A novel validated assay to support the discovery of new anti-malarial gametocytocidal agents.

BACKGROUND: Drugs that kill or inhibit Plasmodium gametocytes in the human host could potentially synergize the impact of other chemotherapeutic interventions by blocking transmission. To develop such agents, reliable methods are needed to study the in vitro activity of compounds against gametocytes. This study describes a novel assay for characterizing the activity of anti-malarial drugs against the later stages of Plasmodium falciparum gametocyte development using real-time PCR (qPCR). METHODS: Genes previously reported to be transcribed at the different sexual stages of the gametocytogenesis were selected for study and their mRNA expression was measured in a gametocytogenesis course by qPCR. Genes mainly expressed in the later stages of gametocyte development were used as a surrogate measurement of drug activity. To distinguish between cidal and static drug effects, two different experiments were performed in parallel, one with constant drug pressure throughout the experiment (144 h), and another in which the gametocyte cultures were exposed to the compound for only 48 h. RESULTS: Four P. falciparum genes coding for proteins Pf77, ROM3, Pfs25, and Pfg377 with transcription specific for late-stage gametocyte development were identified. The in vitro anti-malarial activity of compounds against such gametocytes was assessed by measuring mRNA levels of these genes using qPCR. The assay was validated against standard anti-malarial drugs (epoxomicin, dihydroartemisinin, chloroquine, thiostrepton, and methylene blue) and compounds from the GSK compound library with known anti-gametocyte activity. CONCLUSIONS: This study describes a novel assay for characterizing the activity of anti-malarial drugs against the later stages of P. falciparum gametocyte development using qPCR in genetically unmodified parasites. The method described is a reliable and user-friendly technique with a medium throughput that could be easily implemented in any laboratory.

Transmission-Blocking Potential of MEFAS, a Hybrid Compound Derived from Artesunate and Mefloquine.

Most antimalarial drugs target asexual parasites without reducing gametocyte formation or development. Drugs with dual roles, i.e., those that can target both asexual parasites and gametocytes, would improve the control of malaria. In the current study, MEFAS, a hybrid drug derived from mefloquine and artesunate that has been shown to be an active blood schizonticidal drug, was assessed to determine its ability to block the infectivity of Plasmodium falciparum gametocytes. MEFAS was 280 and 15 times more effective than mefloquine alone and artesunate alone, respectively.

A New Set of Chemical Starting Points with Plasmodium falciparum Transmission-Blocking Potential for Antimalarial Drug Discovery.

The discovery of new antimalarials with transmission blocking activity remains a key issue in efforts to control malaria and eventually eradicate the disease. Recently, high-throughput screening (HTS) assays have been successfully applied to Plasmodium falciparum asexual stages to screen millions of compounds, with the identification of thousands of new active molecules, some of which are already in clinical phases. The same approach has now been applied to identify compounds that are active against P. falciparum gametocytes, the parasite stage responsible for transmission. This study reports screening results for the Tres Cantos Antimalarial Set (TCAMS), of approximately 13,533 molecules, against P. falciparum stage V gametocytes. Secondary confirmation and cytotoxicity assays led to the identification of 98 selective molecules with dual activity against gametocytes and asexual stages. Hit compounds were chemically clustered and analyzed for appropriate physicochemical properties. The TCAMS chemical space around the prioritized hits was also studied. A selection of hit compounds was assessed ex vivo in the standard membrane feeding assay and demonstrated complete block in transmission. As a result of this effort, new chemical structures not connected to previously described antimalarials have been identified. This new set of compounds may serve as starting points for future drug discovery programs as well as tool compounds for identifying new modes of action involved in malaria transmission.

Red blood cells derived from peripheral blood and bone marrow CD34⁺ human haematopoietic stem cells are permissive to Plasmodium parasites infection.

The production of fully functional human red cells in vitro from haematopoietic stem cells (hHSCs) has been successfully achieved. Recently, the use of hHSCs from cord blood represented a major improvement to develop the continuous culture system for Plasmodium vivax. Here, we demonstrated that CD34⁺ hHSCs from peripheral blood and bone marrow can be expanded and differentiated to reticulocytes using a novel stromal cell. Moreover, these reticulocytes and mature red blood cells express surface markers for entrance of malaria parasites contain adult haemoglobin and are also permissive to invasion by P. vivax and Plasmodium falciparum parasites.

Activity of clinically relevant antimalarial drugs on Plasmodium falciparum mature gametocytes in an ATP bioluminescence "transmission blocking" assay.

BACKGROUND: Current anti-malarial drugs have been selected on the basis of their activity against the symptom-causing asexual blood stage of the parasite. Which of these drugs also target gametocytes, in the sexual stage responsible for disease transmission, remains unknown. Blocking transmission is one of the main strategies in the eradication agenda and requires the identification of new molecules that are active against gametocytes. However, to date, the main limitation for measuring the effect of molecules against mature gametocytes on a large scale is the lack of a standardized and reliable method. Here we provide an efficient method to produce and purify mature gametocytes in vitro. Based on this new procedure, we developed a robust, affordable, and sensitive ATP bioluminescence-based assay. We then assessed the activity of 17 gold-standard anti-malarial drugs on Plasmodium late stage gametocytes. METHODS AND FINDINGS: Difficulties in producing large amounts of gametocytes have limited progress in the development of malaria transmission blocking assays. We improved the method established by Ifediba and Vanderberg to obtain viable, mature gametocytes en masse, whatever the strain used. We designed an assay to determine the activity of antimalarial drugs based on the intracellular ATP content of purified stage IV-V gametocytes after 48 h of drug exposure in 96/384-well microplates. Measurements of drug activity on asexual stages and cytotoxicity on HepG2 cells were also obtained to estimate the specificity of the active drugs. CONCLUSIONS: The work described here represents another significant step towards determination of the activity of new molecules on mature gametocytes of any strain with an automated assay suitable for medium/high-throughput screening. Considering that the biology of the forms involved in the sexual and asexual stages is very different, a screen of our 2 million-compound library may allow us to discover novel anti-malarial drugs to target gametocyte-specific metabolic pathways.

Design, synthesis, and in vitro activity of novel 2'-O-substituted 15-membered azalides.

Malaria remains one of the most widespread human infectious diseases, and its eradication will largely depend on antimalarial drug discovery. Here, we present a novel approach to the development of the azalide class of antimalarials by describing the design, synthesis, and characterization of novel 2'-O-substituted-9-deoxo-9a-methyl-9a-aza-9a-homoerythromycin A derivatives consisting of different quinoline moieties covalently liked to a 15-membered azalide scaffold at position 2'. By multistep straightforward synthesis, 19 new, stable, and soluble compounds were created and biologically profiled. Most active compounds from the 4-amino-7-chloroquinoline series showed high selectivity for P. falciparum parasites, and in vitro antimalarial activity improved 1000-fold over azithromycin. Antimalarial potency was equivalent to chloroquine against the sensitive strain (3D7A) and up to 48-fold enhanced over chloroquine against the chloroquine-resistant strain (W2). Concurrently, the antibacterial activity of the compounds was eliminated, thus facilitating the development of malaria-specific macrolide agents.

Characterization of the phospholipidogenic potential of 4(1H)-pyridone antimalarial derivatives.

Drug-induced phospholipidosis (PLD) is characterized by the excessive accumulation of phospholipids in lysosomes. It is accompanied by intracellular retention of drug that could be associated with increased cytotoxicity. Drug-induced PLD is recognized as a significant challenge for drug development, depending on the severity of the effect it could be reversible or caused cell death. Therefore, the identification at early stages of drug discovery of the potential to induce PLD can be advantageous for selecting improved development candidates. PLD has commonly been associated with cationic amphiphilic drugs (CADs) composed by a hydrophobic ring structure and a hydrophilic side chain with a charged amine group. 4(1H)-pyridone derivatives are a family of antimalarial agents that act as potent selective inhibitors of Plasmodium falciparum mitochondrial function and according to their chemical structure might be considered to be CADs. In the present study, the potential of 4(1H)-pyridone derivatives to induce PLD in vitro and their general cytotoxicity properties were investigated. A cell-based fluorescence assay using the fluorescent phospholipid probe NBD-PE [N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt] was established. Five PLD-inducing reference compounds and six negative reference compounds were evaluated in vitro in HepG2 cell line. The pyridones tested were ranked by using a chloroquine-equivalent scale (chloroquine constituting a well-known antimalarial drug that acts as a potent inducer of lysosomal storage of phospholipids in both cell cultures and in vivo studies). The present findings indicate that these novel chemical antimalarial compounds are not PLD inducers despite to be considered structurally as CADs. Furthermore, none of the compounds tested showed significant cytotoxicity at their maximum solubility.