Bestatin-based chemical biology strategy reveals distinct roles for malaria M1- and M17-family

Michael 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 and Molecular 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 while length Hb; however, genetic knockout studies of these , chemotherapy remains an excellent means of malaria control, plasmepsins 1–4 and falcipain 2∕20, have revealed that all are drug-resistant parasites necessitate the discovery of new antima- nonessential and functionally redundant (9–11). While falcipain larials. Peptidases are a promising class of drug targets and per- 3 has not been shown to be dispensable, it is expressed later in the form several important roles during the Plasmodium falciparum parasite lifecycle and may have roles beyond Hb degradation. On erythrocytic life cycle. Herein, we report a multidisciplinary effort the other hand, several , some of which may have combining activity-based protein profiling, biochemical, and pepti- roles in Hb degradation, are likely genetically essential (12, 13). domic approaches to functionally analyze two genetically essential Among these nonredundant enzymes are cysteine dipeptidyl ami- P. falciparum metallo-aminopeptidases (MAPs), PfA-M1 and Pf-LAP. nopeptidase 1 (DPAP1) (13) and three metallo-aminopeptidases Through the synthesis of a suite of activity-based probes (ABPs) (MAPs): N (PfA-M1), aminopeptidase P (PfAPP), based on the general MAP inhibitor scaffold, bestatin, we gener- and , (Pf-LAP). ated specific ABPs for these two enzymes. Specific inhibition of MAPs have been postulated to be important for the parasite PfA-M1 caused swelling of the parasite digestive vacuole and life cycle. Early studies suggested that MAP activities were absent prevented proteolysis of hemoglobin (Hb)-derived oligopeptides, from the DV lumen, leading to the proposal that Hb peptides are likely starving the parasite resulting in death. In contrast, inhibition exported to the cytosol for further degradation by MAPs (14–16). of Pf-LAP was lethal to parasites early in the life cycle, prior to the onset of Hb degradation suggesting that Pf-LAP has an essential However, more recent localization and biochemical evidence sug- role outside of Hb digestion. 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- ∣ chemical-genetics ∣ proteomics ∣ small molecule ∣ drug design posed to act on exported globin peptides (19). A cytosolic aspartyl MAP, PfDAP, has been shown to hydrolyze substrates with an alaria is a global disease causing at least 500 million clinical amino-terminal Asp or Glu residue (20), but its contribution to Mcases and more than 1 million deaths each year (1). While blood-stage peptide catabolism appears to be dispensable as significant efforts to control malaria via insect vector elimination genetic disruption causes no overt phenotype (13). Importantly, have been pursued, chemotherapy remains the principal means of none of these studies provides any biological evidence, most malaria control. Moreover, the emergence of drug resistance in importantly in live parasites, for a direct role for MAPs during Hb Plasmodium falciparum, the causative agent of most malaria-as- peptide catabolism. sociated deaths, necessitates the discovery of novel antimalarials. Unfortunately in P. falciparum, genetic approaches to study P. falciparum has a complex life cycle involving mosquito and essential genes are limited and thus direct evidence for the bio- human hosts. This life cycle involves both sexual and asexual logical roles of these MAPs is still lacking (21). As a complemen- stages of growth, wherein the human asexual erythrocytic phase tary approach, we have developed a MAP-specific chemical (blood stage) is the cause of malaria-associated pathology. The genetics platform that utilizes activity-based protein profiling erythrocytic stage begins when extracellular parasites, initially (ABPP) based on the natural product inhibitor bestatin to study released from the liver, invade red blood cells. Once established the roles of MAPs (22). ABPP is a chemical strategy that utilizes in a specialized vacuole inside the host erythrocyte, parasites mechanism-based, tagged small molecule inhibitors to discover grow from the initial ring stage to the trophozoite stage, wherein new enzymes, profile their activity state in complex proteomes, much of the host hemoglobin (Hb) is proteolyzed. Parasites then and identify potential functions for these enzymes during a spe- replicate during the schizont stage to produce expanded popula- cific biological process (23). ABPP has been utilized for a variety tions of invasive merozoites that then rupture from the host cell approximately 48 h postinvasion and go on to recapitulate the life cycle (2). Peptidases are critical to parasite development through- Author contributions: M.B.H., M.K., and D.C.G. designed research; M.B.H., G.V., S.D., G.R., out the life cycle and therefore are considered to be potential 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; and M.B.H., M.K., and D.C.G. wrote antimalarial drug targets (3–5). the paper. One proteolytic pathway that has received significant attention The authors declare no conflict of interest. is the multistep degradation of host Hb (6). While residing inside This article is a PNAS Direct Submission. B.F.C. is a guest editor invited by the Editorial the host red blood cell, malaria parasites endocytose and proteo- Board. lytically digest host Hb in a specialized lysosomal-like digestive Data deposition: The crystal coordinates have been deposited in the Protein Data Bank, vacuole (DV). This process liberates amino acids that the parasite www.pdb.org [PDB ID codes 3T8V (PfA-M1-BTA) and 3T8W (Pf-LAP-PNAP)]. can utilize for protein synthesis and general metabolism (7) and 1To whom correspondence should be addressed. E-mail: [email protected]. may reduce pressure on the host cell produced by the growing This article contains supporting information online at www.pnas.org/lookup/suppl/ parasite (8). Multiple endoproteases make initial cuts in full- doi:10.1073/pnas.1105601108/-/DCSupplemental.

526 of -1 ∣ PNAS Early Edition www.pnas.org/cgi/doi/10.1073/pnas.1105601108 Downloaded by guest on September 26, 2021 of classes, including serine (24), peptidases MAP inhibitor, kills Plasmodium parasites, and is synthetically PNAS PLUS (25), histone deacetylases (26), and kinases (27). tractable using both solution and solid-phase chemistry (22, 35, (-)-Bestatin is a natural product dipeptide analog of actinomy- 36). To identify the target(s) of bestatin in P. falciparum, we uti- cetes that potently inhibits multiple families of MAPs including lized a previously published bestatin-based ABP, MH01 (Fig. 1B) the M1 and M17 families (28–31) (Fig. 1A). Importantly, bestatin (22). MH01 contains a biotin moiety to allow for monitoring of has been shown to inhibit growth of P. falciparum parasites in protein binding and affinity purification for target identification culture and in mouse models of malaria (32–34). In addition, (22) and also utilizes a benzophenone for irreversible UV cross- a recent study has indirectly implicated aminopeptidases in he- linking to protein targets, as the inhibition of MAPs with bestatin moglobin catabolism showing that parasites treated with bestatin is noncovalent. Asynchronous cultures of 3D7 parasites were had decreased levels of hemazoin formation, the detoxified bio- treated with saponin to lyse the erythrocyte and parasitophorous mineral byproduct of Hb digestion (34); likewise, isoleucine up- vacuole membranes and isolated whole parasites were harvested take was decreased in these bestatin treated parasites. However, by centrifugation. Crude parasite lysates, including both soluble the MAP(s) targeted by bestatin, which are responsible for and membrane proteins, were treated with 5 μM MH01, exposed these processes, were not identified. to UV light, and analyzed by Western blot using streptavidin- Herein, we report on a multidisciplinary effort combining HRP to detect biotinylated proteins. The observed labeling pat- bestatin-based, small molecule ABPs with biochemical and pep- tern consisted of four bands at approximately 100 kDa, 70 kDa, tidomic approaches to functionally analyze two essential amino- 55 kDa, and 25 kDa (Fig. 1C). Western blot analysis with PfA-M1 peptidases, PfA-M1 and Pf-LAP. or Pf-LAP antibodies of parasite lysates labeled with MH01 showed that all four labeled species could be accounted for with Results these two antibodies: Three bands corresponded to different spe- Identification of Bestatin Targets in P. falciparum. Because of the cies of PfA-M1 and one to Pf-LAP (Fig. 1C). PfA-M1 is known to paucity of genetic tools for analysis of essential proteins in be proteolytically processed from a 120 kDa proform to a P. falciparum and a lack of highly specific inhibitors with which 115 kDa intermediate yielding the p96 and p68 forms (16), both to probe the individual roles of MAPs, we chose to study the func- of which contain the catalytic domains and are labeled by MH01. tions of these essential parasite MAPs through the development The labeled p25 band is likely a secondary proteolytic breakdown and application of a MAP-specific ABPP platform. ABPP utilizes product. We further confirmed that MH01 bound PfA-M1 and tagged mechanism-based inhibitors, or activity-based probes Pf-LAP using individual parasite lines expressing YFP-tagged

(ABPs), to characterize families or individual active peptidases versions of these proteins (13). After incubation of each YFP- BIOCHEMISTRY within complex proteomes. ABPs typically possess two main tagged transgenic line with MH01, immunoprecipitation using structural components that contribute to their target specificity: streptavidin and Western blotting for YFP revealed that both (i) a mechanism-based inhibitor scaffold to covalently or nonco- PfA-M1-YFP and Pf-LAP-YFP were targeted by MH01 (Fig. 1 D valently target catalytic residues or the of peptidases and E). Likewise, the reciprocal experiment involving the immu- and (ii) a reporter tag, such as a fluorophore or biotin, for the noprecipitation of YFP and Western blotting for biotin revealed visualization, characterization of labeling events, and eventual that each YFP-tagged peptidase protein was biotinylated by affinity purification of target proteins. The mechanism-based in- MH01 (Fig. 1 D and E). Specificity of the interaction was con- hibitor scaffold ensures that ABPs bind to the enzyme(s) in an firmed by pretreatment with unlabeled bestatin, which blocked activity-dependent manner. labeling. Attempted labelings using a parasite line expressing We decided to use the natural product, bestatin, as the scaffold PfAPP-YFP, the other DV-localized MAP, confirmed that it for the development of ABPs for MAPs because it is a general was not a target of MH01 (Fig. S1B in SI Appendix). These results A B

O O O H2N N CO2H H H H H H H N O O N N N N OH H2N N O O Biotin H OH O OOO N OH Bestatin MH01 2

C DE 3D7 mix stages kDa PfA-M1-YFP PfA-M1-YFP Pf-LAP-YFP Pf-LAP-YFP 100 + + bestatin + + bestatin strip and 75 reprobe blot 50 37 input input 25 IP: biotin IP: YFP IP: biotin IP: YFP WB: YFP WB: biotin WB: YFP WB: biotin anti-biotin anti-PfA-M1 anti-Pf-LAP

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 with 5 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 line expressing a YFP-tagged PfA-M1 protein was incubated with MH01 followed by immunoprecipitation for biotin (MH01) and Western blot analysis for YFP, 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 by Western blot for biotin confirmed that Pf-LAP-YFP is biotinylated by MH01. Lastly, MH01 labeling of YFP-tagged MAPs, 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 analysis using anti-YFP of total parasite lysate just before immunoprecipitation.

Harbut et al. PNAS Early Edition ∣ 527 of 534 Downloaded by guest on September 26, 2021 indicate that PfA-M1 and Pf-LAP are likely the only targets of identification and activity profiling using a reporter tag, without bestatin in P. falciparum parasites. In addition, they highlight any necessity for resynthesis or troubleshooting of tag placement. the difficulty in understanding the mechanism of bestatin toxicity, We initially screened a P1′ diverse library against both recom- which could be due to the inhibition of either PfA-M1 or Pf-LAP binant PfA-M1 and Pf-LAP via standard fluorescence protease or both. activity assays. Results of this experiment are presented as a heat map based on percent inhibition of PfA-M1 and Pf-LAP for each MAP ABP Library Design and in Vitro Analysis Against PfA-M1 and Pf- ABP (Fig. 2A). The S1′ pocket of Pf-LAP tended to favor aro- LAP. To investigate the individual functions of the two MAP tar- matic side chains such as Phe, Tyr, and Naphthyl. One probe, gets of bestatin and to gain insight into the mechanism of how Phe-Naphthyl (PNAP), showed strong specificity for Pf-LAP over bestatin kills parasites, we synthesized several libraries of besta- PfA-M1. In addition, several side chains favored binding toward tin-based ABPs with the intention of generating specific ABPs for PfA-M1 over Pf-LAP, which tended to be either small (Ser, Ala) both PfA-M1 and Pf-LAP. Bestatin has two side chains that can or positively charged (Lys, Arg). The probes Phe-Ala, Phe-Lys, be diversified, which are derived from the constituent α-hydroxy- β α and Phe-Arg showed moderate specificity for PfA-M1; however, -amino acid and a natural -amino acid (Fig. 1A). These side we decided not to pursue further studies with the positively chains straddle the active sites of MAPs where the α-hydroxy- charged probes because of potential issues with cell permeability. β-amino acid side chain (termed P1) fits into the S1 pocket of Although the Phe-Ala ABP was somewhat specific for PfA-M1, the enzyme (N-terminal to the scissile bond) and the adjacent we felt it was not yet suitably specific for further biological stu- natural amino acid side chain (P1′) interacts with the S1′ pocket (C-terminal to the scissile bond) (37, 38). In our initial library, dies; thus we investigated modifications to the P1 side chain to the P1′ leucine residue in bestatin was replaced with a series of achieve higher specificity. To increase specificity for PfA-M1, we synthesized a second natural amino acids (except cysteine and methionine, which are α β prone to oxidation; norleucine was included as an isostere for bestatin-based library that diversified the -hydroxy- -amino acid ′ methionine) and a limited number of nonnatural amino acids side chain (P1 position) using a fixed alanine at the P1 position. (Fig. 2A). Each library member was designed to incorporate a Given structural information indicating that the S1 pocket of the benzophenone to enable covalent attachment of the ABP to its PfA-M1 enzyme was hydrophobic (38), the P1 library was synthe- targets and a terminal alkyne at the C terminus, to allow for the sized with a variety of natural and nonnatural hydrophobic P1 later addition of a variety of reporter tags using the bioorthogonal side chains including: Ala, Leu, Diphenyl, Naphthyl, Biphenyl, copper(I)-catalyzed [3 þ 2] azide/alkyne cycloaddition (“click and (Benzyl)Tyr (Fig. 2B). Again each library member had a click- reaction”) (39–41). This library construction strategy increases able alkyne C-terminal to the benzophenone. This secondary the flexibility of downstream applications as each member has ABP library was profiled against recombinant PfA-M1 and Pf- a “taggable” arm; thus, they can be used to directly treat live cells LAP and from the initial heat map analysis, the (Benzyl)Tyr-Ala (as well as cellular lysates or recombinant enzymes) for target ABP (BTA) gave the highest specificity of PfA-M1 over Pf-LAP.

A

O

O P1 O H H H N O N N H2N N O N H H OH O O O H2N O P1’ ABP - Phe Naph Leu Pro Tyr Asn Ile Nle Asp Glu Gly Lys(Z) His Gln Trp BzlTyr BiP Val Thr Ser Ala Arg Lys BES*

Pf-LAP

PfA-M1

01% Inhibition 00

BCPf-LAP specificity PfA-M1 specificity

O P1 O O H H H N O N N H2N N O N H H OH O O O H2N O Phe-Naph Phe-Leu Phe-Ala BzlTyr-Ala P1 - P1’ P1 - P1’ P1 - P1’ P1 - P1’ P1-ABP - Naph Ala BiP BzlTyr DiP Leu (PNAP) (BTA)

Pf-LAP Pos. Control Neg. Control ABP

PfA-M1

0 % Inhibition 100 BES* Phe-Asp P1 - P1’

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

528 of 534 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1105601108 Harbut et al. Downloaded by guest on September 26, 2021 To determine the morphological effects of inhibition using the B PNAS PLUS A BTA-BODIPY (µM) PNAP-BODIPY (nM) probe libraries, parasite development was monitored throughout C ABP K (PfA-M1) K * (Pf-LAP) K * 0 0.1 0.2 0.4 0.4 010204040C the entire lifecycle by Giemsa staining of thin blood smears i i i / K i kDa (Fig. 2C). Three chemotypes (small molecule-induced morpholo- BTA260 nM ~4000 nM 15 100 gical changes) were observed from this analysis: (i) no overt ef- PNAP 330 nM 29 nM 0.088 fect, (ii) early parasite death at the ring/trophozoite transition 75

marked by pyknotic bodies, and (iii) swelling of the DV with para- O 50 site death occurring at the trophozoite/schizont transition. Impor- O O

H N N H2N N 2 H H OOH tantly, the swollen DV as observed with these bestatin-based OH O 37 PNAP BTA ABPs appeared translucent in Giemsa-stained smears, which dis- 25 tinguishes them from the dark swollen DV containing undigested O O H H H N O N N O N H Hb seen after treating parasites with papain family cysteine pro- O O = PfA-M1 = Pf-LAP O tease inhibitors such as E64 that target DV falcipains 2 and 3 (42). H2N Correlating the in vitro results with the live cell morphological screening results revealed that ABPs that produced the swollen C PfA-M1 Pf-LAP DV chemotype displayed a high degree of specificity for PfA-M1 while ABPs more specific for Pf-LAP produced the early death/ pyknosis chemotype. Compounds that failed to inhibit both MAPs, such as Phe-Asp (P1′-Asp) showed no chemotypes and were useful as negative control compounds (Fig. 2C). The con- trasting chemotypes displayed by parasites treated with the most specific compounds, BTA or PNAP, suggested that PfA-M1 and Pf-LAP have essential yet distinct roles in the parasite. BTA co-crystal BTA model

Evaluation of BTA and PNAP Potency and Specificity. To quantitatively D PfA-M1 Pf-LAP assess 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, binding BIOCHEMISTRY to Pf-LAP was slow, as has been reported for bestatin (see Ma- terials and Methods for more details). Analysis of BTA inhibition revealed that the substitution of the P1′ Leu for Ala shifted the specificity moderately toward PfA-M1. With the substitution of the P1 Phe with (Benzyl)Tyr in BTA, the affinity of the ABP was PNAP model PNAP co-crystal only moderately changed for PfA-M1 (Fig. 3A and Fig. S2 in SI Appendix), while the inhibition constant for Pf-LAP radically Fig. 3. Biochemical and structural characterization of BTA and PNAP speci- dropped nearly 100-fold, resulting in an ABP with an estimated ficity against PfA-M1 and Pf-LAP. (A) Kinetic evaluation of inhibition for micromolar K (an estimate was necessary due to insolubility at PNAP and BTA on recombinant PfA-M1 and Pf-LAP reveal over 15-fold spe- i cificity for PfA-M1 over Pf-LAP by BTA; PNAP showed greater than 10-fold high micromolar concentrations). Thus the overall change in the specificity for Pf-LAP over PfA-M1. Ki for BTA for Pf-LAP was estimated ratio of inhibition constants of PfA-M1 over Pf-LAP from besta- due to solubility issues. (B) Activity-based protein profiling using “click” tin to BTA was approximately 75-fold and the absolute specificity fluorescent versions of BTA or PNAP show that each probe specifically targets difference for PfA-M1 over Pf-LAP was at least 15-fold, making PfA-M1 or Pf-LAP, respectively (as indicated by an asterisk). (The superscript BTA a useful biological tool to study PfA-M1 function. Likewise, “C” in lane 5 of both panels indicates pretreatment with 10x of the nonfluor- the substitution of a naphthyl group for the P1′ leucine of PNAP escent version of the respective ABP). (C) The electrostatic potential surface increased the affinity of PNAP for Pf-LAP, resulting in a 170-fold of the cocrystal of PfA-M1 with BTA (Left) and model of Pf-LAP with BTA change in the specificity of PNAP for Pf-LAP relative to BTA and bound in active site (Right). (D) The electrostatic potential surface of the model of PfA-M1 with PNAP (Left) and surface of the cocrystal of Pf-LAP approximately a 12-fold difference in absolute specificity, creat- with PNAP (Right). The zinc ion is shown as black sphere, the carbon atoms ing a relatively specific inhibitor for Pf-LAP. of inhibitors in green. Residues 755–1090 of PfA-M1 are excluded for clarity Although in vitro data suggested that BTA and PNAP were and a single monomer active site is shown for Pf-LAP. Surfaces were color quite specific for PfA-M1 and Pf-LAP, respectively, these data coded according to electrostatic potential. Arrows show points at which do not rule out the possibility that the ABPs have other targets either PNAP or BTA sterically clash with the enzyme. in parasites. To confirm the specificity of each probe in parasites, we utilized the alkyne on each ABP to “click” on a fluorophore groups (O2/O3) and central nitrogen of the bestatin scaffold (BODIPY) tag, to identify target(s) of BTA and PNAP in crude (Fig. S3A in SI Appendix). The S1 pocket showed a slight move- P. falciparum proteomes. The fluorescent ABPs were incubated ment (approximately 1.2 Å between c-α atoms between the key with parasite lysates, UV-crosslinked and targets analyzed via S1 residue, Glu572, of the two structures) to accommodate the in-gel fluorescent scanning. As predicted from the in vitro kinetic (Benzyl)Tyr at the P1 position. The P1′ Ala moiety did not reach assays, BTA exclusively labeled bands identical in migration on far into the S1′ pocket of PfA-M1 as the remaining probe posi- gels to those recognized by antibodies to PfA-M1 in this complex tioned itself close to the S1 pocket (Fig. 3C, Left). We also mod- proteome, while PNAP was specific for a band that correlated in eled BTA into the X-ray crystal structure of Pf-LAP bound to migration with Pf-LAP (Fig. 3B). bestatin (PDB ID code 3KR4). Superposition of BTA onto the To investigate the structural basis for the specificity of BTA for bestatin core showed that the large (Benzyl)Tyr residue at the PfA-M1 we solved the X-ray cocrystal structure of PfA-M1 bound P1 position clashed with the narrow S1 pocket of the active site to the BTA probe. The cocrystal structure was solved to 1.8 Å, in Pf-LAP (Fig. 3C, Right). and electron density clearly resolved the BTA probe and linker We also solved the cocrystal structure of Pf-LAP bound to but lacked any visible density for the “clickable” alkyne C-term- PNAP (Fig. 3D, Right; see Fig. S4 in SI Appendix for hexameric inal tag (Fig. 3C, Left; see Fig. S3A in SI Appendix for stereoview structure). The 2.0-Å X-ray structure resolved the structural basis and Table S1 in SI Appendix for statistics). The BTA ABP bound for the PNAP specificity and potency for Pf-LAP. As expected the to the essential active site zinc ion via the hydroxyl and carbonyl PNAPABP bound in a similar manner to the parent bestatin (43)

Harbut et al. PNAS Early Edition ∣ 529 of 534 Downloaded by guest on September 26, 2021 dominated by coordination of two Zn2þ ions of the active site more fully characterize the effects of these ABPs on parasites (Fig. S3B in SI Appendix). The P1-Phe ring of PNAP fit neatly through their life cycle. To do this, synchronized parasites were into the small hydrophobic S1 pocket of Pf-LAP, and the P1′ treated at the ring stage and followed by light microscopy evalua- naphthyl group also formed a series of hydrophobic interactions tion of Giemsa-stained thin blood smears (Fig. 4A). We found in the S1′ cleft. The only alteration noted to accommodate the that treating parasites with BTA at its IC99 caused a delay in P1′ naphthyl group was the movement of Ser550 (approximately the life cycle and swelling of the DV at the trophozoite stage 2.9 Å between c-α atoms of the Pf-LAP-PNAP structure versus with eventual parasite death around 60 hr posttreatment. As a the Pf-LAP-bestatin structure). This residue is located in a loop comparison, parasites treated with E-64d, a in- that lines the S1′ cleft, and the movement noted in the Pf-LAP- hibitor that blocks DV falcipains (and initial endoproteolytic PNAP structure effectively flips the serine residue away from the cleavage of Hb) had a similar delay and swollen DV (darkly naphthyl group, dragging the loop and preventing any close con- stained rather than translucent) but remained alive at the 60-hr tacts with the P1′ residue (Fig. S3B in SI Appendix). It was also time point. In contrast, PNAP-treated parasites were arrested at possible to model PNAP into the X-ray crystal structure of the transition to the trophozoite stage; therefore it appeared that Pf-LAP bound to bestatin (PDB ID code 3EBH). Superposition PNAP exerted its effect on parasites significantly earlier than the of PNAP onto the bestatin core showed that the naphthyl side time of major Hb digestion. PNAP treatment caused no promi- chain at the P1′ position clashed with the wall of the S1′ pocket nent morphological features (other than death), thus complicat- of the active site in Pf-LAP (Fig. 3D, Left). ing hypothesis generation as to its mechanism of action. To our knowledge the only other inhibitor that kills rings is artesunate Inhibition of PfA-M1 Kills Parasites via Disruption of Hb Digestion and the other members of the artemisinin family (also shown in Whereas Inhibition of Pf-LAP Kills via a Distinct Mechanism. After con- Fig. 4A for comparative purposes); although the mechanism of firming the specificities of BTA and PNAP, we next wanted to action for artesunate may be different than PNAP it is intriguing A D 12 hpi24 hpi 36 hpi 48 hpi 60 hpi I-Media AA-Media 100 *

Untreated 1.2

0.8

BTA 50 0.4 50 IC value ( µ M) 0.0

% Parasite survival AA I E-64d 0 -8 -7 -6 -5 -4 log[BTA], M PNAP I-Media AA-Media 100

Artesunate 0.25 0.20 BTA PNAP B 0.15 DMSO 0.25 µM20.5 µM .0 µM 0.25 µM 50 0.10

50 0.05 IC value ( µ M) 0.00 AA I % Parasite survival Hoechst YFP/ 0 -8 -7 -6 -5 log[PNAP], M Merge

C P < 0.01 I-Media AA-Media 2.0 P < 0.05 100 4.0

1.5 3.0

2.0 1.0 50 50 1.0 IC value (nM) 0.0 0.5 IAA % Parasite survival

Fold change in DV area 0 0.0 DMSO 0.25 µM 0.5 µM 2.0 µM 0.25 µM -9 -8 -7 -6 log[artesunate], M BTA PNAP

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 treated with 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 staining and 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 treated with increasing concentrations of BTA (and 250 nM PNAP) and imaged 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 twofold decrease in the IC50 of BTA, while parasites treated with PNAP or artesunate show a nonsignificant difference. Shown are representative IC50 plots for each compound in both I-Media (lacking exogenous amino acids except for isoleucine), and AA-Media (containing all natural amino acids). The inlay bar graphs show differences of the mean IC50 of three experiments carried out in triplicate (*P < 0.05, Student’s t test).

530 of 534 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1105601108 Harbut et al. Downloaded by guest on September 26, 2021 that there could be overlap among these two structurally diver- sensitized by approximately 2.4-fold to inhibition by BTA in med- PNAS PLUS gent inhibitor classes (44). ia with only isoleucine (Fig. 4D). In contrast, parasites treated Inhibition of PfA-M1 by BTA treatment of parasites caused a with PNAP or the antimalarial artesunate, which kills ring stage novel swollen DV chemotype. To investigate this phenomenon parasites prior to initiation of large-scale Hb degradation and more closely we visualized the swelling of the DV in live parasites thus acts as a negative control, showed statistically insignificant using a parasite line expressing YFP-tagged plasmepsin II that differences in sensitization to either compound in the isoleucine serves as a DV marker (45). Transgenic parasites were treated media. This evidence suggests a role for PfA-M1 in the Hb diges- with increasing concentrations of BTA and evaluated by fluores- tion pathway and also provides further evidence that the primary cent microscopy (Fig. 4B). From these images, we estimated the role of Pf-LAP is not within the Hb digestion pathway. relative average DV size (measuring 10 DVs) after each treat- Initial proteolytic events are thought to be carried out by the 0 ment. Parasites treated with as little as 250 nM BTA (the Ki of redundant falcipains 2∕2 ∕3, plasmepsins I, II, BTA for PfA-M1) showed a statistically significant increase in DV IV, and HAP (46). Hb-derived oligopeptides are then broken size relative to untreated parasites, with saturation of this swelling down by exopeptidases. Considering that PfA-M1 is an amino- at 1 μM (Fig. 4 B and C). The observation that the degree of peptidase, its likely role in the DV would occur after initial Hb DV swelling is dose-dependent and saturable is consistent with proteolysis by the endopeptidases. To confirm this, we treated the hypothesis that, within this concentration range, PfA-M1 is synchronous cultures of parasites during the trophozoite stage, in likely the sole target and performs a key function in the DV. which the majority of Hb degradation takes place. Fig. S7 in SI In contrast, parasites treated with PNAP at a concentration over Appendix shows that parasites treated with E-64d leads to an ac- eightfold greater than its Ki against Pf-LAP did not show any cumulation of full-length Hb and causes the swelling of the DV significant DV swelling (Fig. 4B and Fig. S5 in SI Appendix). with undigested Hb (42). Conversely, parasites treated with BTA Although we saw no evidence of labeling of any other peptidase showed no inhibition of proteolysis of full-length Hb but still in parasite proteomes (Figs. 1 and 3), we wanted to further con- caused the DV to swell. Treatment of parasites with PNAP was firm that the BTA-derived DV chemotype was not caused by similar to DMSO. inhibition of other ; thus we assayed for inhibition of other DV peptidases: DPAP1, PfAPP and falcipain 2. No inhibi- Inhibition of PfA-M1 Blocks Proteolysis of Specific Hb-Derived Oligo- tion of any enzyme was seen at a concentration up to 30 μM peptides. To obtain direct evidence for the role of PfA-M1 in the BTA, indicating that there is likely no cross-reactivity of BTA proteolysis of Hb-derived oligopeptides, we endeavored to find

with these enzymes in live parasites and that the DV swelling is peptide substrates for this enzyme. We used a mass spectrometry- BIOCHEMISTRY caused solely by inhibition of PfA-M1 (Fig. S6 in SI Appendix). based peptidomics approach to assay the relative abundance of Because disruption in the endocytosis or subsequent catabolic peptides (<10 kDa) in parasites either untreated or treated with breakdown of Hb is thought to be lethal to parasites (9), we hy- the specific PfA-M1 inhibitor BTA. To do this, trophozoite stage pothesized that PfA-M1 inhibition by BTA leads to starvation of parasites were treated with BTA or DMSO for 24 hr. Whole para- the parasite via blockage of proteolysis of Hb peptides. To test site extracts were prepared and peptides were enriched by an acid this idea, we assayed whether parasites forced to rely only on Hb extraction followed by a filtration through a 10 kDa filter column. catabolism are more sensitive to BTA than parasites cultured with Resulting peptide extracts were analyzed by nano LC-MS/MS. exogenous amino acids, by assaying the potency of BTA on para- Mass spectrometry analysis of the peptide peaks against both sites cultured in media lacking all amino acids except isoleucine Hb α and β sequences revealed that the great majority of these (the only amino acid not present in Hb). Indeed, parasites were oligopeptides showed no difference between the treated and un- A Untreated parasites 2 BTA-treated parasites 1 5 4 7 3 6

8 3 x 108 3 x 10

8 2 x 108 2 x 10 /z /z m m 1 x 108 1 x 108 MS Intensity MS Intensity LC Retention Time (min) LC Retention Time (min) B C Peptide: VDPENF Hb peptide Fold change (a) Untreated (1) VDPENF (β) +21.0 (2) VDPVNF (α) +22.6 (a) VDPENF (3) KVNVDEVGGEALG (β) +4.2 (b) DPENF (a)(b) Enzyme: PfA-M1 (4) NAVAHVDDMPNALS (α) +4.3 10 mAu 10 mAu (5) TNVKAAWGKVGAHAGEYGAE (α) +2.7 (6) LTPEEKSAVTALW (α) +1.0 (a) Enzyme: DPAP1 (7) PAETPAVHASLD (α) +0.80 Absorbance (215 nm) 10 mAu

57.5 Retention Time (min)

Fig. 5. Inhibition of PfA-M1 blocks proteolysis of specific Hb oligopeptides. (A) Global peptide profiling of treated parasites identifies a subset of peptides that accumulate in BTA-treated parasites relative to DMSO. Peptides were extracted from either DMSO or BTA-treated parasites and peptides analyzed by LC-MS/ MS. Peptides identified are displayed according to elution time, intensity, and M/Z. Area of the peptide peak corresponds to relative abundance. (B) LC-MS/MS sequencing of the peptides reveals accumulated peptides are derived from Hb. Ratio of peptide abundance was calculated by determining the area of peptide intensity of BTA-treated vs untreated. (C) A synthesized version of an abundantly accumulated peptide, identified in the prior LC-MS/MS analysis, was effi- ciently proteolyzed at the N-terminal valine by PfA-M1, while DPAP1, the other likely essential DV aminopeptidase, failed to catalyze any proteolysis. Shown are HPLC traces displaying the synthetic peptide without enzyme (top trace), with PfA-M1 (middle trace), or with DPAP1 (lower trace). Insets show peptide sequences corresponding to HPLC peaks.

Harbut et al. PNAS Early Edition ∣ 531 of 534 Downloaded by guest on September 26, 2021 treated samples; however, several oligopeptides, from both the α From our peptidomics experiments we showed that Hb oligo- and β chains of Hb, appeared to accumulate after treatment of peptides were substrates for PfA-M1 proteolysis. One issue with parasites with BTA (Fig. 5 A and B). this analysis is that small peptides were not found using our mass Further sequence analysis of the accumulated Hb-derived oli- spectrometry method, which was limited to the identification gopeptides revealed that the majority were likely poor substrates of peptides greater than four amino acids in length. It is likely for DPAP1, the other essential aminopeptidase with broad sub- that the concerted action of DV endo- and exoproteases also strate specificity found in the DV (47). We thus hypothesized that produced smaller tri- and dipeptides, which are substrates for PfA-M1 was necessary for proteolysis of these oligopeptides. To PfA-M1 in the DV. In support of this idea, we also attempted to test this idea, a highly enriched peptide that was identified from identify small peptide species using a Single Quad LC/MS (which the peptidomics study of BTA-treated samples was resynthesized allows for profiling peptides between 200 and 400 kDa) that ac- and shown to be resistant to cleavage by DPAP1, yet efficiently cumulated in BTA-treated parasites (Fig. S8 in SI Appendix). cleaved by PfA-M1 (Fig. 5C). This data provides direct evidence These low molecular weight species all matched to predicted of a role for the genetically essential enzyme, PfA-M1, in the dipeptide molecular weights, and more than half were the mole- digestion of small Hb-derived oligopeptides in P. falciparum. cular weight of dipeptides found in Hb. However, this method precluded the definitive identification of these molecules as Discussion peptides (as opposed to metabolites) and their origin (i.e., Hb). Peptidases likely have many essential functions in P. falciparum, However, we believe these data are suggestive that several dipep- yet the biological roles of the majority of putative proteases en- tides, in addition to oligopeptides, are likely important substrates coded by the parasite genome remain to be characterized. One for the PfA-M1 enzyme. reason for this rests in the difficulty in genetically manipulating Our data using the PNAP ABP for Pf-LAP indicated that this essential parasite genes. To circumvent this deficit in genetic enzyme has an important role quite early in the intraerythrocytic tools, a small molecule approach may be used to perturb and thus life cycle rather than during the major period of Hb digestion. investigate essential protein functions in the parasite. Several is- Formulating a testable hypothesis about a specific role for Pf- sues arise from the use of small molecule probes, including (i) LAP is complicated by the fact that its inhibition did not yield target identification, (ii) specificity, and (iii) permeability in live any overt morphological change in the parasite other than death. cells. To address some of these issues, we generated a library of However, we suspect Pf-LAP may have an essential housekeeping “ ” MAP-specific, clickable ABPs. Replacement of a bulky repor- function in the cytosolic turnover of dipeptides (49) and perhaps ter tag with an alkyne group resulted in smaller, more versatile acts in concert with the parasite proteasome, as has been shown ABPs that allowed for their use in live cells for phenotypic ana- for other neutral cytosolic leucine aminopeptidases pathways lysis along with more tradition lysate-based ABPP. (50). Like PNAP, lethal amounts of proteasome inhibitors exert Using this set of chemical tools, we first identified the targets their effect in the ring-trophozoite transition and parasites do not of bestatin in P. falciparum to be PfA-M1 and Pf-LAP; although progress into the later trophozoite stage (51). Our data does not — there are limitations to this ABPP approach i.e., it is hard to completely rule out the possibility of a minor role for Pf-LAP in determine with absolute certainty that there are no low abun- the Hb degradation pathway via proteolysis of Hb-derived dipep- — dance targets further diversification of the MAP inhibitor scaf- tides that have been transported from the DV into the cytoplasm. fold to generate structure-activity relationship (SAR) trends can However, the fact that PNAP-treated parasites die prior to the strengthen functional conclusions. Thus, through diversification major period of Hb degradation suggests that the essential role of the bestatin scaffold, we were able to create a suite of ABPs for Pf-LAP is not within the Hb digestion pathway. with increasing specificity for both PfA-M1 and Pf-LAP and used Our collective data suggest that these two MAPs are both these ABPs to further understand the functions of these essential potential antiparasitic drug targets. In fact, PNAP is, to our enzymes. knowledge, the most potent parasite MAP inhibitor with an IC50 The aggregate data using the BTA ABP strongly suggests that in the 200 nM range, which gives us hope that these types of in- PfA-M1 plays a key role in oliogopeptide proteolysis in the DV. hibitors could be further developed into more drug-like therapeu- The most prominent morphological feature of PfA-M1 inhibition tics. In addition, P. falciparum MAPs share little homology with was the novel swollen, translucent DV chemotype, which was their human counterparts; less than 35% in the case of the M1 likely due to the accumulation of oligopeptides that created hy- family proteases. It is therefore reasonable to suggest that potent, perosmotic conditions. DV swelling also occurs after E-64d inhi- specific inhibitors of P. falciparum MAPs can be designed over bition of DV falcipains; yet these parasites do not die until they human MAPs. In addition, information gleaned from our preli- attempt to egress, which suggests that swelling alone is not lethal minary SAR and crystallography efforts may provide a jumping to parasites, and that there must be enough amino acids gener- off point for future medicinal chemistry efforts against both en- ated (perhaps by plasmepsins) or obtained from the medium in zymes. Our data here suggest that combination therapy involving the presence of this inhibitor to allow life cycle progression. We inhibitors, such as those for falcipains, and PfA- therefore propose that the likely cause of death upon PfA-M1 M1-specific inhibitors might provide an opportunity for a syner- inhibition is a severe deprivation of the DV production of amino gistic drug combination (52). Ultimately, this strategy may repre- acids in the parasite. (Although we cannot rule out that killing sent a good way to reduce the chance of parasite resistance. may be due to indirect effects distinct to PfA-M1 inhibition, such as the accumulation of small peptides in the DV, which could Materials and Methods potentially be toxic.) To support the hypothesis that inhibition General Methods. See SI Appendix for additional chemical and experimental of PfA-M1 disrupts Hb degradation, we demonstrated that the protocols. A summary of the methods is given below. effects of BTA were enhanced when parasites were forced to rely solely on Hb degradation. Why the amino acids present in com- Parasite Culture and IC50 Determination for Bestatin-Based ABPs. Briefly, 3D7 plete media do not allow for the parasite to completely comple- parasites were cultured in RPMI 1640 (Invitrogen) supplemented with Albu- ment for the genetic or pharmacological loss of PfA-M1 via the max II (Invitrogen). For synchronization, schizont stage parasites were mag- net purified using a SuperMACS™ II Cell Separation Unit (Miltenyi Biotech). utilization of exogenous amino acids is an interesting question. For IC50 determinations, synchronized parasites were plated at 1% parasite- This sensitivity may be explained by the fact that the parasite is mia and 6% hematocrit in 96-well plates at a total volume of 50 μL. Serial particularly dependent on leucine generated in the DV from Hb, dilutions of 2x concentration of the respective compound were added to which is thought to be exchanged for isoleucine via an antiport the wells to bring the total volume up to 100 μL and 0.5% parasitemia mechanism (48). and 3% hematocrit. Compounds were assayed for a 72 h period, after which

532 of 534 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1105601108 Harbut et al. Downloaded by guest on September 26, 2021 2x Vybrant DyeCycle Green DNA (Invitrogen) in PBS was added for a final zinc ion for clarity. Superposition of BTA into the Pf-LAP active site was per- PNAS PLUS concentration of 10 μM and incubated at 37 °C for 30 min. DNA content, formed using the X-ray crystal structure of Pf-LAP-bestatin (3KR4) where the as an indicator of parasitemia, was analyzed on an Accuri C6 Flow Cytometer bestatin scaffold was used to superpose BTA. with C-Sampler. IC50 curves were generated using GraphPad Prism (GraphPad Software). Anti-PfA-M1 Sera. Details of the production of rabbit antisera against recom- binant PfA-M1 have been previously described (53). Labeling of Parasite MAPs with Activity-Based Probes. For parasite labeling, mixed stage parasites were harvested and released from erythrocytes with Screening of P1 and P1′ ABP Libraries. Screens of relative ABP potencies were 1% saponin followed by centrifugation at 1;500 × g for 5 min and 3 washes conducted at single fixed ABP concentrations for the P1 (PfA-M1- 250 nM; in cold PBS. Parasite lysates were prepared by freeze-thaw in 50 mM Tris-HCl Pf-LAP- 750 nM), P1′ natural (PfA-M1- 1 μM; Pf-LAP- 250 nM) P1′ nonnatural μ pH 7.0, 50 mM NaCl, 10 M ZnCl, and protease inhibitor cocktail (EDTA-free) (0.37 μM for both enzymes) libraries. PfA-M1 assays contained 50 mM HEPES 1 100 × (Roche) and extracts were clarified by centrifugation at ; g for 10 min pH 7.5, 100 mM NaCl, 25 mM leucyl-7-amido-4-methylcoumarin (Leu-AMC) at 4 °C. Labeling was performed with indicated concentrations of the ABP for and 0.1% Triton X-100; Pf-LAP assays contained 50 mM HEPES pH 7.5, 1 hr at 37 °C followed by UV crosslinking (365 nm) for 1 hr on ice. Competition 100 μM ZnCl2,50–250 μM Leu-AMC and 0.1% Triton X-100. For Pf-LAP, stea- of labeling was carried out by preincubating lysates for 1 hr at 37 °C. For dy-state rates were approximated by linear fits to the progress curves after a immunoprecipitation, lysates were passed through 7 K MWCO desalting col- 1 hr equilibration period in the presence of substrate and inhibitor. umns (Pierce) after UV crosslinking then incubated overnight with streptavi- din Ultralink Resin (Pierce). Proteins were visualized by standard Western K K blotting and VECTASTAIN ABC kit (Vector Labs) or rabbit anti-GFP (ab6556, Determination of i and i Values. Ki values for inhibition of PfA-M1 were Abcam). For fluorescent probes, labeled proteins were visualized in-gel using determined by Dixon plots and a detailed protocol for determining Pf-LAP 1 a Typhoon flatbed scanner (GE Healthcare). K values may be found in SI Appendix.

Synthesis of Bestatin-Based ABP Libraries. A detailed description of the synth- Mass Spectrometry-Based Peptide Profiling. Briefly, parasites were treated μ esis and characterization of these compounds may be found in SI Appendix. with 2 M BTA or DMSO at the midring stage. Parasites were treated for 24 hr at which point they were harvested by saponin treatment, centrifuged, −80 Recombinant Proteins. Details of the expression in Escherichia coli and puri- and stored at °C in the presence of protease inhibitors. To isolate pep- fication of recombinant PfA-M1 (residues 192 to 1,085) will be described se- tides, parasite samples were boiled in water for 10 min and then centrifuged 18 000 × parately (53). Pf-LAP lacking the N-terminal Asn-rich region (residues 79–605) for 10 min at ; g. The supernatant was saved, and the pellet was re- was expressed with a C-terminal hexahistidine tag in E. coli and purified as suspended in 0.25% acetic acid and disrupted by freeze-thaw and microso- previously described (43).The estimated molecular mass of the purified spe- nicated. All fractions were combined and centrifuged at 20;000 × g at 4 °C

cies from size exclusion chromatography (343 kDa) was in good agreement for 20 min. The supernatant was passed through a 10 kDa molecular weight BIOCHEMISTRY with the predicted mass for the hexameric enzyme (357 kDa). The purifica- cutoff filter (Millipore). tion of recombinant DPAP1 has been published (47). The retention times and m∕z values of the peptides identified were used to map corresponding peptide peaks in the chromatograms generated from X-ray Crystallography. PfA-M1 and Pf_LAP enzymes were purified and crystal- nano-HPLC–ESI-MS (LCQ-DecaXP Plus). These peptide peaks were manually lized as previously described (38). Crystals of the PfA-M1-BTA complex were aligned and then for semiquantitative assessment of the abundances of in- obtained by cocrystallization of BTA with PfA-M1 in mother liquor containing dividual peptides, the total peak areas were determined using the Bioworks 1 mM ligand. Crystals of the Pf-LAP-PNAP complex were obtained by cocrys- algorithm PepQuan (the Area/Height Calculation) with parameters set to tallisation of PNAP with Pf-LAP in mother liquor containing 1 mM ligand. area, mass tolerance of 1.5, minimum threshold of 5,000, five smoothing Prior to data collection, Pf-LAP-PNAP cocrystals were soaked in mother liquor points, and including all proteins. The alignment was based on retention containing 1 mM ligand and 1 mM ZnSO4. Data were collected at 100 K using times, m∕z values, and patterns of peaks in close proximity. synchrotron radiation at the Australian synchrotron micro crystallography beamline 3ID1. A summary of statistics is provided in Table S1 in SI ACKNOWLEDGMENTS. We thank the Australian Synchrotron for beamtime Appendix. Raw data and images will be available from TARDIS (54). and Tom Caradoc-Davis in particular for technical assistance. We thank John The inhibitor complex was initially solved and refined against the un- Dalton, PhD, McGill University, for the Pf-LAP antisera. Authors acknowledge bound PfA-M1 and Pf-LAP structure (protein atoms only) as described the support by Penn Genome Frontiers Institute (D.C.G), Ritter Foundation previously (38) and clearly showed unbiased features in the active site for (D.C.G), R01-AI-076342-01 (D.B.), R01-AI-077638 (M.K.), NIH5T32AI007532 both structures. After placement of inhibitors into unbiased density, CNS (M.B.H.), and 1R56-AI-081770-01A2 (D.C.G.). S.M. is an Australian Research composite omit maps were calculated using all atoms. Fig. S3 in SI Appendix Council (ARC) Future Fellow, J.C.W. is an ARC Federation Fellow and a Na- shows inhibitor density contoured at 1.0σ. Fig. S3 in SI Appendix was gener- tional Health and Medical Research Council (NHMRC) Principal Research Fel- ated using MacPymol and uses a 2.0 carve value around each inhibitor and low. We thank the NHMRC and the ARC for funding support.

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