Cellular and Molecular Physiology of Bloodstream Malaria Parasites

National Institute Of Allergy And Infectious Diseases

Investigators

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Abstract

In 2022, the Apicomplexan Molecular Physiology Section Malaria advanced drug development against the plasmodial surface anion channel (PSAC), an essential nutrient and ion channel at the host membrane of erythrocytes infected with malaria parasites. Both PSAC activity and the ternary RhopH protein complex linked to this channel are conserved in all examined Plasmodium species. Because this channel is absent from other genera, it appears to be an important target for therapy development. In one study, we generated a potent and specific drug lead using Plasmodium falciparum, a virulent human pathogen, and derivatives of MBX-2366, a nanomolar affinity pyridazinone inhibitor from a high-throughput screen. As this screening hit lacks the bioavailability and stability needed for in vivo efficacy, we synthesized 315 derivatives to optimize drug-like properties, establish target specificity, and retain potent activity against the parasite-induced permeability. Using a robust, iterative pipeline, we generated MBX-4055, a derivative active against divergent human parasite strains. MBX-4055 has improved oral absorption with acceptable in vivo tolerability and pharmacokinetics. It also has no activity against a battery of 35 human channels and receptors and was refractory to acquired resistance during extended in vitro selection. Single-molecule and single-cell patch-clamp indicate direct action on PSAC. These studies identify pyridazinone as a novel and tractable antimalarial scaffold with a defined mechanism of action. Mol. Pharmacol., in press (2022). PMID: 35798366 In another study, we developed and used a novel Reporter of Insertion and Surface Exposure (RISE) to track delivery of malaria parasite proteins to the host erythrocyte membrane. The RISE technology permits continuous nondestructive tracking of antigen exposure on infected cells though DNA transfection to insert a small 11-amino acid HiBit fragment of NanoLuc into a target protein; HiBit epitope tag exposure at the cell surface is detected through high-affinity complementation with LgBit to produce luminescence. We tracked the export and surface exposure of CLAG3, a parasite protein linked to PSAC activity and nutrient uptake, throughout the Plasmodium falciparum cycle in human erythrocytes. Our approach revealed key determinants of trafficking and surface exposure. Removal of a C-terminal transmembrane domain aborted export. Unexpectedly, certain increases in the exposed reporter size improved the luminescence signal, but other changes abolished the surface signal, revealing that both size and charge of the extracellular epitope influence membrane insertion. Marked cell-to-cell variation with larger inserts containing multiple HiBit epitopes suggests complex regulation of CLAG3 insertion at the host membrane. Quantitative, continuous tracking of CLAG3 surface exposure thus reveals multiple factors that determine this protein's trafficking and insertion at the host erythrocyte membrane. The RISE assay will not only enable confident identification of surface-exposed antigens from divergent intracellular pathogens, but can inform topology and kinetics of membrane protein delivery and surface exposure. mBio 13:e0040422 (2022). PMID: 35420481