Design, synthesis and biological evaluation of antiparasitic dinitroaniline-ether phospholipid hybrids.

A series of nine novel ether phospholipid-dinitroaniline hybrids were synthesized in an effort to deliver more potent antiparasitic agents with improved safety profile compared to miltefosine. The compounds were evaluated for their in vitro antiparasitic activity against L. infantum, L.donovani, L. amazonensis, L. major and L. tropica promastigotes, L. infantum and L. donovani intracellular amastigotes, Trypanosoma brucei brucei and against different developmental stages of Trypanosoma cruzi. The nature of the oligomethylene spacer between the dinitroaniline moiety and the phosphate group, the length of the side chain substituent on the dinitroaniline and the choline or homocholine head group were found to affect both the activity and toxicity of the hybrids. The early ADMET profile of the derivatives did not reveal major liabilities. Hybrid 3, bearing an 11-carbon oligomethylene spacer, a butyl side chain and a choline head group, was the most potent analogue of the series. It exhibited a broad spectrum antiparasitic profile against the promastigotes of New and Old World Leishmania spp., against intracellular amastigotes of two L. infantum strains and L. donovani, against T. brucei and against T. cruzi Y strain epimastigotes, intracellular amastigotes and trypomastigotes. The early toxicity studies revealed that hybrid 3 showed a safe toxicological profile while its cytotoxicity concentration (CC50) against THP-1 macrophages being >100 µM. Computational analysis of binding sites and docking indicated that the interaction of hybrid 3 with trypanosomatid α-tubulin may contribute to its mechanism of action. Furthermore, compound 3 was found to interfere with the cell cycle in T. cruzi epimastigotes, while ultrastructural studies using SEM and TEM in T. cruzi showed that compound 3 affects cellular processes that result in changes in the Golgi complex, the mitochondria and the parasite's plasma membrane. The snapshot pharmacokinetic studies showed low levels of 3 after 24 h following oral administration of 100 mg/Kg, while, its homocholine congener compound 9 presented a better pharmacokinetic profile.

Effects of the natural antimicrobial peptide aureocin A53 on cells of Staphylococcus aureus and Streptococcus agalactiae involved in bovine mastitis in the excised teat model.

Aureocin A53 is an N-formylated antimicrobial peptide (AMP) produced by Staphylococcus aureus. Aureocin A53 has a broad spectrum of antimicrobial activity against human and animal pathogens. In the present study, its antagonistic activity was investigated towards 30 strains of S. aureus and 30 strains of Streptococcus spp. isolated from bovine mastitis cases in Brazil. Bovine mastitis is a disease that causes a major economic impact worldwide. Aureocin A53 inhibited the growth of all 60 strains tested, including multidrug-resistant streptococcal isolates and strains of S. aureus belonging to different pulsotypes. This AMP proved to be bactericidal against the six target strains randomly selected among staphylococci and streptococci, also exhibiting a lytic mode of action against the staphylococcal cells. Furthermore, it was determined that 2,048 AU/mL of the AMP were required to inhibit 99.99% of the cell growth of the strain less sensitive to aureocin A53. Aureocin A53 was not toxic to bovine mammary gland epithelial cells after a 24-h exposure and maintained its antimicrobial activity when tested in the excised-teat model against strains of S. aureus and Streptococcus agalactiae, the species responsible for most intramammary infections, not only in Brazil but in other countries as well. Therefore, the use of aureocin A53 in the development of new pharmacological products for the prophylaxis and/or treatment of bovine mastitis was considered promising.

Design, Synthesis and Antiparasitic Evaluation of Click Phospholipids.

A library of seventeen novel ether phospholipid analogues, containing 5-membered heterocyclic rings (1,2,3-triazolyl, isoxazolyl, 1,3,4-oxadiazolyl and 1,2,4-oxadiazolyl) in the lipid portion were designed and synthesized aiming to identify optimised miltefosine analogues. The compounds were evaluated for their in vitro antiparasitic activity against Leishmania infantum and Leishmania donovani intracellular amastigotes, against Trypanosoma brucei brucei and against different developmental stages of Trypanosoma cruzi. The nature of the substituents of the heterocyclic ring (tail) and the oligomethylene spacer between the head group and the heterocyclic ring was found to affect the activity and toxicity of these compounds leading to a significantly improved understanding of their structure-activity relationships. The early ADMET profile of the new derivatives did not reveal major liabilities for the potent compounds. The 1,2,3-triazole derivative 27 substituted by a decyl tail, an undecyl spacer and a choline head group exhibited broad spectrum antiparasitic activity. It possessed low micromolar activity against the intracellular amastigotes of two L. infantum strains and T. cruzi Y strain epimastigotes, intracellular amastigotes and trypomastigotes, while its cytotoxicity concentration (CC50) against THP-1 macrophages ranged between 50 and 100 µM. Altogether, our work paves the way for the development of improved ether phospholipid derivatives to control neglected tropical diseases.

In vitro activities of adamantylidene-substituted alkylphosphocholine TCAN26 against Trypanosoma cruzi: Antiproliferative and ultrastructural effects.

Phospholipids are the main component of membranes and are responsible for cell integrity. Alkylphospholipid analogues (APs) were first designed as antitumoral agents and were later tested against different cell types. Trypanosoma cruzi, the Chagas disease etiological agent, is sensitive to APs (edelfosine, miltefosine and ilmofosine) in vitro. We investigated the effect of synthetic ring substituted AP against epimastigotes, amastigotes and trypomastigotes. TCAN26, could inhibit the in vitro growth of epimastigotes and amastigotes with the 50% inhibitory concentrations (IC50) in the nanomolar range. Trypomastigotes lysis was also induced with 24-h treatment and a LC50 of 2.3 µM. Ultrastructural analysis by electron microscopy demonstrated that TCAN26 mainly affected the parasite's membranes leading to mitochondrial and Golgi cisternae swelling, membrane blebs, and autophagic figures in the different parasite developmental stages. While the Golgi of the parasites was significantly affected, the Golgi complex of the host cells remained normal suggesting a specific mechanism of action. In summary, our results suggest that TCAN 26 is a potent and selective inhibitor of T. cruzi growth probably due to disturbances of phospholipid biosynthesis.

3D reconstruction of Trypanosoma cruzi-macrophage interaction shows the recruitment of host cell organelles towards parasitophorous vacuoles during its biogenesis.

Trypanosoma cruzi has a complex life cycle where two infective developmental stages, known as trypomastigote and amastigote, can be found in the vertebrate host. Both forms can invade a large variety of cellular types and induce the formation of a parasitophorous vacuole (PV), that, posteriorly, disassembles and releases the parasites into the host cell cytoplasm. The biogenesis of T. cruzi PVs has not been analyzed in professional phagocytic cells. We investigated the biogenesis of PVs containing trypomastigotes or amastigotes in peritoneal macrophages. We observed the presence of profiles of the endoplasmic reticulum and lysosomes from the host cell near PVs at early stages of interaction in both developmental stages, suggesting that both organelles may participate as possible membrane donors for the formation of the PVs. The Golgi complex, however, was observed only near already formed PVs. Electron microscopy tomography and FIB-SEM microscopy followed by 3D reconstruction of entire PVs containing amastigotes or trypomastigotes confirmed the presence of both endoplasmic reticulum and lysosomes in the initial stages of PV formation. In addition, Golgi complex and mitochondria localize around PVs during their biogenesis. Taken together these observations provide a whole view of the invasion process in a professional phagocytic cell.