Resistance to a Protein Farnesyltransferase Inhibitor in Plasmodium falciparum

Abstract

The post-translational farnesylation of proteins serves to anchor a subset of intracellular proteins to membranes in eukaryotic organisms and also promotes protein-protein interactions. Inhibition of protein farnesyltransferase (PFT) is lethal to the pathogenic protozoa Plasmodium falciparum. Parasites were isolated that were resistant to BMS-388891, a tetrahydroquinoline (THQ) PFT inhibitor. Resistance was associated with a 12-fold decrease in drug susceptibility. Genotypic analysis revealed a single point mutation in the ␤ subunit in resistant parasites. The resultant tyrosine 837 to cysteine alteration in the ␤ subunit corresponded to the binding site for the THQ and peptide substrate. Biochemical analysis of Y837C-PFT demonstrated a 13-fold increase in BMS-388891 concentration necessary for inhibiting 50% of the enzyme activity. These data are consistent with PFT as the target of BMS-388891 in P. falciparum and suggest that PFT inhibitors should be combined with other antimalarial agents for effective therapy.Malaria causes about 300 million infections annually.1 Approximately 90% of the deaths occur in Africa, with falciparum malaria a major contributor. For decades, malaria chemotherapy has relied on a limited number of drugs. Acquisition and spread of resistance to these drugs is largely responsible for a recent increase in malaria-related mortality (2, 3). This increasing burden caused by drug-resistant parasites has led investigators to seek out novel antimalarial drug targets. Among these are enzymes necessary for cellular division and differentiation. Previous work has demonstrated that the enzyme protein farnesyltransferase (PFT) 2 is a viable drug target for pathogenic protozoa, including the malaria parasite Plasmodium falciparum (4 -8).3 PFT inhibitors (PFTIs) have been developed by the pharmaceutical industry because of their anti-cancer properties (9 -11). Utilizing this existing resource, we have been able to demonstrate that low nanomolar concentrations of tetrahydroquinoline (THQ)-based PFTIs inhibit P. falciparum PFT (PfPFT) and are cytotoxic to parasites both in vitro and in vivo. 3PFT in eukaryotic cells catalyzes the transfer of a 15-carbon isoprenoid lipid unit, a farnesyl group, from farnesyl pyrophosphate (FPP) to the C terminus of a specific set of proteins, including the Ras superfamily G-proteins (12). In addition to the Ras superfamily proteins, which are involved in the cell cycle, another protein that has been shown to be farnesylated is CENP-E, a centromere-associating protein, which plays a critical role in cell cycle progression during the M-phase (13). However, the identification of farnesylated proteins in P. falciparum and their role in mediating PFTI toxicity are currently under investigation.Because of the enzymatic nature of the drug target, we have begun to investigate potential P. falciparum resistance to PFTIs. The THQ PFTI used in this work, BMS-388891 (Fig. 1), is a potent inhibitor of parasite proliferation and causes severe defects in maturation.3 In addition, w...

Therapeutic intervention based on protein prenylation and associated modifications

“…[133][134][135][136] ). In the case of malaria, the target of FTI toxicity is almost certainly PFT, given that parasites that have become resistant to FTIs contain mutant PFTs that bind FTIs less tightly 137 . The tetrahydroquinoline series of FTIs, originally developed by Bristol-Myers Squibb as anticancer agents, are the most promising antimalarial FTIs reported so far.…”

Discovery of new targets for antimalarial chemotherapy

“…As well, these studies underlined the important role of farnesylated proteins in P. falciparum life cycle, and the need to identify and characterize these potential new targets. A P. falciparum clone resistant to the THQ PFT inhibitor BMS-388891 was experimentally obtained under drug pressure (Eastman et al, 2005). This resistance was associated with a single point mutation (adenine > guanine) in the gene coding for the β subunit of PFT that changed a tyrosine for a cysteine in position 837, predicted to be in the peptide binding pocket.…”

“…They, as well as all future antimalarials, will need to be used in combination with other drugs to decrease the occurrence of resistance. The Y837C mutation arised independently at least twice in 3 × 10 8 parasites in vitro suggesting that this mutation could arise multiple times in an infected person (Eastman et al, 2005).…”

Protein geranylgeranyltransferase-I of Trypanosoma cruzi

“…This includes a subset of the active site residues, suggesting distinct specificity of binding to CaaX peptide substrates and inhibitors between PFT enzymes from these different species [15,16,18,40]. PGGT-I β orthologs have not been identified in gene databases of T. brucei and Leishmania species.…”

“…These are consistent with the CaaX specificity of the T. cruzi PGGT-I (Table1). Alterations in the residues involving substrate interactions in PFT and PGGT-I subunits between protozoan and mammalian orthologs may cause substantial differences in binding specificity for the CaaX motif and smallmolecule inhibitors in protozoa and mammalian cells [15][16][17][18][19][20][21]40]. This suggests not only different selectivity of inhibitors against parasite PFT and PGGT-I from that of mammalian enzymes but also differential consequences of inhibitory effects of PGGT-I or PFT on cellular events in the parasites from those in mammalian cells.…”