Analysis of the Interactome of the Toxoplasma gondii Tgj1 HSP40 Chaperone.

Toxoplasma gondii is an obligate intracellular apicomplexan that causes toxoplasmosis in humans and animals. Central to its dissemination and pathogenicity is the ability to rapidly divide in the tachyzoite stage and infect any type of nucleated cell. Adaptation to different cell contexts requires high plasticity in which heat shock proteins (Hsps) could play a fundamental role. Tgj1 is a type I Hsp40 of T. gondii, an ortholog of the DNAJA1 group, which is essential during the tachyzoite lytic cycle. Tgj1 consists of a J-domain, ZFD, and DNAJ_C domains with a CRQQ C-terminal motif, which is usually prone to lipidation. Tgj1 presented a mostly cytosolic subcellular localization overlapping partially with endoplasmic reticulum. Protein-protein Interaction (PPI) analysis showed that Tgj1 could be implicated in various biological pathways, mainly translation, protein folding, energy metabolism, membrane transport and protein translocation, invasion/pathogenesis, cell signaling, chromatin and transcription regulation, and cell redox homeostasis among others. The combination of Tgj1 and Hsp90 PPIs retrieved only 70 interactors linked to the Tgj1-Hsp90 axis, suggesting that Tgj1 would present specific functions in addition to those of the Hsp70/Hsp90 cycle, standing out invasion/pathogenesis, cell shape motility, and energy pathway. Within the Hsp70/Hsp90 cycle, translation-associated pathways, cell redox homeostasis, and protein folding were highly enriched in the Tgj1-Hsp90 axis. In conclusion, Tgj1 would interact with a wide range of proteins from different biological pathways, which could suggest a relevant role in them.

Synthesis and biological evaluation of 1-alkylaminomethyl-1,1-bisphosphonic acids against Trypanosoma cruzi and Toxoplasma gondii.

As an extension of our project aimed at the search for new chemotherapeutic agents against Chagas disease and toxoplasmosis, several 1,1-bisphosphonates were designed, synthesized and biologically evaluated against Trypanosoma cruzi and Toxoplasma gondii, the etiologic agents of these diseases, respectively. In particular, and based on the antiparasitic activity exhibited by 2-alkylaminoethyl-1,1-bisphosphonates targeting farnesyl diphosphate synthase, a series of linear 2-alkylaminomethyl-1,1-bisphosphonic acids (compounds 21-33), that is, the position of the amino group was one carbon closer to the gem-phosphonate moiety, were evaluated as growth inhibitors against the clinically more relevant dividing form (amastigotes) of T. cruzi. Although all of these compounds resulted to be devoid of antiparasitic activity, these results were valuable for a rigorous SAR study. In addition, unexpectedly, the synthetic designed 2-cycloalkylaminoethyl-1,1-bisphosphonic acids 47-49 were free of antiparasitic activity. Moreover, long chain sulfur-containing 1,1-bisphosphonic acids, such as compounds 54-56, 59, turned out to be nanomolar growth inhibitors of tachyzoites of T. gondii. As many bisphosphonate-containing molecules are FDA-approved drugs for the treatment of bone resorption disorders, their potential nontoxicity makes them good candidates to control American trypanosomiasis and toxoplasmosis.

Selenium-containing analogues of WC-9 are extremely potent inhibitors of Trypanosoma cruzi proliferation.

The obligate intracellular parasite, Trypanosoma cruzi is the etiologic agent of Chagas disease or American trypanosomiasis, which is the most prevalent parasitic disease in the Americas. The present chemotherapy to control this illness is still deficient particularly in the chronic stage of the disease. The ergosterol biosynthesis pathway has received much attention as a molecular target for the development of new drugs for Chagas disease. Especially, inhibitors of the enzymatic activity of squalene synthase were shown to be effective compounds on T. cruzi proliferation in in vitro assays. In the present study we designed, synthesized and evaluated the effect of a number of isosteric analogues of WC-9 (4-phenoxyphenoxyethyl thiocyanate), a known squalene synthase inhibitor, on T. cruzi growth in tissue culture cells. The selenium-containing derivatives turned out to be extremely potent inhibitors of T. cruzi growth. Certainly, 3-phenoxyphenoxyethyl, 4-phenoxyphenoxyethyl, 4-(3-fluorophenoxy)phenoxyethyl, 3-(3-fluorophenoxy)phenoxyethyl selenocyanates and (±)-5-phenoxy-2-(selenocyanatomethyl)-2,3-dihydrobenzofuran arose as relevant members of this family of compounds, which exhibited effective ED50 values of 0.084 µM, 0.11 µM, 0.083, µM, 0.085, and 0.075 µM, respectively. The results indicate that compounds bearing the selenocyanate moiety are at least two orders of magnitude more potent than the corresponding skeleton counterpart bearing the thiocyanate group. Surprisingly, these compounds exhibited excellent selectively index values ranging from 900 to 1800 making these molecules promising candidates as antiparasitic agents.

Antiparasitic Activity of Sulfur- and Fluorine-Containing Bisphosphonates against Trypanosomatids and Apicomplexan Parasites.

Based on crystallographic data of the complexes 2-alkyl(amino)ethyl-1,1-bisphosphonates-Trypanosoma cruzi farnesyl diphosphate synthase, some linear 1,1-bisphosphonic acids and other closely related derivatives were designed, synthesized and biologically evaluated against T. cruzi, the responsible agent of Chagas disease and against Toxoplasma gondii, the etiologic agent of toxoplasmosis and also towards the target enzymes farnesyl pyrophosphate synthase of T. cruzi (TcFPPS) and T gondii (TgFPPS), respectively. The isoprenoid-containing 1,1-bisphosphonates exhibited modest antiparasitic activity, whereas the linear α-fluoro-2-alkyl(amino)ethyl-1,1-bisphosphonates were unexpectedly devoid of antiparasitic activity. In spite of not presenting efficient antiparasitic activity, these data turned out to be very important to establish a structural activity relationship.

In Vitro and In Vivo Activities of Sulfur-Containing Linear Bisphosphonates against Apicomplexan Parasites.

We tested a series of sulfur-containing linear bisphosphonates against Toxoplasma gondii, the etiologic agent of toxoplasmosis. The most potent compound (compound 22; 1-[(n-decylsulfonyl)ethyl]-1,1-bisphosphonic acid) is a sulfone-containing compound, which had a 50% effective concentration (EC50) of 0.11 ± 0.02 µM against intracellular tachyzoites. The compound showed low toxicity when tested in tissue culture with a selectivity index of >2,000. Compound 22 also showed high activity in vivo in a toxoplasmosis mouse model. The compound inhibited the Toxoplasma farnesyl diphosphate synthase (TgFPPS), but the concentration needed to inhibit 50% of the enzymatic activity (IC50) was higher than the concentration that inhibited 50% of growth. We tested compound 22 against two other apicomplexan parasites, Plasmodium falciparum (EC50 of 0.6 ± 0.01 µM), the agent of malaria, and Cryptosporidium parvum (EC50 of ∼65 µM), the agent of cryptosporidiosis. Our results suggest that compound 22 is an excellent novel compound that could lead to the development of potent agents against apicomplexan parasites.

Activity of Fluorine-Containing Analogues of WC-9 and Structurally Related Analogues against Two Intracellular Parasites: Trypanosoma cruzi and Toxoplasma gondii.

Two obligate intracellular parasites, Trypanosoma cruzi, the agent of Chagas disease, and Toxoplasma gondii, an agent of toxoplasmosis, upregulate the mevalonate pathway of their host cells upon infection, which suggests that this host pathway could be a potential drug target. In this work, a number of compounds structurally related to WC-9 (4-phenoxyphenoxyethyl thiocyanate), a known squalene synthase inhibitor, were designed, synthesized, and evaluated for their effect on T. cruzi and T. gondii growth in tissue culture cells. Two fluorine-containing derivatives, the 3-(3-fluorophenoxy)- and 3-(4-fluorophenoxy)phenoxyethyl thiocyanates, exhibited half-maximal effective concentration (EC50 ) values of 1.6 and 4.9 µm, respectively, against tachyzoites of T. gondii, whereas they showed similar potency to WC-9 against intracellular T. cruzi (EC50 values of 5.4 and 5.7 µm, respectively). In addition, 2-[3- (phenoxy)phenoxyethylthio]ethyl-1,1-bisphosphonate, which is a hybrid inhibitor containing 3-phenoxyphenoxy and bisphosphonate groups, has activity against T. gondii proliferation at sub-micromolar levels (EC50 =0.7 µm), which suggests a combined inhibitory effect of the two functional groups.

New insights into molecular recognition of 1,1-bisphosphonic acids by farnesyl diphosphate synthase.

As part of our project pointed at the search of new antiparasitic agents against American trypanosomiasis (Chagas disease) and toxoplasmosis a series of 2-alkylaminoethyl-1-hydroxy-1,1-bisphosphonic acids has been designed, synthesized and biologically evaluated against the etiologic agents of these parasitic diseases, Trypanosoma cruzi and Toxoplasma gondii, respectively, and also towards their target enzymes, T. cruzi and T. gondii farnesyl pyrophosphate synthase (FPPS), respectively. Surprisingly, while most pharmacologically active bisphosphonates have a hydroxyl group at the C-1 position, the additional presence of an amino group at C-3 resulted in decreased activity towards either T. cruzi cells or TcFPPS. Density functional theory calculations justify this unexpected behavior. Although these compounds were devoid of activity against T. cruzi cells and TcFPPS, they were efficient growth inhibitors of tachyzoites of T. gondii. This activity was associated with a potent inhibition of the enzymatic activity of TgFPPS. Compound 28 arises as a main example of this family of compounds exhibiting an ED50 value of 4.7 µM against tachyzoites of T. gondii and an IC50 of 0.051 µM against TgFPPS.

Design, synthesis and biological evaluation of WC-9 analogs as antiparasitic agents.

As a part of our project pointed at the search of new safe chemotherapeutic and chemoprophylactic agents against parasitic diseases, several compounds structurally related to 4-phenoxyphenoxyethyl thiocyanate (WC-9), which were modified at the terminal aromatic ring, were designed, synthesized and evaluated as antiproliferative agents against Trypanosoma cruzi, the parasite responsible of American trypanosomiasis (Chagas disease) and Toxoplasma gondii, the etiological agent of toxoplasmosis. Most of the synthetic analogs exhibited similar antiparasitic activity being slightly more potent than the reference compound WC-9. For example, the nitro derivative 13 showed an ED50 value of 5.2 µM. Interestingly, the regioisomer of WC-9, compound 36 showed similar inhibitory action than WC-9 indicating that para-phenyl substitution pattern is not necessarily required for biological activity. The biological evaluation against T. gondii was also very promising. The ED50 values corresponding for 13, 36 and 37 were at the very low micromolar level against tachyzoites of T. gondii.

Design, synthesis and biological evaluation of sulfur-containing 1,1-bisphosphonic acids as antiparasitic agents.

As part of our efforts aimed at searching for new antiparasitic agents, 2-alkylmercaptoethyl-1,1-bisphosphonate derivatives were synthesized and evaluated against Trypanosoma cruzi, the etiologic agent of Chagas disease, and Toxoplasma gondii, the responsible agent for toxoplasmosis. Many of these sulfur-containing bisphosphonates were potent inhibitors against the intracellular form of T. cruzi, the clinically more relevant replicative form of this parasite, and tachyzoites of T. gondii targeting T. cruzi or T. gondii farnesyl diphosphate synthases (FPPSs), which constitute valid targets for the chemotherapy of these parasitic diseases. Interestingly, long chain length sulfur-containing bisphosphonates emerged as relevant antiparasitic agents. Taking compounds 37, 38, and 39 as representative members of this class of drugs, they exhibited ED(50) values of 15.8 µM, 12.8 µM, and 22.4 µM, respectively, against amastigotes of T. cruzi. These cellular activities matched the inhibition of the enzymatic activity of the target enzyme (TcFPPS) having IC(50) values of 6.4 µM, 1.7 µM, and 0.097 µM, respectively. In addition, these compounds were potent anti-Toxoplasma agents. They had ED(50) values of 2.6 µM, 1.2 µM, and 1.8 µM, respectively, against T. gondii tachyzoites, while they exhibited a very potent inhibitory action against the target enzyme (TgFPPS) showing IC(50) values of 0.024 µM, 0.025 µM, and 0.021 µM, respectively. Bisphosphonates bearing a sulfoxide unit at C-3 were also potent anti-Toxoplasma agents, particularly those bearing long aliphatic chains such as 43-45, which were also potent antiproliferative drugs against tachyzoites of T. gondii. These compounds inhibited the enzymatic activity of the target enzyme (TgFPPS) at the very low nanomolar range. These bisphosphonic acids have very good prospective not only as lead drugs but also as potential chemotherapeutic agents.

1-(Fluoroalkylidene)-1,1-bisphosphonic acids are potent and selective inhibitors of the enzymatic activity of Toxoplasma gondii farnesyl pyrophosphate synthase.

α-Fluorinated-1,1-bisphosphonic acids derived from fatty acids were designed, synthesized and biologically evaluated against Trypanosoma cruzi, the etiologic agent of Chagas disease, and against Toxoplasma gondii, the agent responsible for toxoplasmosis, and also towards the target parasitic enzymes farnesyl pyrophosphate synthase of T. cruzi (TcFPPS) and T. gondii (TgFPPS). Interestingly, 1-fluorononylidene-1,1-bisphosphonic acid (compound 43) proved to be an extremely potent inhibitor of the enzymatic activity of TgFPPS at the low nanomolar range, exhibiting an IC(50) of 30 nM. This compound was two-fold more potent than risedronate (IC(50) = 74 nM) that was taken as a positive control. This enzymatic activity was associated with a strong cell growth inhibition against tachyzoites of T. gondii, with an IC(50) value of 2.7 µM.

Synthesis and biological evaluation of new 2-alkylaminoethyl-1,1-bisphosphonic acids against Trypanosoma cruzi and Toxoplasma gondii targeting farnesyl diphosphate synthase.

The effect of long-chain 2-alkylaminoethyl-1,1-bisphosphonates against proliferation of the clinically more relevant form of Trypanosoma cruzi, the etiologic agent of American trypanosomiasis (Chagas' disease), and against tachyzoites of Toxoplasma gondii was investigated. Particularly, compound 26 proved to be an extremely potent inhibitor against the intracellular form of T. cruzi, exhibiting IC(50) values at the nanomolar range. This cellular activity was associated with a strong inhibition of the enzymatic activity of T. cruzi farnesyl diphosphate synthase (TcFPPS), which constitutes a valid target for Chagas' disease chemotherapy. Compound 26 was an effective agent against T. cruzi (amastigotes) exhibiting an IC(50) value of 0.67 µM, while this compound showed an IC(50) value of 0.81 µM against the target enzyme TcFPPS. This drug was less effective against the enzymatic activity of T. cruzi solanesyl diphosphate synthase TcSPPS showing an IC(50) value of 3.2 µM. Interestingly, compound 26 was also very effective against T. gondii (tachyzoites) exhibiting IC(50) values of 6.23 µM. This cellular activity was also related to the inhibition of the enzymatic activity towards the target enzyme TgFPPS (IC(50)=0.093 µM) As bisphosphonate-containing compounds are FDA-approved drugs for the treatment of bone resorption disorders, their potential low toxicity makes them good candidates to control different tropical diseases.

Synthesis and biological evaluation of 2-alkylaminoethyl-1,1-bisphosphonic acids against Trypanosoma cruzi and Toxoplasma gondii targeting farnesyl diphosphate synthase.

The effect of a series of 2-alkylaminoethyl-1,1-bisphosphonic acids against proliferation of the clinically more relevant form of Trypanosoma cruzi, the etiologic agent of American trypanosomiasis (Chagas' disease), and against tachyzoites of Toxoplasma gondii has been studied. Most of these drugs exhibited an extremely potent inhibitory action against the intracellular form of T. cruzi, exhibiting IC(50) values at the low micromolar level. This cellular activity was associated with a strong inhibition of the enzymatic activity of T. cruzi farnesyl diphosphate synthase (TcFPPS), which constitutes a valid target for Chagas' disease chemotherapy. Compound 17 was an effective agent against amastigotes exhibiting an IC(50) value of 0.84 microM, while this compound showed an IC(50) value of 0.49 microM against the target enzyme TcFPPS. Interestingly, compound 19 was very effective against both T. cruzi and T. gondii exhibiting IC(50) values of 4.1 microM and 2.6 microM, respectively. In this case, 19 inhibited at least two different enzymes of T. cruzi (TcFPPS and solanesyl diphosphate synthase (TcSPPS); 1.01 microM and 0.25 microM, respectively), while it inhibited TgFPPS in T. gondii. In general, this family of drugs was less effective against the activity of T. cruzi SPPS and against T. gondii growth in vitro. As bisphosphonate-containing compounds are FDA-approved drugs for the treatment of bone resorption disorders, their potential low toxicity makes them good candidates to control tropical diseases.

Fluorine-containing aryloxyethyl thiocyanate derivatives are potent inhibitors of Trypanosoma cruzi and Toxoplasma gondii proliferation.

As a part of our project aimed at developing new safe chemotherapeutic and chemoprophylactic agents against tropical diseases, fluorine-containing drugs structurally related to 4-phenoxyphenoxyethyl thiocyanate (1) were designed, synthesized, and evaluated as antiproliferative agents against Trypanosoma cruzi, the parasite responsible of American trypanosomiasis (Chagas' disease), and Toxoplasma gondii, the etiological agent of toxoplasmosis. This thiocyanate derivative had previously proven to be an effective agent against T. cruzi proliferation. Fluorine-containing thiocyanate derivatives 2 and 3 were threefold more potent than our lead drug 1 against intracellular T. cruzi. The biological evaluation against T. gondii was also very promising. The IC(50) values corresponding to 2 and 3 were at the very low micromolar level against tachyzoites of T. gondii. Both of these drugs are interesting examples of effective antiparasitic agents that have outstanding potential not only as lead drugs but also to be used for further in vivo studies.