A new drug screening method was devised utilizing Trypanosoma cruzi cells that express the Escherichia coli ␤-galactosidase gene. Transfected parasites catalyze a colorimetric reaction with chlorophenol red ␤-D-galactopyranoside as substrate. Parasite growth in the presence of drugs in microtiter plates was quantitated with an enzyme-linked immunosorbent assay reader. The assay was performed with the mammalian form of T. cruzi that requires intracellular growth on a monolayer of fibroblast cells. To determine if selective toxicity to the parasites was occurring, the viability of the host cells in the drug was assayed with AlamarBlue. The drugs benznidazole, fluconazole, and amphotericin B were shown to inhibit the parasites at concentrations similar to those previously reported. Several compounds were tested that are inhibitors of glyceraldehyde-3-phosphate dehydrogenase of the related organisms Leishmania mexicana and Trypanosoma brucei. One of these compounds, 2-guanidino-benzimidazole, had an 50% inhibitory concentration of 10 M in our assay. Two derivatives of this compound were identified with in vitro activity at even lower concentrations. In addition, the assay was modified for testing compounds for lytic activity against the bloodstream form of the parasite under conditions used for storing blood products. Thus, an assay with ␤-galactosidase-expressing T. cruzi greatly simplifies screening drugs for selective anti-T. cruzi activity, and three promising new compounds have been identified.

The protozoans Cryptosporidium parvum and Toxoplasma gondii are parasites of major health concern to humans. Both parasites contain a group of calcium-dependent protein kinases (CDPKs), which are found in plants and ciliates but not in humans or fungi. Here we describe a series of potent inhibitors that target CDPK1 in C. parvum (CpCDPK1) and T. gondii (TgCDPK1). These inhibitors are highly selective for CpCDPK1 and TgCDPK1 over the mammalian kinases SRC and ABL. Furthermore, they are able to block an early stage of C. parvum invasion of HCT-8 host cells, which is similar to their effects on T. gondii invasion of human fibroblasts.

The current rapid growth in the number of known 3-dimensional protein structures is producing a database of structures that is increasingly useful as a starting point for the development of new medically relevant molecules such as drugs, therapeutic proteins, and vaccines. This development is beautifully illustrated in the recent book, Protein structure: New approaches to disease and therapy (Perutz, 1992). There is a great and growing promise for the design of molecules for the treatment or prevention of a wide variety of diseases, an endeavor made possible by the insights derived from the structure and function of crucial proteins from pathogenic organisms and from man.We present here 2 illustrations of structure-based drug design. The first is the prospect of developing antitrypanosoma1 drugs based on crystallographic, ligand-binding, and molecular modeling studies of glycolytic glycosomal enzymes from Trypanosomatidae. These unicellular organisms are responsible for several tropical diseases, including African and American trypanosomiases, as well as various forms of leishmaniasis. Because the target enzymes are also present in the human host, this project is a pioneering study in selective design. The second illustrative case is the prospect of designing anti-cholera drugs based on detailed analysis of the structure of cholera toxin and the closely related Escherichia coli heat-labile enterotoxin. Such potential drugs can be targeted either at inhibiting the toxin's receptor binding site or at blocking the toxin's intracellular catalytic activity.Study of the Vibrio cholerae and E. coli toxins serves at the same time as an example of a general approach to structure-based vaccine design. These toxins exhibit a remarkable ability to stimulate the mucosal immune system, and early results have suggested that this property can be maintained by engineered fusion proteins based on the native toxin structure. The challenge is thus to incorporate selected epitopes from foreign pathogens into the native framework of the toxin such that crucial features of both the epitope and the toxin are maintained.That is, the modified toxin must continue to evoke a strong mucosal immune response, and this response must be directed against an epitope conformation characteristic of the original pathogen.

The structure of triosephosphate isomerase from Trypanosoma brucei complexed with the competitive inhibitor N‐hydroxy‐4‐phosphono‐butanamide was determined by X‐ray crystallography to a resolution of 2.84 å. Full occupancy binding of the inhibitor is observed only at one of the active sites of the homodimeric enzyme where the flexible loop is locked in a completely open conformation by crystal contacts. There is evidence that the inhibitor also binds to the second active site of the enzyme, but with low occupancy. The hydroxamyl group of the inhibitor forms hydrogen bonds to the side chains of Asn 11, Lys 13, and His 95, whereas each of its three methylene units is involved in nonpolar interactions with the side chain of the flexible loop residue Ile 172. Interactions between the hydroxamyl and the catalytic base Glu 167 are absent. The binding of this phosphonate inhibitor exhibits three unusual features: (1) the flexible loop is open, in contrast with the binding mode observed in eight other complexes between triosephosphate isomerase and various phosphate and phosphonate compounds; (2) compared with these complexes the present structure reveals a 1.5‐å shift of the anion‐binding site; (3) this is the first phosphonate inhibitor that is not forced by the enzyme into an eclipsed conformation about the P‐CH2 bond. The results are discussed with respect to an ongoing drug design project aimed at the selective inhibition of glycolytic enzymes of T. brucei.