Parasite Death Research Articles

Albendazole – Ivermectin combination decreases inflammation in experimental neurocysticercosis

Neurocysticercosis (NCC) is a public health issue in endemic regions and is considered the main preventable cause of neurologic disease. It is caused by the presence of Taenia solium cysticercus in the central nervous system. The current treatment is performed with anthelminthic drugs – albendazole (ABZ) or praziquantel – associated with anti-inflammatory and corticosteroids in order to prevent the negative effects of the inflammatory reaction to the parasite's death. Ivermectin (IVM) is an anthelminthic drug that has been shown to present an anti-inflammatory effect. The aim of this study was to was to evaluate the histopathologic aspects of experimental NCC after in vivo treatment with a combination of ABZ-IVM. Balb/c mice were intracranially inoculated with T. crassiceps cysticerci and after 30 days of infection were treated with a single dose of NaCl 0.9% (control group), ABZ monotherapy (40 mg/kg), IVM monotherapy (0.2 mg/kg) or a combination of ABZ-IVM. 24h after the treatment the animals were euthanized and the brain was removed for histopathologic analysis. The IVM monotherapy and ABZ-IVM combination showed more degenerated cysticerci, less inflammatory infiltration, meningitis and hyperemia than the other groups. Therefore, it is possible to recommend the combination of albendazole and ivermectin as alternative chemotherapy for NCC due to its antiparasitic and anti-inflammatory effects, with potential to decrease the negative effects of the inflammatory burst when the parasite is killed within the CNS.

Antileishmanial activity of tetra-cationic porphyrins with peripheral Pt(II) and Pd(II) complexes mediated by photodynamic therapy

Leishmaniasis is a neglected disease that impacts more than one billion people in endemic areas of the globe. Several drawbacks are associated with the currently existing drugs for treatment such as low effectiveness, toxicity, and the emergence of resistant strains that demonstrate the importance of looking for novel therapeutic alternatives. Photodynamic therapy (PDT) is a promising novel alternative for cutaneous leishmaniasis treatment because its topical application avoids potential side effects generally associated with oral/parenteral application. A light-sensitive compound known as photosensitizer (PS) interacts with light and molecular oxygen to generate reactive oxygen species (ROS), which promote cell death by oxidative stress through PDT approaches. Here, for the first time, we demonstrate the antileishmanial effect of tetra-cationic porphyrins with peripheral Pt(II)- and Pd(II)-polypyridyl complexes using PDT. The isomeric tetra-cationic porphyrins in the meta positions, 3-PtTPyP, and 3-PdTPyP, exhibited the highest antiparasitic activity against promastigote (IC50-pro = 41.8 nM and 46.1 nM, respectively) and intracellular amastigote forms (IC50-ama = 27.6 nM and 38.8 nM, respectively) of L. amazonensis under white light irradiation (72 J cm–2) with high selectivity (SI > 50) for both forms of parasites regarding mammalian cells. In addition, these PS induced the cell death of parasites principally by a necrotic process in the presence of white light by mitochondrial and acidic compartments accumulation. This study showed that porphyrins 3-PtTPyP and 3-PdTPyP displayed a promising antileishmanial-PDT activity with potential application for cutaneous leishmaniasis treatment.

Anthelmintic effects of peppermint (Mentha piperita), lemon (Citrus limon), and tea tree (Melaleuca alternifolia) essential oils against Monogenean parasite (Dactylogyrus sp.) on carp (Cyprinus carpio).

Dactylogyrus sp. (Monogenea) is one of the most dangerous pathogens causing parasitic infections in carp (Cyprinus carpio) and other freshwater fish. Due to the adverse effects of conventional chemical treatments on the environment and fish, the use of herbal products in aquaculture against various diseases has increased. In the present study, anthelmintic effects of peppermint (Mentha piperita), lemon (Citrus limon), and tea tree (Melaleuca alternifolia) essential oils against Dactylogyrus sp. found on the gills of carp were studied using in vitro and in vivo experiments. In in vitro experiments, 1, 2.5, 5, and 10 μl/ml concentrations of these essential oils were tested on the parasites and cumulative mortalities were observed to reach 100 % after treatment with peppermint, lemon, and tea tree essential oils of 1 μl/ml concentration in approximately 15, 10 and 2 min, respectively. The in vitro experiments demonstrated that the cumulative mortality of the parasites increased with essential oil concentration and exposure duration. As the concentration of essential oil used increased, the observed mean time to death of parasites decreased. In in vivo experiments, median effective concentrations (EC50) as assessed by in vitro tests for peppermint and lemon essential oils in 5 min exposure and EC50 concentration for tea tree essential oil in 2 min exposure was applied on fish as a single bath and there was a significant decrease in the mean parasite intensities (p<0.05). Antiparasitic efficacies of peppermint, lemon, and tea tree essential oils were determined as 28.23 %, 30.95 %, and 35.31 %, respectively. The tea tree oil was the most effective and peppermint oil was the least effective in both in vitro and in vivo experiments. All three herbal essential oils tested in this study have weak antiparasitic potential against monogenean infections in fish.

Synergistic Antileishmanial Effect of Oregano Essential Oil and Silver Nanoparticles: Mechanisms of Action on Leishmania amazonensis.

American tegumentary leishmaniasis, a zoonotic disease caused by the Leishmania genus, poses significant challenges in treatment, including administration difficulty, low efficacy, and parasite resistance. Novel compounds or associations offer alternative therapies, and natural products such as oregano essential oil (OEO), extracted from Origanum vulgare, have been extensively researched due to biological effects, including antibacterial, antifungal, and antiparasitic properties. Silver nanoparticles (AgNp), a nanomaterial with compelling antimicrobial and antiparasitic activity, have been shown to exhibit potent leishmanicidal properties. We evaluated the in vitro effect of OEO and AgNp-Bio association on L. amazonensis and the death mechanisms of the parasite involved. Our results demonstrated a synergistic antileishmanial effect of OEO + AgNp on promastigote forms and L. amazonensis-infected macrophages, which induced morphological and ultrastructural changes in promastigotes. Subsequently, we investigated the mechanisms underlying parasite death and showed an increase in NO, ROS, mitochondrial depolarization, accumulation of lipid-storage bodies, autophagic vacuoles, phosphatidylserine exposure, and damage to the plasma membrane. Moreover, the association resulted in a reduction in the percentage of infected cells and the number of amastigotes per macrophage. In conclusion, our findings establish that OEO + AgNp elicits a late apoptosis-like mechanism to combat promastigote forms and promotes ROS and NO production in infected macrophages to target intracellular amastigote forms.

In-vitro biological evaluation of 3,3′,5,5′-tetramethoxy-biphenyl-4,4′-diol and molecular docking studies on trypanothione reductase and Gp63 from Leishmania amazonensis demonstrated anti-leishmania potential

Available treatments for leishmaniasis have been widely used since the 1940s but come at a high cost, variable efficacy, high toxicity, and adverse side-effects. 3,3′,5,5′-Tetramethoxy-biphenyl-4,4′-diol (TMBP) was synthesized through laccase-catalysis of 2,6-dimethoxyphenol and displayed antioxidant and anticancer activity, and is considered a potential drug candidate. Thus, this study aimed to evaluate the anti-leishmanial effect of TMBP against promastigote and amastigote forms of Leishmania (L.) amazonensis and investigated the mechanisms involved in parasite death. TMBP treatment inhibited the proliferation (IC50 0.62–0.86 µM) and induced the death of promastigote forms by generating reactive oxygen species and mitochondrial dysfunction. In intracellular amastigotes, TMBP reduced the percentage of infected macrophages, being 62.7 times more selective to the parasite (CC50 53.93 µM). TMBP did not hemolyze sheep erythrocytes; indicative of low cytotoxicity. Additionally, molecular docking analysis on two enzyme targets of L. amazonensis: trypanothione reductase (TR) and leishmanolysin (Gp63), suggested that the hydroxyl group could be a pharmacophoric group due to its binding affinity by hydrogen bonds with residues at the active site of both enzymes. TMBP was more selective to the Gp63 target than TR. This is the first report that TMBP is a promising compound to act as an anti-leishmanial agent.

In vitro and in vivo evaluation of efficacy of berberine chloride: Phyto-alternative approach against Trypanosoma evansi infection

Current chemotherapy against the Surra organism, Trypanosoma evansi has several limitations in terms of efficacy, toxicity, availability and emerging resistance. These reasons make the search of new chemo-preventive and chemo-therapeutic agent with high potency and low toxicity. Alkaloid phyto-molecules, berberine has shown promising anti-kinetoplastid activity against T. cruzi, T. congolense, T. brucei, Leishmania donovani and L. tropica. However, till date, there is no investigation of therapeutic efficacy of berberine chloride (BC) against T. evansi. The IC50 value of BC for growth inhibition of T. evansi at 24 h of culture was calculated as 12.15 µM. The specific selectivity index (SSI) of BC was calculated as 19.01 and 10.43 against Vero cell line and Equine PBMC’s, respectively. Thirteen drug target genes affecting various metabolic pathways were studied to investigate the mode of trypanocidal action of BC. In transcript analysis, the mRNA expression of arginine kinase 1 remained refractory to exposure with BC, which provides metabolic plasticity in adverse environmental conditions. In contrary, rest all the drug target gene were down-regulated, which indicates that drug severely affect DNA replication, cell proliferation, energy homeostasis, redox homeostasis and calcium homeostasis of T. evansi, leading to the death of parasite in low concentrations. It is the first attempt to investigate in vitro anti-trypanosomal activity of BC against T. evansi. These data imply that phytochemicals as alternative strategies can be explored in the future as an alternative treatment for Surra in animal.

Conditional permeabilization of the P. falciparum plasma membrane in infected cells links cation influx to reduced membrane integrity.

The intracellular human malaria parasite, Plasmodium falciparum, uses the PfATP4 cation pump to maintain Na+ and H+ homeostasis in parasite cytosol. PfATP4 is the target of advanced antimalarial leads, which produce many poorly understood metabolic disturbances within infected erythrocytes. Here, we expressed the mammalian ligand-gated TRPV1 ion channel at the parasite plasma membrane to study ion regulation and examine the effects of cation leak. TRPV1 expression was well-tolerated, consistent with negligible ion flux through the nonactivated channel. TRPV1 ligands produced rapid parasite death in the transfectant line at their activating concentrations, but were harmless to the wild-type parent. Activation triggered cholesterol redistribution at the parasite plasma membrane, reproducing effects of PfATP4 inhibitors and directly implicating cation dysregulation in this process. In contrast to predictions, TRPV1 activation in low Na+ media accentuated parasite killing but a PfATP4 inhibitor had unchanged efficacy. Selection of a ligand-resistant mutant revealed a previously uncharacterized G683V mutation in TRPV1 that occludes the lower channel gate, implicating reduced permeability as a mechanism for parasite resistance to antimalarials targeting ion homeostasis. Our findings provide key insights into malaria parasite ion regulation and will guide mechanism-of-action studies for advanced antimalarial leads that act at the host-pathogen interface.

Transcriptome profile of halofuginone resistant and sensitive strains of Eimeria tenella.

The antiparasitic drug halofuginone is important for controlling apicomplexan parasites. However, the occurrence of halofuginone resistance is a major obstacle for it to the treatment of apicomplexan parasites. Current studies have identified the molecular marker and drug resistance mechanisms of halofuginone in Plasmodium falciparum. In this study, we tried to use transcriptomic data to explore resistance mechanisms of halofuginone in apicomplexan parasites of the genus Eimeria (Apicomplexa: Eimeriidae). After halofuginone treatment of E. tenella parasites, transcriptome analysis was performed using samples derived from both resistant and sensitive strains. In the sensitive group, DEGs associated with enzymes were significantly downregulated, whereas the DNA damaging process was upregulated after halofuginone treatment, revealing the mechanism of halofuginone-induced parasite death. In addition, 1,325 differentially expressed genes (DEGs) were detected between halofuginone resistant and sensitive strains, and the DEGs related to translation were significantly downregulated after halofuginone induction. Overall, our results provide a gene expression profile for further studies on the mechanism of halofuginone resistance in E. tenella.

Extrapolating Qualitative Mycochemical Profiling and Therapeutic Potential of Two Mycorrhizal Amanita Species (Agaricomycetes) from Pakistan

The present study was an attempt to evaluate the antimicrobial and anthelmintic potential of two Amanita species, i.e., A. orsonii and A. glarea, along with their qualitative mycochemical screening. Maceration technique was adopted to make crude extracts in non-polar (petroleum ether and chloroform) and polar (ethanol and distilled water) solvents. Qualitative mycochemical screening revealed the presence of a variety of secondary metabolites like terpenoids, flavonoids, tannins, alkaloids, saponins, and cardiac glycosides. Antimicrobial activities were carried out by using agar well diffusing method against four bacterial and one fungal strain. The antibacterial potential ranged between 4.86 ± 0.088 mm and 34.83 ± 0.166 mm with maximum inhibition zone exhibited by petroleum ether extract against Pseudomonas fluorescens and least potential by distilled water macerate of A. orsonii against the Escherichia coli. The antifungal activity ranged between 14.5 ± 0.288 and 24.76 ± 0.145 mm, with the highest potential provided by chloroform extract of A. orsonii and least capability put forward by A. glarea by petroleum ether extract against Fusarium solanii. Antibiotics and antimycotic discs were used as standard and some of the crude extracts showed bigger zone of inhibition as compared to standard. Different concentrations of ethanolic extracts of both mushrooms were subjected to anthelmintic potential against parasite Haemonchus contortus. All the crude extracts were more potent than standard oxfandazole used. Anthelmintic potential ranged between 9.9 ± 0.057 and 54.93 ± 0.033 minutes for death of parasite, while the paralyzed time ranged between 4.86 ± 0.088 and 24.86 ± 0.088 minutes. From the results obtained it was concluded that both mushrooms can be used as potential source of curative antibacterial, antifungal, and anthelmintic agents against several diseases that might be used in pharmaceutical industries for making medicines and to screen out secondary metabolites in future.

Laser-based killing of a macroparasite inside its live invertebrate host

Clearing infection is an essential step to address many issues in host-parasite interactions but is challenging when dealing with endoparasites of large size relative to that of their host. Here, we took advantage of the lethality, contactless and versatility of high-energy laser beam to achieve it, using thorny-headed worms (Acanthocephala) and their amphipod intermediate host as a model system. We show that laser-based de-parasitization can be achieved using 450 nm Blue Diode Laser targeting carotenoid pigments in the bird acanthocephalan Polymorphus minutus. Using proboscis evagination failure and DNA degradation to establish parasite death, we found that 80% P. minutus died from within-host exposure to 5 pulses of 50 ms duration, 1.4 W power. Survival of infected gammarids 11 days after laser treatment was 60%. Preliminary tests were also performed with Nanosecond-Green Laser targeting lipids in Pomphorhynchus tereticollis, another acanthocephalan parasite. We discuss the efficiency and side-effect of laser treatment in this host-parasite system and highlight the perspectives that this technology more generally offers in parasitology.

Antimalarials Targeting the Malaria Parasite Cation ATPase P. falciparum ATP4 (PfATP4).

Malaria, caused by parasites of the Plasmodium species and transmitted through the bites of infected female Anopheles mosquitoes, is still a fatal and dangerous disease in mainly tropical and subtropical regions. The widespread resistance of P. falciparum to antimalarial drugs forces the search for new molecules with activity against this parasite. While a large number of compounds can inhibit P. falciparum growth in vitro, unfortunately, only a limited number of targets have been identified so far. One of the most promising approaches has been the identification of effective inhibitors of P-type cation-transporter ATPase 4 (PfATP4) in P. falciparum. PfATP4 is a Na+ efflux pump that maintains a low cytosolic Na+ in the parasite. Thus, upon treatment with PfATP4 inhibitors, the parasites rapidly accumulate Na+, which triggers processes leading to parasite death. PfATP4 is present in the parasite plasma membrane but is absent in mammals; its exclusivity thus makes it a good antimalarial drug target. The current review presents PfATP4 function in the context of the pharmacological influence of its inhibitors. In addition, compounds with inhibitory activities belonging to spiroindolones, dihydroisoquinolones, aminopyrazoles, pyrazoleamides, and 4-cyano-3-methylisoquinolines, are also reviewed. Particular emphasis is placed on the results of preclinical and clinical studies in which their effectiveness was tested. PfATP4-associated antimalarials rapidly cleared parasites in mouse models and preliminary human trials. These findings highlight a fundamental biochemical mechanism sensitive to pharmacological intervention that can form a medicinal chemistry approach for antimalarial drug design to create new molecules with potent PfATP4 inhibitory activity.

Laser-based killing of a macroparasite inside its live invertebrate host

Whether host phenotypic alterations induced by parasites are reversible or not is a core issue to understand underlying mechanisms as well as the fitness costs of infection and recovery to the host. Clearing infection is an essential step to address this issue, which turns out to be challenging with endoparasites of large size relative to that of their host. Here, we took advantage of the lethality, contactless and versatility of high-energy laser beam to design such tool, using thorny-headed worms and their amphipod intermediate host as a model system. We show that laser-based de-parasitization can be achieved using blue laser targeting carotenoid pigments in Polymorphus minutus but not in the larger and less pigmented Pomphorhynchus tereticollis. Using DNA degradation to establish parasite death, we found that 80% P. minutus died from within-host laser exposure. Survival rate of infected gammarids to laser treatment was higher than uninfected ones. The failure to kill P. tereticollis was also observed with nanosecond-green laser, an alternative laser source targeting lipid. We discuss the possible causes of amphipod death following parasite treatment and highlight the perspectives that this technology offers.

A Postulated Mechanism of the Antimalarial Effect of Free Radicals Generated by Artemisinin on Plasmodium falciparum

Artemisinin and its derivatives, a class of antimalarial drugs, were first isolated from Artemisia annua. Artemisinin can alter the pH of the malaria parasite’s digestive vacuole from acidic to alkaline, leading to parasite death. However, the precise mechanism of artemisinin action in changing the digestive vacuole pH has not yet been confirmed. Previous studies reported that artemisinin and its derivatives could kill the parasites through the generation of oxidative stress by the free radicals they generate. This review aims to provide a better understanding of the possible mechanism of action of artemisinin, focusing on the antimalarial activity caused by the generated free radicals through the induction of mutation in the genes that encode the proton pump of the Plasmodium falciparum digestive vacuole.

Graphene quantum dots induce cascadic apoptosis via interaction with proteins associated with anti-oxidation after endocytosis by Trypanosoma brucei.

Trypanosoma brucei, the pathogen causing African sleeping sickness (trypanosomiasis) in humans, causes debilitating diseases in many regions of the world, but mainly in African countries with tropical and subtropical climates. Enormous efforts have been devoted to controlling trypanosomiasis, including expanding vector control programs, searching for novel anti-trypanosomial agents, and developing vaccines, but with limited success. In this study, we systematically investigated the effect of graphene quantum dots (GQDs) on trypanosomal parasites and their underlying mechanisms. Ultrasmall-sized GQDs can be efficiently endocytosed by T. brucei and with no toxicity to mammalian-derived cells, triggering a cascade of apoptotic reactions, including mitochondrial disorder, intracellular reactive oxygen species (ROS) elevation, Ca2+ accumulation, DNA fragmentation, adenosine triphosphate (ATP) synthesis impairment, and cell cycle arrest. All of these were caused by the direct interaction between GQDs and the proteins associated with cell apoptosis and anti-oxidation responses, such as trypanothione reductase (TryR), a key protein in anti-oxidation. GQDs specifically inhibited the enzymatic activity of TryR, leading to a reduction in the antioxidant capacity and, ultimately, parasite apoptotic death. These data, for the first time, provide a basis for the exploration of GQDs in the development of anti-trypanosomials.

Decoding the anti-Leishmania braziliensis activity of 1,10-phenanthroline-5,6-dione and its silver- and copper-based complexes: In vitro and in vivo approaches

Leishmaniasis is a neglected tropical disease, whose chemotherapy still presents a series of severe side effects for the patient. Recently, our group published the leishmanicidal action of 1,10-phenanthroline (1), 1,10-phenanthroline-5,6-dione (phendione, 2) and its silver(I) [Ag(phendione)2]ClO4 (3) and copper(II) [Cu(phendione)3](ClO4)2.4H2O (4) complexes against Leishmania braziliensis. Herein, we deciphered the mechanisms of anti-L. braziliensis action of these promising compounds. The IC50 values were calculated for L. braziliensis promastigotes and the potency order was: 4 (0.5 ​μM) ​> ​3 (6.1 ​μM) ​> ​2 (21.3 ​μM) ​> ​1 (32.7 ​μM). Treatment of promastigotes with test compounds induced mitochondrial dysfunction with concomitant increment in the production of intracellular reactive oxygen species, culminating in parasite death by apoptosis as judged by the increased percentage of annexin-positive/propidium iodide-negative parasites and DNA cleavage (classical phenotypic markers of apoptotic cells). Subsequently, the effects of these test compounds on in vitro interaction between L. braziliensis and hamster macrophages showed a significant reduction in the number of intracellular amastigotes, and consequently parasite death by apoptosis. The test compounds presented a protective action in in vivo hamster infections without presenting toxicity to the mammalian host. The compounds limited the development of the paw lesion in hamsters infected with L. braziliensis as well as drastically reducing the number of parasites in both infected paw and lymph nodes draining the lesion, with 4 being the most effective compound. Collectively, our results emphasize the high potential of metal-based compounds, particularly 4, as new, safe and effective alternatives for the treatment of L. braziliensis infection.

Dual role of an essential HtrA2/Omi protease in the human malaria parasite: Maintenance of mitochondrial homeostasis and induction of apoptosis-like cell death under cellular stress

Members of the HtrA family of serine proteases are known to play roles in mitochondrial homeostasis as well as in programmed cell death. Mitochondrial homeostasis and metabolism are crucial for the survival and propagation of the malaria parasite within the host. Here we have functionally characterized a Plasmodium falciparum HtrA2 (PfHtrA2) protein, which harbours trypsin-like protease activity that can be inhibited by its specific inhibitor, ucf-101. A transgenic parasite line was generated, using the HA-glmS C-terminal tagging approach, for localization as well as for inducible knock-down of PfHtrA2. The PfHtrA2 was localized in the parasite mitochondrion during the asexual life cycle. Genetic ablation of PfHtrA2 caused significant parasite growth inhibition, decreased replication of mtDNA, increased mitochondrial ROS production, caused mitochondrial fission/fragmentation, and hindered parasite development. However, the ucf-101 treatment did not affect the parasite growth, suggesting the non-protease/chaperone role of PfHtrA2 in the parasite. Under cellular stress conditions, inhibition of PfHtrA2 by ucf-101 reduced activation of the caspase-like protease as well as parasite cell death, suggesting the involvement of protease activity of PfHtrA2 in apoptosis-like cell death in the parasite. Under these cellular stress conditions, the PfHtrA2 gets processed but remains localized in the mitochondrion, suggesting that it acts within the mitochondrion by cleaving intra-mitochondrial substrate(s). This was further supported by trans-expression of PfHtrA2 protease domain in the parasite cytosol, which was unable to induce any cell death in the parasite. Overall, we show the specific roles of PfHtrA2 in maintaining mitochondrial homeostasis as well as in regulating stress-induced cell death.

Curcumin modulates neurogliogenesis and purinergic receptor expression in neural precursor cells infected with Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite that causes toxoplasmosis, and its congenital transmission is of paramount concern. During embryonic development, infection with the parasite causes irreversible damage to the still-forming fetus's central nervous system (CNS). In the pathogenesis of neurotoxoplasmosis, purinergic receptors prejudice neuroprotection, neuroinflammation, and activation of microbicide mechanisms against the parasitic vacuole. This study used curcumin as a treatment for neural precursor cells (NPCs) infected with T. gondii. The congenital toxoplasmosis induction consisted of maternal infection with the VEG strain, and NPCs were obtained from the telencephalon of mouse embryos. Curcumin at increasing concentrations was administered in vitro to analyze NPC metabolic activity, cell number, and size, as well as neurogliogenesis, proving to be effective in recovering the size of infected NPCs. Curcumin partially re-established impaired neurogenesis. Purinergic A1, A2A, and P2X7 receptors may be related to neuroprotection, neuroinflammatory control, and activation of mechanisms for inducing the parasite's death. ERK 1/2 was highly expressed in infected cells, while its expression rates decreased after the addition of the treatment, highlighting the possible anti-inflammatory action of curcumin. These findings suggest that curcumin treats neurological perturbations induced by toxoplasmosis.

Anti-malarial drugs: Mechanisms underlying their proarrhythmic effects.

Malaria remains the leading cause of parasitic death in the world. Artemisinin resistance is an emerging threat indicating an imminent need for novel combination therapy. Given the key role of mass drug administration, it is pivotal that the safety of anti-malarial drugs is investigated thoroughly prior to widespread use. Cardiotoxicity, most prominently arrhythmic risk, has been a concern for anti-malarial drugs. We clarify the likely underlying mechanisms by which anti-malarial drugs predispose to arrhythmias. These relate to disruption of (1) action potential upstroke due to effects on the sodium currents, (2) action potential repolarisation due to effects on the potassium currents, (3) cellular calcium homeostasis, (4) mitochondrial function and reactive oxygen species production and (5) cardiac fibrosis. Together, these alterations promote arrhythmic triggers and substrates. Understanding these mechanisms is essential to assess the safety of these drugs, stratify patients based on arrhythmic risk and guide future anti-malarial drug development.

One-Pot Cascade Annulation-Triggered Synthesis of N-6-Substituted Norcryptotackieine Alkaloids and Evaluation of Their Antileishmanial Activities.

Norcryptotackieine or 6H-indolo[2,3-b]quinoline is an indoloquinoline class of alkaloid isolated from Cryptolepis sanguinolenta that is traditionally used for antimalarial therapy. Additional structural tuning can extend the therapeutic potency of these indoloquinolines as antileishmanial drug leads. Synthesis of N-6-functionalized norcryptotackieines suffers from the necessity of complex pre-synthesized starting materials, restricted scope of functionalization, or tedious processes. Consequently, a straightforward synthetic procedure for accessing non-natural N-6-functionalized 6H-indolo[2,3-b]quinolines with potent antileishmanial activities is highly sought-after. Herein, we report a two-step one-pot synthesis of N-6-functionalized norcryptotackieine through a Pd-catalyzed double annulation reaction of commercially available amphipathic amines, 2-iodobenzyl cyanide, and differently functionalized 2-bromobenzaldehydes. The reported procedure allows a broad flexibility of substitution at the N-6 position and access to diversified scaffolds, including two natural products norcryptotackieine and neocryptolepine. Interestingly, 6d showed potent antileishmanial activities by causing disruption in the cytoskeletal structure and apoptotic-mediated death of parasites. Together, our work manifests the shortest route to N-6-substituted norcryptotackieine-derived antileishmanial agents.

Heterologous expression, purification, and partial characterisation of the apicoplast protein 3-oxoacyl-[acyl-carrier-protein] reductase from Toxoplasma gondii

Recombinant expression and purification of proteins have become a staple of modern drug discovery as it enables more precise in vitro analyses of drug targets, which may help obtain biochemical and biophysical parameters of a known enzyme and even uncover unknown characteristics indicative of novel enzymatic functions. Such information is often necessary to prepare adequate screening assays and drug-discovery experiments in general. Toxoplasma gondii is an obligate protozoan parasite that is a member of the phylum Apicomplexa, can develop several neuro-degenerative symptoms and, in specific cases, certain death for human hosts. Its relict non-photosynthetic plastid, the apicoplast, harbours a unique de novo long-chain fatty acid synthesis pathway of a prokaryotic character, FASII. The FASII pathway shows plasticity and, is essential for many intracellular and membranal components, along with fatty acid uptake via salvaging from the host, therefore, its disruption causes parasite death. TgFabG, a FASII enzyme responsible for a single reduction step in the pathway, was recombinantly expressed, purified and biochemically and biophysically characterised in this study. The bioengineering hurdle of expressing the recombinant gene of a eukaryotic, signal peptide-containing protein in a prokaryotic system was overcome for the apicomplexan enzyme TgFabG, by truncating the N-terminal signal peptide. TgFabG was ultimately recombinantly produced in a plasmid expression vector from its 1131 base pair gene, purified as 260 and 272 amino acid proteins using a hexahistidine (6 × Histag) affinity chromatography and its biochemical (enzyme activity and kinetics) and biophysical characteristics were analysed in vitro.