounds, have also been shown to exhibit anticancer properties (8). Thus, the discovery and development of anticancer compounds with antischistosomal properties should be pursued.

Metal-containing drugs (metallodrugs) have long been used as antitumor drugs (9, 10). In recent laboratory studies, some metal ligands have been designed. For example, copper(II) complexes with isatin or imine ligands derived from isatin were prepared, characterized by analytical and spectroscopic techniques, and verified to have biological activity toward the proliferation of different cell types (e.g., human neuroblastoma and promonocytic cells) (11). Subsequently, two new synthesized isatin-Schiff base copper(II) complexes, obtained from isatin and 1,3-diaminopropane or 2-(2-aminoethyl)pyridine, namely, [Cu(isapn)]²⁺ and [Cu(isaepy)₂]²⁺, respectively, have been reported to exert proapoptotic activity dependent on oxidative stress induction and organelle-selective damage. They were able to transport copper into the cell, producing reactive oxygen species (ROS), and could behave as delocalized, lipophilic, cation-like molecules, thus specifically targeting organelles (12). In addition, the metabolic oxidative stress elicited by the copper(II) complex [Cu(isaepy)₂]²⁺ triggers apoptosis in SH-SY5Y cells through induction of the AMP-activated protein kinase/p38MAPK/p53 signaling axis (13).

In this study, a series of oxindolimine-metal complexes, [Cu(isapn)](ClO₄)₂ (complex 1), [Cu(isaepy)₂](ClO₄)₂ (complex 2), the corresponding oxindolimine-zinc(II) complexes [Zn(isapn)](ClO₄) (complex 3) and [Zn(isaepy)Cl₂] (complex 4), and the vanadyl complex [VO(isapn)]SO₄ (complex 5), were tested for potential schistosomicidal properties. The copper(II) and zinc(II) oxindolimine complexes (compounds 1 to 4 in Fig. 1A), derived from 2,3-dioxoindole and 2-(2-aminoethyl)pyridine or 1,3-diaminopropane, have already been prepared and characterized in previous studies (11, 14). The corresponding vanadyl complex (compound 5 in Fig. 1A) was prepared according to an equivalent procedure. Most of these complexes have been previously described, and their chemical, physical, and biological properties have been characterized (11, 14, 15). They exhibit tautomeric complexes, related to keto-enol equilibria in solution, dependent on the pH (Fig. 1B). In some cases, the keto form was isolated in solid state; in others, the enol form was isolated. In solution at physiological pH 7.4, both complexes can be present. In the case of isaepy complexes, the enol form is leading, while in the case of isapn complexes, the keto-enol form is predominant at this pH.

FIG 1.

Chemical structures of oxindolimine-metal complexes as isolated (A) and tautomeric equilibria exemplified for oxindolimine-copper(II) complexes 1 and 2 (B).

Schistosoma mansoni strain BH was obtained from an experimentally infected rodent host and intermediate snail host Biomphalaria glabrata in accordance with the standard procedures used in our laboratory (16). For in vitro studies, schistosomes were collected from the hepatic portal and mesenteric veins of an infected hamster 7 weeks postinfection (17). Freshly harvested parasites were placed in Roswell Park Memorial Institute (RPMI) 1640 culture medium supplemented with 10% fetal bovine serum (Vitrocell, Campinas, SP, Brazil) and containing 200 IU/ml penicillin and 200 μg/ml streptomycin at 37°C and 5% CO₂ until use. One pair of worms was added to the wells of a 24-well culture plate (TPP, St. Louis, MO, USA) containing the aforementioned RPMI 1640 medium (18, 19). The metal complexes were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO) to obtain final test concentrations of 31.25 to 1,000 μM (31.25, 62.5, 125, 250, and 500 μM) in culture plates with a final volume of 2 ml. Each compound was tested at least in triplicate, and the highest concentration of DMSO (0.5%) served as a control. Cultures were incubated at 37°C and 5% CO₂, and the parasites were kept for 120 h and monitored every 24 h with an inverted microscope. The effect of the drug was assessed with emphasis on viability, alteration of the tegument, and oviposition (18, 20, 21). Moreover, the effects of oxindolimine-metal complexes on the tegument of adult S. mansoni were observed with a confocal laser scanning microscope (LSM 510 META; Carl Zeiss, Göttingen, Germany) (18, 22, 23). Finally, these investigations were also complemented by cytotoxicity studies with Vero cells (CCL-81; ATCC, Manassas, VA) according to a previously described procedure (24). In these experiments, compound toxicity was determined by using a concentration range of 125 to 500 μM (125, 250, and 500 μM).

Oxindolimine-metal complexes exhibit antischistosomal properties dependent on the metal and on the ligand.

The 50% inhibitory concentrations (IC₅₀s) of all of the complexes tested are summarized in Table 1. Anthelmintic activity against adult schistosomes was observed during incubation with complexes 1 to 4. In general, copper complex 1 with the isapn ligand (IC₅₀ of 31.25 μM) and copper complex 2 with the isaepy ligand (IC₅₀ of 46.87 μM) demonstrated higher antischistosomal activity than analogous zinc complexes 3 (IC₅₀ of 156.25 μM) and 4 (IC₅₀ of 187.5 μM) with the same ligands. Because metal complexes with the isapn ligand were the most potent compounds against S. mansoni adults, we further analyzed the effects of this ligand with a vanadyl complex, complex 5, on these worms. However, the vanadyl complex was unable to promote parasite death, even at the highest concentration used (500 μM).

TABLE 1.

In vitro anthelmintic activities of oxindolimine-metal complexes on adult S. mansoni worms

Compound	Incubation period (h)	IC50 (μM)a
Complex 1	24	78.12 (74.46–81.22)
	72	39.06 (37.03–41.58)
	120	31.25 (28.65–33.79)
Complex 2	24	89.28 (86.38–93.03)
	72	93.75 (74.46–81.22)
	120	46.87 (43.24–49.83)
Complex 3	24	NDb
	72	187.5 (177.79–195.84)
	120	156.25 (148.67–164.92)
Complex 4	24	ND
	72	208.33 (193.67–220.03)
	120	187.5 (177.79–195.84)
Complex 5	24	ND
	72	ND
	120	ND

^aIC₅₀s were calculated by using sigmoid dose-response curves; 95% confidence intervals are in parentheses.

^bND, not determined (because the compound did not show antischistosomal activity at the highest concentration tested [500 μM]).

Severe tegumental damage induced in S. mansoni by oxindolimine-metal complexes.

In addition to the in vitro schistosomicidal effects described above, confocal laser scanning microscopy analysis revealed morphological changes when schistosomes were exposed to compounds 1 to 4, compared to the control group. Adult worms belonging to the control group showed an intact surface structure and topography (Fig. 2A). In contrast, with copper complexes as an example, after exposure to compound 1 at a concentration of 62.5 μM, male worms exhibited moderate tegumental disruption, with shortening of the tubercles (Fig. 2B). When the concentration of compound 1 was increased to 125 μM (Fig. 2C) or 250 μM (Fig. 2D), parasites revealed extensive tegumental destruction, resulting in swelling, sloughing, and erosion of the surface. When schistosomes were exposed to compound 2 at a concentration of 62.5 μM, they exhibited minimal morphological changes, but shrinking of the surface was evident (Fig. 2E). The tegument showed a roughened appearance and started to disintegrate when schistosomes were exposed to compound 2 at 125 μM (Fig. 2F). When the concentration of compound 2 was increased to 250 μM, as with compound 1, severe tegumental alterations were observed, resulting in swelling, sloughing, and erosion of the surface (Fig. 2G).

FIG 2.

Confocal laser scanning microscopy investigation of male S. mansoni worms after in vitro incubation with copper(II) complexes. Shown are control worms showing intact tubercles (Tu) (A) and schistosomes treated with complex 1 at 62.5 μM (B), 125 μM (C), or 250 μM (D) or complex 2 at 62.5 μM (E), 125 μM (F), or 250 μM (G). Parasites exhibiting tubercle shortening (Ts), swelling (Sw), sloughing (Sl), erosion (Er), shortening of the surface (Ss), and tubercle disintegration (Td). Bars, 50 μm.

The tegumental outer surface of schistosomes is a unique double-membrane structure that is of crucial importance for parasite survival; thus, it is a prime target for drug studies. In this context, alterations of the tegument of S. mansoni induced by several antischistosomal compounds have also been described (25–29). In this study, we observed extensive tegumental damage in S. mansoni that intensified progressively when the concentrations of the oxindolimine-metal complexes tested increased. The results showed that the schistosomicidal activity of metal complexes is associated with both metals and ligands. Indeed, experiments with oxindole-containing copper complexes have demonstrated the best activity against adult S. mansoni. Additionally, except with the vanadyl complex, which was inactive, schistosomes were more sensitive to isapn ligands than to isaepy ligands. It is known that bioactivity on tumor cells lines is modulated by metal complexes and ligands (12, 30). For example, it has been shown that [Cu(isapn)]²⁺ and [Cu(isaepy)₂]²⁺, as lipophilic cationic complexes, cross the cell membrane and selectively induce oxidative stress. In this case, [Cu(isaepy)₂]²⁺ seems to be incorporated into cells more efficiently than [Cu(isapn)]²⁺. In contrast, [Cu(isapn)]²⁺ is more effective as an upstream ROS inducer (12). With respect to different metals, it has been shown that cells were more susceptible to apoptosis when treated with copper [Cu(isaepy)₂]²⁺ than when treated with zinc [Zn(isaepy)₂]²⁺ (30). The mechanism(s) by which metal complexes exert their antischistosomal activity remains unclear, but taken together, the results show that both metal complexes with copper and those with zinc easily cross the cell membrane and induce severe tegumental damage in schistosomes.

Oxindolimine-metal complexes significantly reduced S. mansoni egg production.

During continuous incubation with all of the oxindolimine-metal complexes at sublethal concentrations, the S. mansoni worms remained coupled. However, the total number of eggs laid by the surviving worms was significantly lower (P < 0.05 to P < 0.001) than that of control worms. All of the helminth couples of the negative-control group showed normal viability, and parasites remained paired and active throughout the treatment period. Interestingly, all of the compounds tested, including the vanadyl complex, were able to reduce oviposition, but there were differences among them. For example, no eggs were seen when parasites were exposed to complex 1 at ≥32.45 μM, complex 2 at ≥62.5 μM, or complex 5 at ≥62.5 μM, whereas the number of eggs was reduced by ∼95% and 30 to 60% by complex 3 at 125 μM and complex 4 at 125 μM, respectively (Fig. 3; data not shown).

FIG 3.

Effects of copper(II) complexes at sublethal concentrations on the daily egg output of paired adult S. mansoni worms. Panels: A, complex 1; B, complex 2. The values are the mean values ± the standard deviations (bars) of 10 paired worms. Statistical tests were performed with the GraphPad Prism software. Significant differences were determined by applying Tukey's test for multiple comparisons. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared with untreated groups).

Similar to the results regarding schistosomicidal effects, our data showed that the reduction of S. mansoni egg production is also associated with the nature of both the metal and the ligand used. In this context, the copper complexes were more effective than the zinc complexes in reducing egg output, whereas metal complexes were more efficient with the isapn ligand than with the isaepy ligand. On the other hand, the vanadyl complex, which had not shown a schistosomicidal effect, also affected oviposition of the parasites and was even more efficient than the zinc complexes. Therefore, these results clearly show the importance of the presence of a metal coordinated with a ligand.

Oxindolimine-metal complexes displayed no cytotoxic effect on Vero cells.

An ideal antiparasitic agent is supposed to selectively kill parasites without harming the host. We thus evaluated the cytotoxic effects of all of our oxindolimine-metal complexes on a mammalian cell line (Vero cells). Interestingly, cells treated with any of the oxindolimine-metal compounds at 125, 250, or 500 μM remained viable and showed no significant toxicity (data not shown). Since compounds 1 and 2 exhibit interesting antischistosomal properties, this finding points toward the existence of appreciable selectivity of oxindolimine-metal complexes against S. mansoni.

Conclusions.

Oxindolimine-metal complexes have already shown proapoptotic properties against neuroblastoma, melanoma, and monocyte tumor cells on the basis of DNA binding and mitochondrial damage. They have also been shown to be able to efficiently inhibit human topoisomerase IB (14). Here, some oxindolimine-metal complexes were investigated for the first time as potential schistosomicidal agents. Metal complexes, especially copper(II) complexes, showed significant antischistosomal properties, inducing severe tegumental damage and a significant decrease in the production of eggs. The observed anthelmintic activity was verified to be very dependent on the nature and concentration of the metal complex. Further, this activity was significantly influenced by the ligand. These findings support the potential of oxindolimine-metal complexes as new lead compounds against schistosomes. Nevertheless, further studies should be launched to evaluate the probable mechanism(s) of action, as well as to verify the in vivo efficacy of some metal complexes by using mice harboring S. mansoni.