{"id":{"repo_id":"utswmed","oai_identifier":"oai:utswmed-ir.tdl.org:2152.5/10716"},"canonical_url":"https://search.dev.ndltd.org/etd/utswmed/oai:utswmed-ir.tdl.org:2152.5/10716","repository":{"repo_id":"utswmed","name":"University of Texas Southwestern Medical Center","base_url":"https://utswmed-ir.tdl.org/server/oai/request"},"display":{"title":"Metabolomic Approaches in Parasitology","abstract":"Metabolic pathways and networks are crucial for maintaining cellular functions and sustaining life. Understanding these pathways in parasites may reveal novel targets for therapeutic application if they are sufficiently different from mammalian systems. My work investigated two such pathways: pyrimidine biosynthesis and the impact of inhibiting the pathway enzyme dihydroorotate dehydrogenase (DHODH) and the polyamine biosynthesis pathway and routes to obtaining the pathway precursor L-ornithine. DHODH has recently been validated as a therapeutic target for malaria through clinical studies on the triazolopyrimidine-based Plasmodium DHODH inhibitor DSM265. Selective toxicity towards Plasmodium species could be achieved because malaria parasites lack pyrimidine salvage pathways, and DSM265 selectively inhibits Plasmodium DHODH over the human enzyme. However, while DSM265 does not inhibit human DHODH, it inhibits DHODH from several preclinical species, including mice, suggesting that any toxicity observed in those species could result from on-target DHODH inhibition. To test this hypothesis, I validated the use of the reaction substrate dihydroorotate as a biomarker for DHODH inhibition in mammalian cells and mice treated with DHODH inhibitors and correlated the biomarker response to the degree of DHODH inhibition. This work represents an important advance to monitor for on-target effects in preclinical and clinical applications of DHODH inhibitors used to treat human disease. Trypanosoma brucei is a parasitic protozoan and the causative agent of human African trypanosomiasis. The polyamine biosynthesis pathway plays a critical role in cell survival by producing putrescine and spermidine, essential for various cellular processes. L-Ornithine, the precursor for putrescine biosynthesis, is believed to be obtained by uptake from the environment, as T. brucei lacks a functional arginase enzyme. I characterized a T. brucei protein, Tb927.4.1300, belonging to the amidinotransferase family, potentially involved in L-arginine to L-ornithine conversion. Knockout of Tb927.4.1300 resulted in a growth deficit without significant changes in intracellular L-arginine or L-ornithine levels. Recombinantly expressed Tb927.4.1300 failed to catalyze L-ornithine synthesis with the availability of various substrates. Structural modeling based on AlphaFold revealed that alteration of key catalytic residues in Tb927.4.1300 rendered them unsuitable for the catalytic mechanism. Using isotope tracing experiments, I confirmed the inability of T. brucei to convert L-arginine to L-ornithine, indicating the absence of this enzymatic pathway, and supporting the hypothesis that the parasite is an L-ornithine auxotroph. Taken together, these findings shed light on the metabolic requirements of L-ornithine in T. brucei and characterize Tb927.4.1300 as a catalytically dead homolog of the amidinotransferase family whose knockout results in a growth deficit of the parasite.","abstract_html":"Metabolic pathways and networks are crucial for maintaining cellular functions and sustaining life. Understanding these pathways in parasites may reveal novel targets for therapeutic application if they are sufficiently different from mammalian systems. My work investigated two such pathways: pyrimidine biosynthesis and the impact of inhibiting the pathway enzyme dihydroorotate dehydrogenase (DHODH) and the polyamine biosynthesis pathway and routes to obtaining the pathway precursor L-ornithine. DHODH has recently been validated as a therapeutic target for malaria through clinical studies on the triazolopyrimidine-based Plasmodium DHODH inhibitor DSM265. Selective toxicity towards Plasmodium species could be achieved because malaria parasites lack pyrimidine salvage pathways, and DSM265 selectively inhibits Plasmodium DHODH over the human enzyme. However, while DSM265 does not inhibit human DHODH, it inhibits DHODH from several preclinical species, including mice, suggesting that any toxicity observed in those species could result from on-target DHODH inhibition. To test this hypothesis, I validated the use of the reaction substrate dihydroorotate as a biomarker for DHODH inhibition in mammalian cells and mice treated with DHODH inhibitors and correlated the biomarker response to the degree of DHODH inhibition. This work represents an important advance to monitor for on-target effects in preclinical and clinical applications of DHODH inhibitors used to treat human disease. Trypanosoma brucei is a parasitic protozoan and the causative agent of human African trypanosomiasis. The polyamine biosynthesis pathway plays a critical role in cell survival by producing putrescine and spermidine, essential for various cellular processes. L-Ornithine, the precursor for putrescine biosynthesis, is believed to be obtained by uptake from the environment, as T. brucei lacks a functional arginase enzyme. I characterized a T. brucei protein, Tb927.4.1300, belonging to the amidinotransferase family, potentially involved in L-arginine to L-ornithine conversion. Knockout of Tb927.4.1300 resulted in a growth deficit without significant changes in intracellular L-arginine or L-ornithine levels. Recombinantly expressed Tb927.4.1300 failed to catalyze L-ornithine synthesis with the availability of various substrates. Structural modeling based on AlphaFold revealed that alteration of key catalytic residues in Tb927.4.1300 rendered them unsuitable for the catalytic mechanism. Using isotope tracing experiments, I confirmed the inability of T. brucei to convert L-arginine to L-ornithine, indicating the absence of this enzymatic pathway, and supporting the hypothesis that the parasite is an L-ornithine auxotroph. Taken together, these findings shed light on the metabolic requirements of L-ornithine in T. brucei and characterize Tb927.4.1300 as a catalytically dead homolog of the amidinotransferase family whose knockout results in a growth deficit of the parasite.","abstract_has_math":false,"creators":["Pontikos, Michael Alexander"],"institution":null,"degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":["Tu, Benjamin","Phillips, Margaret A.","Williams, Noelle S.","Liszczak, Glen","Wetzel, Dawn"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2025,"date_issued":"2025-09-08T21:51:25Z","date_published":"2025-09-08T21:51:25Z","updated_at":"2026-07-24T05:52:20Z","subjects":["Oxidoreductases Acting on CH-CH Group Donors","Plasmodium falciparum","Prodrugs","Trypanosoma brucei brucei","Enzyme Inhibitors"],"languages":["en"],"rights":[],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["1535537228"],"render_values":[{"text":"1535537228","href":null,"code":true}]}]},"links":{"outbound_url":"https://hdl.handle.net/2152.5/10716","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Tu, Benjamin","Phillips, Margaret A.","Williams, Noelle S.","Liszczak, Glen","Wetzel, Dawn"]},{"key":"dc:creator","label":"Author","values":["Pontikos, Michael Alexander"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2025-09-08T21:51:25Z","2023-08","August 2023"]},{"key":"dc:type","label":"Dc Type","values":["Thesis","text"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Oxidoreductases Acting on CH-CH Group Donors","Plasmodium falciparum","Prodrugs","Trypanosoma brucei brucei","Enzyme Inhibitors"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://hdl.handle.net/2152.5/10716","1535537228"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Metabolic pathways and networks are crucial for maintaining cellular functions and sustaining life. Understanding these pathways in parasites may reveal novel targets for therapeutic application if they are sufficiently different from mammalian systems. My work investigated two such pathways: pyrimidine biosynthesis and the impact of inhibiting the pathway enzyme dihydroorotate dehydrogenase (DHODH) and the polyamine biosynthesis pathway and routes to obtaining the pathway precursor L-ornithine. DHODH has recently been validated as a therapeutic target for malaria through clinical studies on the triazolopyrimidine-based Plasmodium DHODH inhibitor DSM265. Selective toxicity towards Plasmodium species could be achieved because malaria parasites lack pyrimidine salvage pathways, and DSM265 selectively inhibits Plasmodium DHODH over the human enzyme. However, while DSM265 does not inhibit human DHODH, it inhibits DHODH from several preclinical species, including mice, suggesting that any toxicity observed in those species could result from on-target DHODH inhibition. To test this hypothesis, I validated the use of the reaction substrate dihydroorotate as a biomarker for DHODH inhibition in mammalian cells and mice treated with DHODH inhibitors and correlated the biomarker response to the degree of DHODH inhibition. This work represents an important advance to monitor for on-target effects in preclinical and clinical applications of DHODH inhibitors used to treat human disease. Trypanosoma brucei is a parasitic protozoan and the causative agent of human African trypanosomiasis. The polyamine biosynthesis pathway plays a critical role in cell survival by producing putrescine and spermidine, essential for various cellular processes. L-Ornithine, the precursor for putrescine biosynthesis, is believed to be obtained by uptake from the environment, as T. brucei lacks a functional arginase enzyme. I characterized a T. brucei protein, Tb927.4.1300, belonging to the amidinotransferase family, potentially involved in L-arginine to L-ornithine conversion. Knockout of Tb927.4.1300 resulted in a growth deficit without significant changes in intracellular L-arginine or L-ornithine levels. Recombinantly expressed Tb927.4.1300 failed to catalyze L-ornithine synthesis with the availability of various substrates. Structural modeling based on AlphaFold revealed that alteration of key catalytic residues in Tb927.4.1300 rendered them unsuitable for the catalytic mechanism. Using isotope tracing experiments, I confirmed the inability of T. brucei to convert L-arginine to L-ornithine, indicating the absence of this enzymatic pathway, and supporting the hypothesis that the parasite is an L-ornithine auxotroph. Taken together, these findings shed light on the metabolic requirements of L-ornithine in T. brucei and characterize Tb927.4.1300 as a catalytically dead homolog of the amidinotransferase family whose knockout results in a growth deficit of the parasite."]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Metabolomic Approaches in Parasitology"]}]}],"canonical_facts":{"dc:contributor":["Tu, Benjamin","Phillips, Margaret A.","Williams, Noelle S.","Liszczak, Glen","Wetzel, Dawn"],"dc:creator":["Pontikos, Michael Alexander"],"dc:date":["2025-09-08T21:51:25Z","2023-08","August 2023"],"dc:description":["Metabolic pathways and networks are crucial for maintaining cellular functions and sustaining life. Understanding these pathways in parasites may reveal novel targets for therapeutic application if they are sufficiently different from mammalian systems. My work investigated two such pathways: pyrimidine biosynthesis and the impact of inhibiting the pathway enzyme dihydroorotate dehydrogenase (DHODH) and the polyamine biosynthesis pathway and routes to obtaining the pathway precursor L-ornithine. DHODH has recently been validated as a therapeutic target for malaria through clinical studies on the triazolopyrimidine-based Plasmodium DHODH inhibitor DSM265. Selective toxicity towards Plasmodium species could be achieved because malaria parasites lack pyrimidine salvage pathways, and DSM265 selectively inhibits Plasmodium DHODH over the human enzyme. However, while DSM265 does not inhibit human DHODH, it inhibits DHODH from several preclinical species, including mice, suggesting that any toxicity observed in those species could result from on-target DHODH inhibition. To test this hypothesis, I validated the use of the reaction substrate dihydroorotate as a biomarker for DHODH inhibition in mammalian cells and mice treated with DHODH inhibitors and correlated the biomarker response to the degree of DHODH inhibition. This work represents an important advance to monitor for on-target effects in preclinical and clinical applications of DHODH inhibitors used to treat human disease. Trypanosoma brucei is a parasitic protozoan and the causative agent of human African trypanosomiasis. The polyamine biosynthesis pathway plays a critical role in cell survival by producing putrescine and spermidine, essential for various cellular processes. L-Ornithine, the precursor for putrescine biosynthesis, is believed to be obtained by uptake from the environment, as T. brucei lacks a functional arginase enzyme. I characterized a T. brucei protein, Tb927.4.1300, belonging to the amidinotransferase family, potentially involved in L-arginine to L-ornithine conversion. Knockout of Tb927.4.1300 resulted in a growth deficit without significant changes in intracellular L-arginine or L-ornithine levels. Recombinantly expressed Tb927.4.1300 failed to catalyze L-ornithine synthesis with the availability of various substrates. Structural modeling based on AlphaFold revealed that alteration of key catalytic residues in Tb927.4.1300 rendered them unsuitable for the catalytic mechanism. Using isotope tracing experiments, I confirmed the inability of T. brucei to convert L-arginine to L-ornithine, indicating the absence of this enzymatic pathway, and supporting the hypothesis that the parasite is an L-ornithine auxotroph. Taken together, these findings shed light on the metabolic requirements of L-ornithine in T. brucei and characterize Tb927.4.1300 as a catalytically dead homolog of the amidinotransferase family whose knockout results in a growth deficit of the parasite."],"dc:format":["application/pdf"],"dc:identifier":["https://hdl.handle.net/2152.5/10716","1535537228"],"dc:language":["en"],"dc:subject":["Oxidoreductases Acting on CH-CH Group Donors","Plasmodium falciparum","Prodrugs","Trypanosoma brucei brucei","Enzyme Inhibitors"],"dc:title":["Metabolomic Approaches in Parasitology"],"dc:type":["Thesis","text"]},"updated_at":"2026-07-24T05:52:20Z"}