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Malaria is a devastating illness caused by multiple species of the Plasmodium genus. The parasite’s food vacuole of falcipain proteases have been extensively studied as potential drug targets. Here we report the testing of two established cysteine protease inhibitor scaffolds against both chloroquine sensitive and chloroquine resistant parasites. A subset of purine derived nitriles killed the parasite with moderate potency, and these inhibitors do not seem to exert their antiproliferative effects as cysteine protease inhibitors. Compound potency was determined to be similar against both parasite strains, indicating a low probability of cross resistance with chloroquine. These compounds represent a novel antimalarial scaffold, and a potential starting point for the development new inhibitors.
Malaria infects 250 million and kills nearly one million people every year, with most fatalities occurring in children.1 The protozoan parasite P. falciparum causes the most deadly form of malaria, and the cost of and developing resistance to currently available drugs requires the development of new therapies. Hemoglobin degradation in the parasite’s food vacuole is an essential function for parasite survival, and P. falciparum depends on hemoglobin degradation by the partially redundant proteolytic activities of the falcipains2,3 and plasmepsins.4,5 Three members of the falcipain family of cysteine proteases (FP-2, FP-2′, and FP-3) are known to be involved in hemoglobin catabolism.6–8 Significant effort has been directed towards developing inhibitors of these proteases to discover new antimalarials. These studies have resulted in potent falcipain inhibitors with promising antimalarial activity both in vitro and in vivo.9–17 The thiosemicarbazone18–20 and purine nitrile21,22 scaffolds have well established activity against both mammalian and protozoan cathepsins. In addition, the thiosemicarbazones have been reported to have antimalarial activity19,23–26, though they may act through alternative targets. We have previously developed series of both classes for use as anti-trypanosomal agents.20,22,27,28
It is known that cysteine protease inhibitors display marked synergy with pepstatin A, a potent inhibitor of plasmepsin activity, in inhibiting parasite growth.29–31 Although pepstatin A is only weakly potent in inhibiting parasite growth, it does produce a shift of approximately 200 fold in the EC50 of the broad spectrum cysteine protease inhibitor e64D. We sought to determine whether antimalarial activity of these inhibitors was due to cysteine protease inhibition. To explore this hypothesis, all inhibitors were screened in both the presence and absence of 10 μM pepstatin A against cultured parasites.
Chloroquine has had a central role as both a frontline treatment and prophylactic since the mid-1940’s. It’s heavy usage lead to the development of resistance starting in the 1960’s,32–34 presumably due to decreased drug accumulation in the parasite food vacuole.35–37 Increased clinical resistance to this drug has contributed to the need for the development of new antimalarials that are not cross resistant with chloroquine. To identify inhibitors with this activity profile, all compounds in this study were tested against both chloroquine sensitive (3D7) and resistant (K1) parasites.
Parasites were grown in presence of fresh group O-positive erythrocytes in Petri dishes at a hematocrit of 4–6% in RPMI 1640 (pH 7.3) supplemented with 0.5% AlbuMAX II, 25 mM HEPES, 25 mM NaHCO3, 100 μg/mL hypoxanthine, and 5 μg/mL gentamycin.
Cultures were incubated at 37°C with 5% O2 and 5% CO2. For EC50 determinations, 20 μl of RPMI 1640 was plated per well in a 384 well assay plate. Compounds were prepared as either 5 mM or 10 mM stock solutions in DMSO and transferred to the assay plate by a 384 head pintool. 20 μl of a synchronized culture suspension (1% rings, 10% hematocrit) was added to each well to make a final hematocrit and parasitemia of 5% and 1% respectively. The final DMSO concentration was 0.65%. For synergy experiments, pepstatin A was added to the cell suspension prior to plating. Assay plates were incubated for 72 hours and parasitemia was determined using a previously reported DNA stain based assay.38
A series of nearly 100 inhibitors from the three scaffolds was tested for antimalarial activity in both parasite strains in the presence and absence of 10 μM pepstatin A (fig. 1, table 1). Predicted cell permeability of a subset of these compounds was previously tested in a parallel artificial membrane permeability assay (PAMPA) and most are predicted to have good cellular availability.20,28 Additionally, the inhibitors in this test set have also been previously shown to be relatively non-toxic in a panel of mammalian cell lines.20,22,28
Although the majority of the inhibitors tested did not have significant activity at the highest activity tested (32.5 μM), these studies identified a subset of inhibitors with good predicted membrane permeability as well as modest growth inhibitory potency against cultured parasites (fig. 2). Weakly active inhibitors were identified from all three scaffolds, with scaffold 2 giving the most active compounds. Scaffolds 1 and 3 did not yield any inhibitors with potencies below 25 μM against either strain, and there is no clearly discernable SAR for either scaffold.
The three most active inhibitors (2w, 2z, and 2aa) displayed potencies in the 10 μM range. 2w was the most active against both parasite strains with EC50 values of 6 ± 3 μM and 15 ± 3 μM against K1 and 3D7 in the presence of 10 μM pepstatin. Moving the acetylamino substituent of 2w from the 3′ to the 4′ position (2j) on the distal phenyl ring resulted in a 3–5 fold decrease in potency against the K1 parasite. It is notable that for scaffold 2, aryl substitutions at the Y position appear to be more effective than at the X position. In the cases of 2z and 2aa, moving the aryl substituent from the Y position to the X position resulted in a 2–3 fold decrease in potency.
Only minor shifts in antimalarial potency were observed in the presence of pepstatin A for any of the active inhibitors against either the K1 or 3D7 parasites. In contrast, the EC50 of the falcipain inhibitor e64D shifted 200–300 fold when combined with pepstatin A. These data suggest that cysteine protease inhibition is not likely to be a significant mediator of antimalarial activity for this inhibitor series.
There does not seem to be a significant difference in inhibitor potency between the K1 and 3D7 parasites. Some inhibitors show minor differences in potency between strains, although overall there is a similar activity profile across all three scaffolds, indicating a low chance of chloroquine cross resistance.
The expected membrane permeability of a subset of inhibitors from all three scaffolds was previously tested by PAMPA. Although a wide range of permeability values was observed, there was no clear structure-property relationship between permeability and antimalarial activity.
We have reported the evaluation of a series of inhibitors based on the purine nitrile and thiosemicarbazone scaffolds for activity against two strains of cultured P. falciparum. There were no significant differences in potency between the two strains, indicating that chloroquine cross resistance is unlikely. Antimalarial activity of the series was modest, although inhibitors were identified with moderate potency against both K1 and 3D7 parasites. Active compounds did not act synergistically with the aspartyl protease pepstatin A, suggesting that they do not exert their antiproliferative effects by cysteine protease inhibition. Previous experiments with other cysteine protease inhibitors including peptidyl aziridines, phenyl urenyl chalcones, and vinyl sulfone derivatives suggest these scaffolds also act at targets other than the falcipains.15–17 This is in contrast to studies of this inhibitor series with Trypanosoma brucei, in which a clear relationship between trypanocidal activity and potency against the target protease was observed.22,28
Although the mechanism of action of these compounds in P. falciparum is unclear, the combination of low cytotoxicity, good membrane permeability, and lack of chloroquine cross resistance suggest the purine nitriles may be a useful starting point for the development of new antimalarials. The thiosemicarbazones tested were generally inactive against the parasite, and do not represent promising antimalarial leads.
This work was supported by the American Lebanese Syrian Associated Charities (ALSAC) and St. Jude Children’s Research Hospital, NIH grant AI35707.
Full data set of antimalarial inhibitor potencies. Experimental details of malaria assay.