Investigating Human Erythrocyte Band 3 Tyrosine Phosphorylation and Its Involvement in the Plasmodium falciparum Infection

Despite intense world-wide effort, malaria is still a major cause of morbidity and mortality, especially in third world countries. Malaria is the result of an intracellular parasite, with Plasmodium falciparum being the most lethal species. The emergence of resistance to current anti-malarial therapies in Plasmodium is spreading, but conceivably can be circumvented by targeting processes within the host red blood cell (RBC) required for pathogenesis. For example, a crucial step in the parasite’s lifecycle is weakening of the host RBC’s membrane via modification of host membrane proteins, including the integral RBC membrane protein band 3. This membrane weakening eventually leads to the cell’s lysis, releasing parasites to infect additional RBCs. The weakening of the RBC membrane represents an intriguing target as the band 3 protein responsible for the weakening is modified via tyrosine phosphorylation and the parasite encodes no classical tyrosine kinases in its genome. Over 75% of band 3 is lost by the end of egress, which is critical since the band 3-ankyrin interaction constitutes the major connection linking the cell membrane to the cytoskeleton. Curiously, phosphorylation of band 3 tyrosines by RBC Syk kinase also induces release of band 3 from the cytoskeleton, leading to membrane destabilization and fragmentation. Since tyrosine phosphorylation of band 3 is prominent during the later stages of intra-erythrocytic parasite development, we evaluated a variety of tyrosine kinase inhibitors (TKIs) to assess their abilities to block parasite egress by inhibiting the tyrosine phosphorylation of band 3. In healthy RBC treated with orthovanadate (OV) to induce band 3 tyrosine phosphorylation, Syk inhibitors were the most effective inhibitors of this phosphorylation followed by inhibitors of Src-family kinases. Results reinforce the prominent role of Syk in this process and illustrate a previously unidentified role of tyrosine kinases Hck, Fgr, and c-Src. With these results in mind, P. falciparum cultures were treated with Syk inhibitors to evaluate any antimalarial activity and corroborate the importance of band 3 tyrosine phosphorylation for the successful escape of parasites from the host RBC. In cell culture studies, when infected RBCs were treated with Syk inhibitors, parasitemia was found to decline by \u3e95% within 48 hours. The majority of infected RBCs still contained entrapped parasites, in all likelihood from the inability of parasites to break through the now strengthened red cell membrane. The concentrations of Syk inhibitors demonstrating complete inhibition of parasitemia with a rupture-arrested phenotype correlated with the ability of these inhibitors to prevent band 3 tyrosine phosphorylation in the OV-stimulated RBCs. P. falciparum cultures treated with the other TKIs, targeted for the other tyrosine kinases present in the RBC, displayed a spectrum of drug sensitivities and parasite phenotypes. However, an inhibitor of Hck potently fulfilled the same inhibitory capabilities similar to that of the Syk inhibitors, further supporting a critical role of band 3 tyrosine phosphorylation in parasite egress and a possible role of this tyrosine kinase in this process. Surprisingly, one of the more effective tyrosine kinase inhibitors to demonstrate antimalarial activity was the FDA-approved inhibitor imatinib (Gleevec). With previously demonstrated Syk inhibitory properties, imatinib effectively inhibited parasitemia of cultures with a mechanism similar to that seen in Syk inhibitor treated cultures. Parasitized cells displayed a rupture-arrested phenotype at concentrations that completely inhibited band 3 tyrosine phosphorylation at multiple stages of parasite development. Moreover, because imatinib is an FDA-approved drug authorized for use in adults and children, translation of the therapy into the clinic can be facilitated. To determine the efficacy of Syk inhibitors and imatinib on field isolates, parasitized blood acquired directly from P. falciparum-infected patients in both Southeast Asia and East Africa were assessed on site in ex vivo assays. Blood analysis following treatment revealed the same reduction in parasitemia seen with laboratory strains of P. falciparum, supporting their ability to eliminate human infections by this parasite. Following this success, a dose-escalation clinical trial with FDA-approved imatinib (Gleevec) was initiated to evaluate its antimalarial efficacy in uncomplicated P. falciparum human infections in Vietnam. Although this study is still in progress, preliminary results are very promising, with complete eradication of the infection observed in some patients at the lowest drug concentration tested. With such successful results thus far, we next partnered with a major pharmaceutical company to create more potent and selective drug entities that inhibit band 3 tyrosine phosphorylation for their use as novel antimalarial therapies. A set of compounds, all with diverse kinase inhibitory properties, was selected from their extensive compound library based on structural similarities to the aforementioned Syk inhibitors, since this class of TKI has consistently been shown to be the most effective tyrosine kinase inhibitor that had antimalarial activity through the rupture-arrested phenotype. These compounds were blindly screened to identify promising hits for lead optimization, based on their ability to fit a number of established criteria including antimalarial activity, rupture-arrested phenotype, ability to inhibit OV-stimulated band 3 tyrosine phosphorylation, and Syk inhibitory activity in DiscoveRx kinase assay system. From this study, we successfully identified Syk tyrosine kinase inhibitors as the most effective compounds with antimalarial activity, validating Syk inhibitors as a compelling new class of novel therapies that will help to facilitate the eradication of malaria. At a time when drug-resistant strains of P. falciparum are continually emerging, a strategy that targets a host enzyme that is resistant to mutations by the parasite should constitute a therapeutic mechanism that will suppress further evolution of resistance. By demonstrating the successful elimination of P. falciparum infection in human subjects treated with imatinib in addition to the continued efficacy of Syk inhibitors against a variety of parasite populations, the use of Syk inhibitors as a class of novel antimalarial therapeutics has been established