β-carbolines RCC and C5 induce death of Leishmania amazonensis intracellular amastigotes

Background: Cutaneous leishmaniasis is caused by Leishmania spp., and its treatment is limited. The β-carbolines have shown activity against kinetoplastids. Aim: To evaluate the activity and effects of the β-carbolines, N-{2-[(4,6-bis(isopropylamino)-1,3,5-triazin-2-yl)amino]ethyl}-1-(4-methoxyphenyl)-β-carboline-3-carboxamide (RCC) and N-benzyl-1-(4-methoxy)phenyl-9H-beta-carboline-3-carboxamide (C5), against L. amazonensis intracellular amastigotes and to suggest their mechanism of action. Methods: We analyzed the activity and cytotoxicity of β-carbolines and the morphological alterations by electron microscopy. Mitochondrial membrane potential, production nitric oxide, reactive oxygen species, lipidic bodies, autophagic vacuoles and ATP were also evaluated. Results & conclusion: The results showed that RCC and C5 are active against intracellular amastigotes and were able to induce oxidative stress and ultrastructural alterations such as accumulation of lipid bodies and autophagic vacuoles, leading to parasite death.

Discovery of New Chemical Tools against Leishmania amazonensis via the MMV Pathogen Box

The protozoan parasite Leishmania causes a spectrum of diseases and there are over 1 million infections each year. Current treatments are toxic, expensive, and difficult to administer, and resistance to them is emerging. In this study, we screened the antileishmanial activity of the Pathogen Box compounds from the Medicine for Malaria Venture against Leishmania amazonensis, and compared their structures and cytotoxicity. The compounds MMV676388 (3), MMV690103 (5), MMV022029 (7), MMV022478 (9) and MMV021013 (10) exerted a significant dose-dependent inhibition effect on the proliferation of L. amazonensis promastigotes and intracellular amastigotes. Moreover, studies on the mechanism of cell death showed that compounds 3 and 5 induced an apoptotic process while the compounds 7, 9 and 10 seem to induce an autophagic mechanism. The present findings underline the potential of these five molecules as novel therapeutic leishmanicidal agents.

Antiparasitic Effect of Stilbene and Terphenyl Compounds against Trypanosoma cruzi Parasites

Background: Chagas disease, also known as American trypanosomiasis, is a potentially life-threatening illness caused by the protozoan parasite Trypanosoma cruzi. No progress in the treatment of this pathology has been made since Nifurtimox was introduced more than fifty years ago, and this drug is considered very aggressive and may cause several adverse effects. This drug currently has severe limitations, including a high frequency of undesirable side effects and limited efficacy and availability, so research to discover new drugs for the treatment of Chagas disease is imperative. Many drugs available on the market are natural products as found in nature or compounds designed based on the structure and activity of these natural products. Methods: This study evaluated the in vitro antiparasitic activity of a series of previously synthesized stilbene and terphenyl compounds in T. cruzi epimastigotes and intracellular amastigotes. The action of the most selective compounds was investigated by flow cytometric analysis to evaluate the mechanism of cell death. The ability to induce apoptosis or caspase-1 inflammasomes was assayed in macrophages infected with T. cruzi after treatment, comparing it with that of Nifurtimox. Results: The stilbene ST18 was the most potent compound of the series. It was slightly less active than Nifurtimox in epimastigotes but most active in intracellular amastigotes. Compared to Nifurtimox, it was markedly less cytotoxic when tested in vitro on normal cells. ST18 was able to induce a marked increase in parasites positive for Annexin V and monodansylcadaverine. Moreover, ST18 induced the activation, in infected macrophages, of caspase-1, a conserved enzyme that plays a major role in controlling parasitemia, host survival and the onset of the adaptive immune response in Trypanosoma infection. Conclusions: The antiparasitic activity of ST18 together with its ability to activate caspase-1 in infected macrophages and its low toxicity toward normal cells makes this compound interesting for further clinical investigation.

Photodynamic Therapy Using Toluidine Blue O (TBO) Dye as a Photosensitizer against Leishmania major

Background: Photodynamic therapy (PDT) is alternative treatment of cutaneous leishmaniasis (CL), and phenolthiazine dyes such as Toluidine Blue O (TBO) have the potential role in PDT and notably affect parasites inactivation. This study aimed to evaluate the effectiveness of PDT by using TBO and a light-emitting diode (LED) in the treatment of zoonotic CL (ZCL). Methods: The study was conducted in Iran University of Medical Sciences, Tehran, Iran in 2018-2020. Different concentration (7.8 µg/ mL up to 500 µg/ mL) of TBO as a photosensitizer and a 630 nm LED light as a source of light were used for antileishmanial activity against both forms of Leishmania major promastigotes and intracellular amastigotes. Effective concentration (EC50) and cell cytotoxicity (CC50) were calculated in both infected and non-infected J774.A1 macrophages, respectively. As well as inhibitory concentration (IC50) was quantified in L. major promastigotes for 2 h, 24 h, and 48 h after incubation using a MTT colorimetric assay. Results: TBO dye in combination with the PDT significantly decreases the L. major promastigotes and intracellular amastigotes viability when compared with TBO alone. Both TBO dye in combination with the PDT and TBO alone had no toxic effects on the mice macrophages; however, it significantly killed the entered parasites inside the cells. Our results in the current study established satisfactory findings in clearing intracellular L. major parasites in in-vitro conditions. Conclusion: TBO dye in combination with the PDT can be considered as a harmless, effective and importantly perfect treatment against L. major, causative agent of ZCL, in an in-vitro situation without any negative toxicity to the mice macrophages.

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.

Boswellic Acids Show In Vitro Activity against Leishmania donovani

In continuation of our search for leads from medicinal plants against protozoal pathogens, we detected antileishmanial activity in polar fractions of a dichloromethane extract from Boswellia serrata resin. 11-keto-β-boswellic acid (KBA) could be isolated from these fractions and was tested in vitro against Leishmania donovani axenic amastigotes along with five further boswellic acid derivatives. 3-O-acetyl-11-keto-β-boswellic acid (AKBA) showed the strongest activity with an IC50 value of 0.88 µM against axenic amastigotes but was inactive against intracellular amastigotes in murine macrophages

In Vitro Susceptibility to Miltefosine of Leishmania infantum (syn. L. chagasi) Isolates from Different Geographical Areas in Brazil

Treatment of visceral leishmaniasis in Brazil still relies on meglumine antimoniate, with less than ideal efficacy and safety, making new therapeutic tools an urgent need. The oral drug miltefosine was assayed in a phase II clinical trial in Brazil with cure rates lower than previously demonstrated in India. The present study investigated the susceptibility to miltefosine in 73 Brazilian strains of Leishmania infantum from different geographic regions, using intracellular amastigote and promastigote assays. The EC50 for miltefosine of 13 of these strains evaluated in intracellular amastigotes varied between 1.41 and 4.57 μM. The EC50 of the 73 strains determined in promastigotes varied between 5.89 and 23.7 μM. No correlation between in vitro miltefosine susceptibility and the presence of the miltefosine sensitive locus was detected among the tested strains. The relatively low heterogeneity in miltefosine susceptibility observed for the 73 strains tested in this study suggests the absence of decreased susceptibility to miltefosine in Brazilian L. infantum and does not exclude future clinical evaluation of miltefosine for VL treatment in Brazil.

New phenoxyacetohydrazones against Trypanosoma cruzi

Herein, we reported the design, synthesis, antitrypanosomal and cytotoxic evaluation of a new phenoxyacetohydrazones series. All derivatives were assayed against bloodstream trypomastigote forms of T. cruzi (Y strain) and intracellular amastigotes using the model of L-929 cells infected with trypomastigotes of the Tulahuen strain. Compound (E)-N'-(3.4-dihydroxybenzylidene)-2-phenoxyacetohydrazide (11) showed an activity against trypomastigotes (IC50/24 h = 10.3 µM) equivalent to that of benznidazole and with selectivity index (SI) = 46. Against infected cultures, (E)-N'-((5-nitrofuran-2-yl) methylene)-2-phenoxyacetohydrazide (19) was active at the nanomolar range (IC50/96 h = 100 nM), being about 15-fold more active than the standard drug and with SI equal to 1000. Thus, derivatives 11 and 19 could be considered good prototype for the development of new candidates for Chagas disease therapy.

Expression analysis of centrin gene in promastigote and amastigote forms of leishmania infantum iranian isolates: a promising target for live attenuated vaccine development against canine leishmaniasis

Background Leishmania parasites express various essential proteins in different growth phases (logarithmic/stationary) and forms (promastigote/amastigote). Targeting the genes encoding such proteins paves the way for controlling these parasites. Centrin is an essential gene, which its protein product seems to be vital for the proliferation of Leishmania parasites. Herein, this study was contrived to analyze the expression level of the centrin gene in different growth phases and forms of Leishmania infantum (L. infantum) parasites isolated from various endemic areas of canine leishmaniasis (CanL) in Iran. Results All three collected isolates were identified as L. infantum using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Real-time reverse transcription (RT)-PCR revealed a statistically significant up-regulation (3.13-fold) in the logarithmic phase promastigotes compared to stationary ones (p < 0.01), whereas centrin was expressed equally in intracellular amastigotes at different time points during cell culture. Also, our finding revealed a slight up-regulation of the centrin gene (1.22-fold) in the intracellular amastigotes compared to logarithmic phase promastigotes, which was found statistically non-significant (p > 0.05). Conclusions Centrin gene in Iranian isolates of L. infantum is more expressed in exponential than stationary phases and seems to be considered as a promising target in the development of a genetically modified live attenuated vaccine for CanL control.