CHARACTERIZATION, STRUCTURAL ANALYSIS, AND IDENTIFICATION OF INHIBITORS OF THE ATG8 AND THE ATG8-ATG3 INTERACTION IN THE MALARIA PARASITE, PLASMODIUM

Autophagy is a catabolic process that normally utilizes the lysosome. With central roles in survival during stress and protein and organelle turnover, the far-reaching implications of this system in disease are being increasingly understood. Studying autophagy is complicated by its role in cell survival and programmed cell death and the involvement of the canonical marker of autophagy, Atg8/LC3, in numerous non- autophagic roles. The malaria parasite, Plasmodium, has conserved certain aspects of the autophagic machinery but for what purpose has long remained a mystery. Major advances have recently been gained and suggest a role for Atg8 in apicoplast maintenance, degradation of heme inside the food vacuole, and possibly trafficking of proteins or organelles outside the parasite membrane. Malaria kills nearly a million people each year, mostly children, representing a global public health problem. With ninety percent of these deaths in Africa, malaria also represents a major obstacle to the stability and economic prosperity of the nation. The essentiality of plasmodial Atg8, and its emerging role at the essential apicoplast organelle implicate Atg8 as a good drug target for antimalarial drug design. Atg8 is an ubiquitin- like protein, requiring an E1-activating and E2-conjugating enzyme for lipidation to membranes, required for its function. An important question is how the conjugation systems differ between Plasmodium and humans and how these differences can be exploited to design specific novel antimalarial drugs. We provide structural information on the P. falciparum Atg8-Atg3 interaction, identify small molecule inhibitors against the interaction, and use these inhibitors to probe novel functions of Atg8 in Plasmodium

Structure-based drug design, synthesis and biological assays of P. falciparum Atg3-Atg8 protein-protein interaction inhibitors

The proteins involved in the autophagy (Atg) pathway have recently been considered promising targets for the development of new antimalarial drugs. In particular, inhibitors of the protein-protein interaction (PPI) between Atg3 and Atg8 of Plasmodium falciparum retarded the blood- and liver-stages of parasite growth. In this paper, we used computational techniques to design a new class of peptidomimetics mimicking the Atg3 interaction motif, which were then synthesized by click-chemistry. Surface plasmon resonance has been employed to measure the ability of these compounds to inhibit the Atg3-Atg8 reciprocal protein-protein interaction. Moreover, P. falciparum growth inhibition in red blood cell cultures was evaluated as well as the cyto-toxicity of the compounds

Biochemical and pharmacological characterization of the Atg8 conjugation system in toxoplasma gondii

Indiana University-Purdue University Indianapolis (IUPUI)Toxoplasma gondii is an important human pathogen that infects millions of people worldwide and causing severe and potentially lethal disease in immunocompromised individuals. Recently, a homologue for the autophagy protein Atg8 (TgAtg8) was identified in Toxoplasma that is required for both canonical and noncanonical processes essential for parasite viability. Importantly, TgAtg8 functionality requires its conjugation to phosphatidylethanolamine through the activity of the Atg8 conjugation system. In this thesis, we characterized the proteins that interact with TgAtg8 and TgAtg3, a component of the Atg8 conjugation system, to further define their functions in Toxoplasma and identify opportunities for targeted inhibition of Atg8-related processes. We previously identified that TgAtg8 is acetylated at lysine 23 (K23) and assessed the role of this modification in this thesis. Using mutagenesis, we showed that K23 acetylation did not modulate the interaction with TgAtg3, but appeared to promote TgAtg8 protein stability. Additionally, endogenous mutation of K23 to the nonacetylatable amino acid arginine resulted in severe impairment of parasite replication and spontaneous differentiation into bradyzoites. To gain insight into the role of TgAtg8 in Toxoplasma biology, we next characterized TgAtg8 and TgAtg3 interacting proteins using affinity purification and mass spectrometry. We identified a novel group of interacting proteins that are unique to Toxoplasma, including the dynamin-related protein DrpC. Functional characterization of DrpC identified a potential role of TgAtg8 in trafficking of membrane from the Golgi to the nascent daughter parasites during replication. Lastly, we examined a group of small molecules recently identified as Atg3-Atg8 inhibitors in Plasmodium falciparum and assessed their activity against Toxoplasma. Although the compounds effectively inhibited Toxoplasma replication, they did so through novel mechanisms of action unrelated to the disruption of the TgAtg3-Atg8 interaction. Together, this work provides insight into the function of the Atg8 conjugation system in Toxoplasma that will help guide the future development of novel therapeutics targeting Atg8-related processes

Protein-Protein Interactions in Malaria: Emerging Arena for Future Chemotherapeutics

Malaria is one of the most deadly diseases infecting humans. Advances in elimination and vector control have reduced the global malaria burden in the past decade; however, the emerging threat of drug resistance and suboptimal vaccine efficacies threaten global eradication efforts. Unlocking novel drug and vaccine targets while simultaneously mitigating spread of resistant strains seems to be the need of the hour. Protein-protein interactions (PPIs), an integral part of host-pathogen cross-talk and parasite survival, have only recently emerged as promising drug targets. Large PPI networks (interactome) are being developed to better our understanding of various parasite biochemical pathways. In this chapter, we throw light on several newly characterized protein-protein interactions between the host (humans) and parasite (plasmodium) in key processes such as hemoglobin degradation, enzyme regulation, protein export, egress, invasion, and drug resistance and further discuss their viability for development as novel chemotherapeutic targets

Autophagy Protein Atg3 is Essential for Maintaining Mitochondrial Integrity and for Normal Intracellular Development of Toxoplasma gondii Tachyzoites

Autophagy is a cellular process that is highly conserved among eukaryotes and permits the degradation of cellular material. Autophagy is involved in multiple survival-promoting processes. It not only facilitates the maintenance of cell homeostasis by degrading long-lived proteins and damaged organelles, but it also plays a role in cell differentiation and cell development. Equally important is its function for survival in stress-related conditions such as recycling of proteins and organelles during nutrient starvation. Protozoan parasites have complex life cycles and face dramatically changing environmental conditions; whether autophagy represents a critical coping mechanism throughout these changes remains poorly documented. To investigate this in Toxoplasma gondii, we have used TgAtg8 as an autophagosome marker and showed that autophagy and the associated cellular machinery are present and functional in the parasite. In extracellular T. gondii tachyzoites, autophagosomes were induced in response to amino acid starvation, but they could also be observed in culture during the normal intracellular development of the parasites. Moreover, we generated a conditional T. gondii mutant lacking the orthologue of Atg3, a key autophagy protein. TgAtg3-depleted parasites were unable to regulate the conjugation of TgAtg8 to the autophagosomal membrane. The mutant parasites also exhibited a pronounced fragmentation of their mitochondrion and a drastic growth phenotype. Overall, our results show that TgAtg3-dependent autophagy might be regulating mitochondrial homeostasis during cell division and is essential for the normal development of T. gondii tachyzoites

Autophagy in Plasmodium falciparum intraerythrocytic stages

No abstract available

The relative rate of kill of the MMV Malaria Box compounds provide links to the mode of antimalarial action and highlight scaffolds of medicinal chemistry interest

Objectives: Rapid rate-of-kill (RoK) is a key parameter in the target candidate profile 1 (TCP1) for the next-generation antimalarial drugs for uncomplicated malaria, termed Single Encounter Radical Cure and Prophylaxis (SERCaP). TCP1 aims to rapidly eliminate the initial parasite burden, ideally as fast as artesunate, but minimally as fast as chloroquine. Here we explore whether the relative RoK of the Medicine for Malaria Venture (MMV) Malaria Box compounds are linked to their mode of action (MoA) and identify scaffolds of medicinal chemistry interest. Methods: We used a Bioluminescence Relative RoK (BRRoK) assay over 6 and 48h, with exposure to equipotent-IC50 concentrations, to compare the cytocidal effects of Malaria Box compounds to benchmark antimalarials. Results: BRRoK assay data demonstrate the following relative RoK from fast to slow: inhibitors of PfATP4 > parasite hemoglobin catabolism > DHFR-TS > DHODH > bc1 complex. Core scaffold clustering analyses reveal intrinsic rapid cytocidal action for diamino-glycerols and 2-(aminomethyl)phenol, but slow action for 2-phenylbenzimidazoles, 8-hydroxyquinolines, and triazolopyrimidines. Conclusion: This study provides proof of principle that a compound’s RoK is related to its MoA, and target’s intrinsic RoK is also modified by factors affecting a drug’s access to it. Our findings highlight that as we use medicinal chemistry to improve potency, we can also improve the RoK for some scaffolds. Our BRRoK assay provides the necessary throughput for drug discovery and a critical decision-making tool to support development campaigns. Finally, two scaffolds, diamino-glycerols, and 2-phenoxybenzylamine, exhibit fast cytocidal action, inviting medicinal chemistry improvements towards TCP1 candidates