Mixed alkyl/aryl phosphonates identify metabolic serine hydrolases as antimalarial targets.

Malaria, caused by Plasmodium falciparum, remains a significant health burden. One major barrier for developing antimalarial drugs is the ability of the parasite to rapidly generate resistance. We previously demonstrated that salinipostin A (SalA), a natural product, potently kills parasites by inhibiting multiple lipid metabolizing serine hydrolases, a mechanism that results in a low propensity for resistance.

Peptidic Boronic Acid SUB1 Inhibitors with Improved Selectivity over Human Proteasome.

subtilisin-like serine protease 1 (PfSUB1) is essential for egress of invasive merozoite forms of the parasite, rendering PfSUB1 an attractive antimalarial target. Here, we report studies aimed to improve drug-like properties of peptidic boronic acid PfSUB1 inhibitors including increased lipophilicity and selectivity over human proteasome (H20S). Structure-activity relationship investigations revealed that lipophilic P amino acid side chains as well as -capping groups were well tolerated in retaining PfSUB1 inhibitory potency.

The malaria parasite egress protease SUB1 is activated through precise, plasmepsin X-mediated cleavage of the SUB1 prodomain.

Background: The malaria parasite Plasmodium falciparum replicates within red blood cells, then ruptures the cell in a process called egress in order to continue its life cycle. Egress is regulated by a proteolytic cascade involving an essential parasite subtilisin-like serine protease called SUB1. Maturation of SUB1 initiates in the parasite endoplasmic reticulum with autocatalytic cleavage of an N-terminal prodomain (p31), which initially remains non-covalently bound to the catalytic domain, p54.

Activation loop phosphorylation and cGMP saturation of PKG regulate egress of malaria parasites.

The cGMP-dependent protein kinase (PKG) is the sole cGMP sensor in malaria parasites, acting as an essential signalling hub to govern key developmental processes throughout the parasite life cycle. Despite the importance of PKG in the clinically relevant asexual blood stages, many aspects of malarial PKG regulation, including the importance of phosphorylation, remain poorly understood. Here we use genetic and biochemical approaches to show that reduced cGMP binding to cyclic nucleotide binding domain B does not affect in vitro kinase activity but prevents parasite egress.

Mixed Alkyl/Aryl Phosphonates Identify Metabolic Serine Hydrolases as Antimalarial Targets.

Malaria, caused by remains a significant health burden. A barrier for developing anti-malarial drugs is the ability of the parasite to rapidly generate resistance. We demonstrated that Salinipostin A (SalA), a natural product, kills parasites by inhibiting multiple lipid metabolizing serine hydrolases, a mechanism with a low propensity for resistance.

A patatin-like phospholipase is important for mitochondrial function in malaria parasites.

For their proliferation within red blood cells, malaria parasites depend on a functional electron transport chain (ETC) within their mitochondrion, which is the target of several antimalarial drugs. Here, we have used gene disruption to identify a patatin-like phospholipase, PNPLA2, as important for parasite replication and mitochondrial function in . Parasites lacking PNPLA2 show defects in their ETC and become hypersensitive to mitochondrion-targeting drugs.

Global analysis of putative phospholipases in reveals an essential role of the phosphoinositide-specific phospholipase C in parasite maturation.

For its replication within red blood cells, the malaria parasite depends on a highly active and regulated lipid metabolism. Enzymes involved in lipid metabolic processes such as phospholipases are, therefore, potential drug targets. Here, using reverse genetics approaches, we show that only 1 out of the 19 putative phospholipases expressed in asexual blood stages of is essential for proliferation , pointing toward a high level of redundancy among members of this enzyme family.

The role of V-ATPase in vacuolar physiology and antimalarial drug uptake.

To ensure their survival in the human bloodstream, malaria parasites degrade up to 80% of the host erythrocyte hemoglobin in an acidified digestive vacuole. Here, we combine conditional reverse genetics and quantitative imaging approaches to demonstrate that the human malaria pathogen employs a heteromultimeric V-ATPase complex to acidify the digestive vacuole matrix, which is essential for intravacuolar hemoglobin release, heme detoxification, and parasite survival. We reveal an additional function of the membrane-embedded V-ATPase subunits in regulating morphogenesis of the digestive vacuole independent of proton translocation.

A malaria parasite phospholipase facilitates efficient asexual blood stage egress.

Malaria parasite release (egress) from host red blood cells involves parasite-mediated membrane poration and rupture, thought to involve membrane-lytic effector molecules such as perforin-like proteins and/or phospholipases. With the aim of identifying these effectors, we disrupted the expression of two Plasmodium falciparum perforin-like proteins simultaneously and showed that they have no essential roles during blood stage egress. Proteomic profiling of parasite proteins discharged into the parasitophorous vacuole (PV) just prior to egress detected the presence in the PV of a lecithin:cholesterol acyltransferase (LCAT; PF3D7_0629300).

A choline-releasing glycerophosphodiesterase essential for phosphatidylcholine biosynthesis and blood stage development in the malaria parasite.

The malaria parasite synthesizes significant amounts of phospholipids to meet the demands of replication within red blood cells. De novo phosphatidylcholine (PC) biosynthesis via the Kennedy pathway is essential, requiring choline that is primarily sourced from host serum lysophosphatidylcholine (lysoPC). LysoPC also acts as an environmental sensor to regulate parasite sexual differentiation.

Integrative Genetic Manipulation of Plasmodium cynomolgi Reveals Multidrug Resistance-1 Y976F Associated With Increased In Vitro Susceptibility to Mefloquine.

The lack of a long-term in vitro culture method has severely restricted the study of Plasmodium vivax, in part because it limits genetic manipulation and reverse genetics. We used the recently optimized Plasmodium cynomolgi Berok in vitro culture model to investigate the putative P. vivax drug resistance marker MDR1 Y976F.

Subtilisin-like Serine Protease 1 (SUB1) as an Emerging Antimalarial Drug Target: Current Achievements in Inhibitor Discovery.

Widespread resistance to many antimalarial therapies currently in use stresses the need for the discovery of new classes of drugs with new modes of action. The subtilisin-like serine protease SUB1 controls egress of malaria parasites (merozoites) from the parasite-infected red blood cell. As such, SUB1 is considered a prospective target for drugs designed to interrupt the asexual blood stage life cycle of the malaria parasite.

CDC50 Orthologues in Plasmodium falciparum Have Distinct Roles in Merozoite Egress and Trophozoite Maturation.

In model organisms, type IV ATPases (P4-ATPases) require cell division control protein 50 (CDC50) chaperones for their phospholipid flipping activity. In the malaria parasite Plasmodium falciparum, guanylyl cyclase alpha (GCα) is an integral membrane protein that is essential for release (egress) of merozoites from their host erythrocytes. GCα is unusual in that it contains both a C-terminal cyclase domain and an N-terminal P4-ATPase domain of unknown function.

Synchrony between daily rhythms of malaria parasites and hosts is driven by an essential amino acid.

Rapid asexual replication of blood stage malaria parasites is responsible for the severity of disease symptoms and fuels the production of transmission forms. Here, we demonstrate that a  schedule for asexual replication can be orchestrated by isoleucine, a metabolite provided to the parasite in a periodic manner due to the host's rhythmic intake of food. We infect female C57BL/6 and Per1/2-null mice which have a disrupted canonical (transcription translation feedback loop, TTFL) clock with 1×10 red blood cells containing (DK genotype).

Peptidic boronic acids are potent cell-permeable inhibitors of the malaria parasite egress serine protease SUB1.

Malaria is a devastating infectious disease, which causes over 400,000 deaths per annum and impacts the lives of nearly half the world's population. The causative agent, a protozoan parasite, replicates within red blood cells (RBCs), eventually destroying the cells in a lytic process called egress to release a new generation of parasites. These invade fresh RBCs to repeat the cycle.

Autocatalytic activation of a malarial egress protease is druggable and requires a protein cofactor.

Malaria parasite egress from host erythrocytes (RBCs) is regulated by discharge of a parasite serine protease called SUB1 into the parasitophorous vacuole (PV). There, SUB1 activates a PV-resident cysteine protease called SERA6, enabling host RBC rupture through SERA6-mediated degradation of the RBC cytoskeleton protein β-spectrin. Here, we show that the activation of Plasmodium falciparum SERA6 involves a second, autocatalytic step that is triggered by SUB1 cleavage.

Ca signals critical for egress and gametogenesis in malaria parasites depend on a multipass membrane protein that interacts with PKG.

Calcium signaling regulated by the cGMP-dependent protein kinase (PKG) controls key life cycle transitions in the malaria parasite. However, how calcium is mobilized from intracellular stores in the absence of canonical calcium channels in is unknown. Here, we identify a multipass membrane protein, ICM1, with homology to transporters and calcium channels that is tightly associated with PKG in both asexual blood stages and transmission stages.

Malaria Parasite Schizont Egress Antigen-1 Plays an Essential Role in Nuclear Segregation during Schizogony.

Malaria parasites cause disease through repeated cycles of intraerythrocytic proliferation. Within each cycle, several rounds of DNA replication produce multinucleated forms, called schizonts, that undergo segmentation to form daughter merozoites. Upon rupture of the infected cell, the merozoites egress to invade new erythrocytes and repeat the cycle.

Malaria parasite egress at a glance.

All intracellular pathogens must escape (egress) from the confines of their host cell to disseminate and proliferate. The malaria parasite only replicates in an intracellular vacuole or in a cyst, and must undergo egress at four distinct phases during its complex life cycle, each time disrupting, in a highly regulated manner, the membranes or cyst wall that entrap the parasites. This Cell Science at a Glance article and accompanying poster summarises our current knowledge of the morphological features of egress across the life cycle, the molecular mechanisms that govern the process, and how researchers are working to exploit this knowledge to develop much-needed new approaches to malaria control.

Guanylyl Cyclase-Alpha and the Activity of Its Appended P4-ATPase Domain Are Essential for cGMP Synthesis and Blood-Stage Egress.

Guanylyl cyclases (GCs) synthesize cyclic GMP (cGMP) and, together with cyclic nucleotide phosphodiesterases, are responsible for regulating levels of this intracellular messenger which mediates myriad functions across eukaryotes. In malaria parasites (), as well as their apicomplexan and ciliate relatives, GCs are associated with a P4-ATPase-like domain in a unique bifunctional configuration. P4-ATPases generate membrane bilayer lipid asymmetry by translocating phospholipids from the outer to the inner leaflet.

The malaria parasite sheddase SUB2 governs host red blood cell membrane sealing at invasion.

Red blood cell (RBC) invasion by malaria merozoites involves formation of a parasitophorous vacuole into which the parasite moves. The vacuole membrane seals and pinches off behind the parasite through an unknown mechanism, enclosing the parasite within the RBC. During invasion, several parasite surface proteins are shed by a membrane-bound protease called SUB2.

cAMP signalling and its role in host cell invasion by malaria parasites.

Cyclic adenosine monophosphate (cAMP) is an important signalling molecule across evolution, but until recently there was little information on its role in malaria parasites. Advances in gene editing - in particular conditional genetic approaches and mass spectrometry have paved the way for characterisation of the key components of the cAMP signalling pathway in malaria parasites. This has revealed that cAMP signalling plays a critical role in invasion of host red blood cells by Plasmodium falciparum merozoites through regulating the phosphorylation of key parasite proteins by the cAMP-dependent protein kinase (PKA).