Identifying the modes and mechanisms of action of transmission-blocking antimalarials against Plasmodium falciparum

Phenotypic drug screening has transformed the antimalarial development pipeline, facilitating the discovery of compounds targeting unexplored parasite proteins at a drastically accelerated rate. Breaking the cycle of malaria transmission is crucial to eradication and centres around inhibition of the sexual stage gametocytes and gametes. Transmissible sexual stages represent a population bottleneck of the Plasmodium life-cycle which face little selective pressure and are hence a desirable target of drug intervention. Recently discovered in a phenotypic screen, the N-((4- hydroxychroman-4-yl)methyl)-sulphonamide (N-4HCS) compounds potently inhibit P. falciparum male gamete formation (microgametogenesis) in the mosquito. By combining study of the remarkable Plasmodium gametocyte cellular biology with medicinal chemistry and chemical proteomics, here we performed a detailed analysis of N-4HCS compound activity. Adhering to a traditional drug developmental pipeline, we first performed N- 4HCS hit-to-lead development and yielded compounds with activity in the nanomolar concentration range and favourable safety profiles. Furthermore, an N-4HCS photoaffinity probe was utilised in a proteome-wide photoaffinity labelling study, identifying the P. falciparum sexual stage-specific protein, Pfs16, as the compound target. Notably, the Cellular Thermal Shift Assay confirmed label-free specificity of N-4HCS compounds to Pfs16. Flow cytometry and phase-contrast, widefield- fluorescence and electron microscopy revealed compounds exclusively inhibited microgametogenesis, with a cellular phenotype consistent with published reports on the targeted gene disruption of Pfs16. We therefore present the 16kDa parasitophorous vacuole membrane protein, Pfs16, as a desirable antimalarial target and the N-4HCS compounds as potent candidates for future development. Additionally, a live-cell fluorescence microscopy workflow was devised to enable visualisation of microgametogenesis from the onset of activation through to exflagellation. Crucially, our imaging approach was applicable to drug discovery and is accessible to the malaria research community, thereby facilitating future study of transmission-blocking drug candidates. This thesis hence addressed the urgent requirement for novel antimalarial interventions, developing and aiding future discovery of transmission-blocking drugs.Open Acces

MalDA, accelerating malaria drug discovery

The Malaria Drug Accelerator (MalDA) is a consortium of 15 leading scientific laboratories. The aim of MalDA is to improve and accelerate the early antimalarial drug discovery process by identifying new, essential, druggable targets. In addition, it seeks to produce early lead inhibitors that may be advanced into drug candidates suitable for preclinical development and subsequent clinical testing in humans. By sharing resources, including expertise, knowledge, materials, and reagents, the consortium strives to eliminate the structural barriers often encountered in the drug discovery process. Here we discuss the mission of the consortium and its scientific achievements, including the identification of new chemically and biologically validated targets, as well as future scientific directions

MalDA, Accelerating Malaria Drug Discovery

© 2021 The Authors The Malaria Drug Accelerator (MalDA) is a consortium of 15 leading scientific laboratories. The aim of MalDA is to improve and accelerate the early antimalarial drug discovery process by identifying new, essential, druggable targets. In addition, it seeks to produce early lead inhibitors that may be advanced into drug candidates suitable for preclinical development and subsequent clinical testing in humans. By sharing resources, including expertise, knowledge, materials, and reagents, the consortium strives to eliminate the structural barriers often encountered in the drug discovery process. Here we discuss the mission of the consortium and its scientific achievements, including the identification of new chemically and biologically validated targets, as well as future scientific directions

Development and validation of a bioluminescence assay for high throughput screening of potent and rapidly cytocidal compounds against intraerythrocytic Plasmodium falciparum

The emergence of P. falciparum resistant to current front-line artemisinin combination therapies underscores the urgent demand for new candidate molecules for a single exposure radical cure and prophylaxis drug. Developing a suitable candidate component that is both potent and effects a rapid rate of kill to replace artemisinins requires a new and innovative in vitro screening assays to support discovery. A standard Bioluminescence Relative Rate of Kill (BRRoK) assay to quickly triage rapid cytocidal antimalarial compounds in vitro has been developed. Recognizing limitations in the BRRoK assay necessitated a subsequent development of a modified-BRRoK assay. This mBRRoK assay explores a compound’s RoK and potency together in a fixed-concentration assay format more amenable to a high throughput screening of a large compound libraries. Proof-of-principle for the mBRRoK assay was developed using the Medicine for Malaria Venture (MMV) Malaria Box compounds for which BRRoK data was available. A subsequent validation of mBRRoK was carried out using the MMV Pathogen Box open source discovery library. Potential new leads were identified, of a particular interest are novel PfeEF2 inhibitors (MMV634140 and MMV667494) that show a rapid initial relative rate of kill. These compounds are suggested for further optimization and characterization. The mBRRoK assay was adapted, miniaturized and optimized for high throughput screening of 12,514 TCAMS library. The results demonstrated that this assay is simple, sensitive (81% true discovery rate), reliable and robust with Z´ value of 0.74-0.98 and S/B ratio of 160 to 475. Predicted fast-acting hits were selected and confirmed using the standard BRRoK assay in both the original Dd2luc reporter strain, but also in a new NF54luc (chloroquine-sensitive) strain. The results demonstrated the utility of mBRRoK assay not only for rapid screening of potent and fast-acting compound, but also to study drug-resistance profiles across different parasite strains. The mBRRoK assay offers significant opportunities during early stage of antimalarial drug discovery and development to triage compound sets through understanding potency and initial rate of kill, but is also an assay system amenable to adaptations such as assays in artemisinin resistant reporter strains and the study of stage-specific action

Discovery of benzamides and triarylimidazoles active against Plasmodium falciparum via haemozoin inhibition : high throughput screening, synthesis and structure-activity relationships

Includes bibliographical referencesNew antimalarials are desperately needed to overcome growing P. falciparum resistance to the current drugs. Successful quinoline-based drugs target haemozoin formation causing a cytotoxic accumulation of free haem (Fe(III)PPIX) in the parasite, a target which remains promising for future treatments. Much research has been undertaken on the quinoline antimalarials, which has led to several hypotheses of haemozoin inhibition and drug accumulation mechanisms, however, relatively few studies have been carried out for haemozoin antimalarials with alternate chemotypes. High throughput screening (HTS) can be used to identify novel scaffolds that inhibit β-haematin (βH - synthetic haemozoin) formation and which have favourable P. falciparum activities. In this project, HTS has been carried out on 43,520 small, organic, drug-like compounds as part of a larger screen of 144,330 Vanderbilt University Institute of Chemical Biology (VU) chemical library compounds and 530 were found to be good inhibitors of βH relative to the chloroquine (CQ) and amodiaquine (AQ) controls. A further 171 compounds were found to inhibit parasite growth, showing improved hit rates from previous HTS efforts. Two scaffolds (A=benzamides and B=triarylimidazoles) were selected for further analysis, whereupon analogues were synthesised