Fighting parasitic disease by blocking protein farnesylation

Protein farnesylation is a form of posttranslational modification that occurs in most, if not all, eukaryotic cells. Inhibitors of protein farnesyltransferase (PFTIs) have been developed as anticancer chemotherapeutic agents. Using the knowledge gained from the development of PFTIs for the treatment of cancer, researchers are currently investigating the use of PFTIs for the treatment of eukaryotic pathogens. This “piggy-back” approach not only accelerates the development of a chemotherapeutic agent for protozoan pathogens but is also a means of mitigating the costs associated with de novo drug design. PFTIs have already been shown to be efficacious in the treatment of eukaryotic pathogens in animal models, including both Trypanosoma brucei, the causative agent of African sleeping sickness, and Plasmodium falciparum, one of the causative agents of malaria. Here, current evidence and progress are summarized that support the targeting of protein farnesyltransferase for the treatment of parasitic diseases.—Eastman, R. T., F. S. Buckner, K. Yokoyama, M. H. Gelb, and W. C. Van Voorhis. Fighting parasitic disease by blocking protein farnesylation. J. Lipid Res. 2006. 47: 233–240. Supplementary key words antiprotozoal drugs . Plasmodium . Trypanosoma . Toxoplasma . Giardia . Entamoeba . malaria . trypanosomiasis Parasitic diseases continue to have a major impact on morbidity and mortality in tropical and subtropical regions. Among these, malaria causes 300 million infections annually, with 1–3 million deaths occurring in Africa (1). The emergence and spread of parasites resistant to existing antimalarial agents is largely responsible for the recent increase in malaria-related mortality. Another reemerging disease is African sleeping sickness (African trypanosomiasis), with an estimated 50,000 deaths in 2002 (1). The increasing burden of these diseases, along with the inadequacies of current drugs for African sleeping sickness in terms of safety, efficacy, and ease of administration, have led investigators to seek new chemotherapeutic agents (2, 3). Among the current drug targets under study are enzymes involved in protein prenylation, or the posttranslational modification of proteins by the covalent modification by isoprenyl lipids, C15 farnesyl and C20 geranylgeranyl (4–7). The isoprenyl lipid modification of proteins has been shown to be critical for various cellular activities in mammals and yeast, including proliferation and apoptosis (8, 9). Growth of the protozoan parasites has been shown to be severely impaired by the inhibition of protein farnesylation compared with mammalian cells, suggesting high potential of the enzyme protein farnesyltransferase (PFT) as an antiparasitic drug target (5, 10–13). The isoprenoid synthesis pathway from mevalonic acid in many eukaryotes, including trypanosomatids (or deoxyxylulose in Apicomplexa, including Plasmodium and Toxoplasma, and plants) is essential for the production of sterols, dolichol, ubiquinone, and other isoprene derivatives in many eukaryotic cells. Indeed, these pathways have been the study of recent efforts to develop other antiparasitic chemotherapeutic agents, especially the targeting of isoprenoid pyrophosphate synthesis by nitrogen-containing bisphosphonates (14–16). Organisms belonging to the group Apicomplexa contain the nonmevalonate pathway of isoprenoid biosynthesis. One enzyme in this pathway is 2C-methyl-D-erythritol 4-phosphate synthase (IspC protein), which is inhibited by fosmidomycin (17). This has led to a clinical trial using fosmidomycin and clindamycin in combinational therapy for the treatment of malaria (18). This review will discuss the current efforts and progress in developing inhibitors of protein farnesyltransferase (PFTIs) as antiparasitic agents. Manuscript received 1 November 2005 and in revised form 6 December 2005. Published, JLR Papers in Press, December 7, 2005. DOI 10.1194/jlr.R500016-JLR200 Abbreviations: ED50, effective dose that inhibits 50% of parasite proliferation; PFT, protein farnesyltransferase; PFTI, protein farnesyltransferase inhibitor; PGGT-I, protein geranylgeranyltransferase type I; THQ, tetrahydroquinoline. 1 To whom correspondence should be addressed. e-mail: [email protected] (M.H.G.); [email protected] (W.C.V.V.) Copyright D 2006 by the American Society for Biochemistry and Molecular Biology, Inc. This article is available online at http://www.jlr.org Journal of Lipid Research Volume 47, 2006 233 by gest, on S etem er 8, 2017 w w w .j.org D ow nladed fom PROTEIN PRENYLATION IN HIGHER EUKARYOTIC CELLS Protein prenylation refers to the posttranslational modification of proteins by the covalent attachment of a 15 carbon farnesyl or a 20 carbon geranylgeranyl group. The structure of both the farnesyl and geranylgeranyl groups appended to proteins was determined in the early 1990s by Glomset, Gelb, and Farnsworth (19). This type of posttranslational modification creates a hydrophobic tail that facilitates membrane association as well as proteinprotein interactions. Among known prenylated proteins are small GTPases, including Ras, Rac, Rho, and Rab, which play a role in cell signal transduction, vesicle trafficking, and cell cycle progression (20). Protein prenylation is mediated by three distinct enzymes: PFT, protein geranylgeranyltransferase type I (PGGT-I), and PGGT-II. PFT recognizes a CaaX motif at the C terminus of specific proteins and transfers a farnesyl from farnesyl pyrophosphate to the thiol group of the cysteine: the CaaX motif is a cysteine followed by two amino acids (typically aliphatic) and a terminal amino acid, X, which is typically a Ser, Met, Ala, or Gln (21). PFT is a zinc-dependent heterodimeric enzyme with an aand a h-subunit. PGGT-I shares the same a-subunit as PFT but has a distinct h-subunit. PGGT-I catalyzes the attachment of geranylgeranyl to proteins with the CaaX motif, in which X is usually a Leu or Phe (22). For both PFT and PGGT-I, other residues may be tolerated in the X position (23). After the action of either PFT or PGGT-I, a prenyl protein-specific protease cleaves the terminal tripeptide from the prenylated protein (24). The final step is methylation of the terminal carboxylic acid by a prenyl protein-specific methyltransferase (25–27). Both of these subsequent enzymatic steps have been shown to be required for the proper localization of certain mammalian proteins and are currently being investigated as additional chemotherapeutic targets (28, 29). The third prenylation enzyme, PGGT-II, catalyzes the addition of two geranylgeranyl groups onto the terminal residues of proteins ending with CC, CCXX, or CXC motifs. To date, proteins modified by PGGT-II have been exclusively members of the Rab low molecular weight G protein family (21). Because of the discoveries that the Ras oncogene is farnesylated and that this modification is required for the proper localization and function of Ras (30–32), protein prenylation has received significant attention as a potential anticancer chemotherapeutic target (33–35). Mutations in Ras are associated with 20–25% of human cancers and 90% of pancreatic carcinomas (34). Numerous pharmaceutical companies have initiated drug discovery programs to generate PFTIs for the treatment of cancer. The first PFTI, which was described in 1993, was found in a chemical library screen based on the ability to inhibit yeast PFT activity (36). There are currently .2,000 primary publications on PFT inhibitors and .300 patents worldwide. Four companies have entered clinical trials for the development of PFTIs as a cancer chemotherapeutic agent: Janssen/Johnson & Johnson, Schering-Plough, Merck, and Bristol-Myers Squibb (37–42). Janssen/Johnson & Johnson and Schering-Plough are advancing to late clinical trials for the use of PFTIs in the treatment of certain leukemias (43). To date, PFTIs have proven to be relatively nontoxic in clinical trials and effective when combined with other chemotherapeutic agents for the treatment of certain cancers in vivo (43, 44). Because of strong interest in the development of PFTIs for the treatment of cancer, there is a wealth of pharmacologic information about PFTIs. This pharmacologic information, the lack of toxicity, and a rich source of small-molecule PFTI libraries provide an excellent opportunity for the “piggy-back” investigation of PFTIs for the treatment of tropical diseases such as malaria and African sleeping sickness. PFT IN PATHOGENIC PROTOZOA Protein prenylation occurs in a wide variety of pathogenic protozoa, including Trypanosoma brucei (6, 45), Trypanosoma cruzi (46), Leishmania species (46), Plasmodium falciparum (4, 5), Toxoplasma gondii (47), Giardia lamblia (48), and Entamoeba histolytica (49). Cloning and characterization of the PFT enzyme from trypanosomatid parasites was originally described by our group (46, 50). PFT enzymatic activity was detected in cytosolic fractions of T. brucei using the yeast Ras1 protein containing the Cterminal CaaX sequence Cys-Val-Ile-Met as a substrate (6). T. brucei PFT was subsequently isolated and purified using affinity chromatography with the CaaX peptide Ser-SerCys-Ala-Leu-Met (51). Similar to mammalian PFT, T. brucei PFT is a heterodimer. However, the subunits are larger, owing to numerous peptide segment insertions. These insertions are predicted, by molecular modeling using the known mammalian PFT structure, to be in loops on the surface of the protein and distant from the active site (46, 51). Insertions are also observed in T. cruzi, Leishmania species, and P. falciparum PFTs (5, 46), but their function is as yet unknown. The CaaX substrate specificity differs in T. brucei PFT compared with mammalian PFT, with a higher preference for substrates with a Met or Gln at the X position. Alteration of four amino acid residues in the putative X binding pocket in the active site of T. brucei could be responsible for the restricted peptide substrate specificity and suggests the potential for developing parasite-specific PFT inhibitors (50). P. falciparum PFT was first characterized by Chakrabarti et al. (4, 5). After partial purification by (NH4)2SO4 precipitation and anion-exchange chromatography, it was shown that the CaaX substrate specificity was similar to that of T. brucei PFT, favoring a Met or Gln in the terminal position. Metabolic radiolabeling of prenylated cellular proteins with [H]farnesol demonstrated the incorporation of H into 50 kDa proteins and some lower molecular mass proteins. The 50 kDa proteins were analyzed and found to be modified by a farnesyl group; the lower molecular mass proteins, however, were found to be geranylgeranylated, presumably after the conversion of farnesol into both farnesyl pyrophosphate and geranylger234 Journal of Lipid Research Volume 47, 2006 by gest, on S etem er 8, 2017 w w w .j.org D ow nladed fom anyl pyrophosphate. Our group later showed that the 50 kDa farnesylated proteins, but not the lower molecular mass geranylgeranylated proteins, were specifically inhibited by PFTIs (11). Using synchronized P. falciparum, Chakrabarti et al. (5) demonstrated the stage-specific incorporation of prenylation precursors, the highest amount of incorporation occurring in the trophozoite (mid erythrocytic stage) to schizont (cell division stage) and schizont to ring (early erythrocytic stage) transition states in the erythrocytic life cycle of the parasite. Using a polyclonal antibody raised against rat PFT, Ibrahim et al. (47) immunoprecipitated and identified the T. gondii PFT enzyme. PFT enzyme activity was confirmed using a CaaX-containing lamin substrate. Incubating tachyzoites (intracellular replicative form) with radiolabeled farnesol or geranylgeraniol demonstrated the in vivo prenylation of proteins, with the geranylgeranylation of proteins of 29 kDa and the farnesylation of proteins of 47 kDa (47). Inhibition of the T. gondii PFT enzyme occurred using hydrophobic the peptidomimetic inhibitors Fig. 1. The structures of selected protein farnesyltransferase inhibitors (PFTIs). Fighting parasitic disease by blocking protein farnesylation 235 by gest, on S etem er 8, 2017 w w w .j.org D ow nladed fom FTase Inhibitor I and II (Fig. 1). However, these inhibitors had no effect against the inhibition of PFT enzyme activity in intact parasites, presumably because of poor cellular penetration, as indicated by normal radiolabeling of proteins with [H]farnesol in inhibitor-treated cultures (47). Although the PFT enzyme of G. lamblia has not been isolated, prenylation of proteins has been demonstrated in this primitive eukaryote (48). [H]mevalonic acid was specifically incorporated into cellular proteins of 50 and 20–30 kDa. After cleavage with methyl iodide, the isoprenoid substituents of these proteins were subjected to HPLC analysis and found to run with the same retention time as farnesol and geranylgeraniol (48). Inhibitors of PFT, such as limonene (Fig. 1), perillic acid, and perillyl alcohol, showed a dose-dependent effect on trophozoite (the replicative form of Giardia) growth in vitro (48); however, these agents are not very potent inhibitors of PFTs, and it is difficult to judge whether inhibition of Giardia PFT is the reason for the growth arrest. Based on a genome search, Kumagai et al. (49) have identified and characterized PFT in E. histolytica. Interestingly, it was found that E. histolytica PFT does not preferentially modify proteins with a terminal Met residue. Instead, E. histolytica PFT favors CaaX substrates with smaller terminal amino acids, Ala and Ser, which suggests an altered binding cleft for the CaaX substrate. In further support of the altered substrate specificity of E. histolytica PFT, this PFT has a higher resistance to the CaaM peptidomimetic FTI-276 (Fig. 1) compared with other PFT enzymes (49).