Radial spoke protein 9 is necessary for axoneme assembly in Plasmodium but not in trypanosomatid parasites.

Flagella are important for eukaryote cell motility, including in sperm, and are vital for life cycle progression of many unicellular eukaryotic pathogens. The '9+2' axoneme in most motile flagella comprises nine outer doublet and two central-pair singlet microtubules. T-shaped radial spokes protrude from the outer doublets towards the central pair and are necessary for effective beating. We asked whether there were radial spoke adaptations associated with parasite lineage-specific properties in apicomplexans and trypanosomatids. Following an orthologue search for experimentally uncharacterised radial spoke proteins (RSPs), we identified and analysed RSP9. Trypanosoma brucei and Leishmania mexicana have an extensive RSP complement, including two divergent RSP9 orthologues, necessary for flagellar beating and swimming. Detailed structural analysis showed that neither orthologue is needed for axoneme assembly in Leishmania. In contrast, Plasmodium has a reduced set of RSPs including a single RSP9 orthologue, deletion of which in Plasmodium berghei leads to failure of axoneme formation, failed male gamete release, greatly reduced fertilisation and inefficient life cycle progression in the mosquito. This indicates contrasting selection pressures on axoneme complexity, likely linked to the different mode of assembly of trypanosomatid versus Plasmodium flagella.

Reaction hijacking of tyrosine tRNA synthetase as a new whole-of-life-cycle antimalarial strategy.

Aminoacyl transfer RNA (tRNA) synthetases (aaRSs) are attractive drug targets, and we present class I and II aaRSs as previously unrecognized targets for adenosine 5'-monophosphate-mimicking nucleoside sulfamates. The target enzyme catalyzes the formation of an inhibitory amino acid-sulfamate conjugate through a reaction-hijacking mechanism. We identified adenosine 5'-sulfamate as a broad-specificity compound that hijacks a range of aaRSs and ML901 as a specific reagent a specific reagent that hijacks a single aaRS in the malaria parasite Plasmodium falciparum, namely tyrosine RS (PfYRS). ML901 exerts whole-life-cycle-killing activity with low nanomolar potency and single-dose efficacy in a mouse model of malaria. X-ray crystallographic studies of plasmodium and human YRSs reveal differential flexibility of a loop over the catalytic site that underpins differential susceptibility to reaction hijacking by ML901.

Design of proteasome inhibitors with oral efficacy in vivo against Plasmodium falciparum and selectivity over the human proteasome.

The Plasmodium falciparum proteasome is a potential antimalarial drug target. We have identified a series of amino-amide boronates that are potent and specific inhibitors of the P. falciparum 20S proteasome (Pf20S) ß5 active site and that exhibit fast-acting antimalarial activity. They selectively inhibit the growth of P. falciparum compared with a human cell line and exhibit high potency against field isolates of P. falciparum and Plasmodium vivax They have a low propensity for development of resistance and possess liver stage and transmission-blocking activity. Exemplar compounds, MPI-5 and MPI-13, show potent activity against P. falciparum infections in a SCID mouse model with an oral dosing regimen that is well tolerated. We show that MPI-5 binds more strongly to Pf20S than to human constitutive 20S (Hs20Sc). Comparison of the cryo-electron microscopy (EM) structures of Pf20S and Hs20Sc in complex with MPI-5 and Pf20S in complex with the clinically used anti-cancer agent, bortezomib, reveal differences in binding modes that help to explain the selectivity. Together, this work provides insights into the 20S proteasome in P. falciparum, underpinning the design of potent and selective antimalarial proteasome inhibitors.

Dissection-independent production of Plasmodium sporozoites from whole mosquitoes.

Progress towards a protective vaccine against malaria remains slow. To date, only limited protection has been routinely achieved following immunisation with either whole-parasite (sporozoite) or subunit-based vaccines. One major roadblock to vaccine progress, and to pre-erythrocytic parasite biology in general, is the continued reliance on manual salivary gland dissection for sporozoite isolation from infected mosquitoes. Here, we report development of a multi-step method, based on batch processing of homogenised whole mosquitoes, slurry, and density-gradient filtration, which combined with free-flow electrophoresis rapidly produces a pure, infective sporozoite inoculum. Human-infective Plasmodium falciparum and rodent-infective Plasmodium berghei sporozoites produced in this way are two- to threefold more infective than salivary gland dissection sporozoites in in vitro hepatocyte infection assays. In an in vivo rodent malaria model, the same P. berghei sporozoites confer sterile protection from mosquito-bite challenge when immunisation is delivered intravenously or 60-70% protection when delivered intramuscularly. By improving purity, infectivity, and immunogenicity, this method represents a key advancement in capacity to produce research-grade sporozoites, which should impact delivery of a whole-parasite based malaria vaccine at scale in the future.

Novel Antimalarial Tetrazoles and Amides Active against the Hemoglobin Degradation Pathway in Plasmodium falciparum.

Malaria control programs continue to be threatened by drug resistance. To identify new antimalarials, we conducted a phenotypic screen and identified a novel tetrazole-based series that shows fast-kill kinetics and a relatively low propensity to develop high-level resistance. Preliminary structure-activity relationships were established including identification of a subseries of related amides with antiplasmodial activity. Assaying parasites with resistance to antimalarials led us to test whether the series had a similar mechanism of action to chloroquine (CQ). Treatment of synchronized Plasmodium falciparum parasites with active analogues revealed a pattern of intracellular inhibition of hemozoin (Hz) formation reminiscent of CQ's action. Drug selections yielded only modest resistance that was associated with amplification of the multidrug resistance gene 1 (pfmdr1). Thus, we have identified a novel chemical series that targets the historically druggable heme polymerization pathway and that can form the basis of future optimization efforts to develop a new malaria treatment.

Actomyosin forces and the energetics of red blood cell invasion by the malaria parasite Plasmodium falciparum.

All symptoms of malaria disease are associated with the asexual blood stages of development, involving cycles of red blood cell (RBC) invasion and egress by the Plasmodium spp. merozoite. Merozoite invasion is rapid and is actively powered by a parasite actomyosin motor. The current accepted model for actomyosin force generation envisages arrays of parasite myosins, pushing against short actin filaments connected to the external milieu that drive the merozoite forwards into the RBC. In Plasmodium falciparum, the most virulent human malaria species, Myosin A (PfMyoA) is critical for parasite replication. However, the precise function of PfMyoA in invasion, its regulation, the role of other myosins and overall energetics of invasion remain unclear. Here, we developed a conditional mutagenesis strategy combined with live video microscopy to probe PfMyoA function and that of the auxiliary motor PfMyoB in invasion. By imaging conditional mutants with increasing defects in force production, based on disruption to a key PfMyoA phospho-regulation site, the absence of the PfMyoA essential light chain, or complete motor absence, we define three distinct stages of incomplete RBC invasion. These three defects reveal three energetic barriers to successful entry: RBC deformation (pre-entry), mid-invasion initiation, and completion of internalisation, each requiring an active parasite motor. In defining distinct energetic barriers to invasion, these data illuminate the mechanical challenges faced in this remarkable process of protozoan parasitism, highlighting distinct myosin functions and identifying potential targets for preventing malaria pathogenesis.

Synthesis and Structure-Activity Relationship of Dual-Stage Antimalarial Pyrazolo[3,4-b]pyridines.

Malaria remains one of the most deadly infectious diseases, causing hundreds of thousands of deaths each year, primarily in young children and pregnant mothers. Here, we report the discovery and derivatization of a series of pyrazolo[3,4-b]pyridines targeting Plasmodium falciparum, the deadliest species of the malaria parasite. Hit compounds in this series display sub-micromolar in vitro activity against the intraerythrocytic stage of the parasite as well as little to no toxicity against the human fibroblast BJ and liver HepG2 cell lines. In addition, our hit compounds show good activity against the liver stage of the parasite but little activity against the gametocyte stage. Parasitological profiles, including rate of killing, docking, and molecular dynamics studies, suggest that our compounds may target the Qo binding site of cytochrome bc1.

Peptide Probes for Plasmodium falciparum MyoA Tail Interacting Protein (MTIP): Exploring the Druggability of the Malaria Parasite Motor Complex.

Malaria remains an endemic tropical disease, and the emergence of Plasmodium falciparum parasites resistant to current front-line medicines means that new therapeutic targets are required. The Plasmodium glideosome is a multiprotein complex thought to be essential for efficient host red blood cell invasion. At its core is a myosin motor, Myosin A (MyoA), which provides most of the force required for parasite invasion. Here, we report the design and development of improved peptide-based probes for the anchor point of MyoA, the P. falciparum MyoA tail interacting protein (PfMTIP). These probes combine low nanomolar binding affinity with significantly enhanced cell penetration and demonstrable competitive target engagement with native PfMTIP through a combination of Western blot and chemical proteomics. These results provide new insights into the potential druggability of the MTIP/MyoA interaction and a basis for the future design of inhibitors.

Structure-Activity Relationship Studies of a Novel Class of Transmission Blocking Antimalarials Targeting Male Gametes.

Malaria is still a leading cause of mortality among children in the developing world, and despite the immense progress made in reducing the global burden, further efforts are needed if eradication is to be achieved. In this context, targeting transmission is widely recognized as a necessary intervention toward that goal. After carrying out a screen to discover new transmission-blocking agents, herein we report our medicinal chemistry efforts to study the potential of the most robust hit, DDD01035881, as a male-gamete targeted compound. We reveal key structural features for the activity of this series and identify analogues with greater potency and improved metabolic stability. We believe this study lays the groundwork for further development of this series as a transmission blocking agent.

A high throughput screen for next-generation leads targeting malaria parasite transmission.

Spread of parasite resistance to artemisinin threatens current frontline antimalarial therapies, highlighting the need for new drugs with alternative modes of action. Since only 0.2-1% of asexual parasites differentiate into sexual, transmission-competent forms, targeting this natural bottleneck provides a tangible route to interrupt disease transmission and mitigate resistance selection. Here we present a high-throughput screen of gametogenesis against a ~70,000 compound diversity library, identifying seventeen drug-like molecules that target transmission. Hit molecules possess varied activity profiles including male-specific, dual acting male-female and dual-asexual-sexual, with one promising N-((4-hydroxychroman-4-yl)methyl)-sulphonamide scaffold found to have sub-micromolar activity in vitro and in vivo efficacy. Development of leads with modes of action focussed on the sexual stages of malaria parasite development provide a previously unexplored base from which future therapeutics can be developed, capable of preventing parasite transmission through the population.

Hundreds of dual-stage antimalarial molecules discovered by a functional gametocyte screen.

Plasmodium falciparum stage V gametocytes are responsible for parasite transmission, and drugs targeting this stage are needed to support malaria elimination. We here screen the Tres Cantos Antimalarial Set (TCAMS) using the previously developed P. falciparum female gametocyte activation assay (Pf FGAA), which assesses stage V female gametocyte viability and functionality using Pfs25 expression. We identify over 400 compounds with activities <2 µM, chemically classified into 57 clusters and 33 singletons. Up to 68% of the hits are chemotypes described for the first time as late-stage gametocyte-targeting molecules. In addition, the biological profile of 90 compounds representing the chemical diversity is assessed. We confirm in vitro transmission-blocking activity of four of the six selected molecules belonging to three distinct scaffold clusters. Overall, this TCAMS gametocyte screen provides 276 promising antimalarial molecules with dual asexual/sexual activity, representing starting points for target identification and candidate selection.

The Actin Filament-Binding Protein Coronin Regulates Motility in Plasmodium Sporozoites.

Parasites causing malaria need to migrate in order to penetrate tissue barriers and enter host cells. Here we show that the actin filament-binding protein coronin regulates gliding motility in Plasmodium berghei sporozoites, the highly motile forms of a rodent malaria-causing parasite transmitted by mosquitoes. Parasites lacking coronin show motility defects that impair colonization of the mosquito salivary glands but not migration in the skin, yet result in decreased transmission efficiency. In non-motile sporozoites low calcium concentrations mediate actin-independent coronin localization to the periphery. Engagement of extracellular ligands triggers an intracellular calcium release followed by the actin-dependent relocalization of coronin to the rear and initiation of motility. Mutational analysis and imaging suggest that coronin organizes actin filaments for productive motility. Using coronin-mCherry as a marker for the presence of actin filaments we found that protein kinase A contributes to actin filament disassembly. We finally speculate that calcium and cAMP-mediated signaling regulate a switch from rapid parasite motility to host cell invasion by differentially influencing actin dynamics.

Quantitative analysis of Plasmodium ookinete motion in three dimensions suggests a critical role for cell shape in the biomechanics of malaria parasite gliding motility.

Motility is a fundamental part of cellular life and survival, including for Plasmodium parasites--single-celled protozoan pathogens responsible for human malaria. The motile life cycle forms achieve motility, called gliding, via the activity of an internal actomyosin motor. Although gliding is based on the well-studied system of actin and myosin, its core biomechanics are not completely understood. Currently accepted models suggest it results from a specifically organized cellular motor that produces a rearward directional force. When linked to surface-bound adhesins, this force is passaged to the cell posterior, propelling the parasite forwards. Gliding motility is observed in all three life cycle stages of Plasmodium: sporozoites, merozoites and ookinetes. However, it is only the ookinetes--formed inside the midgut of infected mosquitoes--that display continuous gliding without the necessity of host cell entry. This makes them ideal candidates for invasion-free biomechanical analysis. Here we apply a plate-based imaging approach to study ookinete motion in three-dimensional (3D) space to understand Plasmodium cell motility and how movement facilitates midgut colonization. Using single-cell tracking and numerical analysis of parasite motion in 3D, our analysis demonstrates that ookinetes move with a conserved left-handed helical trajectory. Investigation of cell morphology suggests this trajectory may be based on the ookinete subpellicular cytoskeleton, with complementary whole and subcellular electron microscopy showing that, like their motion paths, ookinetes share a conserved left-handed corkscrew shape and underlying twisted microtubular architecture. Through comparisons of 3D movement between wild-type ookinetes and a cytoskeleton-knockout mutant we demonstrate that perturbation of cell shape changes motion from helical to broadly linear. Therefore, while the precise linkages between cellular architecture and actomyosin motor organization remain unknown, our analysis suggests that the molecular basis of cell shape may, in addition to motor force, be a key adaptive strategy for malaria parasite dissemination and, as such, transmission.

Mammalian and malaria parasite cyclase-associated proteins catalyze nucleotide exchange on G-actin through a conserved mechanism.

Cyclase-associated proteins (CAPs) are among the most highly conserved regulators of actin dynamics, being present in organisms from mammals to apicomplexan parasites. Yeast, plant, and mammalian CAPs are large multidomain proteins, which catalyze nucleotide exchange on actin monomers from ADP to ATP and recycle actin monomers from actin-depolymerizing factor (ADF)/cofilin for new rounds of filament assembly. However, the mechanism by which CAPs promote nucleotide exchange is not known. Furthermore, how apicomplexan CAPs, which lack many domains present in yeast and mammalian CAPs, contribute to actin dynamics is not understood. We show that, like yeast Srv2/CAP, mouse CAP1 interacts with ADF/cofilin and ADP-G-actin through its N-terminal α-helical and C-terminal ß-strand domains, respectively. However, in the variation to yeast Srv2/CAP, mouse CAP1 has two adjacent profilin-binding sites, and it interacts with ATP-actin monomers with high affinity through its WH2 domain. Importantly, we revealed that the C-terminal ß-sheet domain of mouse CAP1 is essential and sufficient for catalyzing nucleotide exchange on actin monomers, although the adjacent WH2 domain is not required for this function. Supporting these data, we show that the malaria parasite Plasmodium falciparum CAP, which is entirely composed of the ß-sheet domain, efficiently promotes nucleotide exchange on actin monomers. Collectively, this study provides evidence that catalyzing nucleotide exchange on actin monomers via the ß-sheet domain is the most highly conserved function of CAPs from mammals to apicomplexan parasites. Other functions, including interactions with profilin and ADF/cofilin, evolved in more complex organisms to adjust the specific role of CAPs in actin dynamics.

An EGF-like protein forms a complex with PfRh5 and is required for invasion of human erythrocytes by Plasmodium falciparum.

Invasion of erythrocytes by Plasmodium falciparum involves a complex cascade of protein-protein interactions between parasite ligands and host receptors. The reticulocyte binding-like homologue (PfRh) protein family is involved in binding to and initiating entry of the invasive merozoite into erythrocytes. An important member of this family is PfRh5. Using ion-exchange chromatography, immunoprecipitation and mass spectroscopy, we have identified a novel cysteine-rich protein we have called P. falciparumRh5 interacting protein (PfRipr) (PFC1045c), which forms a complex with PfRh5 in merozoites. Mature PfRipr has a molecular weight of 123 kDa with 10 epidermal growth factor-like domains and 87 cysteine residues distributed along the protein. In mature schizont stages this protein is processed into two polypeptides that associate and form a complex with PfRh5. The PfRipr protein localises to the apical end of the merozoites in micronemes whilst PfRh5 is contained within rhoptries and both are released during invasion when they form a complex that is shed into the culture supernatant. Antibodies to PfRipr1 potently inhibit merozoite attachment and invasion into human red blood cells consistent with this complex playing an essential role in this process.

Evidence for diversifying selection on erythrocyte-binding antigens of Plasmodium falciparum and P. vivax.

Malaria parasite antigens involved in erythrocyte invasion are primary vaccine candidates. The erythrocyte-binding antigen 175K (EBA-175) of Plasmodium falciparum binds to glycophorin A on the human erythrocyte surface via an N-terminal cysteine-rich region (termed region II) and is a target of antibody responses. A survey of polymorphism in a malaria-endemic population shows that nucleotide alleles in eba-175 region II occur at more intermediate frequencies than expected under neutrality, but polymorphisms in the homologous domains of two closely related genes, eba-140 (encoding a second erythrocyte-binding protein) and psieba-165 (a putative pseudogene), show an opposite trend. McDonald-Kreitman tests employing interspecific comparison with the orthologous genes in P. reichenowi (a closely related parasite of chimpanzees) reveal a significant excess of nonsynonymous polymorphism in P. falciparum eba-175 but not in eba-140. An analysis of the Duffy-binding protein gene, encoding a major erythrocyte-binding antigen in the other common human malaria parasite P. vivax, also reveals a significant excess of nonsynonymous polymorphisms when compared with divergence from its ortholog in P. knowlesi (a closely related parasite of macaques). The results suggest that EBA-175 in P. falciparum and DBP in P. vivax are both under diversifying selection from acquired human immune responses.