PROTEIN FARNESYLTRANSFERASE FROM TRYPANOSOMA-BRUCEI - A HETERODIMER OF 61-KDA AND 65-KDA SUBUNITS AS A NEW TARGET FOR ANTIPARASITE THERAPEUTICS

We have previously shown that protein prenylation occurs in the Trypan osomatids Trypanosoma brucei (T, brucei), Trypanosoma cruzi, and Leish mania mexicana and that protein farnesyltransferase (PFT) activity can be detected in cytosolic extracts of insect (procyclic) form T, bruce i, A PFT that transfers the farnesyl group from farnesyl pyrophosphate to a cysteine that is 4 residues upstream of the C terminus of the Ra s GTP-binding protein RAS1-CVIM has now been purified 60,000-fold to n ear homogeneity from procyclic T, brucei. By screening a mixture of he xapeptides SSCALX (X is 20 different amino acids), it was found that S SCALM binds to T, brucei PFT with sub-micromolar affinity, and affinit y chromatography using this peptide was a key step in the purification of this enzyme. On SDS-polyacrylamide gel electrophoresis, the enzyme migrates as a pair of bands with apparent molecular masses of 61 and 65 kDa, and thus its subunits are similar to 30% larger than those of the mammalian homolog, The 61-kDa band was identified as the putative beta-subunit by photoaffinity labeling with a P-32-labeled analog of f arnesyl pyrophosphate, Mimetics of the C-terminal tetrapeptide of pren yl accepters have been previously shown to inhibit mammalian PFT, and these compounds also inhibit T. brucei PFT with affinities in the nano molar to micromolar range, although the structure-activity relationshi p is very different for parasite versus mammalian enzyme. Unlike mamma lian cells, the growth of bloodstream T, brucei is completely inhibite d by low micromolar concentrations of two of the PFT inhibitors, and t hese compounds also block protein farnesylation in cultured parasites. These compounds also potently block the growth of the intracellular ( amastigote) form of T, cruzi grown in fibroblast host cells. The resul ts suggest that protein farnesylation is a target for the development of anti-trypanosomatid chemotherapeutics.