Characterisation of the bifunctional dihydrofolate synthase-folylpolyglutamate synthase from Plasmodium falciparum; a potential novel target for antimalarial antifolate inhibition

Abstract

Unusually for a eukaryote, the malaria parasite Plasmodium falciparum expresses dihydrofolate synthase (DHFS) and folylpolyglutamate synthase (FPGS) as a single bifunctional protein. The two activities contribute to the essential pathway of folate biosynthesis and modification. The DHFS activity of recombinant PfDHFS-FPGS exhibited non-standard kinetics at high co-substrate (glutamate and ATP) concentrations, being partially inhibited by increasing concentrations of its principal substrate, dihydropteroate (DHP). Binding of DHP to the catalytic and inhibitory sites exhibited dissociation constants of 0.50microM and 1.25microM, respectively. DHFS activity measured under lower co-substrate concentrations, where data fitted the Michaelis-Menten equation, yielded apparent K(m) values of 0.88microM for DHP, 22.8microM for ATP and 5.97microM for glutamate. Of the substrates tested in FPGS assays, only tetrahydrofolate (THF) was efficiently converted to polyglutamylated forms, exhibiting standard kinetics with an apparent K(m) of 0.96microM; dihydrofolate, folate and the folate analogue methotrexate (MTX) were negligibly processed, emphasising the importance of the oxidation state of the pterin moiety. Moreover, MTX inhibited neither DHFS nor FPGS, even at high concentrations. Conversely, two phosphinate analogues of 7,8-dihydrofolate that mimic tetrahedral intermediates formed during DHFS- and FPGS-catalysed glutamylation were powerfully inhibitory. The K(i) value of an aryl phosphinate analogue against DHFS was 0.14microM and for an alkyl phosphinate against FPGS 0.091microM, with each inhibitor showing a high degree of specificity. This, combined with the absence of DHFS activity in humans, suggests PfDHFS-FPGS might represent a potential new drug target in the previously validated folate pathway of P. falciparum.

Fig. 1

P. falciparum DHFS–FPGS: roles, expression of recombinant protein and product analysis. (a) Position (grey boxes) and roles of DHPS and FPGS activities in the folate pathway of P. falciparum leading to 5,6,7,8-tetrahydrofolate (THF). Polyglutamation of folates is thought to occur on their tetrahydro-forms. The addition of one or more glutamate residues by FPGS to THF or its modified forms, produced de novo or from salvaged host folates, is indicated by nGlu. The dotted arrow indicates sequential steps involving the four enzymes shown. (b) SDS-PAGE analysis of purified PfDHFS–FPGS after Ni-agarose affinity and ion-exchange chromatography; (c) mass spectral analysis of the reaction mix after a standard 1 h incubation of the DHFS assay.

Fig. 2

Dependence of PfDHFS activity on DHP, ATP and glutamate with DHP taken as the variable substrate. (a) ATP is kept at a constant high level of 0.5 mM in all the assays in this panel, with glutamate concentration (Glu) set at three different levels ≥75 μM. (b and c) As for (a), except ATP is kept at a constant low level of 25 μM in (b) and glutamate at a constant low level of 25 μM in (c), with stepped increments of glutamate and ATP, respectively.

Fig. 3

Comparison of DHFS and FPGS activities under identical conditions of high co-substrate concentrations. ATP is at 5 mM, glutamate at 275 μM. The data are fitted to the three-substrate, substrate partial inhibition model of Supplementary Fig. 2, equation a.

Fig. 4

Effect of having both DHFS and FPGS substrates together in the same reaction compared to when present alone. From left to right in (a) 10 μM DHP, 10 μM THF, 10 μM DHP + 10 μM THF; (b) 100 μM DHP, 100 μM THF, 100 μM DHP + 100 μM THF. Glutamylation activity is represented as a percentage of that seen with DHP as substrate in each case. In all cases 5 mM ATP and 275 μM glutamate were present.

Fig. 5

Substrate specificity of PfDHFS–FPGS. Glutamylation of equal concentrations of DHP, folic acid (F), DHF and THF by PfDHFS–FPGS under identical conditions: (a) 25 μM DHP, F, DHF or THF, 25 μM ATP and 18 μM glutamate; (b) 100 μM DHP, F, DHF or THF, 2 mM ATP and 200 μM glutamate. Glutamylation activity is represented as a percentage of that seen with DHP as substrate in each case.

Fig. 6

MTX accumulation in, and inhibition of, P. falciparum. A predominantly ring-stage culture of FCB isolate was divided equally and each half grown for 20 h in the presence of (a) ³H-MTX or (b) ³H-folinic acid in parallel and extracts analysed by HPLC. The arrows indicate the position of MTX and the mono- and polyglutamylated forms (FPG3-5) of folinic acid. (c) Accumulation of MTX (black) in parasite cells compared with folinic acid (white). Counts in the culture supernatant (converted to nM) indicate the (equal) initial label concentrations to which each culture was exposed; ‘extract’ represents the counts extracted from the equal numbers of parasites in each pellet labelled with either MTX or folinic acid after the incubation. (d) Dose–response curves of different strains of cultured malaria parasites to increasing MTX concentration. Values are relative to growth in the absence of drug, taken as 100%. For reasons as yet unclear, extremely low levels of MTX (around 1 nM) have a stimulatory effect on growth.

Fig. 7

Structures of the putative PfDHFS–FPGS inhibitors, compounds 1 and 2. These are folate analogues incorporating aryl- and alkylphosphinic acid moieties [1 and 2, respectively] to simulate postulated unstable tetrahedral intermediates for the two activities . They are shown here in their oxidised forms; they were reduced to their 7,8-dihydropterin forms for the inhibition studies.

Fig. 8

Secondary plots of 1/V_max of PfDHFS and PfFPGS activities against varying concentrations of 1 and 2 for the determination of K_i values of the inhibitors. In order to obtain K_m and V_max data (see Supplementary Fig. 7), DHP was used as the variable substrate in (a) and (c) for DHFS activity; THF in (b) and (d) for FPGS activity. ATP = 50 μM, glutamate = 25 μM. H₂-1 has a K_i of 0.14 μM for DHFS and a K_i of 0.63 μM for FPGS activity. H₂-2 has a K_i of 1.69 μM for DHFS activity and a K_i of 0.091 μM for FPGS activity, as indicated by arrows.