Infections caused by parasitic protozoa, including the organisms Giardia intestinalis and Entamoeba histolytica, are categorized as neglected diseases of poverty. These infections have been associated with more than 50 million cases of intestinal dysentery and an estimated 70-100,000 deaths per year. Additionally, there has been a notable rise in chronic infections from parasites that are refractory to standard antibiotic treatments, further underscoring the need to develop new anti-parasitics with novel targets. The objective of this study is to develop small molecule inhibitors of the two Giardia-specific 5' Methylthioadenosine nucleosidases (MTNs) and demonstrate their in vitro antibiotic efficacy against Giardia intestinalis. MTN is unique to microbe methionine and purine salvage pathways and is not found in humans. This enzyme is responsible for the catabolism of 5' Methylthioadenosine (MTA), a growth inhibitory nucleoside that is the byproduct of S- adenosylmethionine (SAM)-dependent polyamine synthesis and other SAM-dependent reactions. MTN is required to salvage sulfur and purine constituents of MTA that the parasites cannot synthesize de novo. In prior research we characterized the two parasite MTNs and showed that MTN inhibitors are effective anti-parasitic agents in vitro and in vivo. We hypothesize that anti-MTN drugs exert their anti-parasitic effect by depleting essential polyamine and purine pools. To accelerate the rate of drug development, experiments described in Aim 1 of this proposal will synthesize and characterize a library of MTN SMIs based on two recently identified lead compounds. The ability of drug candidates to exhibit anti-MTN activity in vitro will be assessed using an established spectrophotometric enzyme assay. Specificity of the drug candidates will be explored by testing activity against the human enzyme MTAP. Experiments in Aim 2 will further screen compounds showing promising enzyme inhibitory activity for in vitro anti-parasitic activity against cultures of Giardia intestinalis. Synergy studies will be employed to explore the ability of identified drug candidates to promote sensitivity to the established anti-parasitic agent metronidazole. The mechanism of action of MTN SMIs developed as part of this study will be assessed by monitoring global changes in the cellular proteomic and metabolomic composition via LC-MS experiments. All of the experiments described here will involve undergraduate researchers, who have been responsible for generation of most of the preliminary data presented in this proposal. These students are drawn from both Boise State University and the College of Idaho as part of an long term collaboration between the principle investigators that provides innovative multidisciplinary training to 15-20 undergraduates per year in biomedical research. Ultimately, these experiments will help establish MTN as a favorable therapeutic target and aid in the development of a new class of antibiotics to treat parasitic gastrointestinal disease.
The work described in this proposal will establish a fundamentally new family of antibiotics to treat infections caused by the parasitic microorganism Giardia intestinalis, which is responsible for more than 200 million annual cases of diarrheal disease worldwide. Increasing reports of treatment failure with standard antibiotics (e.g. metronidazole) make the development of new drugs to combat these infections critically important. Furthermore, the interdisciplinary nature of this project will provide an opportunity for undergraduate students from the College of Idaho and Boise State University to gain experience in biomedical research and the process of preclinical testing involved in drug development.
