4-Arylthiosemicarbazide derivatives - Pharmacokinetics, toxicity and anti-Toxoplasma gondii activity in vivo.

The increasing resistance of Toxoplasma gondii to drugs and side effects of therapy indicate that specific treatment for these parasites is still needed. The 4-arylthiosemicarbazide derivatives seem to be a solution to this challenge because they have low cytotoxicity against host cells and high anti-T. gondii activity. The molecular mechanism for these compounds is related to the inhibition of tyrosine amino acids involved in the proliferation and parasitophorous vacuole formation. The pharmacokinetic analysis shows that 1-(4-Methylimidazol-5-oyl)-4-(4-nitrophenyl)thiosemicarbazide and 4-(3-Iodophenyl)-1-(4-methylimidazol-5-oyl)thiosemicarbazide administered intragastrically pass into the bloodstream and cross the blood-brain barrier, and the absorption of both compounds is first-order absorption. Toxicity analysis shows that our derivatives possess lower toxicity than the routinely used drugs trimethoprim, sulfadiazine and pyrimethamine, as was observed in the level of liver enzymes and creatinine. Both derivatives are highly potent antiparasitic agents against T. gondii, prolonged survival and cure parasite-infected mice. Additionally, significant reductions in cyst formation in the brain and heart were observed, but the highest decreases were noted in muscle and the level of bradyzoites was similar to these observed in mice treated with commercially used drugs. Collectively, the obtained results support the conclusion that both compounds are highly efficacious in a mouse model of acute and chronic toxoplasmosis.

Structure-activity relationships of Toxoplasma gondii cytochrome bc1 inhibitors.

INTRODUCTION: Toxoplasma gondii is a prolific apicomplexan parasite that infects human and nonhuman animals worldwide and can cause severe brain and eye disease. Safer, more effective therapies for toxoplasmosis are needed. Cytochrome bc1 inhibitors are remarkably effective against toxoplasmosis and other apicomplexan-caused diseases. AREAS COVERED: This work reviews T. gondii cytochrome bc1 inhibitors. Emphasis is placed on the structure-activity relationships of these inhibitors with regard to efficacy, pharmacokinetics, selectivity of T. gondii cytochrome bc1 over host, safety, and potential therapeutic strategies. EXPERT OPINION: Cytochrome bc1 inhibitors are highly promising compounds for toxoplasmosis that have been effective in clinical and preclinical studies. Clinical experience with atovaquone previously validated cytochrome bc1 as a tractable drug target and, over the past decade, optimization of cytochrome bc1 inhibitors has resulted in improved bioavailability, metabolic stability, potency, blood-brain barrier penetration, and selectivity for the T. gondii cytochrome bc1 over the mammalian bc1. Recent studies have demonstrated preclinical safety, identified novel therapeutic strategies for toxoplasmosis using synergistic combinations or long-acting administration and provided insight into their role in chronic infection. This research has identified drug candidates that are more effective than clinically used drugs in preclinical measures of efficacy.

4-Arylthiosemicarbazide Derivatives as Toxoplasmic Aromatic Amino Acid Hydroxylase Inhibitors and Anti-inflammatory Agents.

Approximately one-third of the human population is infected with the intracellular cosmopolitan protozoan Toxoplasma gondii (Tg), and a specific treatment for this parasite is still needed. Additionally, the increasing resistance of Tg to drugs has become a challenge for numerous research centers. The high selectivity of a compound toward the protozoan, along with low cytotoxicity toward the host cells, form the basis for further research, which aims at determining the molecular targets of the active compounds. Thiosemicarbazide derivatives are biologically active organic compounds. Previous studies on the initial preselection of 58 new 4-arylthiosemicarbazide derivatives in terms of their anti-Tg activity and selectivity made it possible to select two promising derivatives for further research. One of the important amino acids involved in the proliferation of Tg and the formation of parasitophorous vacuoles is tyrosine, which is converted by two unique aromatic amino acid hydroxylases to levodopa. Enzymatic studies with two derivatives (R: para-nitro and meta-iodo) and recombinant aromatic amino acid hydroxylase (AAHs) obtained in the E. coli expression system were performed, and the results indicated that toxoplasmic AAHs are a molecular target for 4-arylthiosemicarbazide derivatives. Moreover, the drug affinity responsive target stability assay also confirmed that the selected compounds bind to AAHs. Additionally, the anti-inflammatory activity of these derivatives was tested using THP1-Blue™ NF-κB reporter cells due to the similarity of the thiosemicarbazide scaffold to thiosemicarbazone, both of which are known NF-κB pathway inhibitors.

Bicyclic azetidines target acute and chronic stages of Toxoplasma gondii by inhibiting parasite phenylalanyl t-RNA synthetase.

Toxoplasma gondii commonly infects humans and while most infections are controlled by the immune response, currently approved drugs are not capable of clearing chronic infection in humans. Hence, approximately one third of the world's human population is at risk of reactivation, potentially leading to severe sequelae. To identify new candidates for treating chronic infection, we investigated a series of compounds derived from diversity-oriented synthesis. Bicyclic azetidines are potent low nanomolar inhibitors of phenylalanine tRNA synthetase (PheRS) in T. gondii, with excellent selectivity. Biochemical and genetic studies validate PheRS as the primary target of bicyclic azetidines in T. gondii, providing a structural basis for rational design of improved analogs. Favorable pharmacokinetic properties of a lead compound provide excellent protection from acute infection and partial protection from chronic infection in an immunocompromised mouse model of toxoplasmosis. Collectively, PheRS inhibitors of the bicyclic azetidine series offer promise for treatment of chronic toxoplasmosis.

In Vitro Anti-Toxoplasma gondii Activity Evaluation of a New Series of Quinazolin-4(3H)-one Derivatives.

Toxoplasmosis post serious threaten to human health, leading to severely eye and brain disease, especially for immunocompromised patients and pregnant women. The multiple side effects and long dosing period of current main treatment regiments calls for high effective and low toxicity anti-toxoplasmosis drugs. Herein, we report our efforts to synthesize a series of 2-(piperazin-1-yl)quinazolin-4(3H)-one derivatives and investigate their activity against Toxoplasma gondii tachyzoites in vitro based on cell phenotype screening. Among the 26 compounds, 8w and 8x with diaryl ether moiety at the side chain of piperazine exhibited good efficacy to inhibit T. gondii, with IC50 values of 4 µM and 3 µM, respectively. Structure-activity relationship (SAR) studies implies that hydrophobic aryl at the side chain would be preferred for improvement of activity. Molecular docking study reveals these two compounds appeared high affinity to TgCDPK1 by interaction with the hydrophobic pocket of ATP-binding cleft.

Toxoplasma gondii UBL-UBA shuttle proteins regulate several important cellular processes.

Toxoplasma gondii is an obligate intracellular apicomplexan parasite causing lethal diseases in immunocompromised patients. UBL-UBA shuttle proteins (DDI1, RAD23, and DSK2) are important components of the ubiquitin-proteasome system. By degrading ubiquitinated proteins, UBL-UBA shuttle proteins regulate many cellular processes. However, the specific processes regulated by UBL-UBA shuttle proteins remain elusive. Here, we revealed that the deletion of shuttle proteins results in a selective accumulation of ubiquitinated proteins in the nucleus and aberrant DNA replication. ROP18 was mistargeted and accumulated in the shuttle protein mutant strain, resulting in the recruitment of immunity-related GTPases to the parasitophorous vacuole membrane (PVM). Furthermore, the mistargeting of ROP18 and the recruitment of Irgb6 to the PVM were also observed in the DDI1 mutant strain. DDI1 is a nonclassical UBL-UBA shuttle protein homologous to the HIV-1 protease. Molecular docking showed that DDI1 was a potential target of HIV-1 protease inhibitors. However, these inhibitors blocked the growth of T gondii in vitro but not in vivo. In conclusion, the Toxoplasma UBL-UBA shuttle protein regulates several important cellular processes and the mistargeting of ROP18 may be a representative of the abnormal homeostasis caused by shuttle protein mutation.

Assessment of the Activity of Decoquinate and Its Quinoline-O-Carbamate Derivatives against Toxoplasma gondii In Vitro and in Pregnant Mice Infected with T. gondii Oocysts.

The quinolone decoquinate (DCQ) is widely used in veterinary practice for the treatment of bacterial and parasitic infections, most notably, coccidiosis in poultry and in ruminants. We have investigated the effects of treatment of Toxoplasma gondii in infected human foreskin fibroblasts (HFF) with DCQ. This induced distinct alterations in the parasite mitochondrion within 24 h, which persisted even after long-term (500 nM, 52 days) treatment, although there was no parasiticidal effect. Based on the low half-maximal effective concentration (IC50) of 1.1 nM and the high selectivity index of >5000, the efficacy of oral treatment of pregnant mice experimentally infected with T. gondii oocysts with DCQ at 10 mg/kg/day for 5 days was assessed. However, the treatment had detrimental effects, induced higher neonatal mortality than T. gondii infection alone, and did not prevent vertical transmission. Thus, three quinoline-O-carbamate derivatives of DCQ, anticipated to have better physicochemical properties than DCQ, were assessed in vitro. One such compound, RMB060, displayed an exceedingly low IC50 of 0.07 nM, when applied concomitantly with the infection of host cells and had no impact on HFF viability at 10 µM. As was the case for DCQ, RMB060 treatment resulted in the alteration of the mitochondrial matrix and loss of cristae, but the changes became apparent at just 6 h after the commencement of treatment. After 48 h, RMB060 induced the expression of the bradyzoite antigen BAG1, but TEM did not reveal any other features reminiscent of bradyzoites. The exposure of infected cultures to 300 nM RMB060 for 52 days did not result in the complete killing of all tachyzoites, although mitochondria remained ultrastructurally damaged and there was a slower proliferation rate. The treatment of mice infected with T. gondii oocysts with RMB060 did reduce parasite burden in non-pregnant mice and dams, but vertical transmission to pups could not be prevented.

Cellular and Molecular Targets of Nucleotide-Tagged Trithiolato-Bridged Arene Ruthenium Complexes in the Protozoan Parasites Toxoplasma gondii and Trypanosoma brucei.

Toxoplasma gondii is an apicomplexan parasite that infects and proliferates within many different types of host cells and infects virtually all warm-blooded animals and humans. Trypanosoma brucei is an extracellular kinetoplastid that causes human African trypanosomiasis and Nagana disease in cattle, primarily in rural sub-Saharan Africa. Current treatments against both parasites have limitations, e.g., suboptimal efficacy and adverse side effects. Here, we investigate the potential cellular and molecular targets of a trithiolato-bridged arene ruthenium complex conjugated to 9-(2-hydroxyethyl)-adenine (1), which inhibits both parasites with IC50s below 10-7 M. Proteins that bind to 1 were identified using differential affinity chromatography (DAC) followed by shotgun-mass spectrometry. A trithiolato-bridged ruthenium complex decorated with hypoxanthine (2) and 2-hydroxyethyl-adenine (3) were included as controls. Transmission electron microscopy (TEM) revealed distinct ultrastructural modifications in the mitochondrion induced by (1) but not by (2) and (3) in both species. DAC revealed 128 proteins in T. gondii and 46 proteins in T. brucei specifically binding to 1 but not 2 or 3. In T. gondii, the most abundant was a protein with unknown function annotated as YOU2. This protein is a homolog to the human mitochondrial inner membrane translocase subunit Tim10. In T. brucei, the most abundant proteins binding specifically to 1 were mitochondrial ATP-synthase subunits. Exposure of T. brucei bloodstream forms to 1 resulted in rapid breakdown of the ATP-synthase complex. Moreover, both datasets contained proteins involved in key steps of metabolism and nucleic acid binding proteins.

In vitro activity of N-phenyl-1,10-phenanthroline-2-amines against tachyzoites and bradyzoites of Toxoplasma gondii.

Toxoplasma gondiiis an apicomplexan parasite, the causative agent of toxoplasmosis, a common disease in the world. Toxoplasmosis could be severe, especially in immunocompromised patients. The current therapy is limited, where pyrimethamine and sulfadiazine are the best choices despite being associated with side effects and ineffective against the bradyzoites, the parasitic form present during the chronic phase of the infection. Thus, new therapies against both tachyzoites and bradyzoites from T. gondii are urgent. Herein, we present the anti-T. gondii effect of 1,10-phenanthroline and its N-phenyl-1,10-phenanthroline-2-amine derivatives. The chemical modification of 1,10-phenanthroline tonew derivatives improved the anti-T. gondiiactivity 3.4 fold. The most active derivative presented ED50in the nanomolar range, the smallest value found was for Ph8, 0.1 µM for 96 h of treatment. The host cell viability was maintained after the treatment with the compounds, which were found to be highly selective presenting large selectivity indexes. Treatment with derivatives for 96 h was able to eliminate the T. gondii infection irreversibly. The ultrastructural alterations caused after the treatment with the most effective derivative (Ph8) included signs of cell death, specifically revealed by the Tunel assay for detection of DNA fragmentation. The Phen derivatives were also able to control the growth of the in vitro-derived bradyzoite forms of T. gondii EGS strain, causing its lysis and death. These findings promote the 1,10-phenanthroline derivatives as potential lead compounds for the development of a treatment for acute and chronic phases of toxoplasmosis.

Effect of household bleach on the structure of the sporocyst wall of Toxoplasma gondii.

Toxoplasma gondii oocysts are responsible for food- and water-borne infections in humans worldwide. They are resistant to common chemical disinfectants, including chlorinated products, presumably due to the structure and molecular nature of the oocyst wall but also the sporocyst wall. In this study, we used fluorescence microscopy and transmission electron microscopy to characterise the structure of both the oocyst and sporocyst walls, exposed to household bleach. Bleach removed the outer layer of the oocyst wall and the outer layer of the wall of sporocysts exposed due to rupture of the oocyst wall. The loss of the outer sporocyst wall layer was associated with a decrease in its autofluorescence, which can be linked to the degradation of dityrosine cross-link proteins, and loss of Maclura pomifera lectin-reactive glycoproteins. This study suggests that the inner layers of the oocyst and sporocyst walls are the main structures responsible for the resistance of the parasite to household bleach.

TITLE: Effet de l'eau de Javel à usage domestique sur la structure de la paroi du sporocyste de Toxoplasma gondii. ABSTRACT: Les oocystes de Toxoplasma gondii sont responsables chez l'homme d'infections cosmopolites d'origine alimentaire et hydrique. Ils sont résistants aux désinfectants chimiques usuels, notamment aux produits chlorés, vraisemblablement en raison de la structure et de la nature moléculaire de la paroi de l'oocyste mais aussi de celle du sporocyste. Dans cette étude, nous avons utilisé la microscopie à fluorescence et la microscopie électronique à transmission pour caractériser la structure de la paroi des oocystes et des sporocystes exposés à l'eau de Javel à usage domestique. L'eau de Javel élimine la couche externe de la paroi de l'oocyste et la couche externe de la paroi des sporocystes exposés en raison de la rupture de la paroi de l'oocyste. La perte de la couche externe de la paroi du sporocyste est associée à une diminution de son autofluorescence, qui peut être liée à la dégradation de polymères protéiques de dityrosine, et à une perte des glycoprotéines réactives à la lectine Maclura pomifera. Cette étude suggère que les couches internes des parois de l'oocyste et du sporocyste sont les principales structures responsables de la résistance du parasite à l'eau de Javel à usage domestique.

Lactate dehydrogenase and malate dehydrogenase: Potential antiparasitic targets for drug development studies.

Parasitic diseases remain a major public health concern for humans, claiming millions of lives annually. Although different treatments are required for these diseases, drug usage is limited due to the development of resistance and toxicity, which necessitate alternative therapies. It has been shown in the literature that parasitic lactate dehydrogenases (LDH) and malate dehydrogenases (MDH) have unique pharmacological selective and specificity properties compared to other isoforms, thus highlighting them as viable therapeutic targets involved in aerobic and anaerobic glycolytic pathways. LDH and MDH are important therapeutic targets for invasive parasites because they play a critical role in the progression and development of parasitic diseases. Any strategy to impede these enzymes would be fatal to the parasites, paving the way to develop and discover novel antiparasitic agents. This review aims to highlight the importance of parasitic LDH and MDH as therapeutic drug targets in selected obligate apicoplast parasites. To the best of our knowledge, this review presents the first comprehensive review of LDH and MDH as potential antiparasitic targets for drug development studies.

Natural and Synthetic Naphthoquinones as Potential Anti-Infective Agents.

BACKGROUND: Naphthoquinones are a class of aromatic compounds relevant for their chemical characteristics, structural properties, and biological activity. These compounds are found in nature with a wide range of effects, highlighting their antibacterial, antifungal, and antiprotozoal properties. Additionally, naphthoquinones are used as a scaffold to obtain new derivatives with pharmacological potential, mainly compounds against parasitic diseases. OBJECTIVE: The purpose of this work was to carry out a comprehensive review of naphthoquinones and their derivatives obtained from both natural and synthetic sources, also, to analyze their biological activity against Leishmania spp. (Leishmaniasis), Trypanosoma cruzi (Chagas disease), Plasmodium falciparum (Malaria), Toxoplasma gondii (Toxoplasmosis), and Toxocara canis (Toxocariasis). All of these agents are responsible for relevant diseases worldwide. RESULTS: Natural naphthoquinones, such as plumbagin, diospyrin, burmanin, lapachol, lawsone and psychorubrin, show an antiprotozoal activity similar or enhanced antiprotozoal activity to reference drugs. Some naphthoquinones obtained by synthesis or semi-synthesis showed better biological activity or less toxic effects than natural compounds. CONCLUSION: In this review, natural and synthetic naphthoquinones showed antiparasitic activity, in most cases, with improved results than current drugs currently used in clinical trials. A modification of their structure with different functional groups can enhance their biological effects, improve solubility, and reduce undesirable side effects. Therefore, naphthoquinones are important molecules in the development of new chemotherapeutic agents against parasitic diseases.

In situ ultrastructures of two evolutionarily distant apicomplexan rhoptry secretion systems.

Parasites of the phylum Apicomplexa cause important diseases including malaria, cryptosporidiosis and toxoplasmosis. These intracellular pathogens inject the contents of an essential organelle, the rhoptry, into host cells to facilitate invasion and infection. However, the structure and mechanism of this eukaryotic secretion system remain elusive. Here, using cryo-electron tomography and subtomogram averaging, we report the conserved architecture of the rhoptry secretion system in the invasive stages of two evolutionarily distant apicomplexans, Cryptosporidium parvum and Toxoplasma gondii. In both species, we identify helical filaments, which appear to shape and compartmentalize the rhoptries, and an apical vesicle (AV), which facilitates docking of the rhoptry tip at the parasite's apical region with the help of an elaborate ultrastructure named the rhoptry secretory apparatus (RSA); the RSA anchors the AV at the parasite plasma membrane. Depletion of T. gondii Nd9, a protein required for rhoptry secretion, disrupts the RSA ultrastructure and AV-anchoring. Moreover, T. gondii contains a line of AV-like vesicles, which interact with a pair of microtubules and accumulate towards the AV, leading to a working model for AV-reloading and discharging of multiple rhoptries. Together, our analyses provide an ultrastructural framework to understand how these important parasites deliver effectors into host cells.

Evaluation of Mongolian compound library for potential antimalarial and anti-Toxoplasma agents.

179 compounds in a Mongolian compound library were investigated for their inhibitory effect on the in vitro growth of Plasmodium falciparum and Toxoplasma gondii. Among these compounds, brachangobinan A at a half-maximal inhibition concentration (IC50) of 2.62 µM and a selectivity index (SI) of 27.91; 2-(2'-hydroxy-5'-O-methylphenyl)-5-(2″,5″-dihydroxyphenyl)oxazole (IC50 3.58 µM and SI 24.66); chrysosplenetin (IC50 3.78 µM and SI 15.26); 4,11-di-O-galloylbergenin (IC50 3.87 µM and SI 13.38); and 2-(2',5'-dihydroxyphenyl)-5-(2″-hydroxyphenyl)oxazole (IC50 6.94 µM and SI 11.48) were identified as potential inhibitors of P. falciparum multiplication. Additionally, tricin (IC50 12.94 µM and SI > 23.40) was identified as a potential inhibitor of T. gondii multiplication. Our findings represent a good starting point for developing novel antimalarial and anti-Toxoplasma therapeutics from Mongolian compounds.

A Novel Calcium-Dependent Protein Kinase 1 Inhibitor Potently Prevents Toxoplasma gondii Transmission to Foetuses in Mouse.

Treatments currently used to prevent congenital toxoplasmosis are non-specific of Toxoplasma gondii and have grievous side effects. To develop a more specific and less toxic drug, we have designed SP230, an imidazo[1,2-b]pyridazine salt targeting the Toxoplasma gondii calcium-dependent protein kinase 1 (TgCDPK1) and active against acute toxoplasmosis in mice. Efficiency of SP230 to inhibit foetal transmission of the parasite was evaluated in a mouse model of congenital toxoplasmosis. Swiss mice were infected at mid-pregnancy with tachyzoites or cysts of the ME49 strain of T. gondii by intraperitoneal and oral route, respectively, and treated with SP230 at 50 mg/kg for 5 days by the same routes. Parasite burden in organs of dams and in foetuses was measured by quantitative PCR. Intraperitoneal administration of SP230 drastically reduced the number of parasites (more than 97% of reduction) in the brain and lungs of dams, and led to a reduction of 66% of parasite burden in foetuses. Oral administration of SP230 was particularly efficient with 97% of reduction of parasite burdens in foetuses. SP230 did not impact number and weight of offspring in our conditions. This inhibitor of TgCDPK1 is a promising candidate for the development of alternative therapeutics to treat infected pregnant women.

An Open-Source Toolkit To Expand Bioinformatics Training in Infectious Diseases.

As access to high-throughput sequencing technology has increased, the bottleneck in biomedical research has shifted from data generation to data analysis. Here, we describe a modular and extensible framework for didactic instruction in bioinformatics using publicly available RNA sequencing data sets from infectious disease studies, with a focus on host-parasite interactions. We highlight lessons learned from adapting this course for virtual learners during the coronavirus disease 2019 (COVID-19) pandemic.

Activity of Fluorinated Curcuminoids against Leishmania major and Toxoplasma gondii Parasites.

A new 3,4-difluorobenzylidene analog of curcumin, CDF, was recently reported, which demonstrated significantly enhanced bioavailability and in vivo anticancer activity compared with curcumin. For highlighting the antiparasitic behavior of CDF, we tested this compound together with its new O-methylated analog MeCDF against Leishmania major and Toxoplasma gondii parasites. Both CDF and MeCDF were tested in vitro against L. major and T. gondii. In addition, the in vitro cytotoxicity against Vero cells and macrophages was determined and selectivity indices were calculated. The DPPH radical scavenging activity assay was carried out in order to determine the antioxidant activity of the test compounds. Both compounds showed high activities against both parasite forms with EC50 values in the (sub-)micromolar range (0.35 to 0.8 µM for CDF, 0.31 to 1.2 µM for MeCDF). The higher activity of CDF against L. major amastigotes when compared with MeCDF can in parts be attributed to the antioxidant activity of CDF while MeCDF lacking any antioxidant activity was more active than CDF against T. gondii parasites. In conclusion, CDF and MeCDF are promising antiparasitic drug candidates due to their high activities against L. major and T. gondii parasites.

4-Arylthiosemicarbazide derivatives as a new class of tyrosinase inhibitors and anti-Toxoplasma gondii agents.

We report herein anti-proliferation effects of 4-arylthiosemicarbazides, with a cyclopentane substitution at N1 position, on highly virulent RH strain of Toxoplasma gondii. Among them, the highest in vitro anti-Toxoplasma activity was found with the meta-iodo derivative. Further experiments demonstrated inhibitory effects of thiosemicarbazides on tyrosinase (Tyr) activity, and good correlation was found between percentage of Tyr inhibition and IC50Tg. To confirm the concept that thiosemicarbazides are able to disrupt tyrosine metabolism in Toxoplasma tachyzoites, the most potent Tyr inhibitors were tested for their efficacy of T. gondii growth inhibition. All of them significantly reduced the number of tachyzoites in the parasitophorous vacuoles (PVs) compared to untreated cells, as well as inhibited tachyzoites growth by impeding cell division. Collectively, these results indicate that compounds with the thiosemicarbazide scaffold are able to disrupt tyrosine metabolism in Toxoplasma tachyzoites by deregulation of their crucial enzyme tyrosine hydroxylase (TyrH).

p-Trifluoromethyl- and p-pentafluorothio-substituted curcuminoids of the 2,6-di[(E)-benzylidene)]cycloalkanone type: Syntheses and activities against Leishmania major and Toxoplasma gondii parasites.

A series of the title curcuminoids with structural variance in the heteroatom of the cycloalkanone and the p-substituents of the phenyl rings were tested for their activities against Leishmania major and Toxoplasma gondii parasites. The majority of them showed high activities against both parasite forms with EC50 values in the sub-micromolar concentration range. Bis(p-pentafluorothio)-substituted 3,5-di[(E)-benzylidene]piperidin-4-one 1b was not just noticeable antiparasitic, but also exhibited a considerable selectivity for L. major promastigotes over normal Vero cells. While derivatives differing only in the p-phenyl substituents being CF3 or SF5 showed similar antiparasitic activities, the cyclic ketone hub was more decisive both for the anti-parasitic activities and the selectivities for the parasites vs. normal cells. QSAR calculations confirmed the observed structure-activity relations and suggested structural variations for a further improvement of the antiparasitic activity. Docking studies based on DFT calculations revealed L. major pteridine reductase 1 as a likely molecular target protein of the title compounds.

The quest of the best - A SAR study of trithiolato-bridged dinuclear Ruthenium(II)-Arene compounds presenting antiparasitic properties.

A structure activity relationship (SAR) study of a library of 56 compounds (54 ruthenium and 2 osmium derivatives) based on the trithiolato-bridged dinuclear ruthenium(II)-arene scaffold (general formula [(η6-arene)2Ru2(µ2-SR)3]+, symmetric and [(η6-arene)2Ru2(µ2-SR1)2(µ2-SR2)]+, mixed, respectively) is reported. The 56 compounds (of which 34 are newly designed drug candidates) were synthesized by introducing chemical modifications at the level of bridge thiols, and they were grouped into eight families according to their structural features. The selected fittings were guided by previous results and focused on a fine-tuning of the physico-chemical and steric properties. Newly synthesized complexes were characterized by NMR spectroscopy, mass spectrometry and elemental analysis, and four single-crystal X-ray structures were obtained. The in vitro biological assessment of the compounds was realized by applying a three-step screening cascade: (i) evaluation of the activity against Toxoplasma gondii RH strain tachyzoites expressing ß-galactosidase (T. gondii-ß-gal) grown in human foreskin fibroblast monolayers (HFF) and assessment of toxicity in non-infected HFF host cells; (ii) dose-response assays using selected compound, and (iii) studies on the effects in murine splenocytes. A primary screening was performed at 1 and 0.1 µM, and resulted in the selection of 39 compounds that inhibited parasite proliferation at 1 µM by more than 95% and reduced the viability of HFF by less than 49%. In the secondary screening, dose-response assays showed that the selected compounds exhibited half maximal inhibitory concentration (IC50) values for T. gondii-ß-gal between 0.01 µM and 0.45 µM, with 30 compounds displaying an IC50 lower than 0.1 µM. When applied to non-infected HFF monolayers at 2.5 µM, 8 compounds caused more than 90% and 31 compounds more than 30% viability impairment. The tertiary screening included 14 compounds that did not cause HFF viability loss higher than 50% at 2.5 µM. These derivatives were assessed for potential immunosuppressive activities. First, splenocyte viability was assessed after treatment of cells with concanavalin A (ConA) and lipopolysaccharide (LPS) with compounds applied at 0.1 and 0.5 µM. Subsequently, the 5 compounds exhibiting the lowest splenocyte toxicity were further evaluated for their potential to inhibit B and T cell proliferation. Overall, compound 55 [(η6-p-MeC6H4Pri)2Ru2(µ2-SC6H4-o-CF3)2(µ2-SC6H4-p-OH)]Cl exhibited the most favorable features, and will be investigated as a scaffold for further optimization in terms of anti-parasitic efficacy and drug-like properties.