Article The Antimalarial Natural Product Salinipostin A Identifies Essential a/b Serine Hydrolases Involved in Lipid Metabolism in P. falciparum Parasites Graphical Abstract Authors Euna Yoo, Christopher J. Schulze, Barbara H. Stokes, ..., Eranthie Weerapana, David A. Fidock, Matthew Bogyo Correspondence [email protected] In Brief Using a probe analog of the antimalarial natural product Sal A, Yoo et al. identify its targets as multiple essential serine hydrolases, including a homolog of human monoacylglycerol lipase. Because parasites were unable to generate robust in vitro resistance to Sal A, these enzymes represent promising targets for antimalarial drugs. Highlights d Semi-synthesis of an affinity analog of the antimalarial natural product Sal A d Identification of serine hydrolases as the primary targets of Sal A in P. falciparum d Sal A covalently binds to and inhibits a MAGL-like protein in P. falciparum d Parasites are unable to generate strong in vitro resistance to Sal A Yoo et al., 2020, Cell Chemical Biology 27, 143–157 February 20, 2020 ª 2020 Elsevier Ltd. https://doi.org/10.1016/j.chembiol.2020.01.001 Cell Chemical Biology Article The Antimalarial Natural Product Salinipostin A Identifies Essential a/b Serine Hydrolases Involved in Lipid Metabolism in P. falciparum Parasites Euna Yoo,1,11 Christopher J. Schulze,1,11 Barbara H. Stokes,2 Ouma Onguka,1 Tomas Yeo,2 Sachel Mok,2 Nina F. Gnadig,€ 2 Yani Zhou,3 Kenji Kurita,4 Ian T. Foe,1 Stephanie M. Terrell,1,5 Michael J. Boucher,6,7 Piotr Cieplak,8 Krittikorn Kumpornsin,9 Marcus C.S. Lee,9 Roger G. Linington,4 Jonathan Z. Long,1,5 Anne-Catrin Uhlemann,10 Eranthie Weerapana,3 David A. Fidock,2,10 and Matthew Bogyo1,6,12,* 1Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA 2Department of Microbiology and Immunology, Columbia University Irving Medical Center, New York, NY 10032, USA 3Department of Chemistry, Boston College, Chestnut Hill, MA 02467, USA 4Department of Chemistry, Simon Fraser University, Burnaby, BC V5A 1S6, Canada 5Stanford ChEM-H, Stanford University, Stanford, CA 94305, USA 6Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA 7Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, USA 8Infectious & Inflammatory Disease Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA 9Wellcome Sanger Institute, Hinxton, Cambridgeshire CB10 1SA, UK 10Division of Infectious Diseases, Department of Medicine, Columbia University Irving Medical Center, New York, NY 10032, USA 11These authors contributed equally 12Lead Contact *Correspondence: [email protected] https://doi.org/10.1016/j.chembiol.2020.01.001 SUMMARY laria treatment and control efforts rely on the administration of antimalarial drugs (White et al., 2014). In recent years, artemisi- Salinipostin A (Sal A) is a potent antiplasmodial ma- nin-based combination therapies (ACTs) have been the first- rine natural product with an undefined mechanism line antimalarial drugs and have contributed significantly to a of action. Using a Sal A-derived activity-based reduction in the global malaria burden (Tu, 2011; White, 2008). probe, we identify its targets in the Plasmodium fal- However, emerging resistance to artemisinin and the partner ciparum parasite. All of the identified proteins drugs used in ACTs threatens to reverse the gains that have contain a/b serine hydrolase domains and several been made against this disease (Dondorp et al., 2017; van der Pluijm et al., 2019). Therefore, there is a continuing need to iden- are essential for parasite growth. One of the essential tify novel targets and pathways against which efficacious thera- targets displays a high degree of homology to human peutics can be developed, both to widen the scope of treatment monoacylglycerol lipase (MAGL) and is able to pro- and to overcome existing mechanisms of antimalarial drug cess lipid esters including a MAGL acylglyceride resistance. substrate. This Sal A target is inhibited by the anti- Natural products have evolved to encompass a broad spec- obesity drug Orlistat, which disrupts lipid meta- trum of chemical and functional diversity and structural bolism. Resistance selections yielded parasites that complexity, which enables them to target diverse biological showed only minor reductions in sensitivity and macromolecules in a highly selective fashion. These features of that acquired mutations in a PRELI domain-contain- natural products not only suggest that they may hold much ing protein linked to drug resistance in Toxoplasma promise for the development of therapeutic agents but also gondii. This inability to evolve efficient resistance make them ideal starting points for the development of chemical probes (Harvey, 2000; Li and Vederas, 2009; Rodrigues et al., mechanisms combined with the non-essentiality of 2016; Thomford et al., 2018). Natural product-inspired chemical human homologs makes the serine hydrolases iden- probes have been instrumental in identifying therapeutic targets tified here promising antimalarial targets. and studying poorly understood biological processes and funda- mental biology, yet the targets of most natural products remain unknown (Bottcher et al., 2010; Carlson, 2010; Kreuzer et al., INTRODUCTION 2015; Taunton et al., 1996). Salinipostin A (Sal A) is a natural product produced by a Salinispora sp. bacterium. that was iso- Plasmodium falciparum (Pf) is a protozoan parasite that causes lated from a marine sediment. Sal A displays potent activity the most severe form of human malaria. Malaria threatens 40% against Pf parasites, with a half maximal effective concentration of the world’s population, resulting in an estimated 228 million (EC50) of 50 nM and a selectivity index of >1,000 over a variety of cases and nearly 405,000 deaths annually (WHO, 2018). mammalian cell lines (Schulze et al., 2015). In a previous effort to Because of a lack of effective vaccines against the parasite, ma- identify drug targets of Sal A, selection experiments were Cell Chemical Biology 27, 143–157, February 20, 2020 ª 2020 Elsevier Ltd. 143 attempted but failed to generate resistant parasites. These re- RESULTS sults suggested that Sal A might target multiple proteins or essential pathways for parasite development, and that its po- Salinipostin A Targets Human and Parasite tency is not easily compromised by single point mutations. The Depalmitoylases refractoriness of Sal A to parasite resistance mechanisms com- The Sal A molecule is made up of two parts, namely a reactive bined with its potent and selective activity, and lack of structural cyclic phosphate group that can act as an electrophile for serine similarity to other antimalarials make Sal A an interesting chem- and cysteine nucleophiles and a lipid tail that presumably mimics ical tool to identify druggable pathways in Pf parasites. a native cellular lipid. This saturated 15 carbon lipid tail aligns The a/b hydrolase superfamily is one of the largest groups of with the 16 carbons in palmitic acid such that the reactive phos- structurally related proteins that encompass a diverse range of phorus is located directly at the site of the carbonyl used to form catalytic activities. Members of this family are composed of a thiol ester between palmitate and a cysteine on a palmitoylated largely parallel b sheets flanked on their terminal ends by a he- protein (Couvertier et al., 2014)(Figure 1A). We therefore lices. The a/b hydrolase domain contains a conserved catalytic reasoned that Sal A might act as a substrate mimetic inhibitor dyad or triad formed by a nucleophilic serine residue and an of the serine hydrolases that perform depalmitoylation reactions acid-base residue pair. The archetypal a/b hydrolases are on proteins (Davda and Martin, 2014; Won et al., 2018). We con- esterases, which catalyze two-step nucleophilic substitution re- ducted a competition experiment in which whole Pf parasites actions to mediate hydrolysis of esters, but substrates for a/b were incubated with Sal A before labeling with the broad- hydrolases also include amides and thioesters (Carr and Ollis, spectrum serine hydrolase ABP fluorophosphonate-rhodamine 2009; Holmquist, 2000; Nardini and Dijkstra, 1999; Ollis et al., (FP-Rho) (Simon and Cravatt, 2010). Several hydrolases were in- 1992). In recent years, a/b serine hydrolases have attracted hibited upon treatment with 1 mM Sal A (Figure 1B). Although considerable attention for their critical roles in metabolic pro- palmitoylation is a ubiquitous and dynamic post-translational cesses in humans. These metabolic serine hydrolases have modification in Pf parasites (Corvi et al., 2012; Corvi and Turow- been implicated in neurotransmission, pain sensation, inflam- ski, 2019; Jones et al., 2012), there are no validated depalmitoy- mation, cancer, and bacterial infection (Bachovchin and Cra- lases in this parasite. Therefore, we tested Sal A in the related vatt, 2012; Long and Cravatt, 2011). The Pf genome encodes parasite pathogen, T. gondii. To determine whether Sal A inhibits about 40 putative members of the serine hydrolase superfamily the T. gondii depalmitoylase PPT1 (TgPPT1), we treated wild- based on annotated sequence homologies (Aurrecoechea type (WT) (Dku80) and TgPPT1 knockout (DPPT1) parasites et al., 2009). However, most of these have not been functionally (Child et al., 2013) with Sal A and labeled parasite extracts with characterized. Natural product analogs of cyclipostins and cy-