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In the 100 years since the discovery of Chagas disease, only two drugs have been developed and introduced into clinical practice, and these drugs were introduced over 40 years ago. The tools of drug discovery have improved dramatically in the interim; however, this has not translated into new drugs for Chagas disease. This has been largely because the main practitioners of drug discovery are pharmaceutical companies who are not financially motivated to invest in Chagas disease and other "orphan" diseases. As a result, it has largely been up to academic groups to bring drug candidates through the discovery pipeline and to clinical trials. The difficulty with drug discovery in academia has been the challenge of bringing together the diverse expertise in biology, chemistry, and pharmacology in concerted efforts towards a common goal of developing therapeutics. Funding is often inadequate, but lack of coordination amongst academic investigators with different expertise has also contributed to the slow progress. The purpose of this chapter is to provide an overview of approaches that can be accomplished in academic settings for preclinical drug discovery for Chagas disease. The chapter addresses methods of drug screening against Trypanosoma cruzi cultures and in animal models and includes general topics on compound selection, testing for drug-like properties (including oral bioavailability), investigating the pharmacokinetics and toxicity of compounds, and finally providing parameters to help with triaging compounds., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The 2.1A crystal structure of tryptophanyl-tRNA synthetase (TrpRS) from the diplomonad Giardia lamblia reveals that the N-terminus of this class I aminoacyl-tRNA synthetase forms a 16-residue alpha-helix. This helix replaces a beta-hairpin that is required by human TrpRS for normal activity and has been inferred to play a similar role in all eukaryotic TrpRS. The primary sequences of TrpRS homologs from several basal eukaryotes including Giardia lack a set of three residues observed to stabilize interactions with this beta-hairpin in the human TrpRS. Thus the present structure suggests that the activation reaction mechanism of TrpRS from the basal eukaryote G. lamblia differs from that of higher eukaryotes. Furthermore, the protein as observed in the crystal forms an (alpha(2))(2) homotetramer. The canonical dimer interface observed in all previous structures of tryptophanyl-tRNA synthetases is maintained, but in addition each N-terminal alpha-helix reciprocally interlocks with the equivalent helix from a second dimer to form a dimer of dimers. Although we have no evidence for tetramer formation in vivo, modeling indicates that the crystallographically observed tetrameric structure would be compatible with the tRNA binding mode used by dimeric TrpRS and TyrRS., (Copyright 2010 Elsevier Inc. All rights reserved.)

The great power of protein crystallography to reveal biological structure is often limited by the tremendous effort required to produce suitable crystals. A hybrid crystal growth predictive model is presented that combines both experimental and sequence-derived data from target proteins, including novel variables derived from physico-chemical characterization such as R(30), the ratio between a protein's DSF intensity at 30°C and at T(m). This hybrid model is shown to be more powerful than sequence-based prediction alone - and more likely to be useful for prioritizing and directing the efforts of structural genomics and individual structural biology laboratories., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Trypanosoma cruzi, the protozoan that causes Chagas' disease, was transfected with a fusion gene of hygromycin phosphotransferase and herpes simplex virus-thymidine kinase, HyTK. Transfectants selected in hygromycin had thymidine kinase activity, whereas controls did not. In vitro growth of the mammalian life-stage forms, amastigotes and trypomastigotes, was inhibited 98% by the nucleoside analogue ganciclovir (5 micrograms/ml). Growth of the insect-stage form, epimastigotes, was not inhibited by ganciclovir (up to 250 micrograms/ml) or other nucleoside analogues. Intracellular uptake of ganciclovir by epimastigotes was found to be 10-fold less than that by amastigotes. Mice infected with the HyTK-expressing parasites and treated with ganciclovir had a statistically significant reduction of parasitemia by 57%; however, complete eradication of parasites was not achieved. The parasites recovered from the treated mice continued to be susceptible to ganciclovir in vitro. Parasite clones with higher expression of thymidine kinase were more sensitive to ganciclovir, suggesting that greater expression of the thymidine kinase gene may lead to parasites that can be fully eradicated from infected experimental animals.