Farnesyl pyrophosphate synthase is an essential enzyme in Trypanosoma brucei. In vitro RNA interference and in vivo inhibition studies.
We report the cloning and sequencing of a gene encoding the farnesyl pyrophosphate synthase (FPPS) of Trypanosoma brucei. The protein (TbFPPS) is an attractive target for drug development because the growth of T. brucei has been shown to be inhibited by analogs of its substrates, the nitrogen containing bisphosphonates currently in use in bone resorption therapy. The protein predicted from the nucleotide sequence of the gene has 367 amino acids and a molecular mass of 42 kDa. Several sequence motifs found in other FPPSs are present in TbFPPS, including an 11-mer peptide insertion present also in the Trypanosoma cruzi FPPS. Heterologous expression of TbFPPS in Escherichia coli produced a functional enzyme that was inhibited by several nitrogen-containing bisphosphonates, such as pamidronate and risedronate. Risedronate was active in vivo against T. brucei infection in mice (giving a 60% survival rate), but pamidronate was not effective. The essential nature of TbFPPS was studied using RNA interference (RNAi) to inhibit the expression of the gene. Expression of TbFPPS double-stranded RNA in procyclic trypomastigotes caused specific degradation of mRNA. After 4 days of RNAi, the parasite growth rate declined and the cells subsequently died. Similar results were obtained with bloodstream form trypomastigotes, except that the RNAi system in this case was leaky and mRNA levels and parasites recovered with time. Molecular modeling and structure-activity investigations of enzyme and in vitro growth inhibition data resulted in similar pharmacophores, further validating TbFPPS as the target for bisphosphonates. These results establish that FPPS is essential for parasite viability and validate this enzyme as a target for drug development.
Montalvetti
Andrea
A
Department of Pathobiology and Center for Zoonoses Research, University of Illinois at Urbana-Champaign, Urbana, Illinois 61802, USA.
Fernandez
Alexis
A
Sanders
John M
JM
Ghosh
Subhash
S
Van Brussel
Erin
E
Oldfield
Eric
E
Docampo
Roberto
R
D019883:Alkyl and Aryl Transferases / Q000032:analysis / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D051228:Geranyltranstransferase; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015800:Protozoan Proteins / Q000032:analysis / Q000235:genetics*; D016415:Sequence Alignment; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics
D015800:Protozoan Proteins; D019883:Alkyl and Aryl Transferases; D051228:Geranyltranstransferase