A novel multiple-stage antimalarial agent that inhibits protein synthesis.

There is an urgent need for new drugs to treat malaria, with broad therapeutic potential and novel modes of action, to widen the scope of treatment and to overcome emerging drug resistance. Here we describe the discovery of DDD107498, a compound with a potent and novel spectrum of antimalarial activity against multiple life-cycle stages of the Plasmodium parasite, with good pharmacokinetic properties and an acceptable safety profile. DDD107498 demonstrates potential to address a variety of clinical needs, including single-dose treatment, transmission blocking and chemoprotection. DDD107498 was developed from a screening programme against blood-stage malaria parasites; its molecular target has been identified as translation elongation factor 2 (eEF2), which is responsible for the GTP-dependent translocation of the ribosome along messenger RNA, and is essential for protein synthesis. This discovery of eEF2 as a viable antimalarial drug target opens up new possibilities for drug discovery.

Probability of Transmission of Malaria from Mosquito to Human Is Regulated by Mosquito Parasite Density in Naïve and Vaccinated Hosts.

Over a century since Ronald Ross discovered that malaria is caused by the bite of an infectious mosquito it is still unclear how the number of parasites injected influences disease transmission. Currently it is assumed that all mosquitoes with salivary gland sporozoites are equally infectious irrespective of the number of parasites they harbour, though this has never been rigorously tested. Here we analyse >1000 experimental infections of humans and mice and demonstrate a dose-dependency for probability of infection and the length of the host pre-patent period. Mosquitoes with a higher numbers of sporozoites in their salivary glands following blood-feeding are more likely to have caused infection (and have done so quicker) than mosquitoes with fewer parasites. A similar dose response for the probability of infection was seen for humans given a pre-erythrocytic vaccine candidate targeting circumsporozoite protein (CSP), and in mice with and without transfusion of anti-CSP antibodies. These interventions prevented infection more efficiently from bites made by mosquitoes with fewer parasites. The importance of parasite number has widespread implications across malariology, ranging from our basic understanding of the parasite, how vaccines are evaluated and the way in which transmission should be measured in the field. It also provides direct evidence for why the only registered malaria vaccine RTS,S was partially effective in recent clinical trials.

A novel model fitted to multiple life stages of malaria for assessing efficacy of transmission-blocking interventions.

BACKGROUND: Transmission-blocking interventions (TBIs) aim to eliminate malaria by reducing transmission of the parasite between the host and the invertebrate vector. TBIs include transmission-blocking drugs and vaccines that, when given to humans, are taken up by mosquitoes and inhibit parasitic development within the vector. Accurate methodologies are key to assess TBI efficacy to ensure that only the most potent candidates progress to expensive and time-consuming clinical trials. Measuring intervention efficacy can be problematic because there is substantial variation in the number of parasites in both the host and vector populations, which can impact transmission even in laboratory settings. METHODS: A statistically robust empirical method is introduced for estimating intervention efficacy from standardised population assay experiments. This method will be more reliable than simple summary statistics as it captures changes in parasite density in different life-stages. It also allows efficacy estimates at a finer resolution than previous methods enabling the impact of the intervention over successive generations to be tracked. A major advantage of the new methodology is that it makes no assumptions on the population dynamics of infection. This enables both host-to-vector and vector-to-host transmission to be density-dependent (or other) processes and generates easy-to-understand estimates of intervention efficacy. RESULTS: This method increases the precision of intervention efficacy estimates and demonstrates that relying on changes in infection prevalence (the proportion of infected hosts) alone may be insufficient to capture the impact of TBIs, which also suppress parasite density in secondarily infected hosts. CONCLUSIONS: The method indicates that potentially useful, partially effective TBIs may require multiple infection cycles before substantial reductions in prevalence are observed, despite more rapidly suppressing parasite density. Accurate models to quantify efficacy will have important implications for understanding how TBI candidates might perform in field situations and how they should be evaluated in clinical trials.

The CLIP-domain serine protease homolog SPCLIP1 regulates complement recruitment to microbial surfaces in the malaria mosquito Anopheles gambiae.

The complement C3-like protein TEP1 of the mosquito Anopheles gambiae is required for defense against malaria parasites and bacteria. Two forms of TEP1 are present in the mosquito hemolymph, the full-length TEP1-F and the proteolytically processed TEP1(cut) that is part of a complex including the leucine-rich repeat proteins LRIM1 and APL1C. Here we show that the non-catalytic serine protease SPCLIP1 is a key regulator of the complement-like pathway. SPCLIP1 is required for accumulation of TEP1 on microbial surfaces, a reaction that leads to lysis of malaria parasites or triggers activation of a cascade culminating with melanization of malaria parasites and bacteria. We also demonstrate that the two forms of TEP1 have distinct roles in the complement-like pathway and provide the first evidence for a complement convertase-like cascade in insects analogous to that in vertebrates. Our findings establish that core principles of complement activation are conserved throughout the evolution of animals.

Structure-function analysis of the Anopheles gambiae LRIM1/APL1C complex and its interaction with complement C3-like protein TEP1.

Malaria threatens half the world's population and exacts a devastating human toll. The principal malaria vector in Africa, the mosquito Anopheles gambiae, encodes 24 members of a recently identified family of leucine-rich repeat proteins named LRIMs. Two members of this family, LRIM1 and APL1C, are crucial components of the mosquito complement-like pathway that is important for immune defense against Plasmodium parasites. LRIM1 and APL1C circulate in the hemolymph exclusively as a disulfide-bonded complex that specifically interacts with the mature form of the complement C3-like protein, TEP1. We have investigated the specificity of LRIM1/APL1C complex formation and which regions of these proteins are required for interactions with TEP1. To address these questions, we have generated a set of LRIM1 and APL1C alleles altering key conserved structural elements and assayed them in cell culture for complex formation and interaction with TEP1. Our data indicate that heterocomplex formation is an intrinsic ability of LRIM1 and APL1C and identify key homologous cysteine residues forming the intermolecular disulfide bond. We also demonstrate that the coiled-coil domain is the binding site for TEP1 but also contributes to the specificity of LRIM1/APL1C complex formation. In addition, we show that the LRIM1/APL1C complex interacts with the mature forms of three other TEP proteins, one of which, TEP3, we have characterized as a Plasmodium antagonist. We conclude that LRIM1 and APL1C contain three distinct modules: a C-terminal coiled-coil domain that can carry different TEP protein cargoes, potentially with distinct functions, a central cysteine-rich region that controls complex formation and an N-terminal leucine-rich repeat with a putative role in pathogen recognition.

Fueling Open Innovation for Malaria Transmission-Blocking Drugs: Hundreds of Molecules Targeting Early Parasite Mosquito Stages.

BACKGROUND: Despite recent successes at controlling malaria, progress has stalled with an estimated 219 million cases and 435,000 deaths in 2017 alone. Combined with emerging resistance to front line antimalarial therapies in Southeast Asia, there is an urgent need for new treatment options and novel approaches to halt the spread of malaria. Plasmodium, the parasite responsible for malaria propagates through mosquito transmission. This imposes an acute bottleneck on the parasite population and transmission-blocking interventions exploiting this vulnerability are recognized as vital for malaria elimination. METHODS: 13,533 small molecules with known activity against Plasmodium falciparum asexual parasites were screened for additional transmission-blocking activity in an ex vivo Plasmodium berghei ookinete development assay. Active molecules were then counterscreened in dose response against HepG2 cells to determine their activity/cytotoxicity window and selected non-toxic representative molecules were fully profiled in a range of transmission and mosquito infection assays. Furthermore, the entire dataset was compared to other published screens of the same molecules against P. falciparum gametocytes and female gametogenesis. RESULTS: 437 molecules inhibited P. berghei ookinete formation with an IC50 < 10 µM. of which 273 showed >10-fold parasite selectivity compared to activity against HepG2 cells. Active molecules grouped into 49 chemical clusters of three or more molecules, with 25 doublets and 94 singletons. Six molecules representing six major chemical scaffolds confirmed their transmission-blocking activity against P. falciparum male and female gametocytes and inhibited P. berghei oocyst formation in the standard membrane feeding assay at 1 µM. When screening data in the P. berghei development ookinete assay was compared to published screens of the same library in assays against P. falciparum gametocytes and female gametogenesis, it was established that each assay identified distinct, but partially overlapping subsets of transmission-blocking molecules. However, selected molecules unique to each assay show transmission-blocking activity in mosquito transmission assays. CONCLUSION: The P. berghei ookinete development assay is an excellent high throughput assay for efficiently identifying antimalarial molecules targeting early mosquito stage parasite development. Currently no high throughput transmission-blocking assay is capable of identifying all transmission-blocking molecules.

Synergy in anti-malarial pre-erythrocytic and transmission-blocking antibodies is achieved by reducing parasite density.

Anti-malarial pre-erythrocytic vaccines (PEV) target transmission by inhibiting human infection but are currently partially protective. It has been posited, but never demonstrated, that co-administering transmission-blocking vaccines (TBV) would enhance malaria control. We hypothesized a mechanism that TBV could reduce parasite density in the mosquito salivary glands, thereby enhancing PEV efficacy. This was tested using a multigenerational population assay, passaging Plasmodium berghei to Anopheles stephensi mosquitoes. A combined efficacy of 90.8% (86.7-94.2%) was observed in the PEV +TBV antibody group, higher than the estimated efficacy of 83.3% (95% CrI 79.1-87.0%) if the two antibodies acted independently. Higher PEV efficacy at lower mosquito parasite loads was observed, comprising the first direct evidence that co-administering anti-sporozoite and anti-transmission interventions act synergistically, enhancing PEV efficacy across a range of TBV doses and transmission intensities. Combining partially effective vaccines of differing anti-parasitic classes is a pragmatic, powerful way to accelerate malaria elimination efforts.

Anopheles gambiae blood feeding initiates an anticipatory defense response to Plasmodium berghei.

Mosquitoes have potent innate defense mechanisms that protect them from infection by diverse pathogens. Much remains unknown about how different pathogens are sensed and specific responses triggered. Leucine-Rich repeat IMmune proteins (LRIMs) are a mosquito-specific family of putative innate receptors. Although some LRIMs have been implicated in mosquito immune responses, the function of most family members is largely unknown. We screened Anopheles gambiae LRIMs by RNAi for effects on mosquito infection by rodent malaria and found that LRIM9 is a Plasmodium berghei antagonist with phenotypes distinct from family members LRIM1 and APL1C, which are key components of the mosquito complement-like pathway. LRIM9 transcript and protein levels are significantly increased after blood feeding but are unaffected by Plasmodium or midgut microbiota. Interestingly, LRIM9 in the hemolymph is strongly upregulated by direct injection of the ecdysteroid, 20-hydroxyecdysone. Our data suggest that LRIM9 may define a novel anti-Plasmodium immune defense mechanism triggered by blood feeding and that hormonal changes may alert the mosquito to bolster its defenses in anticipation of exposure to blood-borne pathogens.