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The structure of the glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) from Trypanosoma cruzi complexed with chalepin, a natural product from Pilocarpus spicatus, has been determined by X-ray crystallography to 1.95 A î resolution. The structure is in the apo form without cofactors in the subunits of the tetrameric gGAPDH in the asymmetric unit. Unequivocal density corresponding to the inhibitor was clearly identified in one monomer. The final refined model of the complex shows extensive conformational changes when compared with the native structure. The mode of binding of chalepin to gGAPDH and its implications for inhibitor design are discussed. ß

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Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the reversible oxidative phosphorylation of d-glyceraldehyde 3-phosphate (GAP) into d-glycerate 1,3-bisphosphate (1,3-diPG) in the presence of NAD(+) and inorganic phosphate (P(i)). Within the active site, two anion-binding sites were ascribed to the binding of the C3 phosphate of GAP (P(s)) and to the binding of the attacking phosphate ion (P(i)). The role played by these two sites in the catalytic mechanism in connection with the functional role of coenzyme exchange (NADH-NAD(+) shuttle) has been investigated by several studies leading to the C3 phosphate flipping model proposed by Skarzynski et al. [Skarzynski, T., Moody, P. C., and Wonacott, A. J. (1987) J. Mol. Biol. 193, 171-187]. This model has not yet received direct confirmation. To gain further insight into the role of both sites, we synthesized irreversible inhibitors which form with the essential cysteine residue a thioacyl enzyme analogue of the catalytic intermediate. Here we report the refined glycosomal Trypanosoma cruzi GAPDH in complex with a covalently bound GAP analogue at an improved resolution of 2.0-2.5 A. For this holo-thioacyl enzyme complex, a flip-flop movement is clearly characterized, the change from the P(i) to the P(s) binding site being correlated with the coenzyme exchange step: the weaker interaction of the intermediate when bound at the P(s) site with the cofactor allows its release and also the binding of the inorganic phosphate for the next catalytic step. This result gives strong experimental support for the generally accepted flip-flop model of the catalytic mechanism in GAPDH.

Schistosomiasis is considered the second most important tropical parasitic disease, with severe socioeconomic consequences for millions of people worldwide. Schistosoma mansoni , one of the causative agents of human schistosomiasis, is unable to synthesize purine nucleotides de novo, which makes the enzymes of the purine salvage pathway important targets for antischistosomal drug development. In the present work, we describe the development of a pharmacophore model for ligands of S. mansoni purine nucleoside phosphorylase (SmPNP) as well as a pharmacophore-based virtual screening approach, which resulted in the identification of three thioxothiazolidinones (1-3) with substantial in vitro inhibitory activity against SmPNP. Synthesis, biochemical evaluation, and structure-activity relationship investigations led to the successful development of a small set of thioxothiazolidinone derivatives harboring a novel chemical scaffold as new competitive inhibitors of SmPNP at the low-micromolar range. Seven compounds were identified with IC(50) values below 100 μM. The most potent inhibitors 7, 10, and 17 with IC(50) of 2, 18, and 38 μM, respectively, could represent new potential lead compounds for further development of the therapy of schistosomiasis.