Antiproliferative And Ultrastructural Analysis Triggered by Drugs Contained In The Medicines for Malaria Venture Covid-Box Against Toxoplasma gondii Tachyzoites

1. Introduction

Toxoplasma gondii is the etiologic agent of toxoplasmosis, a zoonosis with a high proportion of seropositive individuals worldwide [1]. The importance of this disease for humans is related to the high morbidity of immunocompromised individuals, such as AIDS patients and newborns [2]. T. gondii infection is asymptomatic in 80% of infected individuals. However, primary infection in AIDS patients can cause the cerebral form of the disease [3].

Congenitally infected newborns can develop neurological problems and eye diseases [4]. In Europe, a risk of eye damage cases of 0.3% to 1% has been observed in adults one or two years after acquiring the infection, and retinochoroiditis results in higher damage in America than in Europe or North America [5,6]. It is believed that 400 to 4.000 children are born with toxoplasmosis each year in the United States [7]. In Brazil, the incidence of congenital transmission can reach 1:770 live births [4], and the estimated prevalence of toxoplasmosis is 42 to 92%, depending on the region of the country [8].

Despite the medical importance of toxoplasmosis, few drugs are available for their treatment [9]. The combination of pyrimethamine (PYR), sulfadiazine (SDZ), and folinic acid has been administered for approximately 70 years. This association is still the first choice for all clinical conditions of the disease [10]. However, this therapeutic scheme has some critical flaws, such as frequent and non-tolerated side effects, which lead to the abandonment of treatment. In addition, there are reports of therapeutic failures with T. gondii strains possibly resistant to these drugs, as well as low absorption and the need for high therapeutic doses or a long-period treatment, making it challenging to manage the treatment [11,12,13].

In this context, drug repositioning has been a promising strategy for new treatments for infectious and neglected diseases. This strategy consists of redirecting an active pharmaceutical compound with commercial use or in development and reusing it for a new therapeutic indication. The development and discovery of medicines are a long and high-investment process [14,15]. Based on this, the Medicines for Malaria Venture (MMV) develops and offers several promising drug libraries with potential repositioning for treating neglected diseases and infections that can cause epidemics. The first drug box provided by MMV was in 2014, Malaria-Box. The anti-T. gondii activity of the compounds available in Malaria-Box has already been described in the literature [16,17,18]. Several other compounds present in other boxes such as, Pathogen Box and Pandemic Box, have also shown anti-T. gondii activity [19,20,21,22].

In 2020, with the advent of the COVID-19 pandemic, MMV made available a new box with 160 drugs and compounds, Covid-Box. The drugs and compounds in this box have structurally different therapeutic classes, selected by experts and initially tested against the new coronavirus (SARS-COV-2). As Covid-Box compounds are in various stages of pharmacological research and development and considering the need to identify new potential treatments for toxoplasmosis and the potential of Covid-Box, this work aimed to expand the study of the activity of these compounds against the tachyzoites of T. gondii and characterize the effect of the best ones on the ultrastructure of the parasite.

2. Materials and Methods

2.1. Drugs and Compounds of Covid-Box

The 160 drugs and compounds were provided free of charge by MMV (https://www.mmv.org/mmv-open/covid-box/covid-box-supporting-information), where the compounds were physically made available as 10 mM solubilized solutions in DMSO in two 96-well plates (A and B) containing 80 compounds each. For storage in this box, the drugs and compounds were dissolved in reserves of 2 mM with 100% DMSO (Merck).

2.2. Parasites

T. gondii tachyzoites of the RH strain were used. Parasite was maintained in vitro through serial passages in 25 cm2 culture flasks of confluent neonatal Normal Human Dermal Fibroblastic Cells (NHDF; Lonza, kindly donated by Dr. Sheila Nardelli, Fiocruz Paraná) in RPMI 1640 medium supplemented with 2% Fetal Bovine Serum (Gibco), Penicillin/Streptomycin, Amphotericin B (Life Technologies, Eugene, OR, USA), and 2 mM glutamine (complete medium). Cells and parasites were maintained at 37°C in a humid atmosphere of 5% CO₂.

2.3. Antiproliferative Assays against Tachyzoite Stage

For preliminary evaluation of the 160 drugs and compounds in the Covid-Box, 6-well plates with monolayers of NHDF cells in complete RPMI-1640 medium were infected with 1.000 newly egressed tachyzoites of T. gondii and treated with 1 µM of each drug. For antiproliferative curves, 12-well plates were used, and cells were infected with 600 tachyzoites. After the addition of the parasites, decreasing concentrations of drugs and compounds (1 µM – 0.0078 µM in two-fold serial dilutions) were added to each well. In the control wells, 0.1% DMSO was added. After 7 days of treatment, the cells were fixed with 70% ethanol and stained with crystal violet. Stained plates were imaged with ChemiDoc MP Imaging System (Bio-Rad), and then plaque areas were analyzed with the ImageJ® software (version 1.52e). Total destruction areas of treated cells were quantified and compared with the untreated (control) to determine the percentage of proliferation and inhibition of T. gondii. For the calculation of the inhibitory concentration of 50% (IC₅₀), the growth inhibition percentage was plotted as a function of drug concentration by fitting the values to the standard curve analysis. The regression analyses were performed using GraphPad Prism 8 Software (GraphPad Inc., San Diego, CA, USA).

2.4. Cytotoxicity Assay in NHDF Cell

The cytotoxic effect of Covid-Box drugs and compounds against NHDF cells was evaluated by the MTS assay [25,26]. For this, 96-well tissue plates containing NHDF cells were treated with different concentrations of drugs and compounds for seven days. At the end of the treatment, the cells were washed with PBS, each well was filled with 100 μL of 10 mM Glucose in PBS and 20 µL of MTS reagent (Promega, Madison, WI, USA) was added. The absorbance was read at 490 nm after 3 h of incubation at 37°C. Cytotoxicity was calculated as the percentage of viable cells versus untreated cells (control). The Cytotoxic Concentration of 50% (CC₅₀) for the host cells was calculated as for IC₅₀, and the Selective Index (SI) was calculated as the ratio of CC₅₀/IC₅₀.

2.5. Druggability Analysis of Drugs and Compounds

For the in silico analysis, the chemical structures of the simplified molecular line input system (SMILES) code from all drugs were obtained from the plate map available at https://www.mmv.org/mmv-open/covid-box/covid-box-supporting-information and loaded into the online programs pkCSM (https://biosig.lab.uq.edu.au/pkcsm/prediction) and SwissADME (http://www.swissadme.ch/) for physicochemical, toxicity and pharmacokinetics properties analysis of the drugs and compounds of Covid-Box. Pyrimethamine (PYR), Sulfadiazine (SDZ), Clindamycin (CLI), Azithromycin (AZT), and Atovaquone (ATO) were analyzed as reference drugs.

2.6 Transmission Electron Microscopy (TEM)

NHDF cultures infected with T. gondii were treated with compounds and fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4). Cells were post-fixed for with 1% osmium tetroxide, 1.25% potassium ferrocyanide, and 5 mM CaCl₂, in 0.1 M sodium cacodylate buffer (pH 7.4). The sample fixation and processing for microscopy were performed as previously described [27]. Samples were dehydrated in alcohol solutions of increasing concentrations (35–100%) and embedded in Epoxy resin (Polybed 812). Ultrathin sections were collected in copper grids, stained with uranyl acetate and lead citrate, and then observed in a Fei TecNai G2 120 kV Spirit Electron Microscope.

2.7. Statistical Analysis

Data were analyzed using GraphPad Prism 8.0 software (GraphPad Inc., San Diego, CA, USA). IC₅₀ and CC₅₀ calculations were done by fitting the values of proliferation/ viability in percentage to a non-linear curve followed by dose-response inhibition analysis through log(inhibitor) vs. normalized response.

3. Results

3.1. Drugs of Covid-Box Showed High Activity and Selectivity against T. gondii Tachyzoites

The antiproliferative effect of 1µM of each 160 drugs and compounds present in the Covid-Box was screened against tachyzoites of the RH strain of T. gondii for seven days of treatment. Thirty compounds inhibited parasite proliferation in at least 70% (Figure 1A-H). Of the 30 best drugs, seven were excluded from the studies. Two (AF03, Amiodarone - MMV001992; BC06, Proscillaridin - MMV001433) were initially excluded because they presented signs of cytotoxicity for the host cells during the initial screening. Other five drugs (Itraconazole (AC06 - MMV637528); Doxycycline (AE05 - MMV000011); Cyclosporine (AF04 - MMV001860); Doxorubicin (BA05 - MMV004066); Digitoxin (BC04 - MMV002436) were excluded as their activity T. gondii have been extensively studied before [23,28,29,30,31,32,33] (See details of the compounds and drugs in https://www.mmv.org/mmv-open/covid-box/covid-box-supporting-information). Thus, 23 drugs were selected for the IC₅₀ and cytotoxicity analysis (Table 1).

The drugs Cycloheximide (MMV000031), Bortezomib (MMV009415), Digoxin (MMV002832), and (-) -Anisomycin (MMV1634116) were the most active, inhibiting the proliferation of T. gondii with IC₅₀s values lower than or equal to 30 nM (Table 1). The drugs Salinomycin, Mycophenolic acid, Abemaciclib, Midostaurin, Emetine, and LY2228820 inhibited T. gondii proliferation with IC₅₀ lower than 100 nM (Table 1). The drugs Ivermectin (MMV672931), Almitrine (MMV1804175), Apilimode (MMV1804187), Bemcentinib (MMV1804190), Niclosamide (MMV003461), Regorafenibe (MMV1804185), and Merimepodib (MMV1804359) were also highly active against T. gondii, presenting IC₅₀ in the range of 0.15 – 0.48 µM (Table 1).

The cytotoxicity assay showed that most compounds were highly selective against T. gondii, and the SI ranged from 3 to 304 (Table 1). Drugs with IC₅₀s less than 30 nM (Cycloheximide and Bortezomib) had the highest SI. Overall, all 23 compounds were selective, and for three of them (Apilimod, Midostaurin, and Salinomycin), this is the first report of activity against T. gondii (Table 1).

3.2. Covid-Box Drugs Show Potential Oral Druggability

Through the SwissADME [42] platform it was possible to obtain information about the physical-chemical properties of the drugs that showed the best activity against T. gondii. From these analyses, it was possible to predict whether these compounds are in accordance with the predictors of Lipinski's rule of 5 (RO5) and Veber (Table 2). We also compared PYR, SDZ, CLI, AZT, and ATO, which are currently used for treating toxoplasmosis. RO5 states that drugs with more than 5H-bond donors, more than 10H-bond acceptors, molecular weight (MW) greater than 500, and calculated LogP (a measure of lipophilicity) greater than 5 are less likely to have good oral absorption and permeation. In addition to the RO5 of Lipinski et al. (1997) [43], the two predictors of Veber et al. (2002) [44] also point that compounds with Total Polar Surface Area (TPSA) equal to or < 140 Å2 and with ten or less rotating bonds have a greater chance of success in oral bioavailability.

The analyses were carried out for the 23 drugs from the Covid-Box selected in the antiproliferative assay and those used as the gold standard (PYR and SDZ) and alternative (AZT, CLI, and ATO) for toxoplasmosis. As expected, PYR and SDZ results agreed with Lipinski's RO5 and Veber's predictors. among the drugs used as alternative treatments, only AZT violates two Lipinski rules (MW and 5H-bond donors) and one Veber criterion (TPSA <140Å2) (Table 2). Of the 23 selected from the Covid-Box, 10 (AA02, AB03, AB04, AE06, AF09, BB10, BD08, BD11, BF06, and BG06) showed compliance with RO5 and Veber`s predictors (Table 2). The other drugs and compounds showed at least one or more non-compliance with RO5 and Veber (Table 2).

Information on the pharmacokinetic properties of selected drugs from the Covid-Box and drugs already used in treating toxoplasmosis were obtained using the platform. Caco-2 permeability values (log Papp at 10-6 cm/s) above 0.90 predict high intestinal permeability. The drugs AA04, AB03, AC10, AG03, AG04, AG08, AG11, and BA07 had log Papp at 10-6 cm/s above 0.90, and AB04, AE06, AF09, AH03, and BB10 showed values of log Papp at 10-6cm/s > 0.70 – < 0.90, which can be inferred that these also have the potential to present high intestinal permeability [45]. The other drugs showed values below 0.60 log Papp at 10-6 cm/s (Table 3). It should also be noted that even the compound BD11, presenting a Caco-2 value below 0.90 (0.553 log Papp at 10-6 cm/s, Intestinal Absorption (Human) = 69.8 %) (Table 3) was the compound selected in the in vitro tests that most inhibited parasite proliferation with an IC₅₀ value = 0.02 μM (Table 1). Volume of distribution values (VDSs) predict drug distribution in tissue. It is known that the lower the interaction of drugs with plasma proteins, the faster they will be absorbed and, therefore, the faster they will be directed to their site of action. Thus, the higher the VDSs value above 0.45 log L/kg, the more the drug is distributed in tissues than in plasma, and values below -0.15 L/kg are considered poorly distributed [45]. Seven drugs or compounds selected from the Covid-Box (AA04, AE06, AF05, AG03, AG04, AG11, and BA07 showed high distribution, and the drugs used for the treatment of toxoplasmosis: SDZ and 0.329), and Covid-Box: AA02, AC10, AD02, AF09, BD02, BD08, BD11, BF06, and BG07 showed VDss higher than -0.15 log L/kg (Table 3). Fraction unbound analyses showed that BF06 and BD11 were the drugs with higher proportion of free state in plasma (Table 3).

The Central Nervous System (CNS) is a common site of infection of T. gondii; thus, we evaluated the predictors for CNS and BBB permeability of the Covid-Box drugs (Table 3). For CNS permeability, compounds with logPS > -2 are predicted to penetrate, but with logPS < -3 are unable to penetrate. From the Covid-Box, six drugs (AA02, AC10, AF09, AG08, BA07, and BG07) showed a prediction of CNS penetration, and 12 presented logPS between -3 and -2 and had the potential for penetration, too (Table 3).

For BBB permeability, LogBB values above 0.3 predict that a compound could readily cross the BBB, and the ones with < -1 are poorly permeable [45]. According to this platform, none of the drugs and compounds selected from Covid-Box and most of the current ones used for toxoplasmosis treatment showed prediction for high crossing into the brain. However, thirteen (AA02, AA04, AE06, AG03, AG04, AG08, AH03, BA07, BA09, BB10, BD11, BF06, and BG07) showed values > -1. Of the drugs already used in the treatment of toxoplasmosis, the only one with a value above 0.3 was ATO (0.401 log BB). PYR presented a value close to the expected value (0.278 log BB) (Table 3).

Using the SwissADME platform, we obtained the Boilled-Egg graph, which also predicts if the drugs have the potential to cross the BBB and have high gastrointestinal absorption (HIA). The BBB permeability data provided by pkCSM was compared with the one provided in the Swiss ADME Boilled-Egg plot [42]. Of the analyzed drugs used for toxoplasmosis treatment, only PYR and ATO showed characteristics with potential BBB permeability at the points drawn above the egg yolk in the graph (yellow color) (Figure 2). The pkCSM program predicted that only ATO could cross the BBB (Table 3). On the same graph, it is possible to predict the compounds' passive gastrointestinal absorption (HIA) properties. Those that were plotted in the egg white region (SDZ and CLI) would be more easily absorbed in the gastrointestinal tract by passive transport than the compounds that were plotted in the gray region of the graph (AZT) (Figure 2A). In addition, the graph provides information such as whether the drugs are glycoprotein inhibitors. Only PYR, SDZ, and ATO drugs are not P-gp substrates (marked with red dots in the graph) (Figure 2A). Compounds that are P-gp inhibitors show increased absorption, while P-gp substrates reduce their absorption [46].

Of Covid-Box, AG04 (Figure 2B), AE06, and BA07 (Figure 2C) presented characteristics with potential permeability to the BBB (points drawn in the upper part of the yolk of the graph) (Figure 2B-C); this information complies with the results obtained for BBB permeability with pKCSM. According to the results obtained in this analysis, none of the others are predictable for readily crossing the natural protections of the CNS [47]. The drugs AA02, AA04, AB03, AC10, AD02, AG03, AG08, AG11, AH03, BA09, BB10, BD08, BD11, BF06, BG06, and BG07 (Figure 2B-C) show potential for an easier absorption in the gastrointestinal tract by passive transport. Of the Covid-Box drugs: AA02, AB03, AB04, AF09, AG04, AH03, BA09, BB10, BD02, BD11, and BF06 are non-P-gp substrate (marked with red dots in graph) (Figure 2B) and AA04, AC10, AD02, AE06, AF05, AG03, AG11, BA07, BD08, BG06, and BG07 are P-gp substrates substrate (marked with blue dots in graph) (Figure 2C).

3.3. Analysis of the Effect on Ultrastructure Induced by Drugs and Compounds of Covid-Box by Transmission Electron Microscopy (TEM)

To confirm that selected Covid-Box drugs and compounds exert a direct effect on T. gondii, we analyzed the ultrastructure by TEM of tachyzoites after the treatment with Cycloheximide (BD11), Bortezomib (BG06), (-)-Anisomycin (BF06), Ivermectin (AF05), Almitrine (AG04), and Merimepodib (BD08) (Figure 3, Figure 4 and Figure 5).

Untreated parasites showed normal morphology and organization (Figure 3A-B), tachyzoites treated with Cycloheximide at a concentration of 62.5 nM show increased endoplasmic reticulum area (stars) and alterations on the plasma membrane structure (arrowhead) (Figure 3C). When tachyzoites were treated with 125 nM Cycloheximide, it was observed that vacuoles containing parasites were completely lysed; the asterisk shows the disruption of the cell membrane (Figure 3D). Parasites treated with the drug Bortezomib at a concentration of 62.5 nM showed Golgi complex alteration (white arrow and star in Figure 3E), disruption of nucleus (N) division, as evidenced by the presence of two and three nucleus profiles in Figure 3E and Figure 3F, respectively. Treatment with Bortezomib also caused vacuolization of tachyzoites cytoplasm (asterisks Figure 3F).

The tachyzoites treated with 100 nM (-)-Anisomycin (BF06) showed disorganization of the endoplasmic reticulum architecture (stars in Figure 4A-B, and inset B). It was also possible to observe drastic changes in the morphology and cell division of the parasite, making it possible to observe a single parasite with two nuclei (N) (Figure 4C). Treatment with 1μM Ivermectin (AF05) (Figure 4D-F) induced the formation of myelin-like structures [48,49] (arrowhead in Figure 4D and inset), resembling a process of cell death by autophagy. In Figure 4E,F, it is possible to observe tachyzoites showing intense vacuolization process (asterisks).

Treatment of the tachyzoites with 1 µM Almitrine (AG04) also induced myelin-like structures (inset in Figure 5A) and impairment of the parasite's cell division (Figure 5B). Figure 5B shows a mass of mother cell in a drastic vacuolization process (asterisks) containing two daughter cells. Figure 5C and inset also show a tachyzoite presenting Golgi complex (GC) disorganization. Changes in the Golgi complex (GC in Figure 5D,E) were also observed in parasites treated with 1.5 μM Merimepodib (BD08). We also observed disorganization of the rhoptries (inset in Figure 5E) and an intense vacuolization process (asterisks in Figure 5F) after treatment with Merimepodib.

Figure 5. A-F. Transmission electron microscopy of the T. gondii after treatment with the drugs AG04 -Almitrine and BD08 – Merimepodib for 48h. (A) Parasites treated with almitrine at a concentration of 1 μM showed an alteration in the cell division process, and it was possible to see two daughter cells in a single parasite; it was also possible to observe a process suggestive of autophagy in the tachyzoites (detail - white arrowhead in insert). (B) After treatment with almitrine, we also observed an intense vacuolization process (asterisks). (C) In addition, 1 μM almitrine also induced fragmentation of the Golgi complex (insert). (D) Parasites treated with 1.5 µM merimepodib induced Golgi complex fragmentation. (E) Another important alteration observed after treatment with 1.5 μM merimepodib was rhoptry disorganization, which can be seen at a higher magnification in the insert. (F) In this figure, it is also possible to observe the intense process of vacuolization in parasites treated with 1.5 μM merimepodib (asterisks). M - Mitochondria; N - Nucleus; GC - Golgi complex; Rp - Rhoptries.

Figure 5. A-F. Transmission electron microscopy of the T. gondii after treatment with the drugs AG04 -Almitrine and BD08 – Merimepodib for 48h. (A) Parasites treated with almitrine at a concentration of 1 μM showed an alteration in the cell division process, and it was possible to see two daughter cells in a single parasite; it was also possible to observe a process suggestive of autophagy in the tachyzoites (detail - white arrowhead in insert). (B) After treatment with almitrine, we also observed an intense vacuolization process (asterisks). (C) In addition, 1 μM almitrine also induced fragmentation of the Golgi complex (insert). (D) Parasites treated with 1.5 µM merimepodib induced Golgi complex fragmentation. (E) Another important alteration observed after treatment with 1.5 μM merimepodib was rhoptry disorganization, which can be seen at a higher magnification in the insert. (F) In this figure, it is also possible to observe the intense process of vacuolization in parasites treated with 1.5 μM merimepodib (asterisks). M - Mitochondria; N - Nucleus; GC - Golgi complex; Rp - Rhoptries.

4. Discussion

In vitro tests were performed to evaluate the potential of applying the drugs and compounds present in the Covid-Box against T. gondii infection and to demonstrate the potential of the antiproliferative effect of the drugs and compounds in the Covid-Box. After these, we selected 23 drugs and compounds that could inhibit the proliferation of the parasite by more than 70%. The discovery of new uses of drugs previously used for other pharmaceutical purposes is a cheaper solution for treating neglected diseases. The antiproliferative analysis showed that the selected compounds inhibited T. gondii proliferation with values of IC₅₀s ranging from 0.02 μM and 0.74 μM, and ten showed IC₅₀ lower than 100 nM (Table 1). Cytotoxicity analysis by the MTS assay also showed that most compounds were highly selective for T. gondii (Table 1). Of the 23 drugs, 11 were recently reported in a study of the anti-T. gondii effect of Covid box compounds, but three (Apilimode, Midostaurin, and Salinomycin) have been reported here for the first time. However, this is the first work to demonstrate the ultrastructural alterations caused by Cycloheximide, Bortezomib, (-)-Anisomycin, Ivermectin, Almitrine, and Merimepodib in tachyzoites through TEM analysis.

Our research identified Cycloheximide, (-)-Anisomycin, and Bortezomib as the most potent drugs against T. gondii tachyzoites, inhibiting parasite proliferation with IC₅₀s in the range of 20-30 nM. This finding is in line with the study by Fichera, Bhopale, and Ross (1995) [41], which found an IC₅₀ value of 0.01 μM after 48 hours of treatment with (-)-Anisomycin, a value in the nanomolar range. In addition, in silico analyses demonstrated that Cycloheximide and (-)-Anisomycin show the predictors of good oral bioavailability according to Lipinski's rule of 5 (RO5) and Veber, while Bortezomib violates only one predictor (Table 2)[50]. In silico analysis also showed that Cycloheximide and (-)-Anisomycin are also non-P-gp substrates, have highly probable gastrointestinal absorption, and potential to cross the BBB (Tables 2, 3 and Figure 2)

Cycloheximide and (-)-Anisomycin are inhibitors of protein synthesis in other pathogens [51,52]. MET analyses showed an increase in the endoplasmic reticulum area of T. gondii after treatment with 62.5 nM cycloheximide (Asterisks in Fig. 3C) and 100 nM (-) Anisomycin (Fig. 4B-C). The endoplasmic reticulum also synthesizes essential lipids to maintain the plasma membrane [53,54]. Interestingly, similar ultrastructural changes were observed when T. gondii was treated with the antifungal drugs itraconazole, eberconazole, and thiolactomycin analogues [21,28]. The treatment T. gondii with this last affected the acylglycerol synthesis by the endoplasmic reticulum [55].

The drug Bortezomib is a proteasome inhibitor. Proteasomes are responsible for the degradation of defective proteins in the cell and are considered a crucial point in the stability of regulatory proteins. Proteasome inhibitors are known to cause cell death [56,57]. Other authors have reported the inhibition of catalytic subunits of the proteasome in Plasmodium falciparum [58,59]. This finding is in line with our data since we observed the formation of structures suggestive of an autophagic process in parasites treated with 62.5 nM Bortezomib by TEM. Adeyemi et al., (2019) [33] found an IC₅₀ value of 0.101μM for Bortezomib against T. gondii tachyzoites after 72 hours of treatment. Cajazeiro et al., (2022) [23] found a different EC₅₀ value of 0.22 μM after 72 hours of treatment, which differs slightly from the IC₅₀ value found in this study after treatment with Bortezomib (0.03 μM). This difference is possible due to the longer treatment time used by us in this study, which suggests a time dependent effect of this drug. Therefore, the sum of the results obtained here supports that Cycloheximide, (-)-Anisomycin, and Bortezomib can be potential drugs for treating the acute phase of toxoplasmosis.

In our study, Ivermectin was another drug with anti-T. gondii activity. The potential activity of Ivermectin against T. gondii and other protozoa, such as Giardia lamblia, Trypanosoma cruzi, Leishmania infantum, and Trypanosoma evansi, has also been reported in the literature [36,60,61,62,63]. However, this is the first study showing the ultrastructural alterations in a parasite from the Apicomplexa phylum. Using TEM, we observed that the treatment with 1 µM Ivermectin for 48 hours induced an intense process of vacuolation and changes in the T. gondii's cell division, confirming a direct effect against this parasite and suggesting death by autophagic induction. A similar effect was observed after the treatment of glioma cells with Ivermectin [64]. Treatment of T. gondii tachyzoites with Almitrine also caused an intense vacuolization and formation of myelin-like structures (Figure 4A-D), also suggestive of autophagic process induction. Almitrine is already used in clinics to treat diseases that affect the respiratory system. It’s in vitro and in vivo effects against T. gondii have been recently reported [23,24] with IC₅₀ values of 0.42 μM and 0.32 μM after 72 hours of treatment, respectively, which are close to the that found in this study.

Mycophenolic acid and Merimepodib are antiviral inhibitors of IMP-dehydrogenase, affecting DNA and RNA synthesis [65]. These drugs affected T. gondii proliferation with IC₅₀ of 0.07 µM and 0.48 µM, respectively. Previous studies showed IC₅₀s of 211 µM (mycophenolic acid) and 0.78 µM (merimepodib) for T. gondii tachyzoites after 24 and 72 h of treatment, respectively [24]. Merimepodib also showed a high selective index for T. gondii compared with HFF cells (human foreskin fibroblasts) [24]. Analyses by TEM showed that merimepodib caused T. gondii Golgi complex fragmentation, rhoptry disorganization, and intense vacuolation (Figure 4D-F). These results suggest that this drug could affect T. gondii, interfering with its secretory pathway.

Developing a new infectious disease treatment is complex because medicines must be absorbed, reach adequate plasma concentrations, and be distributed to tissues and cellular compartments where the infection is present in the body. In the case of toxoplasmosis, this is even more critical, as one of the main sites of infection is the CNS. Based on anti-T. gondii activity assay and to Lipinski's and Veber's predictor's analysis, the 23 drugs and compounds identified in this work are good candidates to become oral drugs since they inhibit T. gondii proliferation at submicromolar range and comply with RO5, showing no more than one violation. We can highlight that in addition to the compounds Cycloheximide, Bortezomib, Anisomycin, Almitrine, and Merimepodib analyzed by TEM, the drug Mycophenolic Acid also presented a micromolar IC₅₀ range lower than 0.10 μM and did not violate Lipinski's rules and the predictors of Veber. In addition, these drugs also demonstrated desirable predictors for oral absorption (Table 2). However, we should not disregard the potential of drugs that did not comply with RO5 or presented values lower than expected for oral absorption or BBB permeability since AZT, a drug already commercialized, presented a value lower than expected (-0.211 log Papp at 10-6 cm/s) and intestinal Absorption (Human) = 45.808%), but despite AZT not having good intestinal absorption, this drug is used to treat toxoplasmosis [9].

5. Conclusions

After Covid-Box screening, we identified three new drugs with anti-T. gondii activity, making this is first study to report their effectiveness against this parasite. In total, 23 drugs were found to be promising candidates for further pre-clinical studies on toxoplasmosis. The discovery of these new drug candidates for the treatment of toxoplasmosis is of great relevance and should be further explored for in vivo analysis in the future. The results presented here have shown that drug repurposing is a potential alternative for the treatment of infectious and neglected diseases and that the boxes provided by MMV are extremely important for solving the problems involved in treating these diseases.