Prodrug activation in malaria parasites mediated by an imported erythrocyte esterase, acylpeptide hydrolase (APEH).

The continued emergence of antimalarial drug resistance highlights the need to develop new antimalarial therapies. Unfortunately, new drug development is often hampered by poor drug-like properties of lead compounds. Prodrugging temporarily masks undesirable compound features, improving bioavailability and target penetration. We have found that lipophilic diester prodrugs of phosphonic acid antibiotics, such as fosmidomycin, exhibit significantly higher antimalarial potency than their parent compounds (1). However, the activating enzymes for these prodrugs were unknown. Here, we show that an erythrocyte enzyme, acylpeptide hydrolase (APEH) is the major activating enzyme of multiple lipophilic ester prodrugs. Surprisingly, this enzyme is taken up by the malaria parasite, Plasmodium falciparum, where it localizes to the parasite cytoplasm and retains enzymatic activity. Using a novel fluorogenic ester library, we characterize the structure activity relationship of APEH, and compare it to that of P. falciparum esterases. We show that parasite-internalized APEH plays an important role in the activation of substrates with branching at the alpha carbon, in keeping with its exopeptidase activity. Our findings highlight a novel mechanism for antimicrobial prodrug activation, relying on a host-derived enzyme to yield activation at a microbial target. Mutations in prodrug activating enzymes are a common mechanism for antimicrobial drug resistance (2-4). Leveraging an internalized host enzyme would circumvent this, enabling the design of prodrugs with higher barriers to drug resistance.

Glutenin is involved in the gluten-driven mucosal T cell response.

Gluten ingestion causes coeliac disease in susceptible individuals. Gluten is a heterogeneous mixture of glutenin and gliadin, the latter of which is considered responsible for disease induction. By combining high-performance liquid chromatography purification steps of gluten with a T cell bioassay and mass spectral analyses, we have identified a glutenin peptide (glt04 707-742) that activates T cells from the small intestine of a coeliac disease patient and results in the secretion of large amounts of IFN-gamma. The minimal T cell stimulatory core of the peptide (residues 724-734) is repetitively present in glutenin molecules. Moreover, it was observed that a large number of naturally occurring variants of this peptide are recognized by the T cells. These data suggest that the large heterogeneity of glutenin proteins dramatically increases the number of available T cell epitopes. Together, the results provide new insight into the nature of the gluten antigens that lead to coeliac disease and suggest that glutenin, next to gliadin-derived antigens, may be involved in the disease process.