Molecular basis of Toxoplasma gondii oryzalin resistance from a novel α-tubulin binding site model.

Oryzalin (ORY) is a dinitroaniline derivative that inhibits the microtubule polymerization in plants and parasitic protozoa by selectively binding to the α-tubulin subunit. This herbicidal agent exhibits good antiprotozoal activity against major human parasites, such as Toxoplasma gondii (toxoplasmosis), Leishmania mexicana (leishmaniasis), and Plasmodium falciparum (malaria). Previous chemical mutagenesis assays on T. gondii α-tubulin (TgAT) have identified key mutations that lead to ORY resistance. Herein, we employed alchemical free energy methods and molecular dynamics simulations to determine if the ORY resistance mutations either decrease the TgAT's affinity of the compound or increase the protein stability. Our results here suggest that L136F and V202F mutations significantly decrease the affinity of ORY to TgAT, while T239I and V252L mutations diminish TgAT's flexibility. On the other hand, protein stability predictors determined that R243S mutation reduces TgAT stability due to the loss of its salt bridge interaction with E27. Interestingly, molecular dynamics simulations confirm that the loss of this key interaction leads to ORY binding site closure. Our study provides a better insight into the TgAT-ORY interaction, further supporting our recently proposed ORY-binding site.

Inhibition of C. albicans Dimorphic Switch by Cobalt(II) Complexes with Ligands Derived from Pyrazoles and Dinitrobenzoate: Synthesis, Characterization and Biological Activity.

Seven cobalt(II) complexes of pyrazole derivatives and dinitrobenzoate ligands were synthesized and characterized. The single-crystal X-ray diffraction structure was determined for one of the ligands and one of the complexes. The analysis and spectral data showed that all the cobalt complexes had octahedral geometries, which was supported by DFT calculations. The complexes and their free ligands were evaluated against fungal strains of Candida albicans and emerging non-albicans species and epimastigotes of Trypanosoma cruzi. We obtained antifungal activity with a minimum inhibitory concentration (MIC) ranging from 31.3 to 250 µg mL-1. The complexes were more active against C. krusei, showing MIC values between 31.25 and 62.5 µg mL-1. In addition, some ligands (L1-L6) and complexes (5 and Co(OAc)2 · 4H2O) significantly reduced the yeast to hypha transition of C. albicans at 500 µg mL-1 (inhibition ranging from 30 to 54%). Finally, the complexes and ligands did not present trypanocidal activity and were not toxic to Vero cells. Our results suggest that complexes of cobalt(II) with ligands derived from pyrazoles and dinitrobenzoate may be an attractive alternative for the treatment of diseases caused by fungi, especially because they target one of the most important virulence factors of C. albicans.

Inducing autotetraploids in cassava using oryzalin and colchicine and their in vitro morphophysiological effects.

Polyploid induction has been used for plant breeding to produce bigger and more robust plants than diploid types. The present study aimed to develop a methodology for in vitro induction of polyploidy in cassava. Apical and lateral microcuttings from the BRS Formosa variety were treated with six oryzalin concentrations for 24 and 48 h. The same methodology was used for colchicine with different concentrations. After 45 days of cultivation and an additional 45 days of subculture, the viability of the explants was assessed and plant acclimatization was performed. Ploidy was determined using flow cytometry. Oryzalin dose and exposure negatively affected cassava explant growth and development compared to untreated explants. Furthermore, apical and lateral explants responded differently to the treatments, showing a diversity in antimitotic sensitivity and effect that is tissue-type specific. In contrast, the doses of 1.25 to 6.25 mM colchicine resulted in high mortality of cassava explants. Therefore, the type of antimitotic affects the morphophysiological behavior of cassava plants in vitro, although apical explants have higher viability and regenerative capacity compared to lateral explants. In addition, the lateral explants have lower mixoploid rates compared to apical explants. Of the 310 plants generated by oryzalin treatments, 277 were diploid, 31 were mixoploid, and 2 were tetraploid. Exposure to oryzalin led to low rate of tetraploids and colchicine caused phytotoxic reactions and death of the explants. The tetraploids were multiplied in vitro to evaluate their yield in the field as well as their behavior against abiotic and biotic stress.

Anthelmintic effect of herbicidal dinitroanilines on the nematode model Caenorhabditis elegans.

Dinitroanilines are known herbicides that impair the polymerization of microtubules. This study investigated the effects of oryzalin and trifluralin on the viability, morphology, and ultrastructure of different life stages of Caenorhabditis elegans. Both drugs reduced the survival of the adult population in 50% after three days of treatment with concentrations of approximately 30 µM and 57 µM, respectively. The development of new adults was monitored for seven days and treatment with both drugs also showed a decrease in the adult population. 25 µM Oryzalin or trifluralin inhibited the hatching of eggs by nearly 100%. Both drugs showed remarkable larvicidal activity at 25 µM against the larvae at first and second stages (L1-L2) and at third and fourth stages (L3-L4) after 24 h. Treatment with dinitroanilines led to incomplete egg embryo development. The oryzalin and trifluralin treatments caused the detachment of cuticular layers of adults and larvae and the formation of a large number of intracellular membrane whirls and lipid bodies in the hypodermal cells and non-contractile muscles of adults. Both drugs also provoked the bagging process, which generated lesions in the uterus of the adults. In addition, trifluralin caused the detachment of certain areas of the cuticle adjacent to the hypodermis in a large number of nematodes. Our results suggest that dinitroanilines are a potentially new alternative for anthelmintic chemotherapy.

Heterogeneity in the sensitivity of microtubules of Giardia lamblia to the herbicide oryzalin.

Giardia lamblia is a protozoan that inhabits the small intestine of vertebrates, attaching to the epithelial cells by means of cytoskeletal elements. G. lamblia trophozoites possess several microtubular structures, namely the adhesive disc, the median body, the funis and the four pairs of flagella. Several drugs that target cytoskeletal proteins have been used in the study of cytoskeletal function and dynamics. In this work, we used oryzalin, which binds to alpha-tubulin, as a tool to study the Giardia cytoskeleton. The trophozoites were treated with oryzalin, and its effects were analysed by immunofluorescency, transmission and scanning electron microscopies. Oryzalin inhibited Giardia proliferation. Treated cells were not able to complete cell division and had flagella showing extensive shortening. Strikingly, the drug did not interfere with the adhesive disc, in contrast to what happens when other drugs are used.

Effect of the anti-microtubule drug oryzalin on growth and differentiation of the parasitic protozoan Leishmania mexicana.

The parasitic protozoan Leishmania mexicana differentiates from a non-motile intracellular amastigote in the mammalian macrophage phagolysosome into a motile, extracellular promastigote in the insect vector gut. This developmental program has been accomplished in vitro, thus providing a useful model for studying changes in the cytoskeleton during cell differentiation. The role of microtubules in leishmania differentiation was demonstrated by using the dinitroaniline herbicide oryzalin, which inhibited both leishmania proliferation and differentiation; 25 microM oryzalin reduced promastigote division by over 95%. Interestingly, at a sublethal dose (5 microM), promastigotes became round and multiflagellated but remained motile. At 50 microM oryzalin, the number of intracellular amastigotes decreased by 50%. However, leishmania differentiation seemed to be the most drug-sensitive stage: there was a 60% reduction in amastigote-to-promastigote differentiation at 0.5 microM oryzalin. The specific action of oryzalin on leishmania microtubules was verified by its inhibition of in vitro polymerization of leishmania microtubules, but not control mammalian microtubules (from rat brain). These findings indicate that microtubules play a major role in leishmania proliferation, maintenance of cell shape, and cytodifferentiation.