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• W3003081294 abstract "•Semi-synthesis of an affinity analog of the antimalarial natural product Sal A•Identification of serine hydrolases as the primary targets of Sal A in P. falciparum•Sal A covalently binds to and inhibits a MAGL-like protein in P. falciparum•Parasites are unable to generate strong in vitro resistance to Sal A Salinipostin A (Sal A) is a potent antiplasmodial marine natural product with an undefined mechanism of action. Using a Sal A-derived activity-based probe, we identify its targets in the Plasmodium falciparum parasite. All of the identified proteins contain α/β serine hydrolase domains and several are essential for parasite growth. One of the essential targets displays a high degree of homology to human monoacylglycerol lipase (MAGL) and is able to process lipid esters including a MAGL acylglyceride substrate. This Sal A target is inhibited by the anti-obesity drug Orlistat, which disrupts lipid metabolism. Resistance selections yielded parasites that showed only minor reductions in sensitivity and that acquired mutations in a PRELI domain-containing protein linked to drug resistance in Toxoplasma gondii. This inability to evolve efficient resistance mechanisms combined with the non-essentiality of human homologs makes the serine hydrolases identified here promising antimalarial targets. Salinipostin A (Sal A) is a potent antiplasmodial marine natural product with an undefined mechanism of action. Using a Sal A-derived activity-based probe, we identify its targets in the Plasmodium falciparum parasite. All of the identified proteins contain α/β serine hydrolase domains and several are essential for parasite growth. One of the essential targets displays a high degree of homology to human monoacylglycerol lipase (MAGL) and is able to process lipid esters including a MAGL acylglyceride substrate. This Sal A target is inhibited by the anti-obesity drug Orlistat, which disrupts lipid metabolism. Resistance selections yielded parasites that showed only minor reductions in sensitivity and that acquired mutations in a PRELI domain-containing protein linked to drug resistance in Toxoplasma gondii. This inability to evolve efficient resistance mechanisms combined with the non-essentiality of human homologs makes the serine hydrolases identified here promising antimalarial targets. Plasmodium falciparum (Pf) is a protozoan parasite that causes the most severe form of human malaria. Malaria threatens 40% of the world's population, resulting in an estimated 228 million cases and nearly 405,000 deaths annually (WHO, 2018WHO World Malaria Report 2018. World Health Organization, 2018Google Scholar). Because of a lack of effective vaccines against the parasite, malaria treatment and control efforts rely on the administration of antimalarial drugs (White et al., 2014White N.J. Pukrittayakamee S. Hien T.T. Faiz M.A. Mokuolu O.A. Dondorp A.M. Malaria.Lancet. 2014; 383: 723-735Abstract Full Text Full Text PDF PubMed Scopus (770) Google Scholar). In recent years, artemisinin-based combination therapies (ACTs) have been the first-line antimalarial drugs and have contributed significantly to a reduction in the global malaria burden (Tu, 2011Tu Y. The discovery of artemisinin (qinghaosu) and gifts from Chinese medicine.Nat. Med. 2011; 17: 1217-1220Crossref PubMed Scopus (821) Google Scholar, White, 2008White N.J. Qinghaosu (artemisinin): the price of success.Science. 2008; 320: 330-334Crossref PubMed Scopus (733) Google Scholar). However, emerging resistance to artemisinin and the partner drugs used in ACTs threatens to reverse the gains that have been made against this disease (Dondorp et al., 2017Dondorp A.M. Smithuis F.M. Woodrow C. Seidlein L.V. How to contain artemisinin- and multidrug-resistant falciparum malaria.Trends Parasitol. 2017; 33: 353-363Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, van der Pluijm et al., 2019van der Pluijm R.W. Imwong M. Chau N.H. Hoa N.T. Thuy-Nhien N.T. Thanh N.V. Jittamala P. Hanboonkunupakarn B. Chutasmit K. Saelow C. et al.Determinants of dihydroartemisinin-piperaquine treatment failure in Plasmodium falciparum malaria in Cambodia, Thailand, and Vietnam: a prospective clinical, pharmacological, and genetic study.Lancet Infect. Dis. 2019; 19: 952-961Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Therefore, there is a continuing need to identify novel targets and pathways against which efficacious therapeutics can be developed, both to widen the scope of treatment and to overcome existing mechanisms of antimalarial drug resistance. Natural products have evolved to encompass a broad spectrum of chemical and functional diversity and structural complexity, which enables them to target diverse biological macromolecules in a highly selective fashion. These features of natural products not only suggest that they may hold much promise for the development of therapeutic agents but also make them ideal starting points for the development of chemical probes (Harvey, 2000Harvey A. Strategies for discovering drugs from previously unexplored natural products.Drug Discov. Today. 2000; 5: 294-300Crossref PubMed Scopus (395) Google Scholar, Li and Vederas, 2009Li J.W. Vederas J.C. Drug discovery and natural products: end of an era or an endless frontier?.Science. 2009; 325: 161-165Crossref PubMed Scopus (1509) Google Scholar, Rodrigues et al., 2016Rodrigues T. Reker D. Schneider P. Schneider G. Counting on natural products for drug design.Nat. Chem. 2016; 8: 531-541Crossref PubMed Scopus (710) Google Scholar, Thomford et al., 2018Thomford N.E. Senthebane D.A. Rowe A. Munro D. Seele P. Maroyi A. Dzobo K. Natural products for drug discovery in the 21st century: innovations for novel drug discovery.Int. J. Mol. Sci. 2018; 19: 1578Crossref PubMed Scopus (513) Google Scholar). Natural product-inspired chemical probes have been instrumental in identifying therapeutic targets and studying poorly understood biological processes and fundamental biology, yet the targets of most natural products remain unknown (Bottcher et al., 2010Bottcher T. Pitscheider M. Sieber S.A. Natural products and their biological targets: proteomic and metabolomic labeling strategies.Angew. Chem. Int. Ed. 2010; 49: 2680-2698Crossref PubMed Scopus (157) Google Scholar, Carlson, 2010Carlson E.E. Natural products as chemical probes.ACS Chem. Biol. 2010; 5: 639-653Crossref PubMed Scopus (164) Google Scholar, Kreuzer et al., 2015Kreuzer J. Bach N.C. Forler D. Sieber S.A. Target discovery of acivicin in cancer cells elucidates its mechanism of growth inhibition.Chem. Sci. 2015; 6: 237-245Crossref Google Scholar, Taunton et al., 1996Taunton J. Collins J.L. Schreiber S.L. Synthesis of natural and modified trapoxins, useful reagents for exploring histone deacetylase function.J. Am. Chem. Soc. 1996; 118: 10412-10422Crossref Scopus (180) Google Scholar). Salinipostin A (Sal A) is a natural product produced by a Salinispora sp. bacterium. that was isolated from a marine sediment. Sal A displays potent activity against Pf parasites, with a half maximal effective concentration (EC50) of 50 nM and a selectivity index of >1,000 over a variety of mammalian cell lines (Schulze et al., 2015Schulze C.J. Navarro G. Ebert D. DeRisi J. Linington R.G. Salinipostins A-K, long-chain bicyclic phosphotriesters as a potent and selective antimalarial chemotype.J. Org. Chem. 2015; 80: 1312-1320Crossref PubMed Scopus (50) Google Scholar). In a previous effort to identify drug targets of Sal A, selection experiments were attempted but failed to generate resistant parasites. These results suggested that Sal A might target multiple proteins or essential pathways for parasite development, and that its potency is not easily compromised by single point mutations. The refractoriness of Sal A to parasite resistance mechanisms combined with its potent and selective activity, and lack of structural similarity to other antimalarials make Sal A an interesting chemical tool to identify druggable pathways in Pf parasites. The α/β hydrolase superfamily is one of the largest groups of structurally related proteins that encompass a diverse range of catalytic activities. Members of this family are composed of largely parallel β sheets flanked on their terminal ends by α helices. The α/β hydrolase domain contains a conserved catalytic dyad or triad formed by a nucleophilic serine residue and an acid-base residue pair. The archetypal α/β hydrolases are esterases, which catalyze two-step nucleophilic substitution reactions to mediate hydrolysis of esters, but substrates for α/β hydrolases also include amides and thioesters (Carr and Ollis, 2009Carr P.D. Ollis D.L. Alpha/beta hydrolase fold: an update.Protein Pept. Lett. 2009; 16: 1137-1148Crossref PubMed Scopus (119) Google Scholar, Holmquist, 2000Holmquist M. Alpha/beta-hydrolase fold enzymes: structures, functions and mechanisms.Curr. Protein Pept. Sci. 2000; 1: 209-235Crossref PubMed Scopus (484) Google Scholar, Nardini and Dijkstra, 1999Nardini M. Dijkstra B.W. Alpha/beta hydrolase fold enzymes: the family keeps growing.Curr. Opin. Struct. Biol. 1999; 9: 732-737Crossref PubMed Scopus (682) Google Scholar, Ollis et al., 1992Ollis D.L. Cheah E. Cygler M. Dijkstra B. Frolow F. Franken S.M. Harel M. Remington S.J. Silman I. Schrag J. et al.The alpha/beta hydrolase fold.Protein Eng. 1992; 5: 197-211Crossref PubMed Scopus (1845) Google Scholar). In recent years, α/β serine hydrolases have attracted considerable attention for their critical roles in metabolic processes in humans. These metabolic serine hydrolases have been implicated in neurotransmission, pain sensation, inflammation, cancer, and bacterial infection (Bachovchin and Cravatt, 2012Bachovchin D.A. Cravatt B.F. The pharmacological landscape and therapeutic potential of serine hydrolases.Nat. Rev. Drug Discov. 2012; 11: 52-68Crossref PubMed Scopus (207) Google Scholar, Long and Cravatt, 2011Long J.Z. Cravatt B.F. The metabolic serine hydrolases and their functions in mammalian physiology and disease.Chem. Rev. 2011; 111: 6022-6063Crossref PubMed Scopus (268) Google Scholar). The Pf genome encodes about 40 putative members of the serine hydrolase superfamily based on annotated sequence homologies (Aurrecoechea et al., 2009Aurrecoechea C. Brestelli J. Brunk B.P. Dommer J. Fischer S. Gajria B. Gao X. Gingle A. Grant G. Harb O.S. et al.PlasmoDB: a functional genomic database for malaria parasites.Nucleic Acids Res. 2009; 37: D539-D543Crossref PubMed Scopus (803) Google Scholar). However, most of these have not been functionally characterized. Natural product analogs of cyclipostins and cyclophostins, which contain the same reactive functional core electrophile as Sal A, target human and mouse α/β serine hydrolases as well as α/β serine hydrolases involved in lipid metabolism in Mycobacterium tuberculosis (Madani et al., 2019Madani A. Ridenour J.N. Martin B.P. Paudel R.R. Abdul Basir A. Le Moigne V. Herrmann J.L. Audebert S. Camoin L. Kremer L. et al.Cyclipostins and cyclophostin analogues as multitarget inhibitors that impair growth of Mycobacterium abscessus.ACS Infect. Dis. 2019; 5: 1597-1608Crossref PubMed Scopus (22) Google Scholar, Malla et al., 2011Malla R.K. Bandyopadhyay S. Spilling C.D. Dutta S. Dupureur C.M. The first total synthesis of (+/–)-cyclophostin and (+/–)-cyclipostin P: inhibitors of the serine hydrolases acetyl cholinesterase and hormone sensitive lipase.Org. Lett. 2011; 13: 3094-3097Crossref PubMed Scopus (29) Google Scholar, Nguyen et al., 2017Nguyen P.C. Delorme V. Benarouche A. Martin B.P. Paudel R. Gnawali G.R. Madani A. Puppo R. Landry V. Kremer L. et al.Cyclipostins and cyclophostin analogs as promising compounds in the fight against tuberculosis.Sci. Rep. 2017; 7: 11751Crossref PubMed Scopus (32) Google Scholar, Nguyen et al., 2018Nguyen P.C. Madani A. Santucci P. Martin B.P. Paudel R.R. Delattre S. Herrmann J.L. Spilling C.D. Kremer L. Canaan S. et al.Cyclophostin and cyclipostins analogues, new promising molecules to treat mycobacterial-related diseases.Int. J. Antimicrob. Agents. 2018; 51: 651-654Crossref PubMed Scopus (23) Google Scholar). These data suggest that α/β hydrolases are promising candidate targets for Sal A in Pf. Activity-based protein profiling (ABPP) has emerged as a powerful and versatile chemical proteomic platform for characterizing the function of enzymes and for rapidly identifying novel targets (Chen et al., 2016Chen B. Ge S.-S. Zhao Y.-C. Chen C. Yang S. Activity-based protein profiling: an efficient approach to study serine hydrolases and their inhibitors in mammals and microbes.RSC Adv. 2016; 6: 113327-113343Crossref Scopus (7) Google Scholar, Cravatt et al., 2008Cravatt B.F. Wright A.T. Kozarich J.W. Activity-based protein profiling: from enzyme chemistry to proteomic chemistry.Annu. Rev. Biochem. 2008; 77: 383-414Crossref PubMed Scopus (888) Google Scholar, Spradlin et al., 2019Spradlin J.N. Hu X. Ward C.C. Brittain S.M. Jones M.D. Ou L. To M. Proudfoot A. Ornelas E. Woldegiorgis M. et al.Harnessing the anti-cancer natural product nimbolide for targeted protein degradation.Nat. Chem. Biol. 2019; 15: 747-755Crossref PubMed Scopus (190) Google Scholar, Wright and Sieber, 2016Wright M.H. Sieber S.A. Chemical proteomics approaches for identifying the cellular targets of natural products.Nat. Prod. Rep. 2016; 33: 681-708Crossref PubMed Google Scholar). Central to ABPP is the use of activity-based probes (ABPs), which covalently modify the active sites of enzymes. The bicyclic phosphotriester core in Sal A is hypothesized to form a covalent bond with target proteins upon nucleophilic attack by a catalytic serine or cysteine residue in the target. We therefore devised an analog of Sal A that contains an alkyne group at the terminus of its lipid tail, thereby enabling its use for affinity isolation of labeled target proteins. Herein, we describe the use of this probe to identify multiple essential α/β serine hydrolases, including an essential parasite ortholog of human monoacylglycerol lipase (MAGL) that appears to be one of several targets. We also report that resistance appears to involve mutations in a separate PRELI domain-containing protein that, in Toxoplasma gondii, functions as a multidrug resistance mediator (Jeffers et al., 2017Jeffers V. Kamau E.T. Srinivasan A.R. Harper J. Sankaran P. Post S.E. Varberg J.M. Sullivan Jr., W.J. Boyle J.P. TgPRELID, a mitochondrial protein linked to multidrug resistance in the parasite Toxoplasma gondii.mSphere. 2017; 2: e00229-16Crossref PubMed Scopus (12) Google Scholar). Collectively, our data provide evidence that Sal A inhibits multiple essential serine hydrolases in Pf, adversely impacting lipid metabolism, and implicate a new set of metabolic targets for future antimalarial drug discovery and development efforts. The Sal A molecule is made up of two parts, namely a reactive cyclic phosphate group that can act as an electrophile for serine and cysteine nucleophiles and a lipid tail that presumably mimics a native cellular lipid. This saturated 15 carbon lipid tail aligns with the 16 carbons in palmitic acid such that the reactive phosphorus is located directly at the site of the carbonyl used to form a thiol ester between palmitate and a cysteine on a palmitoylated protein (Couvertier et al., 2014Couvertier S.M. Zhou Y. Weerapana E. Chemical-proteomic strategies to investigate cysteine posttranslational modifications.Biochim. Biophys. Acta. 2014; 1844: 2315-2330Crossref PubMed Scopus (46) Google Scholar) (Figure 1A). We therefore reasoned that Sal A might act as a substrate mimetic inhibitor of the serine hydrolases that perform depalmitoylation reactions on proteins (Davda and Martin, 2014Davda D. Martin B.R. Acyl protein thioesterase inhibitors as probes of dynamic S-palmitoylation.MedChemComm. 2014; 5: 268-276Crossref PubMed Google Scholar, Won et al., 2018Won S.J. Cheung See Kit M. Martin B.R. Protein depalmitoylases.Crit. Rev. Biochem. Mol. Biol. 2018; 53: 83-98Crossref PubMed Scopus (70) Google Scholar). We conducted a competition experiment in which whole Pf parasites were incubated with Sal A before labeling with the broad-spectrum serine hydrolase ABP fluorophosphonate-rhodamine (FP-Rho) (Simon and Cravatt, 2010Simon G.M. Cravatt B.F. Activity-based proteomics of enzyme superfamilies: serine hydrolases as a case study.J. Biol. Chem. 2010; 285: 11051-11055Crossref PubMed Scopus (221) Google Scholar). Several hydrolases were inhibited upon treatment with 1 μM Sal A (Figure 1B). Although palmitoylation is a ubiquitous and dynamic post-translational modification in Pf parasites (Corvi et al., 2012Corvi M.M. Alonso A.M. Caballero M.C. Protein palmitoylation and pathogenesis in apicomplexan parasites.J. Biomed. Biotechnol. 2012; 2012: 483969Crossref PubMed Scopus (8) Google Scholar, Corvi and Turowski, 2019Corvi M.M. Turowski V.R. Palmitoylation in apicomplexan parasites: from established regulatory roles to putative new functions.Mol. Biochem. Parasitol. 2019; 230: 16-23Crossref PubMed Scopus (4) Google Scholar, Jones et al., 2012Jones M.L. Tay C.L. Rayner J.C. Getting stuck in: protein palmitoylation in Plasmodium.Trends Parasitol. 2012; 28: 496-503Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar), there are no validated depalmitoylases in this parasite. Therefore, we tested Sal A in the related parasite pathogen, T. gondii. To determine whether Sal A inhibits the T. gondii depalmitoylase PPT1 (TgPPT1), we treated wild-type (WT) (Δku80) and TgPPT1 knockout (ΔPPT1) parasites (Child et al., 2013Child M.A. Hall C.I. Beck J.R. Ofori L.O. Albrow V.E. Garland M. Bowyer P.W. Bradley P.J. Powers J.C. Boothroyd J.C. et al.Small-molecule inhibition of a depalmitoylase enhances Toxoplasma host-cell invasion.Nat. Chem. Biol. 2013; 9: 651-656Crossref PubMed Scopus (49) Google Scholar) with Sal A and labeled parasite extracts with FP-Rho. These studies confirmed that Sal A was able to compete with FP-Rho for labeling of TgPPT1, as well as several other serine hydrolases in T. gondii (Figure 1C). Competition studies in human cells (HEK293T) confirmed that Sal A inhibits the human depalmitoylases APT1 and APT2 in a dose-dependent manner, with increased potency against APT2 over APT1 (Figure 1D). In addition, several other less-abundantly labeled human hydrolases were also inhibited by Sal A pretreatment, suggesting that Sal A likely has multiple serine hydrolase targets. Because Sal A likely forms covalent bonds with its targets through reaction at the electrophile phosphorus (Figure 2A), this compound was ideally suited to be converted into a probe that could be used to isolate and identify binding proteins in Pf. Sal A was originally isolated along with ten analogs, which provided important structure-activity relationship information for the design of the chemical probe. Analysis of these analogs revealed a significant drop in potency against parasites as the length of both the northern alkyl chain (C4>C3>C2) and the western alkyl chain (C15>C14>C13) decreased (Schulze et al., 2015Schulze C.J. Navarro G. Ebert D. DeRisi J. Linington R.G. Salinipostins A-K, long-chain bicyclic phosphotriesters as a potent and selective antimalarial chemotype.J. Org. Chem. 2015; 80: 1312-1320Crossref PubMed Scopus (50) Google Scholar). To minimize structural deviations from the natural product scaffold, we designed a chemical probe with a butyl northern alkyl chain and a terminal alkyne on the western alkyl chain (C16), thus introducing a single carbon and 2 degrees of unsaturation to the parent molecule. Although total syntheses of similar natural products have been accomplished (Malla et al., 2011Malla R.K. Bandyopadhyay S. Spilling C.D. Dutta S. Dupureur C.M. The first total synthesis of (+/–)-cyclophostin and (+/–)-cyclipostin P: inhibitors of the serine hydrolases acetyl cholinesterase and hormone sensitive lipase.Org. Lett. 2011; 13: 3094-3097Crossref PubMed Scopus (29) Google Scholar, Zhao et al., 2018Zhao M. Wei X. Liu X. Dong X. Yu R. Wan S. Jiang T. Total synthesis of marine cyclic enol-phosphotriester salinipostin compounds.J. Ocean Univ. China. 2018; 17: 683-689Crossref Scopus (6) Google Scholar), the bicyclic phosphotriester core of Sal A presents numerous synthetic challenges. Therefore, we opted for a semi-synthetic route in which the natural product Sal A was dealkylated at the western chain and the resulting phosphate diester was reacted with an electrophilic alkyne to form two diastereomers of the alkynylated natural product (Sal alk; Figure 2B). Screening of these diastereomers against Pf parasites revealed that the isomer corresponding to the parent natural product retained full potency, whereas the other diastereomer showed a 10-fold drop in potency (Figure S1). In addition, we conducted 72 h growth assays in the presence and absence of isopentenyl pyrophosphate (IPP), which is a product of the apicoplast isoprenoid biosynthesis pathway that is essential for growth during the asexual blood stage. An apicoplast-based killing mechanism can be reversed by supplementation of parasite culture medium with IPP (Yeh and DeRisi, 2011Yeh E. DeRisi J.L. Chemical rescue of malaria parasites lacking an apicoplast defines organelle function in blood-stage Plasmodium falciparum.PLoS Biol. 2011; 9: e1001138Crossref PubMed Scopus (313) Google Scholar). We performed this chemical rescue screen to determine whether the antiplasmodial mechanism of Sal A was related to depletion or disruption of key lipid metabolites derived from the isoprenoid pathway in the apicoplast. The potency of both Sal A and Sal alk was unaffected by IPP, suggesting that these compounds do not target the apicoplast (Figure S1). Having confirmed that Sal alk retained activity equivalent to the parent natural product, we conducted a competition experiment in which parasites were pre-incubated with Sal A for 2 h before Sal alk labeling. CLICK reaction with TAMRA-azide confirmed that Sal alk labeled multiple proteins in Pf and that Sal A competed for Sal alk labeling of several of those proteins in a dose-dependent manner (Figure 2C). Therefore, we next used Sal alk as a probe to affinity purify targets of the natural product using a standard ABPP method. For these studies, we pretreated Pf parasite cultures with either Sal A or a vehicle control and subsequently labeled cultures with Sal alk. Samples were lysed and labeled proteins were conjugated to biotin-azide using CLICK chemistry. Avidin affinity chromatography was performed to enrich for biotin-azide-labeled proteins. The samples were then subjected to tryptic digestion and tandem mass spectrometry analysis (Table S1). Analysis of these data identified ten putative Sal A targets that were enriched in Sal alk-treated samples and that were correspondingly reduced in Sal A-pretreated samples (Figure 2D; Tables 1 and S1). All ten putative target proteins have α/β serine hydrolase domains with either a conserved Ser-His-Asp catalytic triad or a Ser-Asp dyad. Of these, five are annotated as lipases with the characteristic GXSXG motif (http://plasmodb.org/plasmo) (Aurrecoechea et al., 2009Aurrecoechea C. Brestelli J. Brunk B.P. Dommer J. Fischer S. Gajria B. Gao X. Gingle A. Grant G. Harb O.S. et al.PlasmoDB: a functional genomic database for malaria parasites.Nucleic Acids Res. 2009; 37: D539-D543Crossref PubMed Scopus (803) Google Scholar). Based on a piggyBac transposon saturation mutagenesis study (Balu et al., 2005Balu B. Shoue D.A. Fraser Jr., M.J. Adams J.H. High-efficiency transformation of Plasmodium falciparum by the lepidopteran transposable element piggyBac.Proc. Natl. Acad. Sci. U S A. 2005; 102: 16391-16396Crossref PubMed Scopus (134) Google Scholar, Balu et al., 2009Balu B. Chauhan C. Maher S.P. Shoue D.A. Kissinger J.C. Fraser Jr., M.J. Adams J.H. piggyBac is an effective tool for functional analysis of the Plasmodium falciparum genome.BMC Microbiol. 2009; 9: 83Crossref PubMed Scopus (54) Google Scholar, Bushell et al., 2017Bushell E. Gomes A.R. Sanderson T. Anar B. Girling G. Herd C. Metcalf T. Modrzynska K. Schwach F. Martin R.E. et al.Functional profiling of a Plasmodium genome reveals an abundance of essential genes.Cell. 2017; 170: 260-272Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar, Zhang et al., 2018Zhang M. Wang C. Otto T.D. Oberstaller J. Liao X. Adapa S.R. Udenze K. Bronner I.F. Casandra D. Mayho M. et al.Uncovering the essential genes of the human malaria parasite Plasmodium falciparum by saturation mutagenesis.Science. 2018; 360: eaap7847Crossref PubMed Scopus (415) Google Scholar), four of the identified targets are likely to be essential in Pf.Table 1Sal A Target Proteins Identified in P. falciparumProtein IDDescriptionMW (kDa)Essential in P. falciparum piggyBac ScreenSpectral CountsPF3D7_0709700lysophospholipase (PfPARE)42.4no195.0PF3D7_1328500α/β hydrolase115.9no39.7PF3D7_0209100phospholipase A278.3no28.7PF3D7_1001600exported lipase 2 (PfXL2)88.6yes20.7PF3D7_1358000patatin-like phospholipase238.2no19.7PF3D7_1116100serine esterase217.1no13.7PF3D7_1038900esterase, lysophospholipase41.8yes11.3PF3D7_1134500α/β hydrolase210.5yes10.3PF3D7_0818600BEM46-like protein (PBLP)34.9yes8.3PF3D7_0805000α/β hydrolase28.5no4.3 Open table in a new tab One of the proteins identified as a putative Sal A target, PF3D7_1001600 was recently annotated as exported lipase 2 (PfXL2), one of the Plasmodium α/β hydrolases containing the export element (PEXEL) motif that is essential for delivery of most exported proteins (Hiller et al., 2004Hiller N.L. Bhattacharjee S. van Ooij C. Liolios K. Harrison T. Lopez-Estrano C. Haldar K. A host-targeting signal in virulence proteins reveals a secretome in malarial infection.Science. 2004; 306: 1934-1937Crossref PubMed Scopus (658) Google Scholar, Marti et al., 2004Marti M. Good R.T. Rug M. Knuepfer E. Cowman A.F. Targeting malaria virulence and remodeling proteins to the host erythrocyte.Science. 2004; 306: 1930-1933Crossref PubMed Scopus (713) Google Scholar). Studies with trophozoite/early schizont-stage parasites expressing a C-terminal GFP fusion of PfXL2 revealed that the protein is exported into the erythrocyte cytosol of parasitized red blood cells (RBCs) (Spillman et al., 2016Spillman N.J. Dalmia V.K. Goldberg D.E. Exported epoxide hydrolases modulate erythrocyte vasoactive lipids during Plasmodium falciparum infection.MBio. 2016; 7: e01538-16Crossref PubMed Scopus (22) Google Scholar). Nonetheless, the physiological function of PfXL2 remains unknown, although it is considered to be essential for parasite growth (Bushell et al., 2017Bushell E. Gomes A.R. Sanderson T. Anar B. Girling G. Herd C. Metcalf T. Modrzynska K. Schwach F. Martin R.E. et al.Functional profiling of a Plasmodium genome reveals an abundance of essential genes.Cell. 2017; 170: 260-272Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar, Zhang et al., 2018Zhang M. Wang C. Otto T.D. Oberstaller J. Liao X. Adapa S.R. Udenze K. Bronner I.F. Casandra D. Mayho M. et al.Uncovering the essential genes of the human malaria parasite Plasmodium falciparum by saturation mutagenesis.Science. 2018; 360: eaap7847Crossref PubMed Scopus (415) Google Scholar). We also identified a Plasmodium BEM46-like protein (PBLP, PF3D7_0818600) as another potential target of Sal A. A study of PBLP in the rodent malaria parasite Plasmodium yoelii showed that it has an important role in invasion, with deletion of PBLP affecting merozoite formation, sporozoite maturation, and infectivity (Groat-Carmona et al., 2015Groat-Carmona A.M. Kain H. Brownell J. Douglass A.N. Aly A.S. Kappe S.H. A Plasmodium alpha/beta-hydrolase modulates the development of invasive stages.Cell Microbiol. 2015; 17: 1848-1867Crossref PubMed Scopus (8) Google Scholar). The Bud Emergence (BEM) 46 proteins are an evolutionarily conserved class of α/β hydrolases that carry N-terminal signal peptides that can act as transmembrane domains. BEM46 homologs have been implicated in signal transduction and cell polarity (Kumar et al., 2013Kumar A. Kollath-Leiss K. Kempken F. Characterization of bud emergence 46 (BEM46) protein: sequence, structural, phylogenetic and subcellular localization analyses.Biochem. Biophys. Res. Commun. 2013; 438: 526-532Crossref PubMed Scopus (7) Google Scholar, Mercker et al., 2009Mercker M. Kollath-Leiss K. Allgaier S. Weiland N. Kempken F. The BEM46-like protein appears to be essential for hyphal development upon ascospore germination in Neurospora crassa and is targeted to the endoplasmic reticulum.Curr. Genet. 2009; 55: 151-161Crossref PubMed Scopus (14) Google Scholar). All Plasmodium species appear to express a PBLP ortholog, which suggests a conserved albeit as yet unknown function. Based on total spectral counts, the most abundantly labeled potential Sal A target was a putative lysophospholipase (PF3D7_0709700). This protein was recently annotated as PfPARE (P. falciparum Prodrug Activation and Resistance Esterase), an esterase that is responsible for activating some antimalarial prodrug compounds (Istvan et al., 2017Istvan E.S. Mallari J.P. Corey V.C. Dharia N.V. Marshall G.R. Winzeler E.A. Goldberg D.E. Esterase mutation is a mechanism of resistance to antimalarial compounds.Nat. Commun. 2017; 8: 14240Crossref PubMed Scopus (27) Google Scholar). To test whether PfPARE could be a Sal A target, we obtained Pf lines expressing both WT (WT-GFP) and active site mutant (S179T-GFP) PfPARE-GFP fusions replacing the native gene locus. Treatment of these parasites with Sal alk revealed labeling of PfPARE that was dependent on the catalytic serine (Figure S2A). However, parasites expressing the WT or catalytically dead PfPARE were equally sensitive to Sal A treatment (Figure S2B). These results, combined with the predicted lack of essentiality of PfPARE, suggest t" @default.
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• W3003081294 date "2020-02-01" @default.
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• W3003081294 title "The Antimalarial Natural Product Salinipostin A Identifies Essential α/β Serine Hydrolases Involved in Lipid Metabolism in P. falciparum Parasites" @default.
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