Conformational changes in Leishmania mexicana glyceraldehyde-3-phosphate dehydrogenase induced by designed inhibitors.

The glycolytic enzymes of trypanosomes are attractive drug targets, since the blood-stream form of Trypanosoma brucei lacks a functional citric acid cycle and is dependent solely on glycolysis for its energy requirements. Glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from the pathogenic trypanosomatids T. brucei, Trypanosoma cruzi and Leishmania mexicana are quite similar to each other, and yet have sufficient structural differences compared to the human enzyme to enable the structure-based design of compounds that selectively inhibit all three trypanosomatid enzymes but not the human homologue. Adenosine analogs with substitutions on N-6 of the adenine ring and on the 2' position of the ribose moiety were designed, synthesized and tested for inhibition. Two crystal structures of L. mexicana glyceraldehyde-3-phosphate dehydrogenase in complex with high-affinity inhibitors that also block parasite growth were solved at a resolution of 2.6 A and 3.0 A. The complexes crystallized in the same crystal form, with one and a half tetramers in the crystallographic asymmetric unit. There is clear electron density for the inhibitor in all six copies of the binding site in each of the two structures. The L. mexicana GAPDH subunit exhibits substantial structural plasticity upon binding the inhibitor. Movements of the protein backbone, in response to inhibitor binding, enlarge a cavity at the binding site to accommodate the inhibitor in a classic example of induced fit. The extensive hydrophobic interactions between the protein and the two substituents on the adenine scaffold of the inhibitor provide a plausible explanation for the high affinity of these inhibitors for trypanosomatid GAPDHs.

TcRho1, a farnesylated Rho family homologue from Trypanosoma cruzi: cloning, trans-splicing, and prenylation studies.

Rho GTPases are members of the Ras superfamily and are involved in signal transduction pathways, including maintenance of cell morphology and motility, cell cycle progression, and transcription activation. We report the molecular identification in trypanosomatids (Trypanosoma cruzi) of the first member of the Rho family. The cloned Rho protein, TcRho1, shares approximately 40% homology with other members of the Rho family. Southern blot analysis revealed that TcRHO1 is a single copy gene per haploid genome, and Northern blot assays showed a transcript of 1200 nucleotides in length. Mapping the 5'-untranslated region of TcRHO1 transcripts revealed at least five different transcripts derived from differential trans-splicing. Three of the five transcripts contain the trans-splicing site within the coding region of the TcRHO1 gene. TcRho1 also contains the C-terminal sequence CQLF (CAAX motif), which is predicted to direct post-translation prenylation of the cysteine residue. A synthetic peptide containing this C-terminal motif, when tested against Q-Sepharose chromatography fractions from T. cruzi cytosol, was shown to be efficiently farnesylated, but not geranylgeranylated, despite the fact that the CAAX motif with X = Phe specifies geranylgeranylation by mammalian protein geranylgeranyltransferase I. Furthermore, immunoblot analyses of epimastigote protein with anti-S-farnesylcysteine methyl ester and anti-TcRho1 antisera strongly suggested that TcRho1 is farnesylated in vivo. The farnesylation of proteins such as Rho GTPases could be the basis for the selective cytotoxic action of protein farnesyltransferase inhibitors on trypanosomatids versus mammalian cells.