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

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.

Figure 1

Structures of the tetrahydroquinolines BMS-339941 and BMS-388891.

Figure 2

Demonstration of resistance to BMS-339941. (A) Growth inhibition by BMS-339941 of P. falciparum WT and resistant clone 1a. Shown is an in vitro growth inhibition assay, using [³H]-hypoxanthine uptake as a measurement of parasite proliferation. Results are shown as percent inhibition as a function of BMS-339941 inhibitor concentration. P. falciparum Dd2 WT (■), P. falciparum Dd2 G612A clone 1a (▲). (B) BMS-339941 inhibition of PFT enzyme from the parent clone and resistant clone 1a. Using native PFT enzyme isolated from both P. falciparum Dd2 WT (■) and G612A mutant clone 1a (▲), the percent enzyme inhibition as a function of BMS-339941 inhibitor concentration was determined. Both graphs are the mean of multiple replicates and standard deviation. Values were graphed using non-linear regression analysis, X-axes values are log of BMS-339941 drug concentration [nM].

Figure 3

Modeled location of G612A mutation site in Pf PFT. The extra methyl of Ala 612, shown as a white surface mesh, is predicted to push FPP more into the binding site of the inhibitor BMS-339941, thereby perturbing the good fit and reducing the affinity. (The zinc ion bound by PFT and the inhibitor is shown as a small white sphere).