Exploring Kinase Function and Drug Targets in Giardia lamblia

Abstract

Giardia lamblia is the most commonly reported intestinal protozoan parasite and the cause of giardiasis, a gastrointestinal illness resulting in diarrhea, nutrient malabsorption, vomiting, and weight loss 1. It infects approximately 280 million people worldwide annually 2–4. G. lamblia has a simple life cycle consisting of two forms, the binucleate flagellated trophozoites and the tetranucleate infective cysts. Cysts are the environmentally resistant forms responsible for transmission of the disease1. In countries where water quality is compromised, waterborne diseases such as giardiasis are common. This disease contributes to the global health burden of diarrheal diseases that collectively constitute the second-leading cause of death in children under five years old2,4. Infection can also cause developmental delays and failure to thrive5; as few as 3 occurrences (>2 weeks duration) of diarrheal disease per year during the first 2 years of life is associated with reduced height (approximately 10 cm) and intelligence quotient score (10 points) by 7 – 9 years of age6. First choice therapeutic options are limited to metronidazole and chemically related nitroimidazole drugs. Resistance can occur in up to 20% of clinical presentations 7,8. The toxic intermediates cause DNA damage in G. lamblia trophozoites9, and attack protein sulfhydryl groups non-specifically. Even when infection is cleared, pathophysiological changes in the gut may persist, severely impacting quality of life 4,8. Consequently, emergence of metronidazole-resistance strains and adverse reactions to the treatments suggest that alternative therapies against giardiasis are necessary. In summary, this dissertation is an investigation to find targets in G. lamblia that are druggable. We used genetic and molecular techniques to inform us of the biology and of targets essential to the survival of the parasite. Chapter 1 consists of the Introduction, which provides a more detailed explanation of why this research is needed. Chapter 2 of my dissertation focuses on targeting the ATP binding pocket of kinases with small molecule inhibitors. We identified a set of protein kinases in G. lamblia that have small amino acid residues in the gatekeeper position; these small gatekeeper kinases are uncommon in the mammalian kinome, creating opportunity for drug targeting by a class of small molecule inhibitors called “bumped” kinase inhibitor (BKIs). In Chapter 2, we profiled two kinases determined to be essential to cell survival and sensitive to BKIs. Based on phenotypic characteristics, Chapter 3 involves investigating the biological role of one of the two kinases identified during the work done in Chapter 2. Chapter 4 describes the development of a tool that can be used as a high-throughput screening method to assay large drug libraries. This will be essential in profiling small-molecule inhibitors that can be used to target the kinases described in Chapter 2 and 3, as well as future targets in G. lamblia. As a proof of concept, we screened the Pathogen Box available through the Medicines for Malaria Venture. Each of the compounds in the Pathogen Box has confirmed activity against at least one of the key pathogens that cause some of the most neglected diseases on the planet. The Pathogen Box has not been screened against G. lamblia, to our knowledge. Chapter 5 describes an entirely different approach to target G. lamblia. We targeted the Prolyl-tRNA molecule with compounds designed to stall protein synthesis. Recent work identified prolyl-tRNA (ProRS) as a target of halofuginone, a drug derived from a natural product found in traditional herbal treatments for malaria. 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