Disclosure of cinnamic acid/4,9-diaminoacridine conjugates as multi-stage antiplasmodial hits.

4,9-diaminoacridines with reported antiplasmodial activity were coupled to different trans-cinnamic acids, delivering a new series of conjugates inspired by the covalent bitherapy concept. The new compounds were more potent than primaquine against hepatic stages of Plasmodium berghei, although this was accompanied by cytotoxic effects on Huh-7 hepatocytes. Relevantly, the conjugates displayed nanomolar activities against blood stage P. falciparum parasites, with no evidence of hemolytic effects below 100 µM. Moreover, the new compounds were at least 25-fold more potent than primaquine against P. falciparum gametocytes. Thus, the new antiplasmodial hits disclosed herein emerge as valuable templates for the development of multi-stage antiplasmodial drug candidates.

Discovery of harmiprims, harmine-primaquine hybrids, as potent and selective anticancer and antimalarial compounds.

Although cancer and malaria are not etiologically nor pathophysiologically connected, due to their similarities successful repurposing of antimalarial drugs for cancer and vice-versa is known and used in clinical settings and drug research and discovery. With the growing resistance of cancer cells and Plasmodium to the known drugs, there is an urgent need to discover new chemotypes and enrich anticancer and antimalarial drug portfolios. In this paper, we present the design and synthesis of harmiprims, hybrids composed of harmine, an alkaloid of the ß-carboline type bearing anticancer and antiplasmodial activities, and primaquine, 8-aminoquinoline antimalarial drug with low antiproliferative activity, covalently bound via triazole or urea. Evaluation of their antiproliferative activities in vitro revealed that N-9 substituted triazole-type harmiprime was the most selective compound against MCF-7, whereas C1-substituted ureido-type hybrid was the most active compound against all cell lines tested. On the other hand, dimeric harmiprime was not toxic at all. Although spectrophotometric studies and thermal denaturation experiments indicated binding of harmiprims to the ds-DNA groove, cell localization showed that harmiprims do not enter cell nucleus nor mitochondria, thus no inhibition of DNA-related processes can be expected. Cell cycle analysis revealed that C1-substituted ureido-type hybrid induced a G1 arrest and reduced the number of cells in the S phase after 24 h, persisting at 48 h, albeit with a less significant increase in G1, possibly due to adaptive cellular responses. In contrast, N-9 substituted triazole-type harmiprime exhibited less pronounced effects on the cell cycle, particularly after 48 h, which is consistent with its moderate activity against the MCF-7 cell line. On the other hand, screening of their antiplasmodial activities against the erythrocytic, hepatic, and gametocytic stages of the Plasmodium life cycle showed that dimeric harmiprime exerts powerful triple-stage antiplasmodial activity, while computational analysis showed its binding within the ATP binding site of PfHsp90.

Portugal's prominence in malaria research: How a small country became a key player in European research on one of the world's deadliest diseases.

Despite its limited resources, Portugal has gained a prominent position in research on malaria. Several historical and personal factors have contributed to this achievement.

A simple polystyrene microfluidic device for sensitive and accurate SERS-based detection of infection by malaria parasites.

Early and accurate detection of infection by pathogenic microorganisms, such as Plasmodium, the causative agent of malaria, is critical for clinical diagnosis and ultimately determines the patient's outcome. We have combined a polystyrene-based microfluidic device with an immunoassay which utilises Surface-Enhanced Raman Spectroscopy (SERS) to detect malaria. The method can be easily translated to a point-of-care testing format and shows excellent sensitivity and specificity, when compared to the gold standard for laboratorial detection of Plasmodium infections. The device can be fabricated in less than 30 min by direct patterning on shrinkable polystyrene sheets of adaptable three-dimensional microfluidic chips. To validate the microfluidic system, samples of P. falciparum-infected red blood cell cultures were used. The SERS-based immunoassay enabled the detection of 0.0012 ± 0.0001% parasitaemia in a P. falciparum-infected red blood cell culture supernatant, an ∼7-fold higher sensitivity than that attained by most rapid diagnostic tests. Our approach successfully overcomes the main challenges of the current Plasmodium detection methods, including increased reproducibility, sensitivity, and specificity. Furthermore, our system can be easily adapted for detection of other pathogens and has excellent properties for early diagnosis of infectious diseases, a decisive step towards lowering their high burden on healthcare systems worldwide.

Design, synthesis and antiplasmodial evaluation of new amide-, carbamate-, and ureido-type harmicines.

Malaria, one of the oldest parasitic diseases, remains a global health threat, and the increasing resistance of the malaria parasite to current antimalarials is forcing the discovery of new, effective drugs. Harmicines, hybrid compounds in which harmine/ß-carboline alkaloids and cinnamic acid derivatives are linked via an amide bond or a triazole ring, represent new antiplasmodial agents. In this work, we used a multiple linear regression technique to build a linear quantitative structure-activity relationship (QSAR) model, based on a group of 40 previously prepared amide-type (AT) harmicines and their antiplasmodial activities against erythrocytic stage of chloroquine-sensitive strain of P. falciparum (Pf3D7). After analysing the QSAR model, new harmicines were designed and synthesized: six amide-type, eleven carbamate-type and two ureido-type harmicines at the N-9 position of the ß-carboline core. Subsequently, we evaluated the antiplasmodial activity of the new harmicines against the erythrocytic and hepatic stages of the Plasmodium life cycle in vitro and their antiproliferative activity against HepG2 cells. UT harmicine (E)-1-(2-(7-methoxy-1-methyl-9H-pyrido[3,4-b]indol-9-yl)ethyl)-3-(3-(3-(trifluoromethyl)phenyl)allyl)urea at the N-9 position of the ß-carboline ring exhibited pronounced antiplasmodial activity against both the erythrocytic and the hepatic stages of the Plasmodium life cycle, accompanied by good selectivity towards Plasmodium.

The protein aggregation inhibitor YAT2150 has potent antimalarial activity in Plasmodium falciparum in vitro cultures.

BACKGROUND: By 2016, signs of emergence of Plasmodium falciparum resistance to artemisinin and partner drugs were detected in the Greater Mekong Subregion. Recently, the independent evolution of artemisinin resistance has also been reported in Africa and South America. This alarming scenario calls for the urgent development of new antimalarials with novel modes of action. We investigated the interference with protein aggregation, which is potentially toxic for the cell and occurs abundantly in all Plasmodium stages, as a hitherto unexplored drug target in the pathogen. RESULTS: Attempts to exacerbate the P. falciparum proteome's propensity to aggregation by delivering endogenous aggregative peptides to in vitro cultures of this parasite did not significantly affect their growth. In contrast, protein aggregation inhibitors clearly reduced the pathogen's viability. One such compound, the bis(styrylpyridinium) salt YAT2150, exhibited potent antiplasmodial activity with an in vitro IC50 of 90 nM for chloroquine- and artemisinin-resistant lines, arresting asexual blood parasites at the trophozoite stage, as well as interfering with the development of both sexual and hepatic forms of Plasmodium. At its IC50, this compound is a powerful inhibitor of the aggregation of the model amyloid ß peptide fragment 1-40, and it reduces the amount of aggregated proteins in P. falciparum cultures, suggesting that the underlying antimalarial mechanism consists in a generalized impairment of proteostasis in the pathogen. YAT2150 has an easy, rapid, and inexpensive synthesis, and because it fluoresces when it accumulates in its main localization in the Plasmodium cytosol, it is a theranostic agent. CONCLUSIONS: Inhibiting protein aggregation in Plasmodium significantly reduces the parasite's viability in vitro. Since YAT2150 belongs to a novel structural class of antiplasmodials with a mode of action that potentially targets multiple gene products, rapid evolution of resistance to this drug is unlikely to occur, making it a promising compound for the post-artemisinin era.

Translation of liver stage activity of M5717, a Plasmodium elongation factor 2 inhibitor: from bench to bedside.

BACKGROUND: Targeting the asymptomatic liver stage of Plasmodium infection through chemoprevention could become a key intervention to reduce malaria-associated incidence and mortality. METHODS: M5717, a Plasmodium elongation factor 2 inhibitor, was assessed in vitro and in vivo with readily accessible Plasmodium berghei parasites. In an animal refinement, reduction, replacement approach, the in vitro IC99 value was used to feed a Population Pharmacokinetics modelling and simulation approach to determine meaningful effective doses for a subsequent Plasmodium sporozoite-induced volunteer infection study. RESULTS: Doses of 100 and 200 mg would provide exposures exceeding IC99 in 96 and 100% of the simulated population, respectively. CONCLUSIONS: This approach has the potential to accelerate the search for new anti-malarials, to reduce the number of healthy volunteers needed in a clinical study and decrease and refine the animal use in the preclinical phase.

A Histone Deacetylase (HDAC) Inhibitor with Pleiotropic In Vitro Anti-Toxoplasma and Anti-Plasmodium Activities Controls Acute and Chronic Toxoplasma Infection in Mice.

Toxoplasmosis is a highly prevalent human disease, and virulent strains of this parasite emerge from wild biotopes. Here, we report on the potential of a histone deacetylase (HDAC) inhibitor we previously synthesized, named JF363, to act in vitro against a large panel of Toxoplasma strains, as well as against the liver and blood stages of Plasmodium parasites, the causative agents of malaria. In vivo administration of the drug significantly increases the survival of mice during the acute phase of infection by T. gondii, thus delaying its spreading. We further provide evidence of the compound's efficiency in controlling the formation of cysts in the brain of T. gondii-infected mice. A convincing docking of the JF363 compound in the active site of the five annotated ME49 T. gondii HDACs was performed by extensive sequence-structure comparison modeling. The resulting complexes show a similar mode of binding in the five paralogous structures and a quite similar prediction of affinities in the micromolar range. Altogether, these results pave the way for further development of this compound to treat acute and chronic toxoplasmosis. It also shows promise for the future development of anti-Plasmodium therapeutic interventions.

Trypanosoma brucei infection protects mice against malaria.

Sleeping sickness and malaria are parasitic diseases with overlapping geographical distributions in sub-Saharan Africa. We hypothesized that the immune response elicited by an infection with Trypanosoma brucei, the etiological agent of sleeping sickness, would inhibit a subsequent infection by Plasmodium, the malaria parasite, decreasing the severity of its associated pathology. To investigate this, we established a new co-infection model in which mice were initially infected with T. brucei, followed by administration of P. berghei sporozoites. We observed that a primary infection by T. brucei significantly attenuates a subsequent infection by the malaria parasite, protecting mice from experimental cerebral malaria and prolonging host survival. We further observed that an ongoing T. brucei infection leads to an accumulation of lymphocyte-derived IFN-γ in the liver, limiting the establishment of a subsequent hepatic infection by P. berghei sporozoites. Thus, we identified a novel host-mediated interaction between two parasitic infections, which may be epidemiologically relevant in regions of Trypanosoma/Plasmodium co-endemicity.

A crucial role for the C-terminal domain of exported protein 1 during the mosquito and hepatic stages of the Plasmodium berghei life cycle.

Intracellular Plasmodium parasites develop inside a parasitophorous vacuole (PV), a specialised compartment enclosed by a membrane (PVM) that contains proteins of both host and parasite origin. Although exported protein 1 (EXP1) is one of the earliest described parasitic PVM proteins, its function throughout the Plasmodium life cycle remains insufficiently understood. Here, we show that whereas the N-terminus of Plasmodium berghei EXP1 (PbEXP1) is essential for parasite survival in the blood, parasites lacking PbEXP1's entire C-terminal (CT) domain replicate normally in the blood but cause less severe pathology than their wild-type counterparts. Moreover, truncation of PbEXP1's CT domain not only impairs parasite development in the mosquito but also abrogates PbEXP1 localization to the PVM of intrahepatic parasites, severely limiting their replication and preventing their egress into the blood. Our findings highlight the importance of EXP1 during the Plasmodium life cycle and identify this protein as a promising target for antiplasmodial intervention.

Half-Sandwich Cyclopentadienylruthenium(II) Complexes: A New Antimalarial Chemotype.

A small library of "half-sandwich" cyclopentadienylruthenium(II) compounds of the general formula [(η5-C5R5)Ru(PPh3)(N-N)][PF6], a scaffold hitherto absent from the toolbox of antiplasmodials, was screened for activity against the blood stage of CQ-sensitive 3D7-GFP, CQ-resistant Dd2, and artemisinin-resistant IPC5202 Plasmodium falciparum strains and the liver stage of Plasmodium berghei. The best-performing compounds displayed dual-stage activity, with single-digit nanomolar IC50 values against blood-stage malaria parasites, nanomolar activity against liver-stage parasites, and residual cytotoxicity against HepG2 and Huh7 mammalian cells. The parasitic absorption/distribution of 7-nitrobenzoxadiazole-appended fluorescent compounds Ru4 and Ru5 was investigated by confocal fluorescence microscopy, revealing parasite-selective absorption in infected erythrocytes and nuclear accumulation of both compounds. The lead compound Ru2 impaired asexual parasite differentiation, exhibiting fast parasiticidal activity against both ring and trophozoite stages of a synchronized culture of the P. falciparum 3D7 strain. These results point to cyclopentadienylruthenium(II) complexes as a highly promising chemotype for the development of dual-stage antiplasmodials.

Plasmodium berghei EXP-1 interacts with host Apolipoprotein H during Plasmodium liver-stage development.

The first, obligatory replication phase of malaria parasite infections is characterized by rapid expansion and differentiation of single parasites in liver cells, resulting in the formation and release of thousands of invasive merozoites into the bloodstream. Hepatic Plasmodium development occurs inside a specialized membranous compartment termed the parasitophorous vacuole (PV). Here, we show that, during the parasite's hepatic replication, the C-terminal region of the parasitic PV membrane protein exported protein 1 (EXP-1) binds to host Apolipoprotein H (ApoH) and that this molecular interaction plays a pivotal role for successful Plasmodium liver-stage development. Expression of a truncated EXP-1 protein, missing the specific ApoH interaction site, or down-regulation of ApoH expression in either hepatic cells or mouse livers by RNA interference resulted in impaired intrahepatic development. Furthermore, infection of mice with sporozoites expressing a truncated version of EXP-1 resulted in both a significant reduction of liver burden and delayed blood-stage patency, leading to a disease outcome different from that generally induced by infection with wild-type parasites. This study identifies a host-parasite protein interaction during the hepatic stage of infection by Plasmodium parasites. The identification of such vital interactions may hold potential toward the development of novel malaria prevention strategies.

Repurposing Drugs to Fight Hepatic Malaria Parasites.

Malaria remains one of the most prevalent infectious diseases worldwide, primarily affecting some of the most vulnerable populations around the globe. Despite achievements in the treatment of this devastating disease, there is still an urgent need for the discovery of new drugs that tackle infection by Plasmodium parasites. However, de novo drug development is a costly and time-consuming process. An alternative strategy is to evaluate the anti-plasmodial activity of compounds that are already approved for other purposes, an approach known as drug repurposing. Here, we will review efforts to assess the anti-plasmodial activity of existing drugs, with an emphasis on the obligatory and clinically silent liver stage of infection. We will also review the current knowledge on the classes of compounds that might be therapeutically relevant against Plasmodium in the context of other communicable diseases that are prevalent in regions where malaria is endemic. Repositioning existing compounds may constitute a faster solution to the current gap of prophylactic and therapeutic drugs that act on Plasmodium parasites, overall contributing to the global effort of malaria eradication.

Novel Harmicines with Improved Potency against Plasmodium.

Harmicines represent hybrid compounds composed of ß-carboline alkaloid harmine and cinnamic acid derivatives (CADs). In this paper we report the synthesis of amide-type harmicines and the evaluation of their biological activity. N-harmicines 5a-f and O-harmicines 6a-h were prepared by a straightforward synthetic procedure, from harmine-based amines and CADs using standard coupling conditions, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo [4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIEA). Amide-type harmicines exerted remarkable activity against the erythrocytic stage of P. falciparum, in low submicromolar concentrations, which was significantly more pronounced compared to their antiplasmodial activity against the hepatic stages of P. berghei. Furthermore, a cytotoxicity assay against the human liver hepatocellular carcinoma cell line (HepG2) revealed favorable selectivity indices of the most active harmicines. Molecular dynamics simulations demonstrated the binding of ligands within the ATP binding site of PfHsp90, while the calculated binding free energies confirmed higher activity of N-harmicines 5 over their O-substituted analogues 6. Amino acids predominantly affecting the binding were identified, which provided guidelines for the further derivatization of the harmine framework towards more efficient agents.

Primaquine homodimers as potential antiplasmodial and anticancer agents.

Primaquine homodimers, e.g. symmetric PQ-diamides of dicarboxylic acids containing 4 to 8 carbon atoms, were evaluated against Plasmodium berghei hepatic stages and P. falciparum blood stages, as well as against three cancer cell lines. Novel PQ-homodimers exerted much higher activity against hepatic stages, but less pronounced activity against blood stages in comparison to the parent drug. The submicromolar activity of succinic, fumaric and maleic derivatives against P. berghei was determined (IC50 values: 726.2, 198.1 and 358.4 nM, respectively). Our results indicated that the length and type of spacer between two PQ moieties highly modified the antiproliferative activities of PQ-homodimers. The general antiproliferative activity of the adipic and mesaconic derivatives against three cancer cell lines (MCF-7, HCT116, H 460) was observed (GI50 = 1.78-13.7 and 2.36-4.31 µM, respectively), but adipic derivative was less toxic to human embryonic kidney cells (HEK 293). High selectivity of fumaric and suberic derivatives against breast adenocarcinoma cell line MCF-7 was detected. These two compounds have shown no antiproliferative activity against other tumor cells and HEK 293.

Primaquine and Chloroquine Fumardiamides as Promising Antiplasmodial Agents.

This paper describes a continuation of our efforts in the pursuit of novel antiplasmodial agents with optimized properties. Following our previous discovery of biologically potent asymmetric primaquine (PQ) and halogenaniline fumardiamides (1-6), we now report their significant in vitro activity against the hepatic stages of Plasmodium parasites. Furthermore, we successfully prepared chloroquine (CQ) analogue derivatives (11-16) and evaluated their activity against both the hepatic and erythrocytic stages of Plasmodium. Our results have shown that PQ fumardiamides (1-6) exert both higher activity against P. berghei hepatic stages and lower toxicity against human hepatoma cells than the parent drug and CQ derivatives (11-16). The favourable cytotoxicity profile of the most active compounds, 5 and 6, was corroborated by assays performed on human cells (human breast adenocarcinoma (MCF-7) and non-tumour embryonic kidney cells (HEK293T)), even when glucose-6-phosphate dehydrogenase (G6PD) was inhibited. The activity of CQ fumardiamides on P. falciparum erythrocytic stages was higher than that of PQ derivatives, comparable to CQ against CQ-resistant strain PfDd2, but lower than CQ when tested on the CQ-sensitive strain Pf3D7. In addition, both sets of compounds showed favourable drug-like properties. Hence, quinoline fumardiamides could serve as a starting point towards the development of safer and more effective antiplasmodial agents.

GLUT1-mediated glucose uptake plays a crucial role during Plasmodium hepatic infection.

Intracellular pathogens have evolved mechanisms to ensure their survival and development inside their host cells. Here, we show that glucose is a pivotal modulator of hepatic infection by the rodent malaria parasite Plasmodium berghei and that glucose uptake via the GLUT1 transporter is specifically enhanced in P. berghei-infected cells. We further show that ATP levels of cells containing developing parasites are decreased, which is known to enhance membrane GLUT1 activity. In addition, GLUT1 molecules are translocated to the membrane of the hepatic cell, increasing glucose uptake at later stages of infection. Chemical inhibition of GLUT1 activity leads to a decrease in glucose uptake and the consequent impairment of hepatic infection, both in vitro and in vivo. Our results reveal that changes in GLUT1 conformation and cellular localization seem to be part of an adaptive host response to maintain adequate cellular nutrition and energy levels, ensuring host cell survival and supporting P. berghei hepatic development.

Bioluminescence Method for In Vitro Screening of Plasmodium Transmission-Blocking Compounds.

The sporogonic stage of the life cycle of Plasmodium spp., the causative agents of malaria, occurs inside the parasite's mosquito vector, where a process of fertilization, meiosis, and mitotic divisions culminates in the generation of large numbers of mammalian-infective sporozoites. Efforts to cultivate Plasmodium mosquito stages in vitro have proved challenging and yielded only moderate success. Here, we describe a methodology that simplifies the in vitro screening of much-needed transmission-blocking (TB) compounds employing a bioluminescence-based method to monitor the in vitro development of sporogonic stages of the rodent malaria parasite Plasmodium berghei Our proof-of-principle assessment of the in vitro TB activity of several commonly used antimalarial compounds identified cycloheximide, thiostrepton, and atovaquone as the most active compounds against the parasite's sporogonic stages. The TB activity of these compounds was further confirmed by in vivo studies that validated our newly developed in vitro approach to TB compound screening.

Inhibition of Plasmodium Liver Infection by Ivermectin.

Avermectins are powerful endectocides with an established potential to reduce the incidence of vector-borne diseases. Here, we show that several avermectins inhibit the hepatic stage of Plasmodium infection in vitro Notably, ivermectin potently inhibits liver infection in vivo by impairing parasite development inside hepatocytes. This impairment has a clear impact on the ensuing blood stage parasitemia, reducing disease severity and enhancing host survival. Ivermectin has been proposed as a tool to control malaria transmission because of its effects on the mosquito vector. Our study extends the effect of ivermectin to the early stages of mammalian host infection and supports the inclusion of this multipurpose drug in malaria control strategies.

Chemical attenuation of Plasmodium in the liver modulates severe malaria disease progression.

Cerebral malaria is one of the most severe complications of malaria disease, attributed to a complicated series of immune reactions in the host. The syndrome is marked by inflammatory immune responses, margination of leukocytes, and parasitized erythrocytes in cerebral vessels leading to breakdown of the blood-brain barrier. We show that chemical attenuation of the parasite at the very early, clinically silent liver stage suppresses parasite development, delays the time until parasites establish blood-stage infection, and provokes an altered host immune response, modifying immunopathogenesis and protecting from cerebral disease. The early response is proinflammatory and cell mediated, with increased T cell activation in the liver and spleen, and greater numbers of effector T cells, cytokine-secreting T cells, and proliferating, proinflammatory cytokine-producing T cells. Dendritic cell numbers, T cell activation, and infiltration of CD8(+) T cells to the brain are decreased later in infection, possibly mediated by the anti-inflammatory cytokine IL-10. Strikingly, protection can be transferred to naive animals by adoptive transfer of lymphocytes from the spleen at very early times of infection. Our data suggest that a subpopulation belonging to CD8(+) T cells as early as day 2 postinfection is responsible for protection. These data indicate that liver stage-directed early immune responses can moderate the overall downstream host immune response and modulate severe malaria outcome.