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Melatonin acts to synchronize the parasite's intraerythrocytic cycle by triggering the phospholipase C -inositol 1,4,5-trisphosphate (PLC-IP 3 ) signaling cascade. Compounds with an indole scaffold impair in vitro proliferation of blood-stage malaria parasites, indicating that this class of compounds is potentially emerging antiplasmodial drugs. Therefore, we aimed to study the role of the alkyl and aryl thiol moieties of 14 synthetic indole compounds against chloroquine-sensitive (3D7) and chloroquine-resistant (Dd2) strains of Plasmodium falciparum . Four compounds ( 3 , 26 , 18 , 21 ) inhibited the growth of P. falciparum (3D7) by 50% at concentrations below 20 µM. A set of 2-sulfenylindoles also showed activity against Dd2 parasites. Our data suggest that Dd2 parasites are more susceptible to compounds 20 and 28 than 3D7 parasites. These data show that 2-sulfenylindoles are promising antimalarials against chloroquine-resistant parasite strains. We also evaluated the effects of the 14 compounds on the parasitemia of the 3D7 strain and their ability to interfere with the effect of 100 nM melatonin on the parasitemia of the 3D7 strain. Our results showed that compounds 3 , 7 , 8 , 10 , 14 , 16 , 17 , and 20 slightly increased the effect of melatonin by increasing parasitemia by 8-20% compared with that of melatonin-only-treated 3D7 parasites. Moreover, we found that melatonin modulates the expression of kinase-related signaling components giving additional evidence to investigate inhibitors that can block melatonin signaling.

Throughout evolution, the need for single-celled organisms to associate and form a single cluster of cells has had several evolutionary advantages. In complex, multicellular organisms, each tissue or organ has a specialty and function that make life together possible, and the organism as a whole needs to act in balance and adapt to changes in the environment. Sensory organs are essential for connecting external stimuli into a biological response, through the senses: sight, smell, taste, hearing, and touch. The G-protein-coupled receptors (GPCRs) are responsible for many of these senses and therefore play a key role in the perception of the cells' external environment, enabling interaction and coordinated development between each cell of a multicellular organism. The malaria-causing protozoan parasite, Plasmodium falciparum , has a complex life cycle that is extremely dependent on a finely regulated cellular signaling machinery. In this review, we summarize strong evidence and the main candidates of GPCRs in protozoan parasites. Interestingly, one of these GPCRs is a sensor for K + shift in Plasmodium falciparum , PfSR25. Studying this family of proteins in P. falciparum could have a significant impact, both on understanding the history of the evolution of GPCRs and on finding new targets for antimalarials.

The host hormone melatonin is known to modulate the asexual cell-cycle of the human malaria parasite Plasmodium falciparum and the kinase PfPK7 is fundamental in the downstream signaling pathways. The nuclear protein PfMORC displays a histidine kinase domain and is involved in parasite cell cycle control. By using a real-time assay, we show a 24 h (h) rhythmic expression of PfMORC at the parasite asexual cycle and the expression is dramatically changed when parasites were treated with 100 nM melatonin for 17 h. Moreover, PfMORC expression was severely affected in PfPK7 knockout (PfPK7 - ) parasites following melatonin treatment. Parasites expressing 3D7 morc-GFP shows nuclear localization of the protein during the asexual stage of parasite development. Although the PfMORC knockdown had no significant impact on the parasite proliferation in vitro it significantly changed the ratio of the different asexual intraerythrocytic stages of the parasites upon the addition of melatonin. Our data reveal that in addition to the upstream melatonin signaling pathways such as IP 3 generation, calcium, and cAMP rise, a nuclear protein, PfMORC is essential for the hormone response in parasite synchronization.

Half of the world's population lives in countries at risk of malaria infection, which results in approximately 450,000 deaths annually. Malaria parasites infect erythrocytes in a coordinated manner, with cycle durations in multiples of 24 hours, which reflects a behavior consistent with the host's circadian cycle. Interference in cycle coordination can help the immune system to naturally fight infection. Consequently, there is a search for new drugs that interfere with the cycle duration for combined treatment with conventional antimalarials. Melatonin appears to be a key host hormone responsible for regulating circadian behavior in the parasite cycle. In addition to host factors, there are still unknown factors intrinsic to the parasite that control the cycle duration. In this review, we present a series of reports of indole compounds and melatonin derivatives with antimalarial activity that were tested on several species of Plasmodium to evaluate the cytotoxicity to parasites and human cells, in addition to the ability to interfere with the development of the erythrocytic cycle. Most of the reported compounds had an IC50 value in the low micromolar range, without any toxicity to human cells. Triptosil, an indole derivative of melatonin, was able to inhibit the effect of melatonin in vitro without causing changes to the parasitemia. The wide variety of tested compounds indicates that it is possible to develop a compound capable of safely eliminating parasites from the host and interfering with the life cycle, which is promising for the development of new combined therapies against malaria., (© 2020 The Authors. Journal of Pineal Research published by John Wiley & Sons Ltd.)

Melatonin and its indoles derivatives are central in the synchronization of malaria parasites. In this research, we discovered that melatonin is unable to increase the parasitemia in the human malaria Plasmodium falciparum that lacks the kinase PfeIK1. The PfeIK1 knockout strain is a valuable tool in the screening of indol-related compound that blocks the melatonin effect in wild-type (WT) parasite development. The assays were performed by using flow cytometry with simultaneous labeling for mitochondria viability with MitoTracker Deep Red and nucleus staining with SYBR Green. We found that Melatotosil leads to an increase in parasitemia in P. falciparum and blocks melatonin effect in the WT parasite. Using microscopy imaging system, we found that Melatotosil at 500 nM is able to induce cytosolic calcium rise in transgenic PfGCaMP3 parasites. On the contrary, the compound Triptiofen blocks P. falciparum cell cycle with IC 50 9.76 µM ± 0.6, inhibits melatonin action, and does not lead to a cytosolic calcium rise in PfGCaMP3 parasites. We also found that the synthetic indol-related compounds arrested parasite cycle for PfeIK1 knockout and (WT) P. falciparum (3D7) in 72 hours culture assays with the IC 50 values slighting lower for the WT strain. We concluded that the kinase PfeIK1 is central for melatonin downstream signaling pathways involved in parasite cell cycle progression. More importantly, the indol-related compounds block its cycle as an upstream essential mechanism for parasite survival. Our data clearly show that this class of compounds emerge as an alternative for the problem of resistance with the classical antimalarials., (© 2020 The Authors. Journal of Pineal Research published by John Wiley & Sons Ltd.)

The indoleamine compound melatonin has been extensively studied in the regulation of the circadian rhythm in nearly all vertebrates. The effects of melatonin have also been studied in Protozoan parasites, especially in the synchronization of the human malaria parasite Plasmodium falciparum via a complex downstream signalling pathway. Melatonin activates protein kinase A (PfPKA) and requires the activation of protein kinase 7 (PfPK7), PLC-IP 3 , and a subset of genes from the ubiquitin-proteasome system. In other parasites, such as Trypanosoma cruzi and Toxoplasma gondii , melatonin increases inflammatory components, thus amplifying the protective response of the host's immune system and affecting parasite load. The development of melatonin-related indole compounds exhibiting antiparasitic properties clearly suggests this new and effective approach as an alternative treatment. Therefore, it is critical to understand how melatonin confers stimulatory functions in host-parasite biology.

Studying Ca 2+ dynamics in protozoan parasites is not an easy task. Loading of parasites with commonly used Ca 2+ fluorescent dyes (such as Fuo4-AM) remains as the major protocol to measure the Ca 2+ oscillations inside the cell. In this chapter, we describe an alternative method to study Ca 2+ signaling in Plasmodium falciparum parasite. This method employs the construction of transgenic parasites (through standard molecular biology techniques), selection of the transfected population, and use of those parasites in spectrofluorometric Ca 2+ assays.

The malaria parasite Plasmodium falciparum is exposed, during its development, to major changes of ionic composition in its surrounding medium. We demonstrate that the P. falciparum serpentine-like receptor PfSR25 is a monovalent cation sensor capable of modulating Ca 2+ signaling in the parasites. Changing from high (140 mM) to low (5.4 mM) KCl concentration triggers [Ca 2+ ] cyt increase in isolated parasites and this Ca 2+ rise is blocked either by phospholipase C (PLC) inhibition or by depleting the parasite's internal Ca 2+ pools. This response persists even in the absence of free extracellular Ca 2+ and cannot be elicited by addition of Na + , Mg 2+ or Ca 2+ . However, when the PfSR25 gene was deleted, no effect on [Ca 2+ ] cyt was observed in response to changing KCl concentration in the knocked out (PfSR25 - ) parasite. Finally, we also demonstrate that: i) PfSR25 plays a role in parasite volume regulation, as hyperosmotic stress induces a significant decrease in parasite volume in wild type (wt), but not in PfSR25 - parasites; ii) parasites lacking PfSR25 show decreased parasitemia and metacaspase gene expression on exposure to the nitric oxide donor sodium nitroprusside (SNP) and iii), compared to PfSR25 - parasites, wt parasites showed a better survival in albumax-deprived condition.