Resistance mutations at the lipid substrate binding site of Plasmodium falciparum protein farnesyltransferase

doi:10.1016/j.molbiopara.2006.11.012

Molecular and Biochemical Parasitology

Volume 152, Issue 1, March 2007, Pages 66-71

https://doi.org/10.1016/j.molbiopara.2006.11.012 Get rights and content

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. This enzymatic reaction is carried out by protein farnesyltransferase (PFT), which catalyzes the transfer of a 15-carbon isoprenoid lipid unit, a farnesyl group, from farnesyl pyrophosphate to the C-termini of proteins containing a CaaX motif. Inhibition of PFT is lethal to the pathogenic protozoa Plasmodium falciparum. Previously, we have shown that parasites resistant to a tetrahydroquinoline (THQ)-based PFT inhibitor BMS-388891 have mutations leading to amino acid substitutions in PFT that map to the peptide substrate binding domain. We now report the selection of parasites resistant to another THQ PFT inhibitor BMS-339941. In whole cell assays sensitivity to BMS-339941 was reduced by 33-fold in a resistant clone, and biochemical analysis demonstrated a corresponding 33-fold increase in the BMS-339941 K_i for the mutant PFT enzyme. More detailed kinetic analysis revealed that the mutant enzyme required higher concentration of peptide and farnesyl pyrophosphate substrates for optimum catalysis. Unlike previously characterized parasites resistant to BMS-388891, the resistant parasites have a mutation which is predicted to be in a distinct location of the enzymatic pocket, near the farnesyl pyrophosphate binding pocket. This is the first description of a mutation from any species affecting the farnesyl pyrophosphate binding pocket with reduced efficacy of PFT inhibitors. These data provide further support that PFT is the target of THQ inhibitors in P. falciparum and suggest that PFT inhibitors should be combined with other antimalarial agents to minimize the development of resistant parasites.

Introduction

Malaria causes about 300 million infections annually [1]. Approximately 90% of deaths occur in Africa, due to falciparum malaria. For decades, malaria chemotherapy has relied on a limited number of drugs. The emergence and spread of drug resistant Plasmodium falciparum is a cause for grave concern with respect to disease control. The acquisition and spread of resistance to available drugs is largely responsible for a recent increase in malaria-related mortality [2], [3], resulting in about 1–2 million deaths per year [1]. The increasing burden caused by drug resistant parasites has led investigators to seek out novel anti-malarial drug targets. Among these are enzymes necessary for cellular division and differentiation. Previous work has demonstrated that the enzyme protein farnesyltransferase (PFT) is a viable drug target for pathogenic protozoa, including the malaria parasite P. falciparum [4], [5], [6], [7], [8]. PFT inhibitors (PFTIs) have been developed by the pharmaceutical industry owing to their anti-cancer properties [9], [10], [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 [6].

Due to the enzymatic nature of the drug target, we have investigated the potential for P. falciparum to acquire resistance to PFTIs. Upon selection with THQ PFTI BMS-388891 we identified a Y837C mutation of the PFT beta subunit in BMS-388891 resistant parasites, predicted to be in the peptide binding pocket [12]. The corresponding residue in yeast had previously been demonstrated to alter peptide substrate binding and provide resistance to tricyclic inhibitors (Schering-Plough PFT inhibitors SCH44342 and SCH56582) [13].

Now, using a different PFT inhibitor, we have selected resistant P. falciparum which possess a novel mutation in the PFT beta subunit. This mutation is predicted to be near the farnesyl pyrophosphate binding region of the enzyme catalytic site, implicating a unique mechanism of conferring resistance to PFTIs.

Section snippets

Parasites

Experiments described in this study were performed with a clone of P. falciparum Dd2 strain parasites [14]. Parasites were cultured asynchronously in vitro using standard conditions, and media [15]. Parasites from infected erythrocytes were isolated for PFT enzyme extraction by treatment with 0.1% (w/v) saponin.

Selection of BMS-339941-resistant parasites

Selection of resistant parasites was conducted as described previously using a “one-step” selection procedure [14], [16]. Briefly, triplicate 30 ml cultures with a 2% hematocrit were

BMS-339941 resistant P. falciparum clones can be selected

Earlier studies demonstrated that P. falciparum 3D7 was highly sensitive to growth inhibition by the THQ PFTI, BMS-388891 and BMS-339941, with an ED₅₀ of 7.0 and 5.0 nM, respectively [6] (Fig. 1). Previously, we found that using a one-step selection protocol, it was possible to isolate P. falciparum Dd2 parasites that displayed a drug resistant phenotype to BMS-388891. Using the same selection protocol, we wanted to determine if drug resistance parasites could be isolated when selected with

Discussion

In this paper we report the selection of P. falciparum parasites resistant to PFTIs, specifically a THQ BMS-339941. The resulting parasites demonstrated an increased resistance to BMS-339941, which was used to select for resistance, and cross-resistance to other THQs (ED₅₀ values of BMS-388891 listed in Table 1). Similar to previous findings, resistance was associated with single point mutations in the target enzyme, protein farnesyltransferase. Since resistance was generated by continuous

Acknowledgements

The authors would like to thank David M. Floyd, David K. Williams, and Louis J. Lombardo of Bristol-Myers Squibb Co. for providing the original THQ PFTIs, advice on THQ synthetic chemistry, and support of our PFTI program. In addition, we like to acknowledge Corey Strickland, Schering Plough Research Institute, for providing the unpublished structural coordinates cited, Laxman Nallan for synthesis of the inhibitors, and Lynn Barrett, and Kasey L. Rivas for technical support. This work was

References (25)

J. May et al.
Chemoresistance in falciparum malaria
Trends Parasitol
(2003)
R.T. Eastman et al.
Thematic review series: lipid posttranslational modifications. Fighting parasitic disease by blocking protein farnesylation
J Lipid Res
(2006)
D. Chakrabarti et al.
Protein prenyl transferase activities of Plasmodium falciparum
Mol Biochem Parasitol
(1998)
J. Ohkanda et al.
Peptidomimetic inhibitors of protein farnesyltransferase show potent antimalarial activity
Bioorg Med Chem Lett
(2001)
P. Haluska et al.
Farnesyl transferase inhibitors as anticancer agents
Eur J Cancer
(2002)
S.M. Sebti et al.
Farnesyltransferase inhibitors
Semin Oncol
(2004)
R.T. Eastman et al.
Resistance to a protein farnesyltransferase inhibitor in Plasmodium falciparum
J Biol Chem
(2005)
K. Del Villar et al.
A mutant form of human protein farnesyltransferase exhibits increased resistance to farnesyltransferase inhibitors
J Biol Chem
(1999)
D. Chakrabarti et al.
Protein farnesyltransferase and protein prenylation in Plasmodium falciparum
J Biol Chem
(2002)
A.M. Kral et al.
Mutational analysis of conserved residues of the beta-subunit of human farnesyl:protein transferase
J Biol Chem
(1997)

R.W. Snow et al.

The global distribution of clinical episodes of Plasmodium falciparum malaria

Nature

(2005)

N.J. White

Antimalarial drug resistance

J Clin Invest

(2004)

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^☆: Note: Nucleotide sequence data reported in this paper have been submitted to GenBank™ data base with the accession number DQ986323 (P. falciparum Dd2 PFT alpha subunit drug resistant phenotype), and DQ986322 (P. falciparum Dd2 PFT beta subunit drug resistant phenotype).

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Resistance mutations at the lipid substrate binding site of Plasmodium falciparum protein farnesyltransferase☆

Abstract

Introduction

Section snippets

Parasites

Selection of BMS-339941-resistant parasites

BMS-339941 resistant P. falciparum clones can be selected

Discussion

Acknowledgements

Trends Parasitol

J Lipid Res

Mol Biochem Parasitol

Bioorg Med Chem Lett

Eur J Cancer

Semin Oncol

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

The global distribution of clinical episodes of Plasmodium falciparum malaria

Nature

Antimalarial drug resistance

J Clin Invest