1230 representing 10 (0.23 μM), 100 (2.3 μM) and 1,000 (23 μM) times the physiological concentration. The 2.3- μM concentration is the highest concentration of folate that can be achieved in vivo when high doses of folate derivatives are administered (Barredo et al. 1999). Table S1 shows the results of FA addition on the antimalarial activity of PM and CCG using the multidrug- resistant isolate V1/S. At the physiological FA concentra- tion (0.023 μM), the IC50 values of the antimalarial antifolates PM and CCG and the anticancer antifolates MTX, AMP, and TMX were 875, 12, 31, 59, and 15 nM, respectively. Increasing the concentration of FA to 0.23, 2.3 and 23 μM was associated with a significant decrease in PM and CCG activity, and this decrease in activity was proportional to the concentration of FA added to the medium (Table S1). For instance, PM and CCG were reduced by a factor of >45 in the presence of 23 μM of FA. Surprisingly, however, the activities of MTX, AMP, and TMX were virtually unchanged at 0.23 and 2.3 μM of FA, and only a slight decrease was noticed at a concentration of 23 μM of FA, especially with TMX. Likewise, the same trend was observed against the fully sensitive isolate M24. At the physiological FA concentration, the IC50 values of PM, CCG, MTX, AMP, and TMX were 0.3, 0.22, 36, 48, and 4 nM, respectively (Table S2). PM and CCG activities were decreased by 2.5- to 33-fold when 0.023, 2.3, and 2.3 μM of FA were used, while the activities of AMP, MTX, and TMX remained unchanged up to 23 μM of FA (Table S2). We have also tested the effect of THF. The activities of PM and CCG in the presence of THF at physiological folate concentrations were 797 and 10 nM, respectively (Table S1). These activities were decreased by between 5- and 100-fold at the three highest folate concentrations tested. On the other hand, MTX, AMP, and TMX activities remained unchanged (24, 68, and 4 nM, respectively) at all THF concentrations (Table S1). The same trend was observed when M24 was used (Table S2). We tested the effect of FNA, another form of folate that the parasite can salvage. Similar to previous observations, FNA does not affect the activity of MTX, AMP, and TMX, even at concentrations 500 times higher than the physiological folate concentra- tion, unlike the effect on the antimalarials PM and CCG. The use of 5-Me-THF yielded the most surprising results. Indeed, this folate derivative, even when used at high concentration, did not change the activity of any of the tested drugs, including the antimalarials PM and CCG (Tables S1 and S2). Synergy between MTX and sulfa drug The combination of a sulfa drug, an inhibitor of dihy- dropteroate synthase (DHPS) and an inhibitor of DHFR, produces a synergistic inhibition of the folate pathway. To Parasitol Res (2008) 102:1227–1234 explore the mode of action of MTX further, we have tested the synergistic properties of MTX in combination with the DHPS inhibitor dapsone. All FICs were between 1 and 1.82, a clear indication that MTX does not synergize with dapsone, while FICs of the combination CCG–DDS were <0.5 (Table 1). Likewise, we did not observe any synergy between TMX and DDS, and all FICs were in the range 1.2–1.5. Probenecid effect Lastly, we tested the effect of PBN on the activity of anticancers MTX and TMX in P. falciparum. As part of our previous work, we have demonstrated that PBN increases the in vitro activity of antifolates, and this increase is associated with a decrease in folate uptake (Nzila et al. 2003). We assessed the effect of PBN on the activity of MTX and the results are summarized in Fig. 2. PBN alone is a very weak antimalarial with a mean IC50 >1,500 μM against V1/S parasites. We have tested the effect of noninhibitory concentrations of 50, 100, and 150 μM PBN on the activity of MTX, TMX, and PM. The PM IC50 against V1/S was 1,200 nM, and this IC50 decreased by a factor between 2.5 and 5 as PBN concentration increased from 50 to 150 μM; however, MTX and TMX IC50 remained unchanged (at around 30 and 7 nM, respectively). The data clearly show the absence of a PBN effect on MTX and TMX in P. falciparum. Synergy of MTX with other antimalarials We also explored the in vitro interaction of MTX with existing antimalarials by assessing its activity in the presence of (1) inhibitors of malaria dihydrofolate reductase (CCG and Walter Reed compound 99210); (2) triazine- based compounds, synergizers of atovaquone (ATV; PG and CPG), quinoline, and aryl amino-alcohol-related drugs (CQ, PQ, LM, MFQ, HLF, DEAQ, QN, and PRQ); (3) the antifolate anticancer TMX; (4) the sesquiterpene DHA; (5) the benzonaphthyridine PRN; and (6) the naphthoquinone derivative ATV. We used two synergistic combinations as controls: CCG and DDS (see previous section) and ATV and PG. Triazine compounds (PG) synergize with inhibitors of electron transport to the cytochrome bc1 complex (ATV; Jones and Ward 2002; Nzila 2006). The data are summa- rized in Table 1. The combinations of CCG–DDS and ATV–PG were synergistic, as one would predict, with total FIC < 0.5. All of the tested MTX combinations were additive (except two), with the FIC value falling between 0.9 and 1.6, a clear indication that the activity of MTX is neither decreased nor increased in the presence of any of these drugs. However, the total FIC of the combination of MTX with PNR was between 3.14 and 3.52, and two out