Investigating a Novel Erythrocyte Kinase and Its Impact on <i>Plasmodium Falciparum</i> Infection

Plasmodium falciparum has the proclivity to develop drug resistance, raising a need for novel malaria treatments. Host-directed therapeutics are emerging as a new approach to the treatment of several infectious diseases, but their potential utility in malaria infection remains largely unexplored, in part because of our limited understanding of erythrocyte host determinants of malaria infection. To begin to elucidate erythrocyte signaling pathways important for P. falciparum invasion, we performed phospho-antibody microarray experiments using cultured red blood cells (cRBCs) derived ex-vivo from hematopoietic stem cells (HSPCs), enabling the interrogation of both wildtype cells and those with deletion of a critical invasion factor, CD44. Stimulation of these cells with the P. falciparum invasion ligand EBA175 revealed altered phosphorylation of several host kinases, including NUAK1. Using immunoblotting, we confirmed that NUAK1 phosphorylation is increased in EBA175-stimulated cRBCs and in P. falciparum-infected donor RBCs. Together, these data suggest that NUAK1 plays an important role during P. falciparum infection. In other cells, NUAK1 is associated with tumor suppression, proliferation, and oxidative stress, but its function in RBCs is unknown. To determine if NUAK1 controls P. falciparum proliferation in erythrocytes, we performed parasite proliferation assays using two highly selective NUAK1 inhibitors, HTH-01-015 and WZ4003. We found that they inhibited the growth of multiple P. falciparum strains in a dose-dependent manner (IC50 &amp;lt;3 µM), with activity throughout the blood stage. Using egress assays and live microscopy, we found that HTH-01-015 does not inhibit merozoite egress but instead specifically blocks invasion. Importantly, discharge of the P. falciparum microneme organelles was not affected, suggesting that the observed phenotypes were due to an effect of the inhibitor on the host cell. Studies using the immortalized erythroblast cell line BEL-A revealed that the inhibitors disrupted erythroid cell growth (IC50 2.6 µM), consistent with a host cell target. To further examine specificity, we performed kinobead competition assays with HTH-01-015 and WZ4003 in human cell lysates, which identified only NUAK1 and two other human kinases as possible targets. In contrast, our kinobead experiments using P. falciparum lysates revealed that none of the 60 Plasmodium kinases interacted with the inhibitors, underscoring their selectivity for human kinases. Furthermore, we have demonstrated that treatment with HTH-01-015 inhibits phosphorylation of a well-characterized NUAK1 substrate, Myosin phosphatase target subunit 1 (MYPT1), in BEL-A cells. Since myosin is a component of the RBC cytoskeleton, it is tempting to speculate that EBA binding results in NUAK activation, which in turn triggers the mobilization of regulators of RBC cytoskeleton dynamics to promote invasion. In ongoing work to validate this hypothesis, we are using phosphoproteomics to interrogate the system-wide effect of these inhibitors on signaling networks in P. falciparum-infected RBCs, as well as genome editing to generate inhibitor-resistant cRBCs for use in P. falciparum growth assays. We propose that a better understanding of these intricate host-parasite interactions offers opportunities for more effective, resistance-refractory antimalarial therapies, highlighting the crucial role of hematological insights in advancing malaria treatment strategies.