A High-Throughput Inhibitor Screen Targeting CLAG3 Export and Membrane Insertion on Human Erythrocytes Infected with Malaria Parasites

To facilitate intracellular growth and replication, the virulent human malaria parasite <i>P. falciparum</i> remodels its host erythrocyte by exporting many proteins into the host cell cytosol. Along with a few other exported proteins, the parasite CLAG3 protein is then inserted in the host erythrocyte membrane, exposing a small variant loop to host plasma and contributing to essential nutrient acquisition via the plasmodial surface anion channel (PSAC). To explore trafficking mechanisms and develop therapies that block host cell remodeling, we have now used a split NanoLuc reporter and performed a high-throughput screen for inhibitors of parasite CLAG3 trafficking and insertion at the host membrane. We screened ~52,000 small molecules and uncovered 65 chemically diverse hits. Hits that inhibit the NanoLuc reporter without blocking protein export were filtered out by a secondary screen whose signal does not depend on protein export. Because chemicals that interfere with parasite maturation were found to compromise CLAG3 export indirectly, a third screen using a NanoLuc reporter-tagged intracellular protein was used to evaluate nonspecific toxicity. Although our relatively small chemical screen did not identify bona fide inhibitors of CLAG3 host membrane insertion, these studies establish a framework for larger screens to identify novel export inhibitors. Such novel inhibitors will provide important insights into how <i>Plasmodia</i> remodel their host cells and may seed the development of therapies that block the export and membrane insertion of proteins needed for intracellular parasite survival.