Access Type

Open Access Dissertation

Date of Award

January 2017

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Biochemistry and Molecular Biology

First Advisor

Ladislau C. Kovari

Abstract

Noroviruses, which are the leading cause of acute gastroenteritis, cause an estimated 677 million infections and 213,000 deaths each year worldwide. Noroviruses are classified into seven genogroups (GI-GVII); GI, GII, and GIV have been shown to be infectious in humans. However, GII noroviruses cause the majority of outbreaks (89%). No pharmacologic treatment or vaccine currently exists to treat or prevent norovirus infections.

Recently, the development of a norovirus replicon system, a murine model of norovirus infection, and the development of a biochemical protease assay have allowed for the design and development of norovirus inhibitors. However, the replicon and biochemical assay were developed with GI noroviruses. In this work, we have developed a system to design, evaluate, and develop inhibitors against GII noroviruses, which are responsible for the majority of outbreaks.

Using molecular dynamics simulations and other computational tools, we have shown that GII norovirus proteases behave comparably in solution with GI norovirus proteases in terms of protein flexibility as well as binding site solvent exposure, druggability, hydrophobicity, and volume. Therefore, we propose that protease inhibitors designed against either GI or GII norovirus proteases would be cross-reactive with the other genogroup – a broad-spectrum norovirus protease inhibitor is likely feasible. In addition, we have developed a fluorescence based biochemical assay to design and evaluate protease inhibitors against GII norovirus proteases, specifically a GII.4 norovirus protease. Using the biochemical assay and computational techniques, we also show that peptidomimetic inhibitors containing and aldehyde as an electrophilic warhead inhibit a GII.4 norovirus protease more potently than peptidomimetic inhibitors which contain an α,β-unsaturated ethyl ester Michael acceptor moiety as an electrophilic warhead.

Additionally, this work also explored the Zika virus (ZIKV) NS2B-NS3 protease as a potential drug target. ZIKV, an emerging flavivirus, was first discovered in 1947 but only recently caused an infectious outbreak of international concern in the Americas in 2015-2016. ZIKV diverged into two distinct lineages: African and Asian. The recent outbreak in the Americas, which has caused a total of 171,553 confirmed infections over 48 countries, is associated with the Asian ZIKV lineage. Previously thought to be a mild infection, the recent outbreak, which is spread by the Aedes spp. mosquitoes, sexual-transmission, as well as vertical transmission from mother to fetus, is associated with more severe complications, the main concern being the rise in microcephaly cases. Depending on when ZIKV was contracted, 10-15% of pregnancies with laboratory confirmed ZIKV infection result in birth defects.

No vaccine or pharmacologic therapy yet exists to prevent or treat ZIKV infections. However, similar to other flaviviruses, the NS2B/NS3 protease has been proposed as a potential drug target. The NS2B protein, a membrane bound protein, exists acts as a cofactor for the NS3 protease during viral polyprotein cleavage. The structure of the ZIKV NS2B/NS3 protease was recently solved by X-ray crystallography, and a fluorescence based biochemical assay has been proposed in order to design, evaluate, and develop NS2B/NS3 protease inhibitors. The X-ray crystal structure and biochemical assay were developed with the NS2B cofactor covalently linked to the NS3 protease. In this work, we created the linked NS2B/NS3 protease, as well as unlinked NS2B/NS3 protease for comparison. The unlinked NS2B/NS3 protease is roughly five-times more active than the linked NS2B/NS3 protease. Molecular dynamics simulations suggest that covalently linking the NS2B cofactor to the NS3 protease may reduce the substrate binding region flexibility as well as sterically hinder substrate binding. Therefore, we propose that unlinked NS2B/NS3 protease be used to design, evaluate, and develop ZIKV protease inhibitors.

This work has resulted in valuable tools and structural insights that can aid the design and development of both norovirus and Zika virus protease inhibitors. In addition, the techniques described can be used to study other proteins, further understand protease behavior, and design new compounds.

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