Activity-based protein profiling of human and plasmodium serine hydrolases and interrogation of potential antimalarial targets.

Malaria remains a global health issue requiring the identification of novel therapeutic targets to combat drug resistance. Metabolic serine hydrolases are druggable enzymes playing essential roles in lipid metabolism. However, very few have been investigated in malaria-causing parasites. Here, we used fluorophosphonate broad-spectrum activity-based probes and quantitative chemical proteomics to annotate and profile the activity of more than half of predicted serine hydrolases in P. falciparum across the erythrocytic cycle. Using conditional genetics, we demonstrate that the activities of four serine hydrolases, previously annotated as essential (or important) in genetic screens, are actually dispensable for parasite replication. Of importance, we also identified eight human serine hydrolases that are specifically activated at different developmental stages. Chemical inhibition of two of them blocks parasite replication. This strongly suggests that parasites co-opt the activity of host enzymes and that this opens a new drug development strategy against which the parasites are less likely to develop resistance.

Correction: Novel broad-spectrum activity-based probes to profile malarial cysteine proteases.

[This corrects the article DOI: 10.1371/journal.pone.0227341.].

Novel broad-spectrum activity-based probes to profile malarial cysteine proteases.

Clan CA cysteine proteases, also known as papain-like proteases, play important roles throughout the malaria parasite life cycle and are therefore potential drug targets to treat this disease and prevent its transmission. In order to study the biological function of these proteases and to chemically validate some of them as viable drug targets, highly specific inhibitors need to be developed. This is especially challenging given the large number of clan CA proteases present in Plasmodium species (ten in Plasmodium falciparum), and the difficulty of designing selective inhibitors that do not cross-react with other members of the same family. Additionally, any efforts to develop antimalarial drugs targeting these proteases will also have to take into account potential off-target effects against the 11 human cysteine cathepsins. Activity-based protein profiling has been a very useful tool to determine the specificity of inhibitors against all members of an enzyme family. However, current clan CA proteases broad-spectrum activity-based probes either target endopeptidases or dipeptidyl aminopeptidases, but not both subfamilies efficiently. In this study, we present a new series of dipeptydic vinyl sulfone probes containing a free N-terminal tryptophan and a fluorophore at the P1 position that are able to label both subfamilies efficiently, both in Plasmodium falciparum and in mammalian cells, thus making them better broad-spectrum activity-based probes. We also show that some of these probes are cell permeable and can therefore be used to determine the specificity of inhibitors in living cells. Interestingly, we show that the choice of fluorophore greatly influences the specificity of the probes as well as their cell permeability.