The hemoglobin degradation pathway has been pursued as a potential site for intervention with antimalarial drugs for the last 20 years (Francis et al., 1997
). However, most of the efforts to block this essential pathway have focused on inhibition of the FPs (Rosenthal et al., 2002
) and plasmepsins (Ersmark et al., 2006
) in the FV. All of these proteases are important for the initial stages of hemoglobin degradation but their biological roles in this pathway have been shown to be redundant not only within a protease family but also between families (Bonilla et al., 2007a
; Bonilla et al., 2007b
). Single and multiple genetic knockouts of FV plasmepsins (Liu et al., 2005
; Omara-Opyene et al., 2004
), and deletion of FPs 1 and 2 are not lethal (Sijwali et al., 2006
). On the other hand, there is only one DPAP in this pathway, and efforts to knock out this enzyme suggest that it is essential (Klemba et al., 2004
). Therefore, DPAP1 is likely to be a more suitable target for drug development than the FV FPs or plasmepsins.
In this study we show that selective inhibition of DPAP1 by Ala-4(I)Phe-DMK results in parasite death in culture. This inhibitor has at least 1000-fold selectivity towards DPAP1 over the other papain fold cysteine proteases of P. falciparum
(DPAP3 and the FPs). Several lines of evidence suggest that Ala-4(I)Phe-DMK effects on parasites are specifically due to inhibition of DPAP1 rather than the result of off target effects. First, DPAP1 inhibition closely correlates with parasite death (IC50DPAP1,2h
= 2.0; EC50Pot
= 2.8 nM). Second, parasites are 10-times more sensitive to Ala-4(I)Phe-DMK at the trophozoite stage consistent with the role of DPAP1 in hemoglobin degradation, a pathway that is most essential at trophozoite stage. Third, the phenotypic effect observed for this inhibitor, (i.e. formation of an underdeveloped trophozoite), is in agreement with DPAP1 being the primary target since the same phenotype was observed in parasite treated with SAK2, P-hF-AOMK and A-4(I)F-AOMK, which are selective covalent DPAP1 inhibitors whose reactive groups are structurally unrelated to Ala-4(I)Phe-DMK (Figure S1
). Finally, the correlation between DPAP1 inhibition and potency in the replication assay was also observed for our library of non-peptidic inhibitors. The ki
values of show that the stronger an inhibitor is against DPAP1, the more potent it is at killing parasites. More specifically, the dramatic difference in potency between the ML4118S and ML4118R diastereomers indicates that these compounds are not killing parasites by non-specific toxic effects.
Although the original peptidic vinylsulfone inhibitors were not suitable for use in vivo due to their overall lack of stability, they allowed us to study the dynamics of DPAP1 expression/activation in living parasites (). Studies of DPAP1 inhibition over time showed that DPAP1 activity recovers rapidly after it has been irreversibly inhibited. This implies that either DPAP1 is constantly expressed and trafficked into the FV to replenish the DPAP1 activity that is lost due to protease turnover, or that P. falciparum tightly regulates the levels of DPAP1 activity and is able to respond to a drop in activity.
We also provide several lines of evidence to show that sustained inhibition of DPAP1 for 2–3h is required to prevent the recovery of activity and induce parasite death. The potency of Ala-4(I)Phe-DMK in the parasite replication assay corresponds to the IC50DPAP1 after 2h of treatment of parasite lysates (). Furthermore, the IC50DPAP1 for SAK2 reaches a plateau after 3h of treatment that corresponds to its EC50Pot in the replication assay (). Therefore, an effective drug against DPAP1 either needs to have a long half-life in blood or be designed such that it will be retained inside the FV for several hours. In theory this could be achieved by taking advantage of the acidic environment inside the FV or by using a pro-drug strategy to enhance stability in blood.
We were able to overcome the stability problems of peptidic DPAP1 inhibitors by switching to a non-peptidic scaffold. Moreover, homology modeling and computational docking methods allowed us to improve the specificity of our initial hit, HN3019, by more than 25-fold. This suggests that in-silico
methods may be valuable for the design of DPAP inhibitors. Analysis of the structure activity relationship (SAR) of our two classes of compounds, dipeptide vinylsulfone (Table S1
) and non-peptidic TFPAMK () inhibitors indicates that short amino acid side chains are preferred at the S2 pocket (See supplementary material for a detailed SAR analysis). For peptidic inhibitors, Pro in P2 or 4(I)Phe in P1 enhances inhibitor selectivity towards DPAP1 with respect to the other papain fold cysteine proteases (Figure S1
). Interestingly, a Pro in P2 within the context of the non-peptidic scaffold (ML6076) does not result in a DPAP1 selective inhibitor (Figure S1
). Finally, our results with KB16 suggest that secondary amines in P2 might improve the PK and toxicity properties of non-peptidic inhibitors.
In this study, we also showed that DPAP1 inhibitors have effects on parasite growth in vivo. In P. berghei, ML4118S is a much more specific inhibitor of DPAP1 than in P. falciparum. Although this inhibitor has toxicity issues in mice at 20 mg/Kg, we were able to observe a significant decrease in parasitemia 2 days after treatment. Unfortunately, the relatively small sample size that was caused by compound toxicity, coupled with overall animal-to-animal variability in parasite growth makes our data with ML4118S still less than conclusive. However, we did find that the non-toxic KB16 resulted in a significant decrease in parasitemia during the 10 days of treatment. Although these results suggest that DPAP1 inhibition can lead to a decrease in parasitemia in vivo, additional studies using less toxic and more selective inhibitors will be required to confirm our findings. We are currently working towards the development of selective, non-toxic DPAP1-specific compounds that have long blood half-lives in blood. These compounds should allow us to confirm that DPAP1 inhibition can clear parasitemia in vivo.
In general the papain-fold proteases are likely to be interesting as potential anti-malarial drug targets. Members of this family of proteases play essential roles in various aspects of Plasmodium biology, and inhibitors that target multiple family members may be beneficial over compounds that target only one member. Furthermore, the fact that the closest related human homologue to DPAPs can be inhibited without any significant toxic effects suggests that parasite specific targeting may not need to be a priority in drug design for this target family. While the compounds described in this study are certainly not optimal as drug leads due to lack of potency, poor PK and/or toxicity issues, they do provide the first direct evidence that DPAP1 is a viable anti-malarial drug target. Based on these promising results, we have recently completed a screen of more than 100,000 small molecules using a DPAP1 specific fluorogenic substrate assay in parasite lysates. This screen has identified many new inhibitor scaffolds that we are currently evaluating for their viability as potential drug leads.