Next Article in Journal
Bioavailability of Organic Phosphorus Compounds to the Harmful Dinoflagellate Karenia mikimotoi
Previous Article in Journal
Liver Involvement in Children with COVID-19 and Multisystem Inflammatory Syndrome: A Single-Center Bulgarian Observational Study
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Microorganisms
Volume 9
Issue 9
10.3390/microorganisms9091960
microorganisms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Review

Promising Drug Targets and Compounds with Anti-Toxoplasma gondii Activity

by
Marco da Silva
1,
Cátia Teixeira
2,
Paula Gomes
2 and
Margarida Borges
3,4,*
1
Departamento de Ciências Biológicas, Faculdade de Farmácia da Universidade do Porto, 4050-313 Porto, Portugal
2
LAQV-REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências da Universidade do Porto, 4169-007 Porto, Portugal
3
Associate Laboratory i4HB—Institute for Health and Bioeconomy, Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal
4
UCIBIO/REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal
*
Author to whom correspondence should be addressed.
Microorganisms 2021, 9(9), 1960; https://doi.org/10.3390/microorganisms9091960
Submission received: 29 July 2021 / Revised: 9 September 2021 / Accepted: 10 September 2021 / Published: 15 September 2021
(This article belongs to the Special Issue Immunopathology of Toxoplasma gondii Infection)
Browse Figures
Versions Notes

Abstract

:
Toxoplasmosis is a parasitic disease caused by the globally distributed protozoan parasite Toxoplasma gondii, which infects around one-third of the world population. This disease may result in serious complications for fetuses, newborns, and immunocompromised individuals. Current treatment options are old, limited, and possess toxic side effects. Long treatment durations are required since the current therapeutic system lacks efficiency against T. gondii tissue cysts, promoting the establishment of latent infection. This review highlights the most promising drug targets involved in anti-T. gondii drug discovery, including the mitochondrial electron transport chain, microneme secretion pathway, type II fatty acid synthesis, DNA synthesis and replication and, DNA expression as well as others. A description of some of the most promising compounds demonstrating antiparasitic activity, developed over the last decade through drug discovery and drug repurposing, is provided as a means of giving new perspectives for future research in this field.

1. Introduction

The parasite Toxoplasma gondii is an obligate intracellular protozoan that infects most warm-blooded animals [1]. However, its sexual lifecycle can only occur in members of the Felidae family, known as definitive hosts, such as domestic cats and wild felids, whose global T. gondii seroprevalence is estimated to be 35% and 59%, respectively [2]. The rates of T. gondii seroprevalence in humans vary greatly among different geographical areas, ranging from approximately 30% in the American, European, and Asiatic regions, to more than 60% in the African continent. These fluctuations may result from different cultural practices, environmental conditions, or the socioeconomic status of the population. Overall, T. gondii is considered one of the most successful parasites worldwide, infecting about 30% of the world’s population [3]. Despite its ubiquitous distribution, several divergent strains of T. gondii were detected.
Most strains isolated in Europe and North America fall into one of three genotypes, referred to as types I, II and III. Type I are considered the most virulent strains and are lethal in mice, displaying enhanced migratory capacity both in vitro and in vivo, the faster growth rate being in vitro, and being capable of generating high parasite loads in the host organism. Type II and III have intermediate and low virulence, respectively, with type II being the most common strains among human infections, while type III are mostly found in non-human infections. Certain strains are typically predominant in certain geographic locations. The three major clonal lineages are found in Asia and America, in addition to certain specific genotypes for the former area and various recombinant strains for the latter. Type II and III strains are isolated in North Africa, the Middle East, and the Arabic peninsula, while in Europe, type II strains are more common. Several African genotypes, including type II and III strains, are found in sub-Saharan Africa [4,5,6].

2. Toxoplasmosis

In humans, the severity of infection is mainly determined by the host immune system efficacy. In immunocompetent individuals, acute T. gondii infection is usually asymptomatic or may cause, in rare instances, a flu-like disease with mild symptoms. Strain virulence and several host-cell conditions—pH, heat shock, mitochondrial inhibition, and nitric oxide production—may also influence disease outcome [4,7,8]. Under host immune pressure, the parasite naturally becomes dormant. In this process, tachyzoite–bradyzoite conversion occurs, with tissue cyst formation in specific locations, such as cerebral, skeletal, and cardiac muscle tissues, reaching the chronic phase of infection. An intact immune system prevents cyst rupture and reactivation of infection [4,7]. However, in immunocompromised individuals, the parasite generally remains active, causing continuous host-cell infection, leading to acute disease. Moreover, reactivation may also occur in individuals who are initially immunocompetent but later undergo immunosuppression, including individuals with acquired immunodeficiency syndrome (AIDS), or subjected to immunosuppressive therapy, as in the case of autoimmune diseases or organ transplantation. Individuals with AIDS and not initially infected with T. gondii usually develop a severe primary infection, whereas individuals already infected with the parasite who become immunosuppressed are at an increased risk of disease relapse [9,10]. In fact, T. gondii encephalitis was frequently reported in AIDS patients, especially those with low CD4 T lymphocyte cell counts. Therefore, T. gondii may be regarded as an opportunistic parasite that contributes to the death of AIDS patients [10]. In these situations, where T. gondii infection is acquired throughout an individual’s life, the disease is referred to as acquired toxoplasmosis.
Toxoplasmosis may also be potentially dangerous in seronegative pregnant women that become primo-infected during pregnancy, as it may lead to transplacental transmission of the parasite, which may result in congenital toxoplasmosis [11]. However, the incidence of congenital toxoplasmosis varies with the trimester during which primary infection is acquired. The transmission rate is greater in the final stages of pregnancy, as placental irrigation increases, allowing for a greater area of contact with the fetus. On the other hand, the severity of infection is greater in the early stages of pregnancy, due to fetal immunological immaturity [12,13,14]. During the first trimester, there is a higher risk of abortion, stillbirth, or premature birth. In the second trimester, the risk of miscarriage decreases, however, in more severe cases, hydrocephaly, chorioretinitis and cerebral calcification may occur, according to the parasite’s brain and ocular tropism. In the last trimester, although severe clinical manifestations in the newborn are at lower risk, there is an increased probability of congenital infection [11,15,16,17,18].
Regarding cerebral tropism, recent data suggest an association between congenital infection and the development of neurological and psychiatric disorders later in life [9,19,20], including Alzheimer’s disease [19,20], depression [19,20,21], schizophrenia [22,23,24], bipolar disease [24], and even suicidal tendencies [21,24].

3. Current Treatment Options

First-line conventional treatment for acquired and congenital toxoplasmosis generally includes a pyrimethamine-based regimen, which comprises three drugs: pyrimethamine, sulfadiazine and folinic acid (leucovorin; Table 1) [25,26]. Pyrimethamine is a folic acid antagonist as it inhibits the dihydrofolate reductase (DHFR) enzyme, blocking the synthesis of purines and pyrimidines, essential for DNA synthesis and cell multiplication. The action of this drug is enhanced when used in conjunction with sulfonamides, such as sulfadiazine, which is capable of interfering with T. gondii’s folic acid synthesis, by competitively inhibiting the dihydropteroate synthetase (DHPS) enzyme. This combination must not be used during the first trimester of pregnancy due to the teratogenic potential of pyrimethamine, which also causes reversible myelosuppression, forcing combination with folinic acid, to prevent hematologic adverse reactions [25,26,27,28]. Moreover, although rare, different severe complications were reported, such as agranulocytosis, Stevens-Johnson syndrome, toxic epidermal necrolysis and hepatic necrosis, as well as many others [29,30,31,32,33,34]. Although several alternative treatment options are available, including pyrimethamine combined with clindamycin, clarithromycin, azithromycin or atovaquone, and monotherapy using cotrimoxazole (trimethoprim-sulfamethoxazole) or atovaquone, no regimen was found to be more effective than the conventional treatment [25,35]. Despite clinical complications, standard chemotherapy has proven to reduce the risk of development of toxoplasmosis-related sequels and symptoms associated with congenital infection in newborns, if it is administered immediately after diagnosis of either maternal infection or congenital transmission [25,27,36]. Alternatively, when the maternal infection is suspected, but not confirmed, therapy with spiramycin must be implemented. Spiramycin is a potent macrolide antibiotic, and although it does not readily cross the placental barrier, it is greatly accumulated in the placenta, preventing transplacental transmission of T. gondii. Nevertheless, when fetal or neonatal toxoplasmosis is confirmed, spiramycin is discontinued and conventional treatment is applied [36]. The use of steroids is beneficial in the treatment of ocular toxoplasmosis in combination with antimicrobial therapy. However, excessive doses can lead to a minimal response [25]. In fact, phase II clinical trials are currently underway to determine the optimal dose of dexamethasone to be used as adjunctive therapy to reduce brain edema in HIV-infected patients exhibiting cerebral toxoplasmosis [37].
Current treatment options are limited and not optimal regarding the harsh profile of side effects and treatment duration (from 4–6 weeks to over 1 year), which may affect compliance [27,38]. T. gondii-related factors, such as the increasing drug resistance, different drug susceptibility for different strains, and the remaining unknown aspects of the parasite’s pathogenicity, also play an important role in disease progression and treatment failure [39,40,41]. In addition, no current drug can eliminate tissue cysts from the infected host, which remain quiescent, establishing the latent phase of infection, as long as the host’s immune system remains capable enough [27,38]. Although immunization strategies are currently being studied and developed, there is no vaccine available for human administration [42].
Thus, there is an urging need for the development of newer, safer, and more effective treatment alternatives for toxoplasmosis, which consequently relies on rising knowledge in T. gondii pathophysiology and the discovery of promising drug targets.
This review highlights some of the most promising drug targets in anti-T. gondii drug discovery and the compounds discovered, developed, and repurposed over the last decade, while focusing on their relevant features, in vitro and in vivo activity, and future perspectives. A literature search was conducted using PubMed database and the query “((toxoplasmosis) OR (Toxoplasma gondii) OR (anti-Toxoplasma) AND (drug) OR (treatment)). Experimental compounds with established in vitro activity were primarily considered, of which those with in vivo activity or efficacy against bradyzoite-containing cysts were prioritized.

4. Promising Drug Targets and Strategies in Anti-T. gondii Drug Discovery

In the last decade, considerable efforts have been made in the study and development of repurposed drugs and novel compounds with new mechanisms of action. Drug screens involved various parasite targets, including mitochondrial electron transport chain, calcium-dependent protein kinases, type II fatty acid synthesis, DNA synthesis and replication, and DNA expression as well as many others. Drug targets and respective promising inhibitors with interesting mechanisms of action (Figure 1) and efficient in vitro and/or in vivo activity against T. gondii are described and discussed. Relevant experimental in vitro and in vivo results are summarized in Table 2.

4.1. Drug Targets Involved in Parasite Motility and Host-Cell Invasion

T. gondii belongs to the Apicomplexa family, which also includes other relevant protozoans, such as Cryptosporidium spp., a common cause of diarrhea in children, and Plasmodium spp., the etiological agent of malaria. In fact, both T. gondii and Plasmodium spp. share very similar organellar organization [67]. The members of this family possess well-developed structures at the anterior end of the cell—the apical complex—responsible for host-cell invasion [68]. Unlike Plasmodium spp., which specifically infects erythrocytes and hepatocytes, T. gondii does not require a specific host receptor for cell invasion, allowing it to infect all nucleated host cells [67]. The invasion process (Figure 2) requires the participation of specific T. gondii secretory organelles, belonging to the apical complex: the micronemes, small rod-shaped structures accumulated in the apical third of the protozoan body, housing proteins responsible for extracellular motility and invasion; and rhoptries, long club-shaped organelles located at the apical portion of the parasite, which accommodate proteins responsible for the invasion and host-cell manipulation. Proteins segregated by micronemes (named MICs) and rhoptries (named ROPs) allow host-cell entrance while dragging cytoplasmatic membrane around the tachyzoite, forming the Parasitophorous Vacuole (PV)—an intracellular compartment in which T. gondii reproduces asexually. Upon the formation of the PV, the third set of proteins derived from other secretory organelles, the dense granules (GRAs), along with ROPs, decorate the PV membrane [68,69,70,71]. Other ROPs and GRAs accumulate inside PV forming a tubular network of intravacuolar structures that serves various purposes: escape from host-cell aggression, inhibit phagolysosome formation, hinder intravacuolar acidification, metabolic exchange of compounds between PV and host cytoplasm, among others [69,72]. Interaction between T. gondii and host cell endocytic machinery is well described, however, the infection may occur through other mechanisms, such as the chlathrin-mediated endocytosis or micropinocytosis [73]. These processes are essentially controlled by the host cell, revealing a successful interplay between host cell and parasite in T. gondii infection [70].

T. gondii Calcium-Dependent Protein Kinase 1

MICs secretion is essential for parasite motility, host cell invasion, and egress and thus constitutes a potential target for drug development. In fact, the inhibition of T. gondii calcium-dependent protein kinase 1 (TgCDPK1), a member of the serine/threonine-protein kinase family located in the cytosol and regulates the calcium-dependent pathway, which in turn leads to MICs secretion, impairs host-cell invasion capacity [74,75]. TgCDPK1 has thus proven to be an interesting target for drug discovery. In comparison to mammalian kinases, it presents a key structural difference at the “gatekeeper residue” in the ATP-binding pocket. TgCDPK1 contains a small glycine residue, whereas human kinases possess larger residues, providing additional space for extra interactions with the target protein, which resulted in the development of potent and selective ATP-competitive TgCDPK1 inhibitors [75,76,77].
Several bumped kinase inhibitors (BKI) were found to selectively inhibit TgCDPK1, being 15,000-fold more active against the parasite kinase in comparison to human tyrosine kinases [43,78]. BKI-1294, a pyrazolo-pyrimidine based compound, was a promising candidate belonging to this class. Doggett et al. described an in vitro IC50 of 140 nM, leading to a reduction in acute T. gondii infection by 93% when given orally, and high efficiency against established toxoplasmosis [43]. In addition, Müller et al. demonstrated excellent activity of BKI-1294 against congenital toxoplasmosis [57]. However, despite elevated T. gondii specificity, BKI-1294 was found to inhibit the Ether-à-go-go-Related Gene (hERG), which codes for the protein Kv11.1, the alpha subunit of a potassium ion channel that is essential in cardiomyocyte repolarization. Its inhibition may ultimately result in the development of life-threatening cardiac arrhythmias, such as torsades de pointes [44,58,79]. This occurrence halted BKI-1294 development due to the risk of cardiotoxicity. Nevertheless, Sánchez-Sánchez et al. recently demonstrated the safety and significant protection provided by BKI-1294 against abortion and vertical transmission in sheep experimentally infected with T. gondii during pregnancy. Thus, although BKI-1294 advancement for human toxoplasmosis ceased, the reduction of infection rates among other intermediate hosts may be a way to indirectly reduce human infection [59].
Vidadala et al. investigated BKI-1294 modifications that maintained TgCDPK1 selectivity and efficacy while reducing interference with hERG channels. The authors developed a compound (compound 32 in their series of BKIs) with an hERG IC50 > 10 µM, an in vitro IC50 against T. gondii of 60 nM, and high in vivo efficiency regarding brain parasite load when given orally. In fact, compound 32 reduced the number of brain cysts by 88.7% [44].
Recently, Rutaganira et al. tested other pyrazolo-pyrimidine inhibitors of TgCDPK1. The resulting compound (compound 24 in their series of BKIs) exhibited in vitro inhibition of the enzyme and parasite proliferation in the nanomolar and submicromolar range, respectively. In addition, in vivo assays showed this BKI analog to exhibit excellent oral bioavailability, decreased severity of acute infection, reduced cyst burden and delayed chronic reactivation of disease in immunocompromised mice. Noteworthy, compound 24 was able to completely cure some of the immunocompromised animals [45].

4.2. Drug Targets Involved in Fatty Acid Synthesis

The fatty acid synthesis (FAS) pathway can provide attractive approaches in T. gondii drug development, especially since several drug targets include enzymes absent in the host cell. As with other metabolic pathways, FAS takes place in the apicoplast of apicomplexan parasites [80,81]. Whilst T. gondii can effectively scavenge host cell precursors, the fatty acids produced in the apicoplast are essential for parasite development and survival [81,82]. In fact, the parasite can sense lipid availability in the surrounding environment, allowing a proper balance between de novo synthesis and nutrient scavenging pathways, to maintain membrane genesis, which is important for division, growth and overall survival and pathogenesis [83]. Elongation of nascent fatty acids in the FAS II pathway is a process mediated by multiple enzymes located in the endoplasmic reticulum, which are also present in bacteria and plants. In animals and fungi, the elongation process is catalyzed by the type I FAS pathway, leading to a single large multifunctional polypeptide [80,83,84]. Since lipid synthesis is regarded as an essential process, especially during tachyzoite intracellular development, for membrane genesis and lipid homeostasis and signaling, its inhibition is of great interest in anti-T. gondii drug discovery [83].

4.2.1. The FAS II Enzyme Enoyl-Acetyl Carrier Protein Reductase (ENR)

The FAS II apicoplast-located enzyme enoyl-acetyl carrier protein reductase (ENR) catalyzes the last step in fatty acid synthesis [46,82]. ENR is inhibited by triclosan, an antibacterial compound, which inhibits T. gondii in vitro growth at low micromolar to nanomolar concentrations [46]. However, triclosan has poor solubility and oral bioavailability. El-Zawawy et al. described a liposomal-based delivery system for triclosan, which was able to reduce, in vivo, both tachyzoite and cyst burden. In fact, the incorporation of triclosan within the liposomes allowed the use of lower dosages, undermining possible adverse effects, whilst maintaining its antiparasitic activity [47,60]. Stec et al. later reported several promising triclosan analogs with better anti-Toxoplasma activity than the parent compound, such as compound 16c, of this series, presenting an in vitro IC50 of 250 nM, compared to 3 µM of triclosan. In addition, an overall improvement in pharmacokinetics was also observed. However, despite promising in vitro results, in vivo assays only showed decreased T. gondii proliferation in mice at much higher doses (75 mg/kg) when compared to the most effective doses used in triclosan assays (10 mg/kg). Nevertheless, compound 16c was 10–fold less toxic than triclosan in vivo [61]. Overall, the promising in vitro activity and interesting pharmacokinetic profile of compound 16c makes it a potential scaffold for further development.

4.2.2. β-Ketoacyl-Acyl Carrier Protein Synthase I and II (KAS I/II)

The β-ketoacyl-acyl carrier protein synthases I and II (KAS I/II) are other drug targets belonging to the FAS II pathway and that are essential for fatty acid elongation. In fact, mutants that lack these enzymes are deficient in unsaturated fatty acids [48]. KAS I/II are specifically inhibited by thiolactomycin and its analogs. Martins-Duarte et al. determined IC50 values between 1.6 and 29.4 µM, and electron microscopy studies of treated parasites revealed serious morphological alterations in parasite shape and intracellular organelle organization. Treatment with these compounds resulted in swollen mitochondria with disrupted structures, enlarged Golgi complex, and expanded endoplasmic reticulum throughout the whole parasite cytoplasm. The replication process (endodyogeny) was also affected, as uncompleted division processes were observed, resulting in large multinucleated parasites. These findings are indicative of parasite toxicity and death, so thiolactomycin and analogs show a clear impact in parasite development and survival [62]. However, to the best of our knowledge, in vivo testing of these compounds has not yet been reported.

4.2.3. Pantothenate Synthetase

T. gondii is capable of synthesizing FAS precursors outside the FAS II pathway, such as pantothenate, a coenzyme A precursor. The parasite’s pantothenate pathway includes the terminal enzyme pantothenate synthetase that converts pantoate to pantothenate. Unlike humans, T. gondii does not require an external source of pantothenate [49]. Host cell conversion of pantothenate to coenzyme A is rather rapid, preventing T. gondii from scavenging the precursor from the host cell [27]. Thus, de novo pantothenic acid synthesis can be an attractive target for drug discovery, as it avoids interference with host cell FAS. Mageed et al. tested several acylsulfonamides, originally developed to target Mycobacterium tuberculosis pantothenate synthetase. In order to assess parasite growth inhibition, the commonly used drug pyrimethamine served as a positive control. Compounds SW413 and SW404 of this series demonstrated in vitro IC50 values of 20 and 130 nM, respectively, with median toxic doses (TD50) above 1000 µM in human foreskin fibroblasts (HFF). These inhibitors were at least as potent as pyrimethamine regarding parasite growth inhibition. To infer whether pantothenate synthetase is the possible target of the compounds, the EC50 values were assessed in the presence and absence of supplemental pantothenate. The addition of pantothenate increased the EC50 values of both SW413 and SW404, indicating that de novo pantothenate synthesis is essential for T. gondii survival and can be effectively targeted [49]. Regardless, in vivo experiments are needed to examine the efficacy of these candidates in animals.

4.3. Drug Targets Involved in DNA Expression

T. gondii is capable of rapidly differentiating between the active tachyzoite stage and the slow-growing bradyzoite stage. This tachyzoite–bradyzoite interconversion process requires the expression and modulation of stage-specific genes. This modulation may be performed through epigenetic mechanisms, using a post-translational modification (PTM) of histone proteins. PTMs include acetylation or deacetylation of histone residues [85,86]. Acetylation of conserved histone lysine residues by histone acetyltransferases (HATs) generates PTMs that generally lead to increased target gene expression. Instead, deacetylation of these residues by histone deacetylases (HDACs) removes the modification, resulting in decreased target gene expression [50,51]. HATs and HDACs that target stage-specific genes contribute greatly to parasite interconversion between tachyzoite and bradyzoite stages. Modulation of gene expression in this regard may contribute greatly towards the development of treatment options for chronic toxoplasmosis. Preventing bradyzoite differentiation may help to avoid chronic infection, whereas preventing tachyzoite conversion may avoid prompt reactivation of T. gondii acute infection in immunocompromised patients [38]. In addition, this PTM is also present in other drug targets, including several proteins involved in DNA repair, including the chaperone Hsp90 and the ATM serine/threonine-protein kinase [87]. Therefore, epigenetic regulation of gene expression can be considered an attractive idea for T. gondii drug development.

Histone Deacetylase Enzyme TgHDAC3

Histone deacetylase enzyme TgHDAC3 proved to be an effective drug target for T. gondii inhibition [52]. The cyclopeptide FR235222 targets TgHDAC3, causing hyper-acetylation of histone H4 in T. gondii. The compound demonstrated in vitro inhibition of intracellular parasite growth with an IC50 of 9.7 nM, induced in vitro bradyzoite conversion, and was able to reach bradyzoites within ex vivo cysts, preventing tachyzoite conversion. This promising result was further confirmed as FR235222-pretreated bradyzoites were found to be incapable of infecting HFF monolayers, and unable to cause toxoplasmosis in the mouse model. However, host cell toxicity was observed in FR235222 treatment, causing HFF cell inhibition at an IC50 of 128 nM. Further development of FR235222 analogs led to W363 and W399, which demonstrated higher parasite selectivity in comparison to the parent compound while maintaining equivalent parasite IC50 values in vitro [53]. The efficacy of these three compounds in chronically infected mice remains to be described.

4.4. Drug Targets Involved in Mitochondrial Electron Transport Pathway

Mitochondrial Cytochrome bc1 Complex

The cytochrome bc1 complex (bc1), present in the mitochondrial electron transport chain, is a drug target for several apicomplexan parasitic infections, including toxoplasmosis, malaria and babesiosis. Mitochondrial bc1 inhibitors bind to the hydroquinone oxidation (Qo) or quinone reduction (Qi) site of this complex, hindering cell respiration by inhibition of the electron transport pathway [27]. Atovaquone, which is clinically available for alternate treatment and prophylaxis of toxoplasmosis, as well as malaria and babesiosis, is a well-described Qo-site inhibitor. However, due to mutations in the target binding site, the development of resistance limited its use in toxoplasmosis [88].
Endochin-like quinolones (ELQ), which are 4-(1H)-quinolone derivatives, target the Qi site of bc1. The most promising compounds, ELQ-271 and ELQ-316, effectively exhibited low IC50 values for parasite growth inhibition, such as 0.100 and 0.007 nM, respectively. ELQ-271 inhibited human bc1 at 800 nM and ELQ-316 did not show human bc1 inhibition and was not toxic to HFF or human hepatocarcinoma cells (HepG2) at the highest concentration tested (10 µM). A reduction in the number of brain cysts by 88% in mice treated five weeks after infection with an ME49 T. gondii strain (type II strain) was also verified [54,55,56,63]. Due to the broad anti-apicomplexan activity of ELQ-316, combination with atovaquone was suggested to overcome the resistance issues associated with the latter [27]. Recently, McConnell et al. identified the compound ELQ-400 (also known as MMV671636, from Neospora caninum assays) as a promising candidate, capable of inhibiting both Qo and Qi sites of bc1 due to its structural flexibility and favorable substitution pattern. ELQ-400 decreased acute infection with a lethal type I strain of T. gondii in mice. In this assay, all mice survived and presented no signs of infection. ELQ-400 and ELQ-316 were simultaneously evaluated in vivo. Results indicate that both compounds are remarkably effective in decreasing infection in mice, however, they differ in tissue distribution and ability to prevent T. gondii from accessing the brain tissue, due to their distinct blood-brain barrier penetration capacity and half-life. In this regard, ELQ-400 was more effective in preventing the parasite from reaching the brain. Therefore, this compound is thought to effectively act on both acute and chronic phases of T. gondii infection [56,64].

5. Drug Repurposing Approach

Drug repurposing is a strategy for identifying new clinical uses for existing drugs with specific therapeutic indications [89]. This process is becoming an interesting strategy for drug discovery, as it involves potentially lower financial costs in drug development as well as shorter timelines [90].
The Medicines for Malaria Venture (MMV) foundation aims to reduce the burden of malaria by developing and facilitating access to new drug candidates. Thus, an open-access compound library called Malaria Box was made available for academics worldwide, with the purpose of identifying novel bioactive compounds against various pathogens. This library contained 400 blood-stage active anti-Plasmodium compounds. The screening led to the identification of seven potent anti-T. gondii compounds. Among these, the most potent and selective was the piperazine acetamide MMV007791 (compounds provided by the MMV foundation are generally designated by their MMV identifier codes) [91]. In 2015, MMV launched a novel open-access library, modeled after the Malaria Box, known as the Pathogen Box. It consists of 400 drug-like small molecules, contained in 96-well plates, with confirmed bioactivity against at least one of the following pathogens: Plasmodium, Mycobacterium, Kinetoplastids (such as Trypanosoma), Schistosoma, Cryptosporidium, helminths, and dengue virus. Eventually, since T. gondii is an apicomplexan parasite and morphologically similar to some of the parasites contained in this list, many compounds were found to have activity against this protozoan and even shed new insights into its biological pathways [64,65,92,93]. Spalenka et al. screened all 400 compounds for anti-T. gondii activity. Fifteen compounds demonstrated desired efficacy, of which eight presented selectivity and favorable in vitro effects on tachyzoite proliferation. The most active compound, MMV675968, is a diaminoquinazoline with known activity against the enzyme DHFR of Cryptosporidium, potentially targeting the same enzyme in T. gondii. This hypothesis was later assessed, and the compound exhibited an IC50 of 0.02 µM and high selectivity towards parasite DHFR enzyme. Buparvaquone (MMV689480), a well-known hydroxynaphthoquinone with described activity against N. caninum, by inhibiting several enzymes involved in the mitochondrial electron transport pathway, was also identified as having good in vitro anti-T. gondii activity, with an IC50 of 0.10 µM [65].
Recently, Murata et al. screened a chemical compound library, provided by the Drug Discovery Initiative from the University of Tokyo, and two promising compounds with anti-T. gondii activity were identified: tanshinone IIA, a compound with potential cancer cell growth inhibition; and hydroxyzine, a well-known first-generation antihistamine drug. T. gondii targets and mode of action are currently not known for these compounds, however, they were identified as effective in vitro inhibitors of tachyzoite growth, with reduced host cell toxicity. Moreover, tanshinone IIA and hydroxyzine showed inhibitory effects on the growth of bradyzoites. Thus, data indicate that these compounds may represent novel lead compounds to treat acute toxoplasmosis as well as preventing reactivation of latent infection, particularly in immunocompromised individuals [66]. However, in vivo efficacy remains to be reported.

6. Concluding Remarks

Toxoplasmosis is widely distributed worldwide, and the current chemotherapy lacks efficacy and safety. Clinically available options are associated with a relevant spectrum of adverse side effects and generally induce poor compliance within patients. Drug discovery has developed enormously in the last decade, with several drug candidates showing promising results both in vitro and in vivo. This review underlines several promising compounds, drug targets and strategies for anti-T. gondii drug development. Molecular modifications play an important role in this regard, with the aim of improving pharmacokinetic characteristics, including blood–brain barrier access, bioavailability, and half-life. The use of liposomal nanoparticles may also be applied in drugs with promising in vitro results that lack the necessary pharmacokinetic profile. Epigenetics and modulation of gene expression offer vast possibilities in drug discovery, as T. gondii provides a unique HAT/HDAC system that allows various explorative strategies. Drug repurposing has been used for several decades in many different diseases and should be continuously explored.
It is of great importance to invest in the study of novel potent drug candidates. Compounds exhibiting fewer and milder side effects, being overall better tolerated in pregnant women and newborns, specifically, in comparison to current chemotherapy, should be prioritized. Ideally, candidates should be capable of acting on both acute replicating tachyzoite and latent bradyzoite stages, preventing acute disease and reactivation, and even allowing resolution of chronic infection. In addition, the drugs should be bioavailable, capable of reaching therapeutic concentration in all target tissues, such as the brain and eye, as well as concentrate appropriately in the placenta and fetal compartment, while avoiding the generation of drug-resistant strains. Finally, drug costs should be affordable, providing treatment in all world regions. The development of such drugs would revolutionize current T. gondii chemotherapy.

Author Contributions

M.d.S. and M.B. wrote the initial manuscript. M.d.S., M.B., C.T. and P.G. contributed to the article and approved the submitted version. All authors agreed to the published version of the manuscript.

Funding

This research was funded by Fundação para a Ciência e Tecnologia (FCT), Portugal, through projects UIDB/50006/2020, and PTDC/BTM-SAL/29786/2017. This work is financed by national funds from FCT-Fundação para a Ciência e a Tecnologia, I.P., in the scope of the project UIDP/04378/2020 and UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences-UCIBIO and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy-i4HB.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Lindsay, D.S.; Dubey, J.P. Chapter 6—Toxoplasmosis in wild and domestic animals. In Toxoplasma Gondii, 3rd ed.; Weiss, L.M., Kim, K., Eds.; Academic Press: Cambridge, MA, USA, 2020; pp. 293–320. [Google Scholar]
  2. Montazeri, M.; Mikaeili Galeh, T.; Moosazadeh, M.; Sarvi, S.; Dodangeh, S.; Javidnia, J.; Sharif, M.; Daryani, A. The global serological prevalence of Toxoplasma gondii in felids during the last five decades (1967–2017): A systematic review and meta-analysis. Parasit. Vectors 2020, 13, 82. [Google Scholar] [CrossRef] [PubMed]
  3. Molan, A.; Nosaka, K.; Wang, W.; Hunter, M. Global status of Toxoplasma gondii infection: Systematic review and prevalence snapshots. Trop. Biomed. 2019, 36, 898–925. [Google Scholar] [PubMed]
  4. Pittman, K.J.; Knoll, L.J. Long-Term Relationships: The Complicated Interplay between the Host and the Developmental Stages of Toxoplasma gondii during Acute and Chronic Infections. Microbiol. Mol. Biol. Rev. 2015, 79, 387–401. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  5. Saeij, J.P.; Boyle, J.P.; Boothroyd, J.C. Differences among the three major strains of Toxoplasma gondii and their specific interactions with the infected host. Trends Parasitol. 2005, 21, 476–481. [Google Scholar] [CrossRef]
  6. Xiao, J.; Yolken, R.H. Strain hypothesis of Toxoplasma gondii infection on the outcome of human diseases. Acta Physiol. 2015, 213, 828–845. [Google Scholar] [CrossRef] [Green Version]
  7. Kochanowsky, J.A.; Koshy, A.A. Toxoplasma gondii. Curr. Biol. 2018, 28, R770–R771. [Google Scholar] [CrossRef] [Green Version]
  8. Paris, L. 106—Toxoplasmosis. In Hunter’s Tropical Medicine and Emerging Infectious Diseases, 10th ed.; Ryan, E.T., Hill, D.R., Solomon, T., Aronson, N.E., Endy, T.P., Eds.; Content Repository Only: London, UK, 2020; pp. 803–813. [Google Scholar]
  9. Flegr, J.; Escudero, D.Q. Impaired health status and increased incidence of diseases in Toxoplasma-seropositive subjects—An explorative cross-sectional study. Parasitology 2016, 143, 1974–1989. [Google Scholar] [CrossRef] [Green Version]
  10. Wang, Z.-D.; Liu, H.-H.; Ma, Z.-X.; Ma, H.-Y.; Li, Z.-Y.; Yang, Z.-B.; Zhu, X.-Q.; Xu, B.; Wei, F.; Liu, Q. Toxoplasma gondii Infection in Immunocompromised Patients: A Systematic Review and Meta-Analysis. Front. Microbiol. 2017, 8, 389. [Google Scholar] [CrossRef] [Green Version]
  11. McAuley, J.B. Congenital Toxoplasmosis. J. Pediatric Infect. Dis. Soc. 2014, 3 (Suppl. S1), S30–S35. [Google Scholar] [CrossRef]
  12. Foulon, W.; Villena, I.; Stray-Pedersen, B.; Decoster, A.; Lappalainen, M.; Pinon, J.M.; Jenum, P.A.; Hedman, K.; Naessens, A. Treatment of toxoplasmosis during pregnancy: A multicenter study of impact on fetal transmission and children’s sequelae at age 1 year. Am. J. Obs. Gynecol. 1999, 180, 410–415. [Google Scholar] [CrossRef]
  13. Kieffer, F.; Wallon, M. Congenital toxoplasmosis. Handb. Clin. Neurol. 2013, 112, 1099–1101. [Google Scholar] [CrossRef]
  14. Robbins, J.R.; Zeldovich, V.B.; Poukchanski, A.; Boothroyd, J.C.; Bakardjiev, A.I. Tissue barriers of the human placenta to infection with Toxoplasma gondii. Infect. Immun. 2012, 80, 418–428. [Google Scholar] [CrossRef] [Green Version]
  15. Ahmed, M.; Sood, A.; Gupta, J. Toxoplasmosis in pregnancy. Eur J. Obs. Gynecol. Reprod. Biol. 2020, 255, 44–50. [Google Scholar] [CrossRef] [PubMed]
  16. Borges, M.; Magalhaes Silva, T.; Brito, C.; Teixeira, N.; Roberts, C.W. How does toxoplasmosis affect the maternal-foetal immune interface and pregnancy? Parasite Immunol. 2019, 41, e12606. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  17. Garweg, J.G.; Stanford, M.R. Therapy for ocular toxoplasmosis--The future. Ocul. Immunol. Inflamm. 2013, 21, 300–305. [Google Scholar] [CrossRef]
  18. Khan, K.; Khan, W. Congenital toxoplasmosis: An overview of the neurological and ocular manifestations. Parasitol. Int. 2018, 67, 715–721. [Google Scholar] [CrossRef]
  19. Burgdorf, K.S.; Trabjerg, B.B.; Pedersen, M.G.; Nissen, J.; Banasik, K.; Pedersen, O.B.; Sørensen, E.; Nielsen, K.R.; Larsen, M.H.; Erikstrup, C.; et al. Large-scale study of Toxoplasma and Cytomegalovirus shows an association between infection and serious psychiatric disorders. Brain Behav. Immun. 2019, 79, 152–158. [Google Scholar] [CrossRef]
  20. Fond, G.; Capdevielle, D.; Macgregor, A.; Attal, J.; Larue, A.; Brittner, M.; Ducasse, D.; Boulenger, J.P. Toxoplasma gondii: A potential role in the genesis of psychiatric disorders. L’Encéphale 2013, 39, 38–43. [Google Scholar] [CrossRef] [Green Version]
  21. Yalın Sapmaz, Ş.; Şen, S.; Özkan, Y.; Kandemir, H. Relationship between Toxoplasma gondii seropositivity and depression in children and adolescents. Psychiatry Res. 2019, 278, 263–267. [Google Scholar] [CrossRef] [PubMed]
  22. Fuglewicz, A.J.; Piotrowski, P.; Stodolak, A. Relationship between toxoplasmosis and schizophrenia: A review. Adv. Clin. Exp. Med. 2017, 26, 1031–1036. [Google Scholar] [CrossRef]
  23. Khademvatan, S.; Saki, J.; Khajeddin, N.; Izadi-Mazidi, M.; Beladi, R.; Shafiee, B.; Salehi, Z. Toxoplasma gondii Exposure and the Risk of Schizophrenia. Jundishapur J. Microbiol. 2014, 7, e12776. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  24. Sutterland, A.L.; Fond, G.; Kuin, A.; Koeter, M.W.J.; Lutter, R.; van Gool, T.; Yolken, R.; Szoke, A.; Leboyer, M.; de Haan, L. Beyond the association. Toxoplasma gondii in schizophrenia, bipolar disorder, and addiction: Systematic review and meta-analysis. Acta Psychiatr. Scand. 2015, 132, 161–179. [Google Scholar] [CrossRef]
  25. Dunay, I.R.; Gajurel, K.; Dhakal, R.; Liesenfeld, O.; Montoya, J.G. Treatment of Toxoplasmosis: Historical Perspective, Animal Models, and Current Clinical Practice. Clin. Microbiol. Rev. 2018, 31. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  26. Montoya, J.G.; Remington, J.S. Management of Toxoplasma gondii infection during pregnancy. Clin. Infect. Dis. 2008, 47, 554–566. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  27. Alday, P.H.; Doggett, J.S. Drugs in development for toxoplasmosis: Advances, challenges, and current status. Drug Des. Devel. 2017, 11, 273–293. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  28. Paquet, C.; Yudin, M.H. Toxoplasmosis in pregnancy: Prevention, screening, and treatment. J. Obs. Gynaecol. Can. 2013, 35, 78–81. [Google Scholar] [CrossRef]
  29. Ardabili, S.; Kohl, J.; Gül, G.; Hodel, M. What obstetricians should be aware of: Serious side effects of antibiotic toxoplasmosis treatment in pregnancy. BMJ Case Rep. 2021, 14, e240809. [Google Scholar] [CrossRef] [PubMed]
  30. Ben-Harari, R.R.; Goodwin, E.; Casoy, J. Adverse Event Profile of Pyrimethamine-Based Therapy in Toxoplasmosis: A Systematic Review. Drugs R D 2017, 17, 523–544. [Google Scholar] [CrossRef] [Green Version]
  31. Borkowski, P.K.; Brydak-Godowska, J.; Basiak, W.; Olszyńska-Krowicka, M.; Rabczenko, D. Adverse Reactions in Antifolate-Treated Toxoplasmic Retinochoroiditis. Adv. Exp. Med. Biol. 2018, 1108, 37–48. [Google Scholar] [CrossRef]
  32. Guaraldo, L.; Villar, B.B.F.; Durão, N.M.G.; Louro, V.C.; Quintana, M.S.B.; Curi, A.L.L.; Neves, E.S. Ocular toxoplasmosis: Adverse reactions to treatment in a Brazilian cohort. Trans. R Soc. Trop Med. Hyg. 2018, 112, 188–192. [Google Scholar] [CrossRef]
  33. Iaccheri, B.; Fiore, T.; Papadaki, T.; Androudi, S.; Foster, C. Adverse Reactions with Antitoxoplasma Therapy. Investig. Ophthalmol. Vis. Sci. 2003, 44, 1415. [Google Scholar]
  34. Shammaa, A.M.; Powell, T.G.; Benmerzouga, I. Adverse outcomes associated with the treatment of Toxoplasma infections. Sci. Rep. 2021, 11, 1035. [Google Scholar] [CrossRef] [PubMed]
  35. Rajapakse, S.; Chrishan Shivanthan, M.; Samaranayake, N.; Rodrigo, C.; Deepika Fernando, S. Antibiotics for human toxoplasmosis: A systematic review of randomized trials. Pathog. Glob. Health 2013, 107, 162–169. [Google Scholar] [CrossRef] [Green Version]
  36. Paquet, C.; Yudin, M.H. No. 285-Toxoplasmosis in Pregnancy: Prevention, Screening, and Treatment. J. Obs. Gynaecol. Can. 2018, 40, e687–e693. [Google Scholar] [CrossRef] [PubMed]
  37. Dexamethasone for Cerebral Toxoplasmosis-ClinicalTrials.gov Identifier: NCT04341155. Available online: https://ClinicalTrials.gov/show/NCT04341155 (accessed on 6 September 2021).
  38. Konstantinovic, N.; Guegan, H.; Stäjner, T.; Belaz, S.; Robert-Gangneux, F. Treatment of toxoplasmosis: Current options and future perspectives. Food Waterborne Parasitol. 2019, 15, e00036. [Google Scholar] [CrossRef]
  39. Doliwa, C.; Escotte-Binet, S.; Aubert, D.; Velard, F.; Schmid, A.; Geers, R.; Villena, I. Induction of sulfadiazine resistance in vitro in Toxoplasma gondii. Exp. Parasitol. 2013, 133, 131–136. [Google Scholar] [CrossRef]
  40. Meneceur, P.; Bouldouyre, M.A.; Aubert, D.; Villena, I.; Menotti, J.; Sauvage, V.; Garin, J.F.; Derouin, F. In vitro susceptibility of various genotypic strains of Toxoplasma gondii to pyrimethamine, sulfadiazine, and atovaquone. Antimicrob. Agents Chemother. 2008, 52, 1269–1277. [Google Scholar] [CrossRef] [Green Version]
  41. Silva, L.A.; Reis-Cunha, J.L.; Bartholomeu, D.C.; Vítor, R.W.A. Genetic Polymorphisms and Phenotypic Profiles of Sulfadiazine-Resistant and Sensitive Toxoplasma gondii Isolates Obtained from Newborns with Congenital Toxoplasmosis in Minas Gerais, Brazil. PLoS ONE 2017, 12, e0170689. [Google Scholar] [CrossRef]
  42. Barros, M.; Teixeira, D.; Vilanova, M.; Correia, A.; Teixeira, N.; Borges, M. Vaccines in Congenital Toxoplasmosis: Advances and Perspectives. Front. Immunol. 2021, 11, 3900. [Google Scholar] [CrossRef]
  43. Doggett, J.S.; Ojo, K.K.; Fan, E.; Maly, D.J.; van Voorhis, W.C. Bumped Kinase Inhibitor 1294 Treats Established Toxoplasma gondii. Antimicrob. Agents Chemother. 2014, 58, 3547–3549. [Google Scholar] [CrossRef] [Green Version]
  44. Vidadala, R.S.; Rivas, K.L.; Ojo, K.K.; Hulverson, M.A.; Zambriski, J.A.; Bruzual, I.; Schultz, T.L.; Huang, W.; Zhang, Z.; Scheele, S.; et al. Development of an Orally Available and Central Nervous System (CNS) Penetrant Toxoplasma gondii Calcium-Dependent Protein Kinase 1 (TgCDPK1) Inhibitor with Minimal Human Ether-a-go-go-Related Gene (hERG) Activity for the Treatment of Toxoplasmosis. J. Med. Chem. 2016, 59, 6531–6546. [Google Scholar] [CrossRef] [Green Version]
  45. Rutaganira, F.U.; Barks, J.; Dhason, M.S.; Wang, Q.; Lopez, M.S.; Long, S.; Radke, J.B.; Jones, N.G.; Maddirala, A.R.; Janetka, J.W.; et al. Inhibition of Calcium Dependent Protein Kinase 1 (CDPK1) by Pyrazolopyrimidine Analogs Decreases Establishment and Reoccurrence of Central Nervous System Disease by Toxoplasma gondii. J. Med. Chem. 2017, 60, 9976–9989. [Google Scholar] [CrossRef]
  46. McLeod, R.; Muench, S.P.; Rafferty, J.B.; Kyle, D.E.; Mui, E.J.; Kirisits, M.J.; Mack, D.G.; Roberts, C.W.; Samuel, B.U.; Lyons, R.E.; et al. Triclosan inhibits the growth of Plasmodium falciparum and Toxoplasma gondii by inhibition of apicomplexan Fab I. Int. J. Parasitol. 2001, 31, 109–113. [Google Scholar] [CrossRef]
  47. El-Zawawy, L.A.; El-Said, D.; Mossallam, S.F.; Ramadan, H.S.; Younis, S.S. Triclosan and triclosan-loaded liposomal nanoparticles in the treatment of acute experimental toxoplasmosis. Exp. Parasitol. 2015, 149, 54–64. [Google Scholar] [CrossRef] [PubMed]
  48. Jackowski, S.; Murphy, C.M.; Cronan, J.E., Jr.; Rock, C.O. Acetoacetyl-acyl carrier protein synthase. A target for the antibiotic thiolactomycin. J. Biol. Chem. 1989, 264, 7624–7629. [Google Scholar] [CrossRef]
  49. Mageed, S.N.; Cunningham, F.; Hung, A.W.; Silvestre, H.L.; Wen, S.; Blundell, T.L.; Abell, C.; McConkey, G.A. Pantothenic Acid Biosynthesis in the Parasite Toxoplasma gondii: A Target for Chemotherapy. Antimicrob. Agents Chemother. 2014, 58, 6345–6353. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  50. Andrews, K.T.; Haque, A.; Jones, M.K. HDAC inhibitors in parasitic diseases. Immunol. Cell Biol. 2012, 90, 66–77. [Google Scholar] [CrossRef]
  51. Vanagas, L.; Jeffers, V.; Bogado, S.S.; Dalmasso, M.C.; Sullivan, W.J., Jr.; Angel, S.O. Toxoplasma histone acetylation remodelers as novel drug targets. Expert Rev. Anti-Infect. 2012, 10, 1189–1201. [Google Scholar] [CrossRef] [Green Version]
  52. Bougdour, A.; Maubon, D.; Baldacci, P.; Ortet, P.; Bastien, O.; Bouillon, A.; Barale, J.-C.; Pelloux, H.; Ménard, R.; Hakimi, M.-A. Drug inhibition of HDAC3 and epigenetic control of differentiation in Apicomplexa parasites. J. Exp. Med. 2009, 206, 953–966. [Google Scholar] [CrossRef]
  53. Maubon, D.; Bougdour, A.; Wong, Y.-S.; Brenier-Pinchart, M.-P.; Curt, A.; Hakimi, M.-A.; Pelloux, H. Activity of the Histone Deacetylase Inhibitor FR235222 on Toxoplasma gondii: Inhibition of Stage Conversion of the Parasite Cyst Form and Study of New Derivative Compounds. Antimicrob. Agents Chemother. 2010, 54, 4843–4850. [Google Scholar] [CrossRef] [Green Version]
  54. Alday, P.H., Jr.; Bruzal, I.; Pou, S.; Nilsen, A.; Riscoe, M.K.; Doggett, J. Mechanism of Action of ELQ-316 Against Toxoplasma gondii: Evidence for Qi Site Inhibition of Cytochrome bc1. Open Forum Infect. Dis. 2016, 3. [Google Scholar] [CrossRef]
  55. Alday, P.H.; Bruzual, I.; Nilsen, A.; Pou, S.; Winter, R.; Ben Mamoun, C.; Riscoe, M.K.; Doggett, J.S. Genetic Evidence for Cytochrome b Qi Site Inhibition by 4(1H)-Quinolone-3-Diarylethers and Antimycin in Toxoplasma gondii. Antimicrob. Agents Chemother. 2017, 61, e01866-16. [Google Scholar] [CrossRef] [Green Version]
  56. McConnell, E.V.; Bruzual, I.; Pou, S.; Winter, R.; Dodean, R.A.; Smilkstein, M.J.; Krollenbrock, A.; Nilsen, A.; Zakharov, L.N.; Riscoe, M.K.; et al. Targeted Structure-Activity Analysis of Endochin-like Quinolones Reveals Potent Qi and Qo Site Inhibitors of Toxoplasma gondii and Plasmodium falciparum Cytochrome bc(1) and Identifies ELQ-400 as a Remarkably Effective Compound against Acute Experimental Toxoplasmosis. ACS Infect. Dis. 2018, 4, 1574–1584. [Google Scholar] [CrossRef]
  57. Müller, J.; Aguado-Martínez, A.; Ortega-Mora, L.-M.; Moreno-Gonzalo, J.; Ferre, I.; Hulverson, M.A.; Choi, R.; McCloskey, M.C.; Barrett, L.K.; Maly, D.J.; et al. Development of a murine vertical transmission model for Toxoplasma gondii oocyst infection and studies on the efficacy of bumped kinase inhibitor (BKI)-1294 and the naphthoquinone buparvaquone against congenital toxoplasmosis. J. Antimicrob. Chemother. 2017, 72, 2334–2341. [Google Scholar] [CrossRef] [Green Version]
  58. Schaefer, D.A.; Betzer, D.P.; Smith, K.D.; Millman, Z.G.; Michalski, H.C.; Menchaca, S.E.; Zambriski, J.A.; Ojo, K.K.; Hulverson, M.A.; Arnold, S.L.M.; et al. Novel Bumped Kinase Inhibitors Are Safe and Effective Therapeutics in the Calf Clinical Model for Cryptosporidiosis. J. Infect. Dis. 2016, 214, 1856–1864. [Google Scholar] [CrossRef] [PubMed]
  59. Sánchez-Sánchez, R.; Ferre, I.; Re, M.; Ramos, J.J.; Regidor-Cerrillo, J.; Pizarro Díaz, M.; González-Huecas, M.; Tabanera, E.; Benavides, J.; Hemphill, A.; et al. Treatment with Bumped Kinase Inhibitor 1294 Is Safe and Leads to Significant Protection against Abortion and Vertical Transmission in Sheep Experimentally Infected with Toxoplasma gondii during Pregnancy. Antimicrob. Agents Chemother. 2019, 63, e02527-18. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  60. El-Zawawy, L.A.; El-Said, D.; Mossallam, S.F.; Ramadan, H.S.; Younis, S.S. Preventive prospective of triclosan and triclosan-liposomal nanoparticles against experimental infection with a cystogenic ME49 strain of Toxoplasma gondii. Acta Trop. 2015, 141, 103–111. [Google Scholar] [CrossRef]
  61. Stec, J.; Fomovska, A.; Afanador, G.A.; Muench, S.P.; Zhou, Y.; Lai, B.-S.; El Bissati, K.; Hickman, M.R.; Lee, P.J.; Leed, S.E.; et al. Modification of triclosan scaffold in search of improved inhibitors for enoyl-acyl carrier protein (ACP) reductase in Toxoplasma gondii. ChemMedChem 2013, 8, 1138–1160. [Google Scholar] [CrossRef] [Green Version]
  62. Martins-Duarte, E.S.; Jones, S.M.; Gilbert, I.H.; Atella, G.C.; de Souza, W.; Vommaro, R.C. Thiolactomycin analogues as potential anti-Toxoplasma gondii agents. Parasitol. Int. 2009, 58, 411–415. [Google Scholar] [CrossRef]
  63. Doggett, J.S.; Nilsen, A.; Forquer, I.; Wegmann, K.W.; Jones-Brando, L.; Yolken, R.H.; Bordón, C.; Charman, S.A.; Katneni, K.; Schultz, T.; et al. Endochin-like quinolones are highly efficacious against acute and latent experimental toxoplasmosis. Proc. Natl. Acad. Sci. USA 2012, 109, 15936–15941. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  64. Secrieru, A.; Costa, I.C.C.; O’Neill, P.M.; Cristiano, M.L.S. Antimalarial Agents as Therapeutic Tools Against Toxoplasmosis-A Short Bridge between Two Distant Illnesses. Molecules 2020, 25, 1574. [Google Scholar] [CrossRef] [Green Version]
  65. Spalenka, J.; Escotte-Binet, S.; Bakiri, A.; Hubert, J.; Renault, J.-H.; Velard, F.; Duchateau, S.; Aubert, D.; Huguenin, A.; Villena, I. Discovery of New Inhibitors of Toxoplasma gondii via the Pathogen Box. Antimicrob. Agents Chemother. 2018, 62, e01640-17. [Google Scholar] [CrossRef] [Green Version]
  66. Murata, Y.; Sugi, T.; Weiss, L.M.; Kato, K. Identification of compounds that suppress Toxoplasma gondii tachyzoites and bradyzoites. PLoS ONE 2017, 12, e0178203. [Google Scholar] [CrossRef] [Green Version]
  67. Seeber, F.; Steinfelder, S. Recent advances in understanding apicomplexan parasites. F1000Research 2016, 5, 1369. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  68. Dubremetz, J.F.; Garcia-Réguet, N.; Conseil, V.; Fourmaux, M.N. Apical organelles and host-cell invasion by Apicomplexa. Int. J. Parasitol 1998, 28, 1007–1013. [Google Scholar] [CrossRef]
  69. Lüder, C.G.; Stanway, R.R.; Chaussepied, M.; Langsley, G.; Heussler, V.T. Intracellular survival of apicomplexan parasites and host cell modification. Int. J. Parasitol. 2009, 39, 163–173. [Google Scholar] [CrossRef]
  70. Portes, J.; Barrias, E.; Travassos, R.; Attias, M.; de Souza, W. Toxoplasma gondii Mechanisms of Entry Into Host Cells. Front. Cell Infect. Microbiol. 2020, 10, 294. [Google Scholar] [CrossRef]
  71. Portes, J.A.; de Souza, W. Development of an in vitro system to study the developmental stages of Toxoplasma gondii using a genetically modified strain expressing markers for tachyzoites and bradyzoites. Parasitol. Res. 2019, 118, 3479–3489. [Google Scholar] [CrossRef] [PubMed]
  72. Plattner, F.; Soldati-Favre, D. Hijacking of host cellular functions by the Apicomplexa. Annu. Rev. Microbiol. 2008, 62, 471–487. [Google Scholar] [CrossRef] [PubMed]
  73. Mayor, S.; Parton, R.G.; Donaldson, J.G. Clathrin-independent pathways of endocytosis. Cold Spring Harb. Perspect. Biol. 2014, 6, a016758. [Google Scholar] [CrossRef] [Green Version]
  74. Cardew, E.M.; Verlinde, C.L.M.J.; Pohl, E. The calcium-dependent protein kinase 1 from Toxoplasma gondii as target for structure-based drug design. Parasitology 2018, 145, 210–218. [Google Scholar] [CrossRef] [Green Version]
  75. Ojo, K.K.; Larson, E.T.; Keyloun, K.R.; Castaneda, L.J.; DeRocher, A.E.; Inampudi, K.K.; Kim, J.E.; Arakaki, T.L.; Murphy, R.C.; Zhang, L.; et al. Toxoplasma gondii calcium-dependent protein kinase 1 is a target for selective kinase inhibitors. Nat. Struct. Mol. Biol. 2010, 17, 602–607. [Google Scholar] [CrossRef] [Green Version]
  76. Lourido, S.; Zhang, C.; Lopez, M.S.; Tang, K.; Barks, J.; Wang, Q.; Wildman, S.A.; Shokat, K.M.; Sibley, L.D. Optimizing small molecule inhibitors of calcium-dependent protein kinase 1 to prevent infection by Toxoplasma gondii. J. Med. Chem. 2013, 56, 3068–3077. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  77. Murphy, R.C.; Ojo, K.K.; Larson, E.T.; Castellanos-Gonzalez, A.; Perera, B.G.K.; Keyloun, K.R.; Kim, J.E.; Bhandari, J.G.; Muller, N.R.; Verlinde, C.L.M.J.; et al. Discovery of Potent and Selective Inhibitors of CDPK1 from C. parvum and T. gondii. ACS Med. Chem. Lett. 2010, 1, 331–335. [Google Scholar] [CrossRef] [PubMed]
  78. Winzer, P.; Müller, J.; Aguado-Martínez, A.; Rahman, M.; Balmer, V.; Manser, V.; Ortega-Mora, L.M.; Ojo, K.K.; Fan, E.; Maly, D.J.; et al. In Vitro and In Vivo Effects of the Bumped Kinase Inhibitor 1294 in the Related Cyst-Forming Apicomplexans Toxoplasma gondii and Neospora caninum. Antimicrob. Agents Chemother. 2015, 59, 6361–6374. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  79. Vandenberg, J.I.; Perry, M.D.; Perrin, M.J.; Mann, S.A.; Ke, Y.; Hill, A.P. hERG K(+) channels: Structure, function, and clinical significance. Physiol. Rev. 2012, 92, 1393–1478. [Google Scholar] [CrossRef] [Green Version]
  80. Mazumdar, J.; Striepen, B. Make It or Take It: Fatty Acid Metabolism of Apicomplexan Parasites. Eukaryot. Cell 2007, 6, 1727–1735. [Google Scholar] [CrossRef] [Green Version]
  81. Mazumdar, J.; Wilson, E.H.; Masek, K.; Hunter, C.A.; Striepen, B. Apicoplast fatty acid synthesis is essential for organelle biogenesis and parasite survival in Toxoplasma gondii. Proc. Natl. Acad. Sci. USA 2006, 103, 13192–13197. [Google Scholar] [CrossRef] [Green Version]
  82. Ramakrishnan, S.; Docampo, M.D.; MacRae, J.I.; Ralton, J.E.; Rupasinghe, T.; McConville, M.J.; Striepen, B. The intracellular parasite Toxoplasma gondii depends on the synthesis of long-chain and very long-chain unsaturated fatty acids not supplied by the host cell. Mol. Microbiol. 2015, 97, 64–76. [Google Scholar] [CrossRef] [Green Version]
  83. Dubois, D.; Fernandes, S.; Amiar, S.; Dass, S.; Katris, N.J.; Botté, C.Y.; Yamaryo-Botté, Y. Toxoplasma gondii acetyl-CoA synthetase is involved in fatty acid elongation (of long fatty acid chains) during tachyzoite life stages. J. Lipid Res. 2018, 59, 994–1004. [Google Scholar] [CrossRef] [Green Version]
  84. Ramakrishnan, S.; Docampo, M.D.; MacRae, J.I.; Pujol, F.M.; Brooks, C.F.; van Dooren, G.G.; Hiltunen, J.K.; Kastaniotis, A.J.; McConville, M.J.; Striepen, B. Apicoplast and Endoplasmic Reticulum Cooperate in Fatty Acid Biosynthesis in Apicomplexan Parasite Toxoplasma gondii. J. Biol. Chem. 2012, 287, 4957–4971. [Google Scholar] [CrossRef] [Green Version]
  85. Sullivan, W.J., Jr.; Hakimi, M.A. Histone mediated gene activation in Toxoplasma gondii. Mol. Biochem. Parasitol. 2006, 148, 109–116. [Google Scholar] [CrossRef] [PubMed]
  86. Sullivan, W.J., Jr.; Jeffers, V. Mechanisms of Toxoplasma gondii persistence and latency. FEMS Microbiol. Rev. 2012, 36, 717–733. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  87. Angel, S.O.; Vanagas, L.; Ruiz, D.M.; Cristaldi, C.; Saldarriaga Cartagena, A.M.; Sullivan, W.J. Emerging Therapeutic Targets Against Toxoplasma gondii: Update on DNA Repair Response Inhibitors and Genotoxic Drugs. Front. Cell. Infect. Microbiol. 2020, 10, 289. [Google Scholar] [CrossRef]
  88. McFadden, D.C.; Tomavo, S.; Berry, E.A.; Boothroyd, J.C. Characterization of cytochrome b from Toxoplasma gondii and Qo domain mutations as a mechanism of atovaquone-resistance. Mol. Biochem. Parasitol. 2000, 108, 1–12. [Google Scholar] [CrossRef]
  89. Ashburn, T.T.; Thor, K.B. Drug repositioning: Identifying and developing new uses for existing drugs. Nat. Rev. Drug Discov. 2004, 3, 673–683. [Google Scholar] [CrossRef] [PubMed]
  90. Pushpakom, S.; Iorio, F.; Eyers, P.A.; Escott, K.J.; Hopper, S.; Wells, A.; Doig, A.; Guilliams, T.; Latimer, J.; McNamee, C.; et al. Drug repurposing: Progress, challenges and recommendations. Nat. Rev. Drug Discov. 2019, 18, 41–58. [Google Scholar] [CrossRef]
  91. Boyom, F.F.; Fokou, P.V.T.; Tchokouaha, L.R.Y.; Spangenberg, T.; Mfopa, A.N.; Kouipou, R.M.T.; Mbouna, C.J.; Donfack, V.F.D.; Zollo, P.H.A. Repurposing the Open Access Malaria Box To Discover Potent Inhibitors of Toxoplasma gondii and Entamoeba histolytica. Antimicrob. Agents Chemother. 2014, 58, 5848–5854. [Google Scholar] [CrossRef] [Green Version]
  92. Patra, A.T.; Hingmire, T.B.; Belekar, M.; Xiong, A.; Subramanian, G.; Bozdech, Z.; Preiser, P.; Shanmugam, D.; Chandramohanadas, R. Whole Cell Phenotypic Screening Of MMV Pathogen Box identifies Specific Inhibitors of Plasmodium falciparum merozoite maturation and egress. bioRxiv 2019, 772434. [Google Scholar] [CrossRef]
  93. Radke, J.B.; Burrows, J.N.; Goldberg, D.E.; Sibley, L.D. Evaluation of Current and Emerging Antimalarial Medicines for Inhibition of Toxoplasma gondii Growth in Vitro. ACS Infect. Dis. 2018, 4, 1264–1274. [Google Scholar] [CrossRef]
Microorganisms 09 01960 g001 550
Figure 1. Graphical representation of a Toxoplasma gondii tachyzoite. Organelle and pathway targets of several experimental and repurposed compounds [43,44,45,46,47,48,49,50,51,52,53,54,55,56]. AR: apical ring; Mic: microneme; R: rhoptry; DG: dense granule; A: apicoplast; M: mitochondrion, N: nucleus; BKI: bumped-kinase inhibitor; ELQ: endochin-like quinolone.
Figure 1. Graphical representation of a Toxoplasma gondii tachyzoite. Organelle and pathway targets of several experimental and repurposed compounds [43,44,45,46,47,48,49,50,51,52,53,54,55,56]. AR: apical ring; Mic: microneme; R: rhoptry; DG: dense granule; A: apicoplast; M: mitochondrion, N: nucleus; BKI: bumped-kinase inhibitor; ELQ: endochin-like quinolone.
Microorganisms 09 01960 g001
Microorganisms 09 01960 g002 550
Figure 2. Toxoplasma gondii host-cell invasion and establishment of intracellular parasitic compartments [68,69,70,71,72]. (A) To invade the host cell, the parasite requires the secretion of proteins from two organelles, micronemes and rhoptries. These proteins allow the parasite to enter the host cell while coating itself with the host cytoplasmatic membrane, forming the Parasitophorous Vacuole (PV). (B) Upon entry, the PV is decorated with rhoptry proteins (ROPs) and proteins derived from the third set of organelles, known as dense granules (GRAs). (C) When the parasite is established inside the PV, it replicates asexually, generating a large enough number of tachyzoites capable of rupturing the host cell and infecting surrounding cells or, if in specific tissues or under certain conditions, convert into metabolically less competent bradyzoites and form tissue cysts.
Figure 2. Toxoplasma gondii host-cell invasion and establishment of intracellular parasitic compartments [68,69,70,71,72]. (A) To invade the host cell, the parasite requires the secretion of proteins from two organelles, micronemes and rhoptries. These proteins allow the parasite to enter the host cell while coating itself with the host cytoplasmatic membrane, forming the Parasitophorous Vacuole (PV). (B) Upon entry, the PV is decorated with rhoptry proteins (ROPs) and proteins derived from the third set of organelles, known as dense granules (GRAs). (C) When the parasite is established inside the PV, it replicates asexually, generating a large enough number of tachyzoites capable of rupturing the host cell and infecting surrounding cells or, if in specific tissues or under certain conditions, convert into metabolically less competent bradyzoites and form tissue cysts.
Microorganisms 09 01960 g002
Table
Table 1. Current drugs used for toxoplasmosis treatment.
Table 1. Current drugs used for toxoplasmosis treatment.
CompoundChemical Structure *Mechanism of ActionReferences
Pyrimethamine Microorganisms 09 01960 i001Antifolate[25,27]
Sulfadiazine Microorganisms 09 01960 i002Antifolate[25,27]
Folinic acid Microorganisms 09 01960 i003Reduction of pyrimethamine side effects[25,27]
Spiramycin Microorganisms 09 01960 i004Protein synthesis inhibitor[25,35]
Clindamycin Microorganisms 09 01960 i005Protein synthesis inhibitor[25,35]
Clarithromycin Microorganisms 09 01960 i006Protein synthesis inhibitor[25,35]
Azithromycin Microorganisms 09 01960 i007Protein synthesis inhibitor[25,35]
Atovaquone Microorganisms 09 01960 i008Mitochondrial electron transport chain inhibitor[25,27]
Cotrimoxazole Microorganisms 09 01960 i009Antifolate[25,27]
* Chemical structures were designed using ChemDraw 17.0 software (PerkinElmer Informatics, Waltham, United States of America).
Table
Table 2. Experimental compounds with anti-Toxoplasma gondii activity.
Table 2. Experimental compounds with anti-Toxoplasma gondii activity.
CompoundChemical Structure *Drug TargetAffected T. gondii PathwayIn vitro IC50/T. gondii Strain/Host CellIn vivo Results/T. gondii Strain/Animal Model/Infection RouteReferences
BKI-1294 Microorganisms 09 01960 i010TgCDPK1Parasite microneme secretion140 nM/
RH/
HFF		93% reduction of parasite burden at 30 mg/kg. No T. gondii detected in peritoneal fluid of half the mice at 100 mg/kg/
RH/
CF-1 mice/
Intraperitoneal
Protection against abortion and vertical transmission in sheep experimentally infected with T. gondii tachyzoites during pregnancy		[43,44,57,58,59]
Compound 32 a Microorganisms 09 01960 i011TgCDPK1Parasite microneme secretion60 nM/
ME49/
HFF		88.7% reduction in the number of brain cysts/
ME49/
CBA/J mice/
Oral gavage		[44]
Compound 24 a Microorganisms 09 01960 i012TgCDPK1Parasite microneme secretionTgCDPK1 inhibition at 10.9 nM (enzyme activity assay)
Inhibition of parasite proliferation at 0.264 µM/
RH/
HFF		Decreased severity of acute infection. Delayed chronic reactivation of disease. Completely cured part of the animals/
ME49/
BALB/c mice and mice lacking IFN-γ receptor/
Oral gavage		[45]
Triclosan Microorganisms 09 01960 i013ENRFAS II3 µM/
RH/
HFF		Reduction in mice mortality, parasite burden and viability. Poor solubility and oral bioavailability/
RH/
Swiss albino mice/
Intraperitoneal		[46,47,60]
Triclosan-liposomal Microorganisms 09 01960 i014
Liposomal		ENRFAS IINDReduction in host mortality and T. gondii brain burden by 98%/
ME49/
Swiss albino mice/
Oral gavage		[46,47]
Compound 16c b Microorganisms 09 01960 i015ENRFAS II250 nM/
RH/
HFF		Improvement in pharmacokinetics in comparison to triclosan
Decreased peritoneal burden of T. gondii/
RH/
Swiss albino mice/
Intraperitoneal		[61]
Thiolactomycin (and analogs) Microorganisms 09 01960 i016KAS I/IIFAS II1.6–29.4 µM/
RH/
LLCMK2
Serious morphological alterations in treated parasites (electron microscopy)		ND[62]
SW413 c Microorganisms 09 01960 i017Pantothenate synthetaseFatty acid chain elongation
(within FAS II)		20 nM/
RH/
HFF		ND[49]
SW404 c Microorganisms 09 01960 i018Pantothenate synthetaseFatty acid chain elongation
(within FAS II)		130 nM/
RH/
HFF		ND[49]
FR235222 Microorganisms 09 01960 i019TgHDAC3DNA expression9.7 nM//
RH/
HFF
Dramatic morphological alterations in in vitro-induced bradyzoites at 30 nM
Prevented bradyzoite-tachyzoite conversion (ex vivo cysts) at 200 nM		Reduced tachyzoite infection in mice. No brain cysts detected. No detectable humoral response/
PRU/
Outbred female Swiss mice/
Intraperitoneal		[53]
W363 d Microorganisms 09 01960 i020TgHDAC3DNA expression10.2 nM/
RH/
HFF		ND[53]
W399 d Microorganisms 09 01960 i021TgHDAC3DNA expression11.3 nM/
RH/
HFF		ND[53]
ELQ-271 e Microorganisms 09 01960 i022Cytochrome bc1 complex (Qi site)Cell respiration0.1 nM/
RH-2F/
HFF
Human bc1 inhibition at 800 nM		ND[54,55,56,63]
ELQ-316 e Microorganisms 09 01960 i023Cytochrome bc1 complex (Qi site)Cell respiration0.007 nM/
2F/
HFF
No human bc1 inhibition
Not toxic to HFF/HepG2 cells at 10 µM		88% reduction in the number of brain cysts/
ME49/
CBA/J mice/
Intraperitoneal		[54,55,56,63]
ELQ-400 e Microorganisms 09 01960 i024Cytochrome bc1 complex (Qi and Qo site)Cell respiration5 µM/
RH/
HFF		Increased mice survival and reduction in brain and spleen parasite load/
RH/
CF-1 mice/
Intraperitoneal		[56,64]
MMV675968 f Microorganisms 09 01960 i025DHFRFolate synthesis0.02 µM/
RH/
Vero
High selectivity towards parasite DHFR		ND[65]
Buparvaquone
(MMV689480) f		 Microorganisms 09 01960 i026Mitochondrial electron transport chain enzymes (dehydrogenase enzymes)Cell respiration0.10 µM/
RH/
Vero		ND[65]
Tanshinone IIA g Microorganisms 09 01960 i027NKNK2.5 µM/
PLK/
HFF
No host cell toxicity at 25 µM
Reduced number of in vitro-induced bradyzoites at 1 µM		ND[66]
Hydroxyzine g Microorganisms 09 01960 i028NKNK1.0 µM/
PLK h/
HFF
No host cell toxicity at 25 µM
Reduced number of in vitro-induced bradyzoites at 2 µM		ND[66]
Abbreviations: ND: not done; NK: not known; IC50: half-maximal inhibitory concentration; TgCDPK1: T. gondii calcium-dependent protein kinase 1; ENR: enoyl-acetyl carrier protein reductase; KAS I/II: β-ketoacyl-acyl carrier protein synthase; FAS II: type II fatty acid synthesis; TgHDAC3: T. gondii histone deacetylase 3; DHFR: dihydrofolate reductase; HFF: human foreskin fibroblasts; IFN: interferon; LLCMK2: rhesus monkey kidney epithelial cells; HepG2: human hepatocarcinoma cells. *: Chemical structures were designed using ChemDraw 17.0 software. a: Bumped-kinase inhibitors derived from BKI-1294. b: Triclosan analogue. c: Acylsulfonamides originally developed for Mycobacterium tuberculosis. d: FR235222 derivatives. e: Endochin-like quinolones. f: Repurposed compounds from the pathogen box (initiative of the Medicines for Malaria Venture). g: Repurposed compounds from the compound library provided by the Drug Discovery Initiative (University of Tokyo). h: Type II strain of T. gondii, derived from the ME49 strain.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Silva, M.d.; Teixeira, C.; Gomes, P.; Borges, M. Promising Drug Targets and Compounds with Anti-Toxoplasma gondii Activity. Microorganisms 2021, 9, 1960. https://doi.org/10.3390/microorganisms9091960

AMA Style

Silva Md, Teixeira C, Gomes P, Borges M. Promising Drug Targets and Compounds with Anti-Toxoplasma gondii Activity. Microorganisms. 2021; 9(9):1960. https://doi.org/10.3390/microorganisms9091960

Chicago/Turabian Style

Silva, Marco da, Cátia Teixeira, Paula Gomes, and Margarida Borges. 2021. "Promising Drug Targets and Compounds with Anti-Toxoplasma gondii Activity" Microorganisms 9, no. 9: 1960. https://doi.org/10.3390/microorganisms9091960

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop