Trypanosoma cruzi is the protozoan trypanosomatid parasite that causes Chagas disease, which affects 10-12 million overwhelmingly poor people in 22 Latin American countries and an estimated 300,000 in the United States. It is transmissible even in non-endemic countries by blood or organ donation, and mother-to-child infection kills 1 in 20 affected children. Often undetected, chronic infection frequently causes fatal cardiomyopathy via insidious destruction of the myocardium and ion conduction systems of the heart, and also causes severe visceral organ disease. Existing therapies have major toxicity problems, require lengthy treatment, and are believed to be effective only against the acute stage of the disease. Developing safe and effective new therapies for Chagas disease requires improving knowledge of valid drug targets in T. cruzi and obtaining rational bases for new drug leads. Polyamine biosynthesis is essential and druggable in trypanosomatid parasites related to T. cruzi. Therefore, I hypothesize (1) that disruption of the polyamine biosynthetic pathway in T. cruzi is lethal or severely detrimental to virulence and (2) that this pathway contains enzymes with distinct features from their human counterparts sufficient to permit their selective inhibitio. To test these hypotheses, I propose to evaluate and characterize promising drug targets in T. cruzi polyamine biosynthesis: ADOMETDC, SPDSYN, SPMSYN, and TRYSYN. To test the essentiality of polyamine biosynthesis in T. cruzi, I will: 1.1) generate deletion mutants of ADOMETDC, SPDSYN and SPMSYN in strains of T. cruzi by homologous recombination and isolate clones bearing these potentially conditionally lethal deletions by supplementation with spermidine and spermine, which can be scavenged by the parasite;1.2) evaluate the effects of these deletions by measuring levels of polyamines and trypanothione (a metabolite immediately downstream of polyamine pathway) as well as parasite growth rates in culture with various polyamine supplementation regimens;1.3) determine infectivity of each deletion strain in mammalian cells. To identify distinct features of polyamine biosynthetic enzymes as a basis for rational drug design, I will: 2.1) develop efficient recombinant ADOMETDC, SPDSYN, SPMSYN and TRYSYN expression systems, and develop and refine protocols to obtain highly purified ADOMETDC, SPDSYN, SPMSYN and TRYSYN;and finally, 2.2) solve and analyze crystal structures of ADOMETDC, SPMSYN, SPDSYN, and TRYSYN alone and in complex with substrates and substrate analogs. In addition to validating new therapeutic approaches to T. cruzi infection, new fundamental insights into polyamine metabolism arising from this study may be applicable to the biology and treatment of other protozoan parasite infections and non-infectious diseases such as cancer.
Chagas disease is a devastating parasitic disease affecting 8-12 million predominantly poor people across Latin America and the United States. Despite the widespread and devastating nature of the disease, only two medicines are available to treat it and both suffer from high toxicity, lengthy courses of treatment, and low efficacy. We believe that several specific parasite genes encode proteins that might be weak points that can be exploited by developing new drugs to target these proteins and kill the parasite, and so the goal of this work is to identify these and find key differences between them and related human genes to help design drugs that kill the parasite and leave the patient unharmed, making an effective drug without harmful side-effects.
