Interrogating the Structural Landscape of Malaria Biomarkers with Epitope Targeted Peptide Capture Agents

Citation

Liang, JingXin (2018) Interrogating the Structural Landscape of Malaria Biomarkers with Epitope Targeted Peptide Capture Agents. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/rxtr-6152. https://resolver.caltech.edu/CaltechTHESIS:06112018-181603942

Abstract

Antibodies have conventionally been used as molecular recognition agents against epitopes, or antigenic regions, for protein capture and detection. The ability of monoclonal and polyclonal antibodies to selectively bind their targets with high affinities makes them excellent agents for specific protein recognition. However, as large proteins themselves (~150 kDa), antibodies are susceptible to changes in pH, temperature, and biochemical environment, particularly proteolytic cleavage. Additionally, epitope binding on antibodies is reliant on their rigid tertiary structure to position key functional groups that facilitation antigen recognition. Retaining the integrity of the protein structure creates rigid limitations against chemical modifications of antibodies to suit unique needs.

Protein-catalyzed capture agents (PCCs) developed within the Heath group at Caltech address the limitation of antibodies as affinity agents. Using epitope-targeted in situ click screening methodology, the Heath group has developed peptidomimetic molecules that offer an alternative solution to antibodies. These PCCs exhibit high affinity and selectivity for their protein targets. As peptide-based molecules, PCCs can be engineered to be biochemically stable and resistant to changes in their chemical environment. Their peptide-based structures are readily amenable to chemical modifications and allow for adaptation to a range of applications.

This thesis describes the development of PCCs against unique protein biomarkers for the detection of the most lethal species of malaria infection, Plasmodium falciparum. Malaria is a global health epidemic and its eradication is reliant on rapid and accurate diagnostics for prompt treatment. We targeted the P. falciparum specific biomarkers lactate dehydrogenase (LDH) and Histidine-rich protein 2 (HRP2), both of which present unique challenges for protein capture. The LDH biomarker is homologous across malaria species, whereas HRP2 is highly polymorphic and lacks distinct secondary structure. The variation in sensitivity of HRP2 detection by antibody-based tests has been attributed to the genetic polymorphism of the biomarker.

In Chapter 1, we describe the development of high affinity PCCs that bind selectively to the LDH biomarker. We targeted an epitope that was highly homologous across LDH species. This chapter also details the expansion of mono-valent PCC agents into bivalent ligands using the protein architecture to select secondary ligands for binding improvement. For the HRP2 biomarker, we developed a multiple epitope targeting strategy to address protein polymorphism. We targeted for epitopes in HRP2 and developed PCCs that bind in the range of monoclonal antibodies.

Chapter 2 details the expansion of PCC agents developed against HRP2 into multivalent molecules for improved binding. The development of bivalent ligands from combinatorial screening of linker libraries is presented. The optimal linker lengths determined by the screens are described.

In Chapter 3, a general strategy for targeting the protein landscape to inhibit formation of a protein and biomolecule complex with PCCs against HRP2 is demonstrated. Specifically, the inhibition of heme sequestration by HRP2 is shown. A bivalent ligand that targets two epitopes on HRP2 is shown to have enhanced inhibitory potency over any single or cocktail combination of PCCs.

Altogether, the studies herein demonstrate the utility of peptidomimetic molecules as agents for protein capture and detection as well as a generalizable strategy of functional inhibition through epitope-targeting.

Thesis Files