Abstract Malaria is one of the world’s most common and deadly infectious diseases. Approximately 627,000 people, mostly African children, die each year due to complications of malaria (WHO estimate for 2021). A unique feature of Plasmodium falciparum malaria is the adhesion of parasite-infected erythrocytes (iRBCs) to vascular endothelium. Sequestration iRBCs correlates with the sequela of cerebral malaria, a devastating and often fatal consequence of the disease. The iRBCs develop specialized surface protrusions called knobs mediating their attachment to blood vessels in multiple organs. Formation of knobs on the surface of iRBCs mediates cytoadherence to endothelial cells. A critical component of knobs is knob-associated histidine rich protein (KAHRP) that anchors multiple host and parasite derived proteins to the RBC cytoskeleton. We have identified specific segments of KAHRP that mediate its self-assembly into spiral-shaped structures by sequential participation of head-to-tail interactions. Domain mapping revealed that the amino terminal histidine-rich segment of KAHRP functions as a critical determinant of self-assembly. Reconstitution of the recombinant histidine-rich segment of KAHRP in knobby iRBCs disrupted the knob morphology and abrogated adhesion of iRBCs in vitro. These results suggest a novel mechanism for disrupting the self-association of KAHRP as a potential strategy against cytoadhesion in malaria. Guided by these preliminary data, we propose to test the hypothesis that disrupting specific interactions within KAHRP inhibits knob formation, thus blocking the adhesion of iRBCs to endothelial cells. This hypothesis will be tested as follows: Aim 1: Mechanism of KAHRP self-assembly and knob formation. Our results demonstrate that KAHRP self-assembly is mediated through interactions between its histidine rich region and the C-terminal 5’ repeats. We have identified a specific KAHRP polypeptide that attenuated cytoadherence of iRBCs as effectively as the KAHRP gene knockdown studies. Moreover, using phage display cDNA technology, we identified novel KAHRP-binding peptides as potential inhibitors of the knob self-assembly process. We propose to use erythrocyte loading of KAHRP segments as well as cell permeable peptides to quantify the formation, composition, and dispersion of knobs using established biochemical and microscopy approaches. Aim 2: Disruption of RBC knob- endothelial interactions. Using existing recombinant KAHRP segments and newly identified peptides by the phage display screens, we will evaluate the disruption of iRBCs adhesion to a variety of substrates including endothelial cells, platelets, and purified receptors. State-of-the-art imaging tools will be used to monitor real time kinetics of cytoadherence ex vivo. Together, these studies will elucidate the mechanism of knob assembly in the Plasmodium falciparum infected RBCs and reveal new therapeutic approaches for mitigating the adhesion of infected human RBCs to e...