Inhibitory action of phenothiazinium dyes against Neospora caninum

Abstract

Neospora caninum is an Apicomplexan parasite related to important losses in livestock, causing abortions and decreased fertility in affected cows. Several chemotherapeutic strategies have been developed for disease control; however, no commercial treatment is available. Among the candidate drugs against neosporosis, phenothiazinium dyes, offer a low cost-efficient approach to parasite control. We report the anti-parasitic effects of the phenothiaziums Methylene Blue (MB), New Methylene Blue (NMB), 1,9-Dimethyl Methylene Blue (DMMB) and Toluidine Blue O (TBO) on N. caninum, using in vitro and in vivo models. The dyes inhibited parasite proliferation at nanomolar concentrations (0.019-1.83 μM) and a synergistic effect was achieved when Methylene Blue was combined with New Methylene Blue (Combination Index = 0.84). Moreover, the phenothiazinium dyes improved parasite clearance when combined with Pyrimethamine (Pyr). Combination of Methylene Blue + 1,9-Dimethyl Methylene Blue demonstrated superior efficacy compared to Pyrimethamine based counterparts in an in vivo model of infection. We also observed that Methylene Blue, New Methylene Blue and 1,9-Dimethyl Methylene Blue increased by 5000% the reactive oxygen species (ROS) levels in N. caninum tachyzoites. Phenothiazinium dyes represent an accessible group of candidates with the potential to compound future formulations for neosporosis control. Neospora caninum, an obligate intracellular protozoan, belongs to Apicomplexa phylum, such as Plasmodium spp and Toxoplasma gondii. It causes neosporosis, a disease strongly correlated to abortion and decreased fertility in cattle, costing billions of dollars worldwide 1-3. There is no effective commercial treatment for neosporosis, despite the efforts of several chemotherapy-focused groups 4-6. For example, Artemisinins 7-10 , Miltefosine 11 , plant extracts 12-14 , Diamidines 15,16 , Buparvaquone 4,17 , organometallic ruthenium complexes 18 , Thiazolides 19,20 were evaluated in in vitro and in vivo models with encouraging results. Moreover, polyether ionophore antibiotics 21 , Triazinones 22-24 , bumped kinase inhibitors 25 have also been tested in farm ruminants 26. Based on the promising effects of Methylene Blue (MB) against Plasmodium spp, the etiologic agent of malaria, our group determined the efficacy of this molecule on N. caninum, either alone or combined with Pyrimethamine (Pyr) 27. MB, a phenothiazinium dye, was the first synthetic drug described to cure a patient affected by malaria in the XIX century, pioneering work performed by Paul Ehrlich 28,29. Indeed, MB was used against Plasmodium until the use of Chloroquine and other drugs (massively utilized after the Second World War), which lack some of the reversible MB side effects (blue urine and sclera) 30,31. During the later 20 th /early 21 st Centuries, the intense use of Chloroquine, Artemisinin, and Pyrimethamine has led to proven cases of Plasmodium resistance, demanding novel therapeutic candidates 32. Interestingly, no reports of Plasmodium resistance hav...

Atovaquone, chloroquine, primaquine, quinine and tetracycline: antiproliferative effects of relevant antimalarials on Neospora caninum

“…There is no commercial strategy to control neosporosis, despite the recent advances (Anghel et al, 2018;Harmse et al, 2017;Pereira et al, 2020;Sánchez-Sánchez et al, 2018a, b) and the development of management control measures designed to reduce parasite transmission (Reichel et al, 2014). Likewise, there are few options for the treatment of human toxoplasmosis, which is usually treated with compounds (antifolates, clindamycin, and atovaquone) that are toxic, especially to pregnant women (Neville et al, 2015).…”

“…Thus, the application of anti-malarial drugs indicates an interesting source for drug repurposing against N. caninum. For example, methylene blue and analogues, pyrimethamine and artemisinin formulations have been successfully tested on in vitro (Kim et al, 2002;Lindsay & Dubey, 1989;Pereira et al, 2017Pereira et al, , 2018 and in vivo (Pereira et al, 2020) models of N. caninum infection. Likewise, several novel candidates with anti-N. caninum activity were identified from the Malaria Venture (MMV) Pathogen Box, with promising results (Müller et al, 2017(Müller et al, , 2020.…”

The potential of phenothiazinium dyes as cytotoxicity markers in cisplatin-treated cells

“…The supernatant was discarded, cells were washed with phosphate-buffered saline (PBS), and the cultures were incubated with 100 µM of the phenothiazinium dyes (MB, NMB, TBO, and DMMB), MTT (0.5 mg/ml) or NR (50 µg/ml; 173 µM) for 3 h at 37 °C and 5% CO 2 in duplicate. After treatment with DMSO and cisplatin, labeling with MTT or NR was performed as described previously 27 , 28 with modifications 29 , 30 . Cells incubated with phenothiazinium dyes were processed by the same protocol used for NR.…”

Arylamine Analogs of Methylene Blue: Substituent Effect on Aggregation Behavior and DNA Binding

“…There are many reports on the synthesis of new phenothiazine derivatives, MB analogs, for the needs of modern medicine, including the design of new drugs, as well as the development of new generation photodynamic agents [ 13 , 14 , 15 , 16 ]. There are several reports on the synthesis of 3,7-bis(aryl-amino)phenothiazine derivatives [ 17 , 18 , 19 , 20 ].…”