Discovery of spirooxadiazoline oxindoles with dual-stage antimalarial activity.

Malaria remains a prevalent infectious disease in developing countries. The first-line therapeutic options are based on combinations of fast-acting artemisinin derivatives and longer-acting synthetic drugs. However, the emergence of resistance to these first-line treatments represents a serious risk, and the discovery of new effective drugs is urgently required. For this reason, new antimalarial chemotypes with new mechanisms of action, and ideally with activity against multiple parasite stages, are needed. We report a new scaffold with dual-stage (blood and liver) antiplasmodial activity. Twenty-six spirooxadiazoline oxindoles were synthesized and screened against the erythrocytic stage of the human malaria parasite P. falciparum. The most active compounds were also tested against the liver-stage of the murine parasite P. berghei. Seven compounds emerged as dual-stage antimalarials, with IC50 values in the low micromolar range. Due to structural similarity with cipargamin, which is thought to inhibit blood-stage P. falciparum growth via inhibition of the Na + efflux pump PfATP4, we tested one of the most active compounds for anti-PfATP4 activity. Our results suggest that this target is not the primary target of spirooxadiazoline oxindoles and further studies are ongoing to identify the main mechanism of action of this scaffold.

Excreted Trypanosoma brucei proteins inhibit Plasmodium hepatic infection.

Malaria, a disease caused by Plasmodium parasites, remains a major threat to public health globally. It is the most common disease in patients with sleeping sickness, another parasitic illness, caused by Trypanosoma brucei. We have previously shown that a T. brucei infection impairs a secondary P. berghei liver infection and decreases malaria severity in mice. However, whether this effect requires an active trypanosome infection remained unknown. Here, we show that Plasmodium liver infection can also be inhibited by the serum of a mouse previously infected by T. brucei and by total protein lysates of this kinetoplastid. Biochemical characterisation showed that the anti-Plasmodium activity of the total T. brucei lysates depends on its protein fraction, but is independent of the abundant variant surface glycoprotein. Finally, we found that the protein(s) responsible for the inhibition of Plasmodium infection is/are present within a fraction of ~350 proteins that are excreted to the bloodstream of the host. We conclude that the defence mechanism developed by trypanosomes against Plasmodium relies on protein excretion. This study opens the door to the identification of novel antiplasmodial intervention strategies.

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.

African trypanosomes expressing multiple VSGs are rapidly eliminated by the host immune system.

Trypanosoma brucei parasites successfully evade the host immune system by periodically switching the dense coat of variant surface glycoprotein (VSG) at the cell surface. Each parasite expresses VSGs in a monoallelic fashion that is tightly regulated. The consequences of exposing multiple VSGs during an infection, in terms of antibody response and disease severity, remain unknown. In this study, we overexpressed a high-mobility group box protein, TDP1, which was sufficient to open the chromatin of silent VSG expression sites, to disrupt VSG monoallelic expression, and to generate viable and healthy parasites with a mixed VSG coat. Mice infected with these parasites mounted a multi-VSG antibody response, which rapidly reduced parasitemia. Consequently, we observed prolonged survival in which nearly 90% of the mice survived a 30-d period of infection with undetectable parasitemia. Immunodeficient RAG2 knock-out mice were unable to control infection with TDP1-overexpressing parasites, showing that the adaptive immune response is critical to reducing disease severity. This study shows that simultaneous exposure of multiple VSGs is highly detrimental to the parasite, even at the very early stages of infection, suggesting that drugs that disrupt VSG monoallelic expression could be used to treat trypanosomiasis.

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.

Coupling the cell-penetrating peptides transportan and transportan 10 to primaquine enhances its activity against liver-stage malaria parasites.

Novel primaquine-cell penetrating peptide conjugates were synthesised and tested in vitro against liver stage Plasmodium berghei parasites. Generally, the conjugates were more active than the parent peptides and, in some cases, than the parent drug. These are unprecedented findings that may open a new route towards antimalarial drug rescuing.

Multistage Antiplasmodium Activity of Astemizole Analogues and Inhibition of Hemozoin Formation as a Contributor to Their Mode of Action.

A drug repositioning approach was leveraged to derivatize astemizole (AST), an antihistamine drug whose antimalarial activity was previously identified in a high-throughput screen. The multistage activity potential against the Plasmodium parasite's life cycle of the subsequent analogues was examined by evaluating against the parasite asexual blood, liver, and sexual gametocytic stages. In addition, the previously reported contribution of heme detoxification to the compound's mode of action was interrogated. Ten of the 17 derivatives showed half-maximal inhibitory concentrations (IC50s) of <0.1 µM against the chloroquine (CQ)-sensitive Plasmodium falciparum NF54 ( PfNF54) strain while maintaining submicromolar potency against the multidrug-resistant strain, PfK1, with most showing low likelihood of cross-resistance with CQ. Selected analogues ( PfNF54-IC50 < 0.1 µM) were tested for cytotoxicity on Chinese hamster ovarian (CHO) cells and found to be highly selective (selectivity index > 100). Screening of AST and its analogues against gametocytes revealed their moderate activity (IC50: 1-5 µM) against late stage P. falciparum gametocytes, while the evaluation of activity against P. berghei liver stages identified one compound (3) with 3-fold greater activity than the parent AST compound. Mechanistic studies showed a strong correlation between in vitro inhibition of ß-hematin formation by the AST derivatives and their antiplasmodium IC50s. Analyses of intracellular inhibition of hemozoin formation within the parasite further yielded signatures attributable to a possible perturbation of the heme detoxification machinery.

Structure-Activity Relationship Studies and Plasmodium Life Cycle Profiling Identifies Pan-Active N-Aryl-3-trifluoromethyl Pyrido[1,2- a]benzimidazoles Which Are Efficacious in an in Vivo Mouse Model of Malaria.

Structure-activity relationship studies involving N-aryl-3-trifluoromethyl pyrido[1,2- a]benzimidazoles (PBI) identified several compounds possessing potent in vitro activities against the asexual blood, liver, and gametocyte stages of the Plasmodium parasite with no cross-resistance to chloroquine. Frontrunner lead compounds with good in vitro absorption, distribution, metabolism, and excretion (ADME) profiles were subjected to in vivo proof-of-concept studies in NMRI mice harboring the rodent P. berghei infection. This led to the identification of compounds 10 and 49, effecting 98% and 99.93% reduction in parasitemia with mean survival days of 12 and 14, respectively, at an oral dose of 4 × 50 mg/kg. In vivo pharmacokinetics studies on 10 revealed slow absorption, low volume of distribution, and low clearance profiles. Furthermore, this series displayed a low propensity to inhibit the human ether-a-go-go-related gene (hERG) potassium ion channel whose inhibition is associated with cardiotoxicity.

Endoperoxide-8-aminoquinoline hybrids as dual-stage antimalarial agents with enhanced metabolic stability.

Hybrid compounds may play a critical role in the context of the malaria eradication agenda, which will benefit from therapeutic tools active against the symptomatic erythrocytic stage of Plasmodium infection, and also capable of eliminating liver stage parasites. To address the need for efficient multistage antiplasmodial compounds, a small library of 1,2,4,5-tetraoxane-8- aminoquinoline hybrids, with the metabolically labile C-5 position of the 8-aminoquinoline moiety blocked with aryl groups, was synthesized and screened for antiplasmodial activity and metabolic stability. The hybrid compounds inhibited development of intra-erythrocytic forms of the multidrug-resistant Plasmodium falciparum W2 strain, with EC50 values in the nM range, and with low cytotoxicity against mammalian cells. The compounds also inhibited the development of P. berghei liver stage parasites, with the most potent compounds displaying EC50 values in the low µM range. SAR analysis revealed that unbranched linkers between the endoperoxide and 8-aminoquinoline pharmacophores are most beneficial for dual antiplasmodial activity. Importantly, hybrids were significantly more potent than a 1:1 mixture of 8-aminoquinoline-tetraoxane, highlighting the superiority of the hybrid approach over the combination therapy. Furthermore, aryl substituents at C-5 of the 8-aminoquinoline moiety improve the compounds' metabolic stability when compared with their primaquine (i.e. C-5 unsubstituted) counterparts. Overall, this study reveals that blocking the quinoline C-5 position does not result in loss of dual-stage antimalarial activity, and that tetraoxane-8- aminoquinoline hybrids are an attractive approach to achieve elimination of exo- and intraerythrocytic parasites, thus with the potential to be used in malaria eradication campaigns.

Sleeping sickness is a circadian disorder.

Sleeping sickness is a fatal disease caused by Trypanosoma brucei, a unicellular parasite that lives in the bloodstream and interstitial spaces of peripheral tissues and the brain. Patients have altered sleep/wake cycles, body temperature, and endocrine profiles, but the underlying causes are unknown. Here, we show that the robust circadian rhythms of mice become phase advanced upon infection, with abnormal activity occurring during the rest phase. This advanced phase is caused by shortening of the circadian period both at the behavioral level as well as at the tissue and cell level. Period shortening is T. brucei specific and independent of the host immune response, as co-culturing parasites with explants or fibroblasts also shortens the clock period, whereas malaria infection does not. We propose that T. brucei causes an advanced circadian rhythm disorder, previously associated only with mutations in clock genes, which leads to changes in the timing of sleep.

Inhibition of Plasmodium Hepatic Infection by Antiretroviral Compounds.

Recent WHO guidelines on control of human immunodeficiency virus (HIV) call for the widespread use of antiretroviral (AR) therapy (ART) for people living with HIV. Given the considerable overlap between infections by HIV and Plasmodium, the causative agent of malaria, it is important to understand the impact of AR compounds and ART regimens on infections by malaria parasites. We undertook a systematic approach to identify AR drugs and ART drug combinations with inhibitory activity against the obligatory hepatic stage of Plasmodium infection. Our in vitro screen of a wide array of AR drugs identified the non-nucleoside reverse transcriptase inhibitors efavirenz and etravirine (ETV), and the protease inhibitor nelfinavir, as compounds that significantly impair the development of the rodent malaria parasite P. berghei in an hepatoma cell line. Furthermore, we show that WHO-recommended ART drug combinations currently employed in the field strongly inhibit Plasmodium liver infection in mice, an effect that may be significantly enhanced by the inclusion of ETV in the treatment. Our observations are the first report of ETV as an anti-Plasmodial drug, paving the way for further evaluation and potential use of ETV-containing ARTs in regions of geographical overlap between HIV and Plasmodium infections.

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.