Functional annotation of serine hydrolases in the asexual erythrocytic stage of Plasmodium falciparum

Abstract

Enzymes of the serine hydrolase superfamily are ubiquitous, highly versatile catalysts that mediate a wide variety of metabolic reactions in eukaryotic cells, while also being amenable to selective inhibition. We have employed a fluorophosphonate-based affinity capture probe and mass spectrometry to explore the expression profile and metabolic roles of the 56-member P. falciparum serine hydrolase superfamily in the asexual erythrocytic stage of P. falciparum. This approach provided a detailed census of active serine hydrolases in the asexual parasite, with identification of 21 active serine hydrolases from α/β hydrolase, patatin, and rhomboid protease families. To gain insight into their functional roles and substrates, the pan-lipase inhibitor isopropyl dodecylfluorophosphonate was employed for competitive activity-based protein profiling, leading to the identification of seven serine hydrolases with potential lipolytic activity. We demonstrated how a chemoproteomic approach can provide clues to the specificity of serine hydrolases by using a panel of neutral lipase inhibitors to identify an enzyme that reacts potently with a covalent monoacylglycerol lipase inhibitor. In combination with existing phenotypic data, our studies define a set of serine hydrolases that likely mediate critical metabolic reactions in asexual parasites and enable rational prioritization of future functional characterization and inhibitor development efforts.

Defining the Essential Exportome of the Malaria Parasite

“…Although direct fatty acid import and de novo synthesis of lipids may be possible, our comparative review revealed the presence of an essential P. falciparum exported lipase (PF3D7_1001600; Table S1), hinting that lipid import and its catabolism inside the host RBC before import into the parasite may be important for intraerythrocytic growth (Figure 1C). PF3D7_1001600 has recently been shown to exhibit lipolytic activity in vitro and thus is likely to be functional [66,67]. Membrane biogenesis is also potentially mediated, at least partially, by essential exported proteins.…”

Resistance to Some But Not Other Dimeric Lindenane Sesquiterpenoid Esters Is Mediated by Mutations in a Plasmodium falciparum Esterase

“…PfPARE belongs to a plasmodial subfamily of α/β hydrolases, which are enzymes with very diverse specificities . Although its physiological substrates remain unknown, current evidence suggests that PfPARE plays a role in compound detoxification, as illustrated by the diversity of reported antiplasmodial compounds that have induced PfPARE mutations such as pepstatin esters, MMV011438, AN13762, and now a subset of lindenane sesquiterpenoid dimers. , Ester prodrugs are commonly used to mask polar functionalities to improve pharmacological properties and alleviate unwanted side effects by taking advantage of the ubiquity of esterases within pathogenic targets . However, resistance mediated by PfPARE inactivation has developed readily in vitro , highlighting the importance of identifying compounds that are susceptible to PfPARE activity early during drug discovery.…”

The Impact of Activity‐Based Protein Profiling in Malaria Drug Discovery

“…FP probes have been extensively used to study human hydrolases and a couple of examples for malarial serine hydrolases have also been described. [11,29] Elahi et al [30] used FP probes (Figure 4C) to profile the activity of serine hydrolases in the asexual erythrocytic stage of P. falciparum. A combination of gel-and mass spectrometrybased ABPP using a FP-desthiobiotin ABP identified 26 enriched P. falciparum proteins in schizont-stage parasites, of which 21 were serine hydrolase superfamily homologs.…”

“…Elahi et al [30] . used FP probes (Figure 4C) to profile the activity of serine hydrolases in the asexual erythrocytic stage of P. falciparum .…”