Biochemical and Structural Approaches to Anthelmintic Drug Discovery: From Caenorhabditis elegans to Necator americanus

Author

• Walter McReadyFollow

Semester of Graduation

Spring 2026

Degree Type

Thesis

Degree Name

Master in Integrative Biology

Department

Department of Cellular and Molecular Biology

Committee Chair/First Advisor

Dr. Soon Goo Lee

Second Advisor

Dr. Martin Hudson

Third Advisor

Dr. Brandon Carpenter

Abstract

Parasitic infections, including hookworm disease, remain a major global health problem, and the emergence of drug resistance is an increasing concern. Phosphoethanolamine N-methyltransferase (PMT) is a promising drug target catalyzing a key step in phosphatidylcholine biosynthesis that is essential for parasite survival but absent in humans. This thesis characterizes PMT2 enzymes from the human hookworm Necator americanus (NaPMT2) and Ancylostoma ceylanicum (AcPMT2) and establishes the foundation for both in vitro and in vivo drug screening platforms using the model organism Caenorhabditis elegans. The biochemical, biophysical, and structural properties of NaPMT2 and AcPMT2 were characterized, confirming both enzymes as functional S-adenosylmethionine (SAM)-dependent methyltransferases through steady-state kinetic analyses and substrate binding studies. Alanine scanning mutagenesis of eight conserved active site residues identified Arg175, Tyr183, and His299 as essential for catalytic function. Structural analysis using X-ray crystallography determined the first crystal structures of a PMT2 enzyme from a human-infecting hookworm, including an apo structure at 3.05 Å resolution and a product-bound structure with S-adenosylhomocysteine (SAH) and phosphocholine at 2.31 Å resolution, providing a framework for the development of anthelmintic therapeutics. In parallel, transgenic C. elegans lines expressing NaPMT2 and PfPMT under the elt-3 hypodermal promoter were generated and validated by sequencing. Rescue of the CePMT2 knockout phenotype was achieved with the endogenous CePMT2 gene, confirming the validity of the expression platform, although NaPMT2 did not rescue the knockout phenotype, likely due to insufficient expression levels or differences in substrate specificity. Overall, this work provides a comprehensive understanding of hookworm PMT2 enzymes from the atomic to the physiological level and lays the groundwork for the development of novel PMT-targeted antiparasitic therapeutics.

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