SENPs play an essential role in the regulation of SUMOylation, a posttranslational modification that modulates numerous biological processes. The essential role of SENPs in other eukaryotes suggests these proteases may also be essential for P. falciparum. In this study, we demonstrated that P. falciparum has at least one active SENP protease, PfSENP1. Using a positional scanning substrate library, we demonstrated that PfSENP1 has distinct cleavage specificity relative to its human homologs. By screening a library of cysteine protease inhibitors we identified one compound, JCP-666, that inhibited SUMO processing by parasite lysates and recombinant PfSENP1. Because of the instability in the epoxide electrophile of JCP-666, we focused our efforts on a novel analog VEA-260 that showed increased potency and overall stability compared to JCP-666. This compound inhibited parasite replication in a dose-dependent manner, suggesting that PfSENP activity is essential for parasite survival.
The ability of VEA-260 to inhibit PfSUMO processing by both recombinant PfSENP1 and soluble parasite lysates, the selectivity of VEA-260 for hSENP1/2-like proteases over PfSENP2 human homologs, and the ability of VEA-260 to block parasite replication all suggest that PfSENP1 is the major SENP protease in P. falciparum
lysates. Although the precise effect of VEA-260 on PfSENP2 is unknown, these data suggest one of three possible scenarios: VEA-260 inhibits both PfSENP1 and 2, inhibition of PfSENP1 alone is sufficient to block parasite replication, or PfSENP2 is not an active protease. P. falciparum
, like yeast, has only two predicted SENP proteases. PfSENP1 is more closely related to yeast Ulp1 (hSENP1/2-like) and PfSENP2 is more closely related to yeast Ulp2 (hSENP6/7-like). Ulp1 is cytosolic, lethal upon deletion, and more highly related to the hSENP1/2 that are susceptible to VEA-260 inhibition. In contrast, Ulp2 is localized to the nucleus and can be genetically disrupted (Kroetz, et al., 2009
; Li and Hochstrasser, 2003
). Defects resulting from Ulp2 disruption can be partially overcome by over-expression of the catalytic domain of Ulp1. Ulp2 is more closely related to the subset of human SENPs that includes hSENP6 and 7. As demonstrated here, VEA-260 is not an effective inhibitor of hSENP6, suggesting PfSENP2 is a less likely to be a target of this inhibitor. Therefore, we propose that PfSENP1 is the primary protease responsible for the SENP activity in P. falciparum
soluble lysates and that inhibition of this protease alone is sufficient to block parasite replication.
Although we cannot rule out the possibility that VEA-260 inhibits PfSENP2, we speculate that PfSENP2 may not be an active protease. The catalytic domain of PfSENP2 has undergone a significant expansion relative to human SENPs, with nearly 200 additional amino acids relative to hSENP6 and 7, which themselves contain a 200 amino acid insertion relative to hSENP1 and 2 (Table S1
; Lima and Reverter, 2008
). Furthermore, the expansion within PfSENP2 contains long stretches of amino acid repeat sequences that may have disrupted protease function and may have also prevented expression of PfSENP2 in E. coli
. In addition, PfSENP2 homologs (yeast Ulp2 and hSENP6 and 7) primarily function in the processing of poly-SUMO chains (Kroetz, et al., 2009
; Lima and Reverter, 2008
). We demonstrated here that PfSUMO cannot form poly-SUMO chains; therefore, it is unclear what role, if any, PfSENP2 plays in SUMO regulation in the parasite.
Regardless of whether or not PfSENP2 is a functional protease, VEA-260 is an inhibitor of PfSENP1. Most classes of protease inhibitors that target cysteine proteases in P. falciparum
show some level of cross-reactivity with the falcipains (Arastu-Kapur, et al., 2008
; Greenbaum, et al., 2002
). Our lead SENP inhibitor, VEA-260, did not cross-react with the other major parasite cysteine proteases, including the falcipains, at nearly double its EC50
value in the replication assay. Furthermore, JCP-668 showed dose dependent inhibition of parasite growth proportional to the decreased potency of this compound for PfSENP1, and JCP-667 showed no toxicity to parasite growth correlating with its inability to inhibit PfSENP1. Although JCP-665 showed toxic effects not related to PfSENP1 inhibition, these effects were not sufficient to fully block parasite replication. These data suggest that the complete block in parasite replication by VEA-260 and JCP-668 was due to inhibition of the target SENP. Modulation of PfSENP1 expression would be useful to validate that VEA-260 susceptibility is proportional to the level of PfSENP1. However, previous studies in yeast have demonstrated that Ulp1 overexpression exerts a dominant-negative effect and is therefore likely to result in lethality in P. falciparum
(Mossessova and Lima, 2000
). Although VEA-260 also inhibits hSENP1 and 2, we demonstrated that it does not inhibit all SENPs nor does it inhibit highly related cysteine proteases, such as the DUB UCH-L3. These data indicate that VEA-260 may be a promising lead compound with a high degree of selectivity for PfSENP1 over other cysteine proteases.
Identification of a small molecule SENP-selective inhibitor also provides a new tool for the analysis of the SUMOylation pathway in P. falciparum
. Recent proteomic analysis of SUMO modified proteins in P. falciparum
identified a preliminary list of 23 proteins (Issar, et al., 2008
). This is a surprisingly small number compared to proteomic studies in other organisms including yeast (>200 proteins identified) and the highly related parasite Toxoplasma gondii
(>100 putative modified protein identified; (Braun, et al., 2009
; Denison, et al., 2005
). For example, SUMOylation of PfSir2 was not observed by mass spectrometry in P. falciparum
, despite western blot and co-immunoprecipitation evidence that this protein is in fact modified by SUMO. This is likely due to the low levels of SUMO modified protein at any given time and the high level of activity of SENPs in P. falciparum
lysates. With the discovery of small, cell permeable inhibitors of PfSENPs it should be possible to generate more complete profiles of SUMOylated proteins by mass spectrometry analysis. Furthermore, these inhibitors can be used to specifically block removal of SUMO at specific stages of the parasite lifecycle to provide a more complete picture of the dynamics of SUMOylation.