Leveraging the<i>Plasmodium falciparum</i> esterase repertoire to design parasite-specific prodrugs

Abstract Background Antimalarial drug resistance remains a constant threat, highlighting the need to identify new antimalarials with novel modes of action. 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. However, many prodrugs are activated by non-specific host enzymes, losing these beneficial features before they reach their target site. Designing prodrugs that are activated by enzymes specific to a target pathogen, “pathogen specific prodrugs,” would allow prodrugs to maintain their beneficial pharmacokinetic and pharmacodynamic effects until they reach their target. We have previously shown that lipophilic ester prodrug modifications substantially increase the potency of antimalarial phosphonates both in vitro and in vivo. However, ester prodrug activation in the malaria parasite, Plasmodium falciparum is poorly understood, as only a single P. falciparum esterase has been characterized. Methods In this study, we use a novel fluorogenic ester substrate library, to characterize the substrate specificity of the entire esterase repertoire of P. falciparum. We compare this substrate specificity to that of host erythrocytes, serum, liver and intestinal epithelium. We employ a combination of substrate activity profiling, enzymatic inhibition assays, and liquid chromatography mass spectrometry, to identify the activating enzyme of highly potent antimalarial phosphonate prodrugs. Results We find that structurally similar substrates exhibit varying degrees of activation by human and parasite esterases. We identify a group of long unbranched alkyl substrates that are specifically activated by P. falciparum esterases, suggesting an important structure activity relationship. We also identify an erythrocyte serine hydrolase (APEH), which is imported by the parasite and activates multiple antimalarial phosphonate prodrugs. Conclusion This study identifies ester prodrug modifications that are specifically activated by P. falciparum, and highlights candidate enzymes responsible for their activation. Our data reveals that an imported human esterase is involved in the activation of antimalarial phosphonate ester prodrugs. Our method can be applied to other organisms or genetic mutants to rapidly characterize esterase activity. Ongoing work will use recombinantly expressed enzymes to specifically characterize their substrate specificities, allowing for the identification of substrates and prodrugs targeted towards those enzymes.