Malaria is an infectious disease caused by Plasmodium parasites and transferred by Anopheles mosquitos. Due to Artemisinin resistance, new druggable targets identification and new drug development are urgently needed. Serine hydrolases (SHs) are one of the largest classes of enzymes having important roles in life processes. The deadliest malaria parasite, P. falciparum, encodes more than 50 SHs including proteases, lipases, esterase and others, while only several of them have been characterized. The study of uncharacterized SHs will shed light on future drug development to treat malaria. In this study, we applied chemical biology and genetic approaches to identify SHs important for the pathogenic asexual stage growth of P. falciparum parasites. We mainly focused on a depalmitoylase essential for merozoite invasion and lysophospholipases (LPLs) essential for acquiring fatty acids (FAs) from the host. Identifying essential metabolic enzymes will benefit the treatment to malaria. We focused on metabolic SHs and identified two SHs were refractory to knock out. We studied a likely essential SH named PfABHD17A, which is a human depalmitoylase homolog. PfABHD17A is localized on the rhoptry, an organelle essential for invasion. We expressed the recombinant PfABHD17A, conducted inhibitor screen and discovered that human depalmitoylase inhibitor ML211 inhibits PfABHD17A in vitro. ML211 inhibits merozoite invasion but not egress, which together with the localization of PfABHD17A on the rhoptries, suggested that PfABHD17A is essential in merozoite invasion. We also purified PfABHD17A and verified that PfABHD17A may exhibit depalmitoylase activity in vitro. LPLs are important for asexual stage parasites acquiring FAs from the host. The P. falciparum genome includes 17 putative LPLs while LPLs responsible for hydrolyzing FA from lysophosphatidylcholine (LPC) in the asexual stage are currently unknown. Using a chemical biology approach, we identified serine hydrolase inhibitor AKU-010 inhibits LPC hydrolysis effectively. Using activity-based protein profiling (ABPP) and genetic approaches, we identified that AKU-010 inhibits a series of SHs including Exported Lipases (XLs), Exported Lipases Homolog (XLH) and Plasmodium falciparum prodrug activation and resistance esterase (PfPARE). We generated a series of knockout parasite lines on the AKU-010 targets and identified that red blood cell (RBC)-localized XL2 and cytosolic XLH4 contribute to most LPC hydrolysis activity in the asexual stage. XLs and XLHs are important for parasites using LPC for growth and contribute to detoxification from accumulated LPC. XL2 and XL4 together are essential for parasite growth under high LPC concentration medium, such as human serum. XL/XLH-deficient parasites could still acquire FA from LPC, which is mainly contributed by parasite membrane- localized PfPARE. PfPARE has little impact on parasite growth and LPC metabolism with the existence of XLs and XLHs but is important after the loss of XLs and XLHs. Parasites deficient in PfPARE, XLs and XLHs have little ability to release FA from LPC and cannot use LPC as FAs source for growth. In summary, we identified metabolic SHs mediating protein depalmitoylation and lipid metabolism and in asexual stage Plasmodium falciparum, which may benefit future drug development to treat malaria. / Doctor of Philosophy / Malaria is an infectious disease caused by Plasmodium parasites and transferred by mosquitos. New druggable target identification and drug development are urgently needed to deal with the malaria issue. We focused on an understudied enzyme superfamily termed serine hydrolase (SHs), which includes more than 50 members in the deadliest malaria parasite, P. falciparum. We identified that several druggable enzymes, which can mediate protein depalmitoylation and lipid metabolism, are important for parasite growth in the pathogenic stage. Identifying essential metabolic enzymes will benefit the treatment to malaria. We screened eleven SHs and discovered that two of them are likely essential in the pathogenic stage. We focused on one human depalmitoylase homolog termed PfABHD17A. We screened the inhibitors on PfABHD17A and used the inhibitor to suggest that PfABHD17A is essential for the growth of pathogenic stage parasites. We also identified lipases important for acquiring fatty acids (FAs) from the host. Using chemical biology and genetic approaches, we discovered that three lipases are important for acquiring FAs form the host in the pathogenic stage. Inhibiting these enzymes may kill the parasite in the host.

<p>Molecular modelling strategies for evaluation of enzyme selectivity wereinvestigated with a focus on principles of how molecular interactionscould be evaluated to provide information about selectivity. Althoughmolecular modelling provides tools for evaluation of geometrical andenergy features of molecular systems, no general strategies for evaluationof enzyme selectivity exist. Geometrical analyses can be based uponinspection and reasoning about molecular interactions, which provide aneasily accessible way to gain information, but suffer from the risk of biasput in by the modeller. They can also be based on geometrical features ofmolecular interactions such as bond lengths and hydrogen-bond formation.Energy analyses are appealing for their modeller independenceand for the possibility to predict not only stereopreference, but also itsmagnitude.In this thesis, four examples of enantio- or regioselective serinehydrolase-catalysed reaction systems are presented together with developedmodelling protocols for explanation, prediction or enhancement ofselectivity. Geometrical as well as energy-based methodology were used,and provided an understanding of the structural basis of enzymeselectivity. In total, the protocols were successful in making qualitative explanationsand predictions of stereoselectivity, although quantitative determinationswere not achieved.</p>

Hepatitis C virus (HCV) is a hepatotropic positive-sense RNA virus of the Flaviviridae virus family and is a major cause of chronic liver disease worldwide. Like all obligate parasites, HCV relies on host pathways to enable its pathogenesis. HCV, in particular, has a clear link with hepatic lipid metabolism, promoting a lipid-rich environment for its proliferation. This manifests as liver steatosis in many patients harboring chronic HCV infection. Based on our recent findings regarding an immunometabolic and HCV antiviral microRNA (miRNA), miRNA-185 targeting and down regulating serine hydrolases (SH) involved in lipid and endocannabinoid metabolism, here we investigate HCV and its dependency on certain metabolic serine hydrolases involved in lipid and endocannabinoid metabolism. Serine hydrolases are one of the largest and most diverse enzyme families. This enzyme family has emerged as a center of therapeutic potential due to its implications in many metabolic roles. Here, we demonstrate that pharmacological inhibition of metabolic serine hydrolases alpha-beta hydrolyzing domain 6 (ABHD6), carboxylesterase 1 (CES1), and monoacylglycerol lipase (MGLL), enzymes involved in the hydrolysis of the endogenous cannabinoid receptor 1 (CB1) agonist 2-arachidonoyl glycerol (2-AG) are potently antiviral against HCV. Serine hydrolase inhibition with the MGLL inhibitor MJN110 paired with endocannabinoid signaling antagonization led to additive antiviral effects against HCV and has revealed modulation of the viral pathogenic phenotype to be its key course of action. MGLL inhibitor MJN110 transcriptomic characterization revealed modulations in humoral immunity and phagocytosis and acts antiviraly against HCV independent of CB1 antagonization. This provides an avenue for future investigation, assessing the viability of CB1 antagonization, and MGLL as a key host targeted antiviral factor in affecting HCV viral life cycle.

Molecular modelling strategies for evaluation of enzyme selectivity wereinvestigated with a focus on principles of how molecular interactionscould be evaluated to provide information about selectivity. Althoughmolecular modelling provides tools for evaluation of geometrical andenergy features of molecular systems, no general strategies for evaluationof enzyme selectivity exist. Geometrical analyses can be based uponinspection and reasoning about molecular interactions, which provide aneasily accessible way to gain information, but suffer from the risk of biasput in by the modeller. They can also be based on geometrical features ofmolecular interactions such as bond lengths and hydrogen-bond formation.Energy analyses are appealing for their modeller independenceand for the possibility to predict not only stereopreference, but also itsmagnitude.In this thesis, four examples of enantio- or regioselective serinehydrolase-catalysed reaction systems are presented together with developedmodelling protocols for explanation, prediction or enhancement ofselectivity. Geometrical as well as energy-based methodology were used,and provided an understanding of the structural basis of enzymeselectivity. In total, the protocols were successful in making qualitative explanationsand predictions of stereoselectivity, although quantitative determinationswere not achieved.

As obligatory intracellular parasites, viruses are in a constant battle with their host to establish infection. They can facilitate their propagation by modulating host immune or metabolic pathways. This modulation involves targeting various molecular factors such as microRNAs (miRNA), enzymes, or small molecules. Understanding how viruses alter the chemical makeup of a cell is crucial to identifying what pathways are being targeted, furthering our understanding of the virus life cycle, and may aid in identifying biomarkers of disease. Here, we examine host-virus interactions in the context of two viruses, hepatitis c virus (HCV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). First, the modulation of serine hydrolases by a pro-viral microRNA, miRNA-122, is investigated using activity-based protein profiling (ABPP). This study identifies a downstream target of miRNA-122 that is differentially activated during HCV infection which can be targeted pharmacologically to reduce HCV infectivity. Second, we apply similar techniques to identify serine hydrolase changes associated with SARS-CoV-2 infection. Results point towards enrichment of endocannabinoid metabolism which may offer an alternative therapeutic avenue for combating SARS-CoV-2 infection. Together, the work presented in this thesis provides avenues for further investigation into miRNA-122 interactions during HCV infection and endocannabinoid metabolism in SARS-CoV-2 infection.

The serine hydrolase (SH) enzyme superfamily is one of the largest and most diverse enzyme classes in eukaryotes and prokaryotes. The most virulent human malaria parasite Plasmodium falciparum has over 40 predicted serine hydrolases (SH). Prior investigation on a few of these have suggested their critical role in parasite biology. The majority of the SHs in P. falciparum have not been functionally characterized. Investigation of these uncharacterized SHs will provide new insights into essential features of parasite metabolism and possibly lead to new antimalarial targets. In this study, we have employed activity-based protein profiling (ABPP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to functionally characterize SHs. In our effort to profile plasmodial SHs using ABPP, we have identified a human erythrocyte SH, acylpeptide hydrolase (APEH) in the developing parasites. This finding is the first report of internalization of host hydrolytic enzyme by the parasite. Treatment of parasites with an APEH specific triazole urea inhibitor, AA74-1, caused growth inhibition in parasites with poor potency in the first replication cycle, however, the potency dramatically increased in the second cycle. We show that this unique growth inhibition profile is due to the inability of AA74-1 to inhibit parasite-internalized APEH in vivo. These findings suggest that internalization of active APEH by the parasite is essential for parasite survival. Lipases catalyze the hydrolysis of ester bonds of lipid species such as neutral lipids and phospholipids. Although roles of lipases in propagation, as well as virulence in various organisms, have been acknowledged, in P. falciparum lipases remain understudied. We combined LC-MS/MS with the SH-directed ABPP to identify lipases of SH superfamily in P. falciparum. We have identified 16 plasmodial SHs with putative lipase activity. Bioinformatics analysis of our identified lipases is consistent with our findings. We have screened a panel of various classes of SH inhibitors in a competitive ABPP. A plasmodial putative lipase was potently and specifically inhibited by human monoacylglycerol lipase inhibitor. This inhibition profile suggests it as a monoacylglycerol lipase which plays a role in releasing fatty acids from neutral lipid. This finding shows that how inhibitor screening can aid in building hypotheses on biological roles of an enzyme. Altogether, in this dissertation, we have presented a robust strategy of identifying and functionally characterizing SHs in P. falciparum, which opens the door to the discovery of new biological processes. / Doctor of Philosophy / Malaria contributed to nearly a half a million deaths in 2017. The vast majority of malaria-related deaths are due to the parasite Plasmodium falciparum. This parasite resides inside human red blood cells (erythrocytes) and grows rapidly during a 48 hour cycle. There are over 40 serine hydrolase (SH) superfamily proteins in the parasite. Biological functions of the majority of SHs in the parasite remains unknown. Study on these SHs will provide new insights into parasite biology, and possibly present new antimalarial drug targets. We used chemical biology techniques to identify and functionally characterize parasite SHs. In one study, we show the parasite intenalized a human erythrocyte SH, acylpeptide hydrolase (APEH). We used an APEH-specific inhibitor to investigate the biological significance of internalized APEH in parasite biology. Treatment of the parasite with the inhibitor resulted in parasite growth inhibition suggesting internalization of APEH is essential for parasite survival. Lipases are enzymes that aid in break down of lipids and have shown to be crucial for growth and pathogenicity in various organisms. Lipases and lipid catabolism remain understudied in the malaria parasite. We used mass spectrometry in our approach to identify 16 lipases in asexual parasites. We have also shown that screening with highly specific inhibitors can help in predicting biological function of a particular enzyme. In summary, in this body of work, we have presented an approach of studying SHs in the malaria parasite, which will provide new insights into parasite biology.

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