The rise of antibiotic resistant microbes (bacteria, fungi, and parasites), combined with the current void of new drugs entering the clinical setting, has created an urgent need for the discovery of new antimicrobials. High-throughput screening (HTS) assays represent a fast and cost-efficient method for identifying new therapeutic compounds and have been the longstanding gold standard for drug discovery. The focus of this dissertation is on the development and implementation of novel methodologies to increase the throughput of target-based HTS by designing assays that allow multiple drug targets to be probed simultaneously. During my graduate studies, I developed three distinct HTS assays. In each of these assays, drug targets were incorporated into synthetic pathways obeying various reaction topologies (e.g., cyclical, parallel, or linear). Each of these reaction topologies conferred specific advantages and limitations to the individual assays. The first assay reconstitutes the bacterial tRNA-dependent pathway for lipid aminoacylation. This two-step pathway combines a tRNA aminoacylation step catalyzed by an aminoacyl-tRNA synthetase (aaRS), and a transferase step, which transfers the amino acid born by the tRNA onto membrane lipids. aaRSs are essential enzymes in all domains of life and represent longstanding drug targets in pathogenic species. The transferase reaction in the pathway is also an appealing drug target since it impacts the cellular permeability of antibiotics. Inhibitors of this reaction could dramatically increase the efficacy of existing therapeutics. The second assay I developed also targets aaRSs, but utilizes a parallel topology that permits the probing of the synthetic and editing activities of up to four aaRSs simultaneously. The third assay utilizes a linear topology that reconstitutes the entire purine salvage pathway from Plasmodium falciparum. Because parasites are unable to synthesize purines de novo, this pathway represents an appealing target for novel antimalarials. Pilot screens using this assay revealed inhibitors for multiple enzymes in the pathway, validating the design of the system. This body of work aims to shift the current paradigm of single-target systems that have historically dominated the HTS field, toward multi-target designs that can be used to more efficiently screen compound libraries against essential pathways in pathogenic microbes.


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Graduation Date





Roy, Herve


Doctor of Philosophy (Ph.D.)


College of Medicine


Biomedical Sciences

Degree Program

Biomedical Sciences









Release Date

August 2024

Length of Campus-only Access

5 years

Access Status

Doctoral Dissertation (Campus-only Access)

Restricted to the UCF community until August 2024; it will then be open access.