With 429 000 deaths per year, malaria remains the most devastating parasitic disease for humans. It is caused by Plasmodium parasites and transmitted by Anopheles mosquitoes. The efficiency of artemisinin-based combination therapies (ACTs), the spearhead of malarial treatments, is now threatened by the appearance and spreading of artemisinin-resistant parasites. Moreover, the development of control strategies to block parasite transmission is a priority of WHO. In this project, we propose to study the mode of action of two antimalarial drugs, primaquine (PQ) and plasmodione (PD) that are active against gametocytes, the parasite stages responsible for human to mosquito transmission. PQ is the only available antimalarial medicine with established activity against mature gametocytes and is currently under intense clinical validation for widespread use in combination with ACTs. PD and derivatives are new early leads displaying fast-acting antimalarial activity and potent transmission-blocking properties. These drugs kill parasites most likely through pleiotropic redox-mediated mechanisms that remain poorly understood. While having distinct bioactivations, some of their modes of action seem to share common features. Indeed, while PQ is transformed by human cytochromes cytP450 into presumably highly active hydroxylated metabolites, the antimalarial activity of PD comes largely from its specific bioactivation within the infected erythrocytes and subsequent redox cycling properties. The existence of putative targets proteins for these compounds also remains an opened question. To decipher their complex mode of action, we propose to set up a multidisciplinary approach combining different expertise in organic synthesis, chemical proteomics, biochemistry, cell biology, and genetics, and to use the yeast as a model in parallel to Plasmodium studies. Our project has three main objectives: 1) to monitor the oxidative damages caused by PQ/PD, 2) to identify target proteins and redox enzymes controlling drug sensitivity and resistance in yeast and parasites, 3) to confirm the contribution of these candidate genes to drug sensitivity and resistance in Plasmodium. We expect that this proposal will allow us to shed light and understand the biochemical pathways and genes implicated in the modes of action of these antimalarials, and will allow their drug targeting as essential components in asexual and sexual parasites, responsible for malaria physiopathology and transmission, respectively.

Mosquito vectors of human disease are still responsible for the death of about 750,000 people annually. Genetic resistance to insecticides is spreading in mosquito populations, new arthropod-borne viral diseases emerge continuously and drugs against malaria stimulate the emergence of drug-resistant parasites. In our fight against mosquito-borne disease, the gene drive technology shows great promise to complement existing vector control methods. Gene drives are transgenic constructs designed to invade populations of a target species, based on the activity of molecular scissors such as the CRISPR/Cas9 system. Gene drive could be deployed either for population suppression, which may cause a loss in biodiversity and ecosystem imbalance, or for genetic modification at the scale of a species. In confined laboratory conditions, we will develop gene drive constructs carrying anti-pathogen molecules to render Anopheles gambiae and Aedes aegypti mosquitoes unable to transmit malaria parasites and viruses (dengue, Zika). We will optimize and test several innovative gene drive approaches, with particular attention to avoid the emergence of drive-refractory mosquitoes: multiplex drives; anti-Plasmodium dual-effect drive; drive-or-die constructs; indirect drives. We will monitor the efficiency of each of these strategies in caged mosquitoes, the possible undesired effects arising from on and off-target mutations induced by the drive, develop “recall strains” able to block the drive, and test the constructs’ propensity for horizontal transfer towards other insect species and microorganisms. Project partners have complementary expertise in Anopheles and Aedes biology, in gene engineering and in mosquito infection assays with human pathogens. This project aims both to optimize gene drive and to address some of the problems and concerns raised by this research field. We hope it will foster the emergence of alternative approaches to insecticide-based control interventions, which are reaching their limits.

Malaria (MAL) and schistosomiasis (SCH) are infectious diseases of poverty of global importance. No vaccines are available and new chemotypes are urgently needed for the development of drugs to fight those two diseases. For MAL, caused by Plasmodium spp. parasites and transmitted by Anopheles spp. mosquitoes, the problem is the spread of resistance to the currently available drugs, including artemisinin-based therapies. Female genital SCH caused by Schistosoma spp, is one of the most common gynecologic conditions of women who live in poverty in Africa and has emerged as one of Africa's most important cofactors in its AIDS epidemic. For SCH, only one drug, praziquantel, is available; but resistant parasites jeopardize the efficacy of cure rates at high infection intensities. To overcome these gaps, we have established a French-Swiss academic consortium, ROSaction with complementary expertise, which includes chemistry, parasitology and yeast genetics, with the aim of developing preclinical candidates of the 3-benzylmenadione (bMD) series: plasmodione (against MAL) and schistodiones (against SCH). Depending on their chemical substitution pattern, bMDs have highly specific and potent antiparasitic activities on Plasmodium spp., or Schistosoma mansoni worms, both in vitro and in vivo, with no obvious signs of toxicity in mice and in G6PD-deficient red blood cells. The goal of this application is to optimize the discovered early bMD-based leads for each disease model, to improve their pharmacokinetic properties, to validate the respective antiparasitic properties of the optimized compounds in vivo, and to identify the sensitive parasite stages, the drug modes of action and their protein targets. Mode of action studies conducted by the French teams with P. falciparum and Saccharomyces cerevisiae have indicated that the bMD derivatives are substrates of mitochondrial NADH-dehydrogenases, and that the chemical reduction of the bMD derivatives initiates a redox cycling process that results in the production of reactive oxygen species (ROS). The Swiss applicants of the project have established a strong expertise in antiparasitic drug efficacy testing. They have developed various in vitro and in vivo models for Plasmodium and S. mansoni, which are being used in hit discovery and hit-to-lead development of new antiparasitic agents. The proposed research program is divided into three work packages (WP). In WP1, we will synthesize novel 3-benzylmenadione analogues of plasmodione and schistodiones with improved antiparasitic activitiy, selectivity, and pharmacokinetics, and a second generation of activity-based protein profiling (ABPP) probes based on the 3-bMD scaffold. In WP2, the antiparasitic properties and selectivity profile of the molecules synthesized in WP1 will be assessed on P. falciparum and S. mansoni. The results will guide further optimization of the compounds (WP1) and enable mechanistic studies that will be performed in WP3. In WP3, we will investigate the mode of action of the antimalarial plasmodione and search for drug targets and possible mechanism of resistance. Using high throughput approaches in the yeast model, we will identify new targets by extending our screens for PD-resistant and hypersensitive yeast mutants. In parallel, activity-based protein profiling (ABPP) probes (synthesized in WP1) will enable us to capture and identify plasmodione targets in P. falciparum. Candidate drug targets and resistance genes/mutations will be validated in the parasite by genetic constructions. We will study the mode of action of schistodiones using yeast as a model and on the basis of the knowledge acquired in Plasmodium. We aim at providing a molecular understanding on how bMDs kill parasites and what is the basis of their selectivity.

Dengue viruses (DENV-1, -2, -3 and -4) and their main mosquito vector Aedes aegypti are responsible for the most rapidly advancing arboviral disease of humans. Mosquito susceptibility to DENV infection is a quantitative trait that varies between and within species, however the genes that make mosquitoes resistant or susceptible to DENV in nature have remained elusive. This project aims to elucidate the genetic basis of DENV resistance/susceptibility in Ae. aegypti using genome-wide gene mapping and functional validation by genetic engineering. It will take advantage of the natural resistance phenotype of a wild-type Ae. aegypti strain recently isolated from Bakoumba (Gabon). The Bakoumba strain displays strong resistance against DENV-1 and intermediate resistance against DENV-2 and DENV-4. We propose to carry out a genetic cross between the Bakoumba strain and a DENV-1 susceptible strain from Cairns (Australia) to map the underlying genomic loci. Our specific aims are to (1) identify mosquito candidate genes by genome-wide genetic mapping and (2) functionally validate the most promising candidate genes by genetic engineering in vivo. Our genetic mapping strategy is based on bulk segregant analysis, whereby individuals from a segregating population are genotyped in large pools with contrasted phenotypes (resistant vs. susceptible). In a pilot study, we obtained preliminary evidence that segregating single-nucleotide polymorphisms associated with DENV-1 infection can be detected at the gene level in a laboratory cross between the Bakoumba and Cairns strains. We will use reverse genetics to functionally test the candidate genes in the Bakoumba and Cairns genetic backgrounds. Our approach of reverse genetics includes transgenic overexpression and CRISPR/Cas9-mediated swapping of putative resistance/susceptible alleles between their parental genetic backgrounds of origin. Unlike gene knockout or gene knockdown, our reverse genetics strategy will be able to capture allelic effects. Moreover, it will be possible to extend the functional characterization of the allelic variants identified with DENV-1 to the other DENV types. Partners of the project combine expertise in quantitative genetics and mosquito transgenesis, respectively. Overall, this project will shed light on the molecular determinants of mosquito vector competence for arboviruses by identifying and characterizing novel restriction factors and antiviral mechanisms. In addition, it will contribute to expand the molecular toolbox to genetically engineer mosquitoes. Ultimately, results from the project will support the development of novel entomological strategies to fight emerging arboviral diseases.

The tiger mosquito Aedes albopictus can vector dengue, Zika and chikungunya viruses and is highly invasive in France and the world. Current vector control techniques fail to contain its expansion, as insecticides cannot reach all larval breeding sites, favor the emergence of genetic resistance, while being noxious to biodiversity and human health. We have established several transgenic lines that ectopically express the Nix gene, which converts females into functional males. The genetic construct also comprises a docking site in which additional transgenes can be inserted. We propose to exploit this collection of docking sites by adding components of the CRISPR/Cas9 system to (i) confer to Nix a gene drive property, in order to massively convert mosquito females into inoffensive males, with the aim to suppress invasive populations; (ii) associate Nix to a female sterilizing system independent of masculinisation and effective in subsequent generations; (iii) combine the two processes to prevent the emergence of mutant females that would be genetically resistant to the transgenes’ masculinizing effects. This project is expected to provide a new vector control tool specifically targeting Aedes albopictus, without collateral damage on the rest of the environment. It pertains to a preparation strategy to face the threat of future epidemics favored by the expansion and proliferation of this important vector of viruses.