Bestatin-based chemical biology strategyreveals distinct roles for malaria M1- andM17-family aminopeptidasesMichael B. Harbuta, Geetha Velmourouganea, Seema Dalalb, Gilana Reissa, James C. Whisstockc, Ozlem Onderd,Dustin Brissond, Sheena McGowanc, Michael Klembab, and Doron C. Greenbauma,1

aDepartment of Pharmacology, University of Pennsylvania, 433 South University Avenue, 304G Lynch Laboratories, Philadelphia, PA 19104-6018;bDepartment of Biochemistry, Virginia Polytechnic Institute and State University, 306 Engel Hall, Blacksburg, VA 24061; cDepartment of Biochemistry andMolecular Biology and Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton,Victoria 3800, Australia; and dDepartment of Biology, University of Pennsylvania, 326 Leidy Laboratory, Philadelphia, PA 19104

Edited by Benjamin F. Cravatt, The Scripps Research Institute, La Jolla, CA, and accepted by the Editorial Board July 19, 2011 (received for review May 4, 2011)

Malaria causes worldwide morbidity and mortality, and whilechemotherapy remains an excellent means of malaria control,drug-resistant parasites necessitate the discovery of new antima-larials. Peptidases are a promising class of drug targets and per-form several important roles during the Plasmodium falciparumerythrocytic life cycle. Herein, we report a multidisciplinary effortcombining activity-based protein profiling, biochemical, and pepti-domic approaches to functionally analyze two genetically essentialP. falciparummetallo-aminopeptidases (MAPs), PfA-M1 and Pf-LAP.Through the synthesis of a suite of activity-based probes (ABPs)based on the general MAP inhibitor scaffold, bestatin, we gener-ated specific ABPs for these two enzymes. Specific inhibition ofPfA-M1 caused swelling of the parasite digestive vacuole andprevented proteolysis of hemoglobin (Hb)-derived oligopeptides,likely starving the parasite resulting in death. In contrast, inhibitionof Pf-LAP was lethal to parasites early in the life cycle, prior to theonset of Hb degradation suggesting that Pf-LAP has an essentialrole outside of Hb digestion.

protease ∣ chemical-genetics ∣ proteomics ∣ small molecule ∣ drug design

Malaria is a global disease causing at least 500 million clinicalcases and more than 1 million deaths each year (1). Whilesignificant efforts to control malaria via insect vector eliminationhave been pursued, chemotherapy remains the principal means ofmalaria control. Moreover, the emergence of drug resistance inPlasmodium falciparum, the causative agent of most malaria-as-sociated deaths, necessitates the discovery of novel antimalarials.

P. falciparum has a complex life cycle involving mosquito andhuman hosts. This life cycle involves both sexual and asexualstages of growth, wherein the human asexual erythrocytic phase(blood stage) is the cause of malaria-associated pathology. Theerythrocytic stage begins when extracellular parasites, initiallyreleased from the liver, invade red blood cells. Once establishedin a specialized vacuole inside the host erythrocyte, parasitesgrow from the initial ring stage to the trophozoite stage, whereinmuch of the host hemoglobin (Hb) is proteolyzed. Parasites thenreplicate during the schizont stage to produce expanded popula-tions of invasive merozoites that then rupture from the host cellapproximately 48 h postinvasion and go on to recapitulate the lifecycle (2). Peptidases are critical to parasite development through-out the life cycle and therefore are considered to be potentialantimalarial drug targets (3–5).

One proteolytic pathway that has received significant attentionis the multistep degradation of host Hb (6). While residing insidethe host red blood cell, malaria parasites endocytose and proteo-lytically digest host Hb in a specialized lysosomal-like digestivevacuole (DV). This process liberates amino acids that the parasitecan utilize for protein synthesis and general metabolism (7) andmay reduce pressure on the host cell produced by the growingparasite (8). Multiple endoproteases make initial cuts in full-

length Hb; however, genetic knockout studies of these enzymes,plasmepsins 1–4 and falcipain 2∕20, have revealed that all arenonessential and functionally redundant (9–11). While falcipain3 has not been shown to be dispensable, it is expressed later in theparasite lifecycle and may have roles beyond Hb degradation. Onthe other hand, several exopeptidases, some of which may haveroles in Hb degradation, are likely genetically essential (12, 13).Among these nonredundant enzymes are cysteine dipeptidyl ami-nopeptidase 1 (DPAP1) (13) and three metallo-aminopeptidases(MAPs): aminopeptidase N (PfA-M1), aminopeptidase P (PfAPP),and leucyl aminopeptidase, (Pf-LAP).

MAPs have been postulated to be important for the parasitelife cycle. Early studies suggested that MAP activities were absentfrom the DV lumen, leading to the proposal that Hb peptides areexported to the cytosol for further degradation by MAPs (14–16).However, more recent localization and biochemical evidence sug-gest that PfA-M1 and PfAPP are located inside the DV (17, 18),while Pf-LAP is located in the parasite cytosol and has been pro-posed to act on exported globin peptides (19). A cytosolic aspartylMAP, PfDAP, has been shown to hydrolyze substrates with anamino-terminal Asp or Glu residue (20), but its contribution toblood-stage peptide catabolism appears to be dispensable asgenetic disruption causes no overt phenotype (13). Importantly,none of these studies provides any biological evidence, mostimportantly in live parasites, for a direct role for MAPs during Hbpeptide catabolism.

Unfortunately in P. falciparum, genetic approaches to studyessential genes are limited and thus direct evidence for the bio-logical roles of these MAPs is still lacking (21). As a complemen-tary approach, we have developed a MAP-specific chemicalgenetics platform that utilizes activity-based protein profiling(ABPP) based on the natural product inhibitor bestatin to studythe roles of MAPs (22). ABPP is a chemical strategy that utilizesmechanism-based, tagged small molecule inhibitors to discovernew enzymes, profile their activity state in complex proteomes,and identify potential functions for these enzymes during a spe-cific biological process (23). ABPP has been utilized for a variety

Author contributions: M.B.H., M.K., and D.C.G. designed research; M.B.H., G.V., S.D., G.R.,and S.M. performed research; D.B. contributed new reagents/analytic tools; M.B.H., G.V.,S.D., J.C.W., O.O., S.M., M.K., and D.C.G. analyzed data; andM.B.H., M.K., and D.C.G. wrotethe paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. B.F.C. is a guest editor invited by the EditorialBoard.

Data deposition: The crystal coordinates have been deposited in the Protein Data Bank,www.pdb.org [PDB ID codes 3T8V (PfA-M1-BTA) and 3T8W (Pf-LAP-PNAP)].1To whom correspondence should be addressed. E-mail: dorong@mail.med.upenn.edu.

See Author Summary on page 13885.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1105601108/-/DCSupplemental.

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of enzyme classes, including serine hydrolases (24), peptidases(25), histone deacetylases (26), and kinases (27).

(-)-Bestatin is a natural product dipeptide analog of actinomy-cetes that potently inhibits multiple families of MAPs includingthe M1 and M17 families (28–31) (Fig. 1A). Importantly, bestatinhas been shown to inhibit growth of P. falciparum parasites inculture and in mouse models of malaria (32–34). In addition,a recent study has indirectly implicated aminopeptidases in he-moglobin catabolism showing that parasites treated with bestatinhad decreased levels of hemazoin formation, the detoxified bio-mineral byproduct of Hb digestion (34); likewise, isoleucine up-take was decreased in these bestatin treated parasites. However,the MAP(s) targeted by bestatin, which are responsible forthese processes, were not identified.

Herein, we report on a multidisciplinary effort combiningbestatin-based, small molecule ABPs with biochemical and pep-tidomic approaches to functionally analyze two essential amino-peptidases, PfA-M1 and Pf-LAP.

ResultsIdentification of Bestatin Targets in P. falciparum. Because of thepaucity of genetic tools for analysis of essential proteins inP. falciparum and a lack of highly specific inhibitors with whichto probe the individual roles of MAPs, we chose to study the func-tions of these essential parasite MAPs through the developmentand application of a MAP-specific ABPP platform. ABPP utilizestagged mechanism-based inhibitors, or activity-based probes(ABPs), to characterize families or individual active peptidaseswithin complex proteomes. ABPs typically possess two mainstructural components that contribute to their target specificity:(i) a mechanism-based inhibitor scaffold to covalently or nonco-valently target catalytic residues or the active site of peptidasesand (ii) a reporter tag, such as a fluorophore or biotin, for thevisualization, characterization of labeling events, and eventualaffinity purification of target proteins. The mechanism-based in-hibitor scaffold ensures that ABPs bind to the enzyme(s) in anactivity-dependent manner.

We decided to use the natural product, bestatin, as the scaffoldfor the development of ABPs for MAPs because it is a general

MAP inhibitor, kills Plasmodium parasites, and is syntheticallytractable using both solution and solid-phase chemistry (22, 35,36). To identify the target(s) of bestatin in P. falciparum, we uti-lized a previously published bestatin-based ABP, MH01 (Fig. 1B)(22). MH01 contains a biotin moiety to allow for monitoring ofprotein binding and affinity purification for target identification(22) and also utilizes a benzophenone for irreversible UV cross-linking to protein targets, as the inhibition of MAPs with bestatinis noncovalent. Asynchronous cultures of 3D7 parasites weretreated with saponin to lyse the erythrocyte and parasitophorousvacuole membranes and isolated whole parasites were harvestedby centrifugation. Crude parasite lysates, including both solubleand membrane proteins, were treated with 5 μM MH01, exposedto UV light, and analyzed by Western blot using streptavidin-HRP to detect biotinylated proteins. The observed labeling pat-tern consisted of four bands at approximately 100 kDa, 70 kDa,55 kDa, and 25 kDa (Fig. 1C). Western blot analysis with PfA-M1or Pf-LAP antibodies of parasite lysates labeled with MH01showed that all four labeled species could be accounted for withthese two antibodies: Three bands corresponded to different spe-cies of PfA-M1 and one to Pf-LAP (Fig. 1C). PfA-M1 is known tobe proteolytically processed from a 120 kDa proform to a115 kDa intermediate yielding the p96 and p68 forms (16), bothof which contain the catalytic domains and are labeled by MH01.The labeled p25 band is likely a secondary proteolytic breakdownproduct. We further confirmed that MH01 bound PfA-M1 andPf-LAP using individual parasite lines expressing YFP-taggedversions of these proteins (13). After incubation of each YFP-tagged transgenic line with MH01, immunoprecipitation usingstreptavidin and Western blotting for YFP revealed that bothPfA-M1-YFP and Pf-LAP-YFP were targeted by MH01 (Fig. 1 Dand E). Likewise, the reciprocal experiment involving the immu-noprecipitation of YFP and Western blotting for biotin revealedthat each YFP-tagged peptidase protein was biotinylated byMH01 (Fig. 1 D and E). Specificity of the interaction was con-firmed by pretreatment with unlabeled bestatin, which blockedlabeling. Attempted labelings using a parasite line expressingPfAPP-YFP, the other DV-localized MAP, confirmed that itwas not a target of MH01 (Fig. S1B in SI Appendix). These results

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Fig. 1. Identification of PfA-M1 and Pf-LAP as the targets of the antiparasitic MAP inhibitor bestatin. (A) Structure of bestatin. (B) Structure of MH01.(C) Identification of PfA-M1 and Pf-LAP as the parasite targets of bestatin. Parasite lysates were prepared by freeze-thaw lysis and subsequently labeled with5 μM MH01, UV crosslinked, and analyzed by Western blot for biotin. Four proteins were labeled by MH01. The same blot was stripped and reprobed se-quentially with antibodies for PfA-M1 and Pf-LAP; three MH01 labeled proteins were accounted for by PfA-M1 and fourth by Pf-LAP. (D) A parasite lineexpressing a YFP-tagged PfA-M1 protein was incubated with MH01 followed by immunoprecipitation for biotin (MH01) and Western blot analysis forYFP, which confirmed that PfA-M1 is targeted by MH01 (first panel). The reciprocal experiment involving the immunoprecipitation of PfA-M1-YFP (after in-cubation of parasites with MH01) using a YFP-specific antibody confirmed PfA-M1-YFP is biotinylated and thus labeled by MH01 (second panel). (E) Likewise,the same analysis was performed using a parasite line expressing a YFP-tagged Pf-LAP protein; incubation of these parasites with MH01, followed by im-munoprecipitation of biotin (MH01) and Western blot analysis for YFP confirmed that MH01 labels Pf-LAP. The reciprocal experiment involving immunopre-cipitation of Pf-LAP-YFP and analysis byWestern blot for biotin confirmed that Pf-LAP-YFP is biotinylated byMH01. Lastly, MH01 labeling of YFP-taggedMAPs,in both cases, is blocked by pretreatment with unbiotinylated bestatin as seen as a lack of labeling. Input lanes below each panel show Western blot analysisusing anti-YFP of total parasite lysate just before immunoprecipitation.

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indicate that PfA-M1 and Pf-LAP are likely the only targets ofbestatin in P. falciparum parasites. In addition, they highlightthe difficulty in understanding the mechanism of bestatin toxicity,which could be due to the inhibition of either PfA-M1 or Pf-LAPor both.

MAP ABP Library Design and in Vitro Analysis Against PfA-M1 and Pf-LAP. To investigate the individual functions of the two MAP tar-gets of bestatin and to gain insight into the mechanism of howbestatin kills parasites, we synthesized several libraries of besta-tin-based ABPs with the intention of generating specific ABPs forboth PfA-M1 and Pf-LAP. Bestatin has two side chains that canbe diversified, which are derived from the constituent α-hydroxy-β-amino acid and a natural α-amino acid (Fig. 1A). These sidechains straddle the active sites of MAPs where the α-hydroxy-β-amino acid side chain (termed P1) fits into the S1 pocket ofthe enzyme (N-terminal to the scissile bond) and the adjacentnatural amino acid side chain (P1′) interacts with the S1′ pocket(C-terminal to the scissile bond) (37, 38). In our initial library,the P1′ leucine residue in bestatin was replaced with a series ofnatural amino acids (except cysteine and methionine, which areprone to oxidation; norleucine was included as an isostere formethionine) and a limited number of nonnatural amino acids(Fig. 2A). Each library member was designed to incorporate abenzophenone to enable covalent attachment of the ABP to itstargets and a terminal alkyne at the C terminus, to allow for thelater addition of a variety of reporter tags using the bioorthogonalcopper(I)-catalyzed [3þ 2] azide/alkyne cycloaddition (“clickreaction”) (39–41). This library construction strategy increasesthe flexibility of downstream applications as each member hasa “taggable” arm; thus, they can be used to directly treat live cells(as well as cellular lysates or recombinant enzymes) for target

identification and activity profiling using a reporter tag, withoutany necessity for resynthesis or troubleshooting of tag placement.

We initially screened a P1′ diverse library against both recom-binant PfA-M1 and Pf-LAP via standard fluorescence proteaseactivity assays. Results of this experiment are presented as a heatmap based on percent inhibition of PfA-M1 and Pf-LAP for eachABP (Fig. 2A). The S1′ pocket of Pf-LAP tended to favor aro-matic side chains such as Phe, Tyr, and Naphthyl. One probe,Phe-Naphthyl (PNAP), showed strong specificity for Pf-LAP overPfA-M1. In addition, several side chains favored binding towardPfA-M1 over Pf-LAP, which tended to be either small (Ser, Ala)or positively charged (Lys, Arg). The probes Phe-Ala, Phe-Lys,and Phe-Arg showed moderate specificity for PfA-M1; however,we decided not to pursue further studies with the positivelycharged probes because of potential issues with cell permeability.Although the Phe-Ala ABP was somewhat specific for PfA-M1,we felt it was not yet suitably specific for further biological stu-dies; thus we investigated modifications to the P1 side chain toachieve higher specificity.

To increase specificity for PfA-M1, we synthesized a secondbestatin-based library that diversified the α-hydroxy-β-amino acidside chain (P1 position) using a fixed alanine at the P1′ position.Given structural information indicating that the S1 pocket of thePfA-M1 enzyme was hydrophobic (38), the P1 library was synthe-sized with a variety of natural and nonnatural hydrophobic P1side chains including: Ala, Leu, Diphenyl, Naphthyl, Biphenyl,and (Benzyl)Tyr (Fig. 2B). Again each library member had a click-able alkyne C-terminal to the benzophenone. This secondaryABP library was profiled against recombinant PfA-M1 and Pf-LAP and from the initial heat map analysis, the (Benzyl)Tyr-AlaABP (BTA) gave the highest specificity of PfA-M1 over Pf-LAP.

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Fig. 2. Bestatin-based ABP libraries reveal distinct chemotypes produced by ABPs with increased specificity for either PfA-M1 or Pf-LAP. (A) Representativestructure of the bestatin-based ABP scaffold showing the point of diversification at the P1′ position. This ABP library was screened against recombinant PfA-M1and Pf-LAP in single fixed concentrations. Results of the assay are displayed as a heat map: Red indicates higher potency; blue indicates lower potency. ThePhe-Naph ABP showed high specificity for Pf-LAP. (BES* indicates parental bestatin) (B) A library of ABPs was synthesized to identify a probe with increasedspecificity for PfA-M1. Representative structure of the bestatin-based ABP scaffold showing the point of diversification at the P1 position. All compounds hadan Ala at the P1′ position. Results of the assay are displayed as a heat map: Red indicates higher potency; blue indicates lower potency. The (Benzyl)Tyr-Ala ABPshowed high specificity for PfA-M1. (C) Synchronized parasites were treated with each compound at 1 μM and assayed for morphological changes by lightmicroscopy of Giemsa-stained blood smears through the erythrocytic lifecycle. Scale bar, 5 μm. ABPs more specific for PfA-M1 showed swelling of the DV, whileprobes more specific for Pf-LAP showed an early death chemotype.

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To determine the morphological effects of inhibition using theprobe libraries, parasite development was monitored throughoutthe entire lifecycle by Giemsa staining of thin blood smears(Fig. 2C). Three chemotypes (small molecule-induced morpholo-gical changes) were observed from this analysis: (i) no overt ef-fect, (ii) early parasite death at the ring/trophozoite transitionmarked by pyknotic bodies, and (iii) swelling of the DV with para-site death occurring at the trophozoite/schizont transition. Impor-tantly, the swollen DV as observed with these bestatin-basedABPs appeared translucent in Giemsa-stained smears, which dis-tinguishes them from the dark swollen DV containing undigestedHb seen after treating parasites with papain family cysteine pro-tease inhibitors such as E64 that target DV falcipains 2 and 3 (42).

Correlating the in vitro results with the live cell morphologicalscreening results revealed that ABPs that produced the swollenDV chemotype displayed a high degree of specificity for PfA-M1while ABPs more specific for Pf-LAP produced the early death/pyknosis chemotype. Compounds that failed to inhibit bothMAPs, such as Phe-Asp (P1′-Asp) showed no chemotypes andwere useful as negative control compounds (Fig. 2C). The con-trasting chemotypes displayed by parasites treated with the mostspecific compounds, BTA or PNAP, suggested that PfA-M1 andPf-LAP have essential yet distinct roles in the parasite.

Evaluation of BTA and PNAP Potency and Specificity.To quantitativelyassess the relative specificity of BTA and PNAP, inhibition con-stants against recombinant PfA-M1 and Pf-LAP were determined(Fig. 3A). All ABPs bound rapidly to PfA-M1; in contrast, bindingto Pf-LAP was slow, as has been reported for bestatin (see Ma-terials and Methods for more details). Analysis of BTA inhibitionrevealed that the substitution of the P1′ Leu for Ala shifted thespecificity moderately toward PfA-M1. With the substitution ofthe P1 Phe with (Benzyl)Tyr in BTA, the affinity of the ABP wasonly moderately changed for PfA-M1 (Fig. 3A and Fig. S2 in SIAppendix), while the inhibition constant for Pf-LAP radicallydropped nearly 100-fold, resulting in an ABP with an estimatedmicromolar K�i (an estimate was necessary due to insolubility athigh micromolar concentrations). Thus the overall change in theratio of inhibition constants of PfA-M1 over Pf-LAP from besta-tin to BTA was approximately 75-fold and the absolute specificitydifference for PfA-M1 over Pf-LAP was at least 15-fold, makingBTA a useful biological tool to study PfA-M1 function. Likewise,the substitution of a naphthyl group for the P1′ leucine of PNAPincreased the affinity of PNAP for Pf-LAP, resulting in a 170-foldchange in the specificity of PNAP for Pf-LAP relative to BTA andapproximately a 12-fold difference in absolute specificity, creat-ing a relatively specific inhibitor for Pf-LAP.

Although in vitro data suggested that BTA and PNAP werequite specific for PfA-M1 and Pf-LAP, respectively, these datado not rule out the possibility that the ABPs have other targetsin parasites. To confirm the specificity of each probe in parasites,we utilized the alkyne on each ABP to “click” on a fluorophore(BODIPY) tag, to identify target(s) of BTA and PNAP in crudeP. falciparum proteomes. The fluorescent ABPs were incubatedwith parasite lysates, UV-crosslinked and targets analyzed viain-gel fluorescent scanning. As predicted from the in vitro kineticassays, BTA exclusively labeled bands identical in migration ongels to those recognized by antibodies to PfA-M1 in this complexproteome, while PNAP was specific for a band that correlated inmigration with Pf-LAP (Fig. 3B).

To investigate the structural basis for the specificity of BTA forPfA-M1 we solved the X-ray cocrystal structure of PfA-M1 boundto the BTA probe. The cocrystal structure was solved to 1.8 Å,and electron density clearly resolved the BTA probe and linkerbut lacked any visible density for the “clickable” alkyne C-term-inal tag (Fig. 3C, Left; see Fig. S3A in SI Appendix for stereoviewand Table S1 in SI Appendix for statistics). The BTA ABP boundto the essential active site zinc ion via the hydroxyl and carbonyl

groups (O2/O3) and central nitrogen of the bestatin scaffold(Fig. S3A in SI Appendix). The S1 pocket showed a slight move-ment (approximately 1.2 Å between c-α atoms between the keyS1 residue, Glu572, of the two structures) to accommodate the(Benzyl)Tyr at the P1 position. The P1′ Ala moiety did not reachfar into the S1′ pocket of PfA-M1 as the remaining probe posi-tioned itself close to the S1 pocket (Fig. 3C, Left). We also mod-eled BTA into the X-ray crystal structure of Pf-LAP bound tobestatin (PDB ID code 3KR4). Superposition of BTA onto thebestatin core showed that the large (Benzyl)Tyr residue at theP1 position clashed with the narrow S1 pocket of the active sitein Pf-LAP (Fig. 3C, Right).

We also solved the cocrystal structure of Pf-LAP bound toPNAP (Fig. 3D, Right; see Fig. S4 in SI Appendix for hexamericstructure). The 2.0-ÅX-ray structure resolved the structural basisfor the PNAP specificity and potency for Pf-LAP. As expected thePNAPABP bound in a similar manner to the parent bestatin (43)

* (Pf-LAP)K i (PfA-M1)K iABP K iK i */

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Fig. 3. Biochemical and structural characterization of BTA and PNAP speci-ficity against PfA-M1 and Pf-LAP. (A) Kinetic evaluation of inhibition forPNAP and BTA on recombinant PfA-M1 and Pf-LAP reveal over 15-fold spe-cificity for PfA-M1 over Pf-LAP by BTA; PNAP showed greater than 10-foldspecificity for Pf-LAP over PfA-M1. K�i for BTA for Pf-LAP was estimateddue to solubility issues. (B) Activity-based protein profiling using “click”fluorescent versions of BTA or PNAP show that each probe specifically targetsPfA-M1 or Pf-LAP, respectively (as indicated by an asterisk). (The superscript“C” in lane 5 of both panels indicates pretreatment with 10x of the nonfluor-escent version of the respective ABP). (C) The electrostatic potential surfaceof the cocrystal of PfA-M1 with BTA (Left) and model of Pf-LAP with BTAbound in active site (Right). (D) The electrostatic potential surface of themodel of PfA-M1 with PNAP (Left) and surface of the cocrystal of Pf-LAPwith PNAP (Right). The zinc ion is shown as black sphere, the carbon atomsof inhibitors in green. Residues 755–1090 of PfA-M1 are excluded for clarityand a single monomer active site is shown for Pf-LAP. Surfaces were colorcoded according to electrostatic potential. Arrows show points at whicheither PNAP or BTA sterically clash with the enzyme.

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dominated by coordination of two Zn2þ ions of the active site(Fig. S3B in SI Appendix). The P1-Phe ring of PNAP fit neatlyinto the small hydrophobic S1 pocket of Pf-LAP, and the P1′naphthyl group also formed a series of hydrophobic interactionsin the S1′ cleft. The only alteration noted to accommodate theP1′ naphthyl group was the movement of Ser550 (approximately2.9 Å between c-α atoms of the Pf-LAP-PNAP structure versusthe Pf-LAP-bestatin structure). This residue is located in a loopthat lines the S1′ cleft, and the movement noted in the Pf-LAP-PNAP structure effectively flips the serine residue away from thenaphthyl group, dragging the loop and preventing any close con-tacts with the P1′ residue (Fig. S3B in SI Appendix). It was alsopossible to model PNAP into the X-ray crystal structure ofPf-LAP bound to bestatin (PDB ID code 3EBH). Superpositionof PNAP onto the bestatin core showed that the naphthyl sidechain at the P1′ position clashed with the wall of the S1′ pocketof the active site in Pf-LAP (Fig. 3D, Left).

Inhibition of PfA-M1 Kills Parasites via Disruption of Hb DigestionWhereas Inhibition of Pf-LAP Kills via a Distinct Mechanism.After con-firming the specificities of BTA and PNAP, we next wanted to

more fully characterize the effects of these ABPs on parasitesthrough their life cycle. To do this, synchronized parasites weretreated at the ring stage and followed by light microscopy evalua-tion of Giemsa-stained thin blood smears (Fig. 4A). We foundthat treating parasites with BTA at its IC99 caused a delay inthe life cycle and swelling of the DV at the trophozoite stagewith eventual parasite death around 60 hr posttreatment. As acomparison, parasites treated with E-64d, a cysteine protease in-hibitor that blocks DV falcipains (and initial endoproteolyticcleavage of Hb) had a similar delay and swollen DV (darklystained rather than translucent) but remained alive at the 60-hrtime point. In contrast, PNAP-treated parasites were arrested atthe transition to the trophozoite stage; therefore it appeared thatPNAP exerted its effect on parasites significantly earlier than thetime of major Hb digestion. PNAP treatment caused no promi-nent morphological features (other than death), thus complicat-ing hypothesis generation as to its mechanism of action. To ourknowledge the only other inhibitor that kills rings is artesunateand the other members of the artemisinin family (also shown inFig. 4A for comparative purposes); although the mechanism ofaction for artesunate may be different than PNAP it is intriguing

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Fig. 4. Inhibition of PfA-M1 kills parasites via disruption of Hb digestion whereas inhibition of Pf-LAP kills via a distinct mechanism. (A) Parasites were treatedwith BTA (10 μM), E-64d (10 μM), PNAP (3 μM), and artesunate (10 nM) at concentrations roughly equivalent to their IC99, and followed by Giemsa stainingand light microscopy throughout the lifecycle. Scale bar, 5 μm. (B) DV swelling was confirmed by the dose-dependent enlargement of the average DV area.Parasites expressing YFP-tagged plasmepsin II (PMII-YFP), which localizes to the DV, were treatedwith increasing concentrations of BTA (and 250 nM PNAP) andimaged by fluorescence microscopy. (C) DV swelling was determined to be saturable and quantified by treating the PMII-YFP parasites with increasing con-centrations of BTA (PNAP was also used at 250 nM) at midring stage and measuring fluorescent DV area 20 hrs later using a minimum of 10 parasites(þ∕ − standard deviation). (D) Treatment of parasites with BTA in media lacking exogenous amino acids, except for isoleucine, results in a more than twofolddecrease in the IC50 of BTA, while parasites treated with PNAP or artesunate show a nonsignificant difference. Shown are representative IC50 plots for eachcompound in both I-Media (lacking exogenous amino acids except for isoleucine), and AA-Media (containing all natural amino acids). The inlay bar graphsshow differences of the mean IC50 of three experiments carried out in triplicate (*P < 0.05, Student’s t test).

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that there could be overlap among these two structurally diver-gent inhibitor classes (44).

Inhibition of PfA-M1 by BTA treatment of parasites caused anovel swollen DV chemotype. To investigate this phenomenonmore closely we visualized the swelling of the DV in live parasitesusing a parasite line expressing YFP-tagged plasmepsin II thatserves as a DV marker (45). Transgenic parasites were treatedwith increasing concentrations of BTA and evaluated by fluores-cent microscopy (Fig. 4B). From these images, we estimated therelative average DV size (measuring 10 DVs) after each treat-ment. Parasites treated with as little as 250 nM BTA (the Ki ofBTA for PfA-M1) showed a statistically significant increase in DVsize relative to untreated parasites, with saturation of this swellingat 1 μM (Fig. 4 B and C). The observation that the degree ofDV swelling is dose-dependent and saturable is consistent withthe hypothesis that, within this concentration range, PfA-M1 islikely the sole target and performs a key function in the DV.In contrast, parasites treated with PNAP at a concentration overeightfold greater than its Ki against Pf-LAP did not show anysignificant DV swelling (Fig. 4B and Fig. S5 in SI Appendix).Although we saw no evidence of labeling of any other peptidasein parasite proteomes (Figs. 1 and 3), we wanted to further con-firm that the BTA-derived DV chemotype was not caused byinhibition of other proteases; thus we assayed for inhibition ofother DV peptidases: DPAP1, PfAPP and falcipain 2. No inhibi-tion of any enzyme was seen at a concentration up to 30 μMBTA, indicating that there is likely no cross-reactivity of BTAwith these enzymes in live parasites and that the DV swelling iscaused solely by inhibition of PfA-M1 (Fig. S6 in SI Appendix).

Because disruption in the endocytosis or subsequent catabolicbreakdown of Hb is thought to be lethal to parasites (9), we hy-pothesized that PfA-M1 inhibition by BTA leads to starvation ofthe parasite via blockage of proteolysis of Hb peptides. To testthis idea, we assayed whether parasites forced to rely only on Hbcatabolism are more sensitive to BTA than parasites cultured withexogenous amino acids, by assaying the potency of BTA on para-sites cultured in media lacking all amino acids except isoleucine(the only amino acid not present in Hb). Indeed, parasites were

sensitized by approximately 2.4-fold to inhibition by BTA in med-ia with only isoleucine (Fig. 4D). In contrast, parasites treatedwith PNAP or the antimalarial artesunate, which kills ring stageparasites prior to initiation of large-scale Hb degradation andthus acts as a negative control, showed statistically insignificantdifferences in sensitization to either compound in the isoleucinemedia. This evidence suggests a role for PfA-M1 in the Hb diges-tion pathway and also provides further evidence that the primaryrole of Pf-LAP is not within the Hb digestion pathway.

Initial proteolytic events are thought to be carried out by theredundant endopeptidases falcipains 2∕20∕3, plasmepsins I, II,IV, and HAP (46). Hb-derived oligopeptides are then brokendown by exopeptidases. Considering that PfA-M1 is an amino-peptidase, its likely role in the DV would occur after initial Hbproteolysis by the endopeptidases. To confirm this, we treatedsynchronous cultures of parasites during the trophozoite stage, inwhich the majority of Hb degradation takes place. Fig. S7 in SIAppendix shows that parasites treated with E-64d leads to an ac-cumulation of full-length Hb and causes the swelling of the DVwith undigested Hb (42). Conversely, parasites treated with BTAshowed no inhibition of proteolysis of full-length Hb but stillcaused the DV to swell. Treatment of parasites with PNAP wassimilar to DMSO.

Inhibition of PfA-M1 Blocks Proteolysis of Specific Hb-Derived Oligo-peptides. To obtain direct evidence for the role of PfA-M1 in theproteolysis of Hb-derived oligopeptides, we endeavored to findpeptide substrates for this enzyme. We used a mass spectrometry-based peptidomics approach to assay the relative abundance ofpeptides (

treated samples; however, several oligopeptides, from both the αand β chains of Hb, appeared to accumulate after treatment ofparasites with BTA (Fig. 5 A and B).

Further sequence analysis of the accumulated Hb-derived oli-gopeptides revealed that the majority were likely poor substratesfor DPAP1, the other essential aminopeptidase with broad sub-strate specificity found in the DV (47). We thus hypothesized thatPfA-M1 was necessary for proteolysis of these oligopeptides. Totest this idea, a highly enriched peptide that was identified fromthe peptidomics study of BTA-treated samples was resynthesizedand shown to be resistant to cleavage by DPAP1, yet efficientlycleaved by PfA-M1 (Fig. 5C). This data provides direct evidenceof a role for the genetically essential enzyme, PfA-M1, in thedigestion of small Hb-derived oligopeptides in P. falciparum.

DiscussionPeptidases likely have many essential functions in P. falciparum,yet the biological roles of the majority of putative proteases en-coded by the parasite genome remain to be characterized. Onereason for this rests in the difficulty in genetically manipulatingessential parasite genes. To circumvent this deficit in genetictools, a small molecule approach may be used to perturb and thusinvestigate essential protein functions in the parasite. Several is-sues arise from the use of small molecule probes, including (i)target identification, (ii) specificity, and (iii) permeability in livecells. To address some of these issues, we generated a library ofMAP-specific, “clickable” ABPs. Replacement of a bulky repor-ter tag with an alkyne group resulted in smaller, more versatileABPs that allowed for their use in live cells for phenotypic ana-lysis along with more tradition lysate-based ABPP.

Using this set of chemical tools, we first identified the targetsof bestatin in P. falciparum to be PfA-M1 and Pf-LAP; althoughthere are limitations to this ABPP approach—i.e., it is hard todetermine with absolute certainty that there are no low abun-dance targets—further diversification of the MAP inhibitor scaf-fold to generate structure-activity relationship (SAR) trends canstrengthen functional conclusions. Thus, through diversificationof the bestatin scaffold, we were able to create a suite of ABPswith increasing specificity for both PfA-M1 and Pf-LAP and usedthese ABPs to further understand the functions of these essentialenzymes.

The aggregate data using the BTA ABP strongly suggests thatPfA-M1 plays a key role in oliogopeptide proteolysis in the DV.The most prominent morphological feature of PfA-M1 inhibitionwas the novel swollen, translucent DV chemotype, which waslikely due to the accumulation of oligopeptides that created hy-perosmotic conditions. DV swelling also occurs after E-64d inhi-bition of DV falcipains; yet these parasites do not die until theyattempt to egress, which suggests that swelling alone is not lethalto parasites, and that there must be enough amino acids gener-ated (perhaps by plasmepsins) or obtained from the medium inthe presence of this inhibitor to allow life cycle progression. Wetherefore propose that the likely cause of death upon PfA-M1inhibition is a severe deprivation of the DV production of aminoacids in the parasite. (Although we cannot rule out that killingmay be due to indirect effects distinct to PfA-M1 inhibition, suchas the accumulation of small peptides in the DV, which couldpotentially be toxic.) To support the hypothesis that inhibitionof PfA-M1 disrupts Hb degradation, we demonstrated that theeffects of BTA were enhanced when parasites were forced to relysolely on Hb degradation. Why the amino acids present in com-plete media do not allow for the parasite to completely comple-ment for the genetic or pharmacological loss of PfA-M1 via theutilization of exogenous amino acids is an interesting question.This sensitivity may be explained by the fact that the parasite isparticularly dependent on leucine generated in the DV from Hb,which is thought to be exchanged for isoleucine via an antiportmechanism (48).

From our peptidomics experiments we showed that Hb oligo-peptides were substrates for PfA-M1 proteolysis. One issue withthis analysis is that small peptides were not found using our massspectrometry method, which was limited to the identificationof peptides greater than four amino acids in length. It is likelythat the concerted action of DV endo- and exoproteases alsoproduced smaller tri- and dipeptides, which are substrates forPfA-M1 in the DV. In support of this idea, we also attempted toidentify small peptide species using a Single Quad LC/MS (whichallows for profiling peptides between 200 and 400 kDa) that ac-cumulated in BTA-treated parasites (Fig. S8 in SI Appendix).These low molecular weight species all matched to predicteddipeptide molecular weights, and more than half were the mole-cular weight of dipeptides found in Hb. However, this methodprecluded the definitive identification of these molecules aspeptides (as opposed to metabolites) and their origin (i.e., Hb).However, we believe these data are suggestive that several dipep-tides, in addition to oligopeptides, are likely important substratesfor the PfA-M1 enzyme.

Our data using the PNAPABP for Pf-LAP indicated that thisenzyme has an important role quite early in the intraerythrocyticlife cycle rather than during the major period of Hb digestion.Formulating a testable hypothesis about a specific role for Pf-LAP is complicated by the fact that its inhibition did not yieldany overt morphological change in the parasite other than death.However, we suspect Pf-LAPmay have an essential housekeepingfunction in the cytosolic turnover of dipeptides (49) and perhapsacts in concert with the parasite proteasome, as has been shownfor other neutral cytosolic leucine aminopeptidases pathways(50). Like PNAP, lethal amounts of proteasome inhibitors exerttheir effect in the ring-trophozoite transition and parasites do notprogress into the later trophozoite stage (51). Our data does notcompletely rule out the possibility of a minor role for Pf-LAP inthe Hb degradation pathway via proteolysis of Hb-derived dipep-tides that have been transported from the DV into the cytoplasm.However, the fact that PNAP-treated parasites die prior to themajor period of Hb degradation suggests that the essential rolefor Pf-LAP is not within the Hb digestion pathway.

Our collective data suggest that these two MAPs are bothpotential antiparasitic drug targets. In fact, PNAP is, to ourknowledge, the most potent parasite MAP inhibitor with an IC50in the 200 nM range, which gives us hope that these types of in-hibitors could be further developed into more drug-like therapeu-tics. In addition, P. falciparum MAPs share little homology withtheir human counterparts; less than 35% in the case of the M1family proteases. It is therefore reasonable to suggest that potent,specific inhibitors of P. falciparum MAPs can be designed overhuman MAPs. In addition, information gleaned from our preli-minary SAR and crystallography efforts may provide a jumpingoff point for future medicinal chemistry efforts against both en-zymes. Our data here suggest that combination therapy involvingendopeptidase inhibitors, such as those for falcipains, and PfA-M1-specific inhibitors might provide an opportunity for a syner-gistic drug combination (52). Ultimately, this strategy may repre-sent a good way to reduce the chance of parasite resistance.

Materials and MethodsGeneral Methods. See SI Appendix for additional chemical and experimentalprotocols. A summary of the methods is given below.

Parasite Culture and IC50 Determination for Bestatin-Based ABPs. Briefly, 3D7parasites were cultured in RPMI 1640 (Invitrogen) supplemented with Albu-max II (Invitrogen). For synchronization, schizont stage parasites were mag-net purified using a SuperMACS™ II Cell Separation Unit (Miltenyi Biotech).For IC50 determinations, synchronized parasites were plated at 1% parasite-mia and 6% hematocrit in 96-well plates at a total volume of 50 μL. Serialdilutions of 2x concentration of the respective compound were added tothe wells to bring the total volume up to 100 μL and 0.5% parasitemiaand 3% hematocrit. Compounds were assayed for a 72 h period, after which

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2x Vybrant DyeCycle Green DNA (Invitrogen) in PBS was added for a finalconcentration of 10 μM and incubated at 37 °C for 30 min. DNA content,as an indicator of parasitemia, was analyzed on an Accuri C6 Flow Cytometerwith C-Sampler. IC50 curves were generated using GraphPad Prism (GraphPadSoftware).

Labeling of Parasite MAPs with Activity-Based Probes. For parasite labeling,mixed stage parasites were harvested and released from erythrocytes with1% saponin followed by centrifugation at 1;500 × g for 5 min and 3 washesin cold PBS. Parasite lysates were prepared by freeze-thaw in 50 mM Tris-HClpH 7.0, 50 mM NaCl, 10 μM ZnCl, and protease inhibitor cocktail (EDTA-free)(Roche) and extracts were clarified by centrifugation at 1;100 × g for 10 minat 4 °C. Labeling was performed with indicated concentrations of the ABP for1 hr at 37 °C followed by UV crosslinking (365 nm) for 1 hr on ice. Competitionof labeling was carried out by preincubating lysates for 1 hr at 37 °C. Forimmunoprecipitation, lysates were passed through 7 K MWCO desalting col-umns (Pierce) after UV crosslinking then incubated overnight with streptavi-din Ultralink Resin (Pierce). Proteins were visualized by standard Westernblotting and VECTASTAIN ABC kit (Vector Labs) or rabbit anti-GFP (ab6556,Abcam). For fluorescent probes, labeled proteins were visualized in-gel usinga Typhoon flatbed scanner (GE Healthcare).

Synthesis of Bestatin-Based ABP Libraries. A detailed description of the synth-esis and characterization of these compounds may be found in SI Appendix.

Recombinant Proteins. Details of the expression in Escherichia coli and puri-fication of recombinant PfA-M1 (residues 192 to 1,085) will be described se-parately (53). Pf-LAP lacking the N-terminal Asn-rich region (residues 79–605)was expressed with a C-terminal hexahistidine tag in E. coli and purified aspreviously described (43).The estimated molecular mass of the purified spe-cies from size exclusion chromatography (343 kDa) was in good agreementwith the predicted mass for the hexameric enzyme (357 kDa). The purifica-tion of recombinant DPAP1 has been published (47).

X-ray Crystallography. PfA-M1 and Pf_LAP enzymes were purified and crystal-lized as previously described (38). Crystals of the PfA-M1-BTA complex wereobtained by cocrystallization of BTAwith PfA-M1 inmother liquor containing1 mM ligand. Crystals of the Pf-LAP-PNAP complex were obtained by cocrys-tallisation of PNAP with Pf-LAP in mother liquor containing 1 mM ligand.Prior to data collection, Pf-LAP-PNAP cocrystals were soaked in mother liquorcontaining 1 mM ligand and 1 mM ZnSO4. Data were collected at 100 K usingsynchrotron radiation at the Australian synchrotron micro crystallographybeamline 3ID1. A summary of statistics is provided in Table S1 in SIAppendix. Raw data and images will be available from TARDIS (54).

The inhibitor complex was initially solved and refined against the un-bound PfA-M1 and Pf-LAP structure (protein atoms only) as describedpreviously (38) and clearly showed unbiased features in the active site forboth structures. After placement of inhibitors into unbiased density, CNScomposite omit maps were calculated using all atoms. Fig. S3 in SI Appendixshows inhibitor density contoured at 1.0σ. Fig. S3 in SI Appendix was gener-ated using MacPymol and uses a 2.0 carve value around each inhibitor and

zinc ion for clarity. Superposition of BTA into the Pf-LAP active site was per-formed using the X-ray crystal structure of Pf-LAP-bestatin (3KR4) where thebestatin scaffold was used to superpose BTA.

Anti-PfA-M1 Sera. Details of the production of rabbit antisera against recom-binant PfA-M1 have been previously described (53).

Screening of P1 and P1′ ABP Libraries. Screens of relative ABP potencies wereconducted at single fixed ABP concentrations for the P1 (PfA-M1- 250 nM;Pf-LAP- 750 nM), P1′ natural (PfA-M1- 1 μM; Pf-LAP- 250 nM) P1′ nonnatural(0.37 μM for both enzymes) libraries. PfA-M1 assays contained 50 mM HEPESpH 7.5, 100 mM NaCl, 25 mM leucyl-7-amido-4-methylcoumarin (Leu-AMC)and 0.1% Triton X-100; Pf-LAP assays contained 50 mM HEPES pH 7.5,100 μM ZnCl2, 50–250 μM Leu-AMC and 0.1% Triton X-100. For Pf-LAP, stea-dy-state rates were approximated by linear fits to the progress curves after a1 hr equilibration period in the presence of substrate and inhibitor.

Determination of Ki and K�i Values. Ki values for inhibition of PfA-M1 weredetermined by Dixon plots and a detailed protocol for determining Pf-LAPK1� values may be found in SI Appendix.

Mass Spectrometry-Based Peptide Profiling. Briefly, parasites were treatedwith 2 μM BTA or DMSO at the midring stage. Parasites were treated for24 hr at which point they were harvested by saponin treatment, centrifuged,and stored at −80 °C in the presence of protease inhibitors. To isolate pep-tides, parasite samples were boiled in water for 10 min and then centrifugedfor 10 min at 18;000 × g. The supernatant was saved, and the pellet was re-suspended in 0.25% acetic acid and disrupted by freeze-thaw and microso-nicated. All fractions were combined and centrifuged at 20;000 × g at 4 °Cfor 20 min. The supernatant was passed through a 10 kDa molecular weightcutoff filter (Millipore).

The retention times and m∕z values of the peptides identified were usedto map corresponding peptide peaks in the chromatograms generated fromnano-HPLC–ESI-MS (LCQ-DecaXP Plus). These peptide peaks were manuallyaligned and then for semiquantitative assessment of the abundances of in-dividual peptides, the total peak areas were determined using the Bioworksalgorithm PepQuan (the Area/Height Calculation) with parameters set toarea, mass tolerance of 1.5, minimum threshold of 5,000, five smoothingpoints, and including all proteins. The alignment was based on retentiontimes, m∕z values, and patterns of peaks in close proximity.

ACKNOWLEDGMENTS. We thank the Australian Synchrotron for beamtimeand Tom Caradoc-Davis in particular for technical assistance. We thank JohnDalton, PhD, McGill University, for the Pf-LAP antisera. Authors acknowledgethe support by Penn Genome Frontiers Institute (D.C.G), Ritter Foundation(D.C.G), R01-AI-076342-01 (D.B.), R01-AI-077638 (M.K.), NIH5T32AI007532(M.B.H.), and 1R56-AI-081770-01A2 (D.C.G.). S.M. is an Australian ResearchCouncil (ARC) Future Fellow, J.C.W. is an ARC Federation Fellow and a Na-tional Health and Medical Research Council (NHMRC) Principal Research Fel-low. We thank the NHMRC and the ARC for funding support.

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