Available online at www.sciencedirect.com

Working in concert: the metalloaminopeptidases from §

Plasmodium falciparum

Sheena McGowan

Malaria remains the world’s most prevalent human parasitic Parasites of the genus Plasmodium are the causative agents

disease. Because of the rapid spread of drug resistance in of malaria. While four Plasmodium species commonly infect

parasites, there is an urgent need to identify diverse new drug humans, Plasmodium falciparum (Pf) causes the most

targets. One group of that are emerging as targets deaths [3–5]. To date, nine MAPs have been identified

for novel antimalarials are the metalloaminopeptidases. These in Pf (3D7) [6]. Four of these are aminopepti-

catalyze the removal of the N-terminal amino acids dases. The other five enzymes comprise a prolyl imino-

2

from and . Given the restricted specificities of peptidase (or post-prolyl ), a prolyl

each of these enzymes for different N-terminal amino acids, it is aminopeptidase (see footnote 2), a aminopepti-

thought that they act in concert to facilitate turnover. dase, an and an aspartyl amino-

Here we review recent structure and functional data relating to peptidase. Given the restricted specificities of each of

the development of the Plasmodium falciparum these enzymes for particular N-terminal amino acids, it

metalloaminopeptidases as drug targets is thought that they act in concert to facilitate hemoglobin

Addresses (Hb) catabolism [7,8], a process essential to the survival of

Department of and Molecular Biology, Monash University, Pf. However, recent studies show that the PfMAPs may

Clayton Campus, Melbourne, Victoria 3800, Australia

have additional roles to that of Hb digestion, including

important housekeeping functions and in conjunction with

Corresponding author: McGowan, Sheena

([email protected]) the parasite proteosome [9 ,10]. The essential nature of

the PfMAP activity has sparked interest in using them as

targets for the development of novel antimalarials [11,12].

Current Opinion in Structural Biology 2013, 23:828–835

This review comes from a themed issue on and regulation Drug campaigns have successfully targeted proteases; for

Edited by Ben M Dunn and Alexander Wlodawer example, the angiotensin-converting enzymes (hypoten-

sion drugs including the original Captopril, Bristol Myers

For a complete overview see the Issue and the Editorial

Squibb) and HIV (HIV/AIDs anti-retrovirals

Available online 13th August 2013

including the original Saquinavir, Hoffman-La Roche).

0959-440X/$ – see front matter, # 2013 The Author. Published by

However, developing drugs for new protease targets has

Elsevier Ltd. All rights reserved.

proven to be particularly challenging, in part due to the

http://dx.doi.org/10.1016/j.sbi.2013.07.015

importance of achieving selectivity [13]. Since the para-

site and host MAPs show limited sequence identity

(<35%) and have differing substrate specificities, rational

Introduction design and delivery of selective parasite-specific inhibi-

The metalloaminopeptidases (MAPs) constitute a diverse tors is an exciting possibility. A general approach for

set of protease enzymes with essential roles in cell main- therapeutically targeting proteases is to identify specific

tenance, growth and development, and defense. Their inhibitors — generally small molecules — that can block

active sites coordinate essential metal ion(s) that activate the . The search for inhibitors can be acceler-

a water molecule to form an hydroxide nucleophile which ated with knowledge of both the active site structure and

attacks the scissile bond, releasing the P1 pos- the intrinsic subsite occupancy. If rational drug design

1

ition [1]. The are classified platforms are going to succeed, it will be absolutely

into families based on structural patterns and substrate essential that precise structure–activity-relationship and

specificity in particular by the preference shown for mechanistic data are available that can be used to exploit

specific amino acids at the P1 position [2]. selective intricacies of the parasite over its mam-

malian homologs.

§ The methionine aminopeptidases

This is an open-access article distributed under the terms of the

The methionine aminopeptidases (MetAP) perform the

Creative Commons Attribution-NonCommercial-No Derivative Works

essential housekeeping role of catalyzing the removal of

License, which permits non-commercial use, distribution, and repro-

duction in any medium, provided the original author and source are credited.

1 2

The nomenclature of Schechter and Berger [1] is used here. P1 and To date, there is no structural information for either prolyl amino-

0 0

P1 refer to substrate residues while the S1, S1 refer to the correspond- peptidase and as such, these two proteases will not be discussed further

ing enzyme subsites. in this review.

Current Opinion in Structural Biology 2013, 23:828–835 www.sciencedirect.com

The Plasmodium falciparum metalloaminopeptidases McGowan 829

the N-terminal initiator methionine during protein syn- and these compounds are now being considered as anti-

thesis [14]. Two distinct types of MetAP enzymes exist, malarial agents targeting PfMetAP2 [16].

MetAP1 and MetAP2, having mutually redundant yet

essential functions within all organisms [15]. In compari- There are three isoforms of MetAP1 in Pf; PfMetAP1a,

son to MetAP1, MetAP2 has a 65 amino acid insertion in PfMetAP1b and PfMetAP1c [6] (Figure 1a). The three

its catalytic domain and an additional N-terminal domain MetAP1 isoforms are active and have comparable activi-

(in eukaryotes) [14]. The PfMetAP2 enzyme contains a ties to other members of the mammalian MetAP1 family

274 residue N-terminal domain and a conserved catalytic [17]. PfMetAP1a contains the minimal catalytic domain

C-terminal domain [16] (Figure 1a). Recombinant PfMe- and PfMetAP1c has a signal peptide in its longer N-

tAP2 binds fumagillin derivatives with high selectivity terminal for targeting the enzyme to the apicoplast [16]

(Figure 1a). PfMetAP1b is the most characterized of the

three MetAP1s. It is expressed in the early intra-erythro-

Figure 1

cytic stage of the Pf lifecycle [18]. Inhibition of PfMe-

tAP1b with the highly selective compound XC11 that

(a)

contains a 2-(2-pyridinyl)-pyrimidine core resulted in

PfMetAP1a MetAP1 inhibition of Pf proliferation in vitro and was active in

mouse malaria models that included drug-resistant Pf

strains [17]. It has been suggested that PfMetAP1b is a

PfMetAP1b MetAP1

promising target for the development of novel antimalar-

ial drugs [17]. There are currently no published structures

PfMetAP1c SP T MetAP1 of the PfMetAPs; however, the Structural Genomics

Consortium solved the structure of PfMetAP1b, the

target of the antimalarial XC11 compound (3S6B, depos-

PfMetAP2 NTE MetAP2

ited RCSB 2011). The structure shows a typical MetAP1

core fold of pseudo twofold-related N-terminal and C-

terminal domains, containing two a-helices and two anti-

(b)

parallel b-strands (Figure 1b). Unlike characterized mam-

PfMetAP1b

malian homologs, the PfMetAP1b active site contains a

single FeIII ion rather than a di-metal core. The bio-

logical relevance of this difference remains unknown.

Structural alignment of 3S6B with other known MetAP1

D204 folds identifies the human MetAP1b as the closest struc-

H270

D193 tural homolog (and 4FLI the closest structural homolog)

E334

[19]. Structural alignment of the human enzyme with

E303

PfMetAP1b identifies that the FeIII ion in PfMetAP1b is

equivalent to one metal ion site present in the human

MetAP1 structures and that a water molecule occupies

the second metal site (Figure 1b).

Current Opinion in Structural Biology The M1 alanyl aminopeptidase, PfA-M1

The PfA-M1 alanyl aminopeptidase enzyme is responsible

for the final stages of hemoglobin (Hb) digestion [9 ], a

The structure of the PfMetAPs. (a) The domain structure of the

PfMetAPs. The three isoforms of PfMetAP1 all contain a canonical process that provides a source of amino acids for incorp-

MetAP1 catalytic domain (shown in ruby). PfMetAP1c contains an N- oration into parasite proteins [20–22]. The PfA-M1

terminal signal peptide (in blue, SP) followed by a transit peptide domain

enzyme has a broad specificity for the P1 amino acid

(in green, T) to target the enzyme to the apicoplast [16]. PfMetAP1c also

and includes most basic and hydrophobic amino acids

contains a 210 residue insertion in the MetAP1 catalytic domain

(shown as black line). The PfMetAP2 enzyme has a 274 residue N- [23,24,25 ] but preferentially cleaves leucine and meth-

terminal extension (in green, NTE) before a canonical MetAP2 domain. ionine [25 ]. PfA-M1 also has subsite specificity require-

˚

(b) The 1.95 A X-ray crystal structure of PfMetAP1b shows a canonical 0 0

ments for activity [26 ]. Engagement of the S1, S1 and S2

MetAP1 fold (in ruby) (3S6B, MolProbity Quality Score 1.42 = 97th

subsites can contribute to the catalytic efficiency [26 ].

percentile). The PfMetAP1b enzyme has an N-terminal extension of 56

Proteolytic activity of PfA-M1 has been identified in both

amino acids when compared to the human enzyme representatives (in

gray in a and b), extra 10 unstructured amino acids at its C-terminus (in the parasitic cytosol and the digestive vacuole, the mem-

gray). A single FeIII ion (orange sphere) is coordinated by residues H270, brane vesicle where Hb digestion begins [7,9 ,23,24,27].

E303 and E334 (shown in sticks). A water molecule (light blue sphere) is

Whether PfA-M1 digests Hb peptides in the digestive

located in the second metal site in other MetAP1 structures and is

vacuole or the cell cytosol has been the topic of debate

coordinated by conserved active site residues D193 and D204 (shown in

sticks). The water molecule contributes to an apparent pentavalent [7,9 ,23,24,27–29,30 ]. The PfA-M1 enzyme shows a

coordination of the FeIII ion (metal bonds shown as black dashes). 100-fold decrease (kcat/Km) in overall efficiency in an acidic

www.sciencedirect.com Current Opinion in Structural Biology 2013, 23:828–835

830 Catalysis and regulation

pH (representing the digestive vacuole environment), of an active isoform of only 68 kDa that was reported earlier

arguing for a cytosolic function [23,24,29,31]. However, [31]. Further, recombinant work using PfA-M1 supports

specific inhibition of the enzyme results in a change in the the propensity for cleavage, creating two fragments that

digestive vacuole morphology [9 ] and Ragheb et al. [30 ] remain tightly associated ([24] and McGowan unpub-

convincingly argue that, although the enzyme is less effi- lished). A nuclear species of PfA-M1 has also been ident-

cient at the vacuole pH (acidic), the high concentration of ified; however, its role in the parasite nucleus remains

Hb peptide substrates (low mM in the vacuole) drives the unknown [30 ]. The N-terminal extension of the protease

substrate binding to PfA-M1 and leads to sufficient amino- has been shown to contribute to the targeting and distri-

peptidase activity [30 ]. bution of PfA-M1 in the parasite [30 ].

PfA-M1 is encoded on a single gene that encodes a Inhibition of PfA-M1 results in antiplasmodial activity

monomeric protein of 1085 amino acids in length [32–34] and as such is considered an attractive candidate

(122 kDa) [23,31]. The full length species possesses a to initiate screening and lead optimization in a search for

unique 196 amino acid N-terminal extension that is novel antimalarial agents. To date, inhibitors of PfA-M1

removed to form the proteolytically active form of the have targeted the active site of the enzyme and recent

protein (103 kDa) [23,24,27,30 ]. This proteolytically inhibitor scaffolds described include phosphonopeptides

active species is processed to a stable complex of 68 and [24,25 ,33], peptide-based bestatin and analogs

35 kDa fragments [30 ] and argues against the existence [9 ,24,35,36 ], and hydroxamate derivatives [37,38 ].

Figure 2

(a) Bestatin (b) O

H2N N COOH H E461 H496 H500 OH D459 hPheP[CH2]Phe E519 H2N O K374 D379 P OH D399

OH O

CHR-2863 O O N H O O O HN OH

BDM14471 F H H 1 196 391 649 743 1085 N N OH NTE O O 1 85 276 301 605 Active N

Current Opinion in Structural Biology

The X-ray crystal structure of PfA-M1 (a) and PfA-M17 (b) and examples of recent dual inhibitors of the two enzymes. PfA-M1 (a) adopts a canonical

bacterial aminopeptidase fold and is divided into four domains (cartoon is colored by domain). A simplified domain architecture with residue numbers

of domain boundaries is shown underneath cartoon model. The active site is located in domain II and is completely buried within the protein’s core

(domain II in green). The active site (inset) contains a single catalytic zinc ion (shown as black sphere, inset a) and is coordinated by a classic catalytic

triad of H496, H500, E519 (shown in sticks, inset a) and a water molecule (in yellow) in its unbound form. Short molecular dynamic simulations of the

PfA-M1 (75 ns of apo-bound and inhibitor-bound enzyme) [50] suggest the enzyme uses a two-step catalytic mechanism, in which the conformation of

the active site is altered between the Michaelis complex and the transition state [50]. PfA-M17 is a biological homohexamer and is shown as a cartoon

model (b). Each PfA-M17 monomer contains an N-terminal regulatory domain (N-terminal domain shown in green) and a C-terminal catalytic domain

(shown in red, Figure 4a) that together adopt the leucine aminopeptidase (LAP) family fold [51]. A helical linker connects the N-terminal and C-terminal

(shown in yellow). A simplified domain architecture with residue numbers of domain boundaries is shown underneath cartoon model. The active site of

M17 contains two metal-binding sites (inset b), a readily exchangeable site (site 1) and a tight (site 2) [44 ,45]. The LAP enzyme family

members also have a well-coordinated carbonate ion in their active site (shown as sticks, inset b). The metal ions are coordinated by residues K374,

D379, D399, D459 and E461 (shown as sticks, inset b). The divalent metal cation binding at site 1 of PfA-M17 can be functionally exchanged for other

2+ 2+ 2+ 2+

metal cations, the enzyme displaying a preference in the order Zn > Mn > Co > Mg [45]. The type of metal cation in the active site of these

enzymes can therefore influence the catalytic efficiency against peptide substrates as well as the binding of inhibitors. The X-ray crystal structures of

PfA-M1 and PfA-M17 have both been solved in their bound and unbound states. Two potent inhibitors of the enzymes, Bestatin and hPheP[CH2]Phe,

have been structurally analyzed when bound to both PfA-M1 and PfA-M17 [24,44 ]. The two inhibitors, CHR-2863 and BDM14471, have both shown

antiplasmodial activity against drug resistance strains of malaria [38 ,40 ].

Current Opinion in Structural Biology 2013, 23:828–835 www.sciencedirect.com

The Plasmodium falciparum metalloaminopeptidases McGowan 831

The active site of PfA-M1 contains a single catalytic zinc additional function(s) since specific inhibition of its

ion (Figure 2). The inhibitor scaffolds all act to chelate activity in Pf parasite cultures causes growth retardation

the zinc ion and mimic the transition state of the early in the intra-erythrocytic life cycle before Hb diges-

molecule. Targeting the zinc ion using phosphonopep- tion is initiated [8,9 ].

tides and bestatin derivatives produced potent inhibitors

of the enzyme [9 ,24,32,33,36 ]; however, creating a PfA-M17 possesses a severely restricted specificity for N-

parasite specific inhibitor is likely to require the engage- terminally exposed bulky, hydrophobic amino acids

ment of specific elements of the PfA-M1 architecture. Like [25 ] and a preference for leucine [41]. A similar sub-

many proteases, PfA-M1 has an ability to accommodate strate and biochemical profile was obtained when the

organic molecules even when its active site is buried within M17 aminopeptidase from Plasmodium vivax was charac-

a large protein. Velmouragane et al. showed that the S1 terized [42]. A possible explanation for the essential role

pocket is capable of substantial inhibitor-induced re- of the PfA-M17 (and indeed PfA-M1) is that, in addition

arrangement due to the presence of a flexible polar glu- to regulating the general intracellular pool of amino acids,

tamic acid (Glu572) in the pocket [36 ]. One of the best both PfA-M1 and PfA-M17 are critical in the of

cleaved P1 residues is Arg or homoArg [25 ] and recent dipeptides that release free leucine. The only amino acid

work has shown that derivatives of these amino acids are

also able to induce changes in the backbone position of S1

Figure 3

pocket that are not associated with Glu572 [39].

The cellular location of the PfA-M1 enzyme also

represents significant challenges for drug design and E381

H535

delivery; however, recent advances show that compounds

can be delivered and reach the site of action. An orally D325

H87

bioavailable hydroxamate-containing ester, CHR-2863, D435

has been shown to be efficacious against murine malaria

(Plasmodium chaubaudi) [40 ]. The compound is, however,

a close relative of Tosedostat, a potent inhibitor of a

number of intracellular mammalian aminopeptidases.

The authors conclude that CHR-2863 still requires

toxicity testing but represents an important step forward

in showing that the PfMAPs can be targeted by oral

administration of inhibitors [40 ]. In a separate study, a

malono-hydroxamic lead compound [37], with good phy-

sicochemical properties, in vitro plasma stability and

promising antiplasmodial activity in vitro, was assessed

in mice using the malaria strain Plasmodium berghei [38 ].

The in vivo results did not match the potency noted in

vitro and Deprez-Poulain et al., then assessed the plasma 1 92 306 577

concentration and distribution of BDM14471 after a

single dose in mice [38 ]. The results showed that the Current Opinion in Structural Biology

compound was capable of penetrating the erythrocyte,

indicating that this was not the cause of the gap between

The X-ray crystal structure of PfM18AAP reveals a dodecameric

the in vitro and in vivo results. The authors suggested, assembly. The model of PfM18AAP is shown as a space-filled molecule,

given these results that the proteolytic activity of PfA-M1 except for chain A which is shown as cartoon. The molecule is colored

by chain and not all 12 chains are visible in this view. Each PfM18AAP

is not critical for survival of intra-erythrocytic parasites,

monomer (chain A is shown as cartoon to show detail of monomer)

but does concede that the compound may be trapped in

contains two domains — one predominantly used for protein-protein

the red blood cell and not actually enter the parasite interactions (in cyan, regulatory domain) and a second catalytic domain

[38 ]. A further question, not highlighted in the article, is (in magenta, Figure 4). Together the two domains adopt a typical

fold [46 ]. A simplified domain architecture

whether the compound can penetrate the digestive

with residue numbers of domain boundaries is shown underneath

vacuole where the proteolytic activity of PfA-M1 is

cartoon model. Three PfM18AAP catalytic domains interact to form a

thought to predominate [9 ].

cone-shaped trimer and regulatory domains act as packing buffers

between the trimer of catalytic domains. The dodecameric enzyme is

The , PfA-M17 built from the interactions of four PfM18AAP trimers with an interior

active site cavity of M18 comprising four trimer cones, each with their

The leucyl Aminopeptidase PfA-M17 is postulated to be

three active sites [46 ]. The active site of PfM18AAP also has a di-cation

involved in the Hb digestion pathway, generating free

core as shown in the inset (zinc ions shown as black spheres) and the

amino acids at the final stages of this process [41]. How- metals are coordinated by residues H87, D325, E381, D435 and H535

ever, in contrast to PfA-M1, PfA-M17 may have (shown as sticks, inset).

www.sciencedirect.com Current Opinion in Structural Biology 2013, 23:828–835

832 Catalysis and regulation

not found in human Hb and which cannot be synthesized tight binding site (site 2) [45]. While M17 retains residual

by the parasite is isoleucine. This amino acid must be catalytic activity when the metal ion from site 1 is

obtained from the external environment [20]. The uptake removed, the removal of metal ions from both sites results

of isoleucine by parasites is mediated by a two-step in an inactive apo-enzyme that cannot be re-activated by

process involving an ATP-independent transported addition of divalent metal cations [45].

capable of exchanging intra-cellular leucine for isoleu-

cine, and the ATP-dependent storage or accumulation of The aspartyl aminopeptidase, PfM18AAP

this amino acid within parasites [43]. Despite annotation as an aspartyl aminopeptidase, the

PfM18AAP enzyme has highly restricted specificity for

The PfA-M17 enzyme forms a functional homohexamer glutamate as well as aspartate [46 ,47] and reduced

[41,44 ]. The X-ray crystal structure of the M17 revealed activity against asparagine [46 ]. The precise role of

the complex detail of the biological hexamer (Figure 3) PfM18AAP in Pf remains unknown and will require potent

[44 ]. PfA-M17 uses self-association to restrict access to and specific inhibitors/activity-based probes to identify its

its active sites and as such is subject to spatial function in vivo. Gene disruption/truncation experiments

and temporal control. Access to the enclosed active-site of PfM18AAP indicated that the enzyme was dispensable

cavity has been hypothesized to occur via channels for blood-stage replication but had an associated fitness

formed by the N-terminal domains of each monomer cost to the parasite [7]. Anti-sense RNA knockdown of

[44 ]. Each PfA-M17 active site contains two metal- PfM18AAP, however, resulted in parasite growth abnorm-

binding sites, a readily exchangeable site (site 1) and a alities, vascularization, and cellular damage [47]. The

Figure 4

RBC Cytoplasm Hb

pfPost-ProMAP PfM18AAP PfProMAP E PfMetAP1a-c ? M D P

Rb L/W Nucleus Digestive vacuole PfA-M17 M PffA-M1A (nuclear)(nuc F T/Y

L/M PfA-M1 A (cytosol) R Protein synthesis K PfA-M1(DV) F/W/Y Hb digestion Q/G

Current Opinion in Structural Biology

The current status of the role of the PfMAPs in the intra-erythrocytic parasites. All three PfMetAP1 isoforms are detectable in the early erythrocytic

lifecycle; however, microarray analysis indicates that PfMetAP2 is not expressed in this stage. PfMetAP1a has continual expression throughout this

stage of the lifecycle. The role of the PfMetAP1a–c is thought to be in protein synthesis, removing the initiator methionine from newly synthesized

proteins. The X-ray crystal structure of PfMetAP1b is depicted in ruby. The location of the enzyme is generally considered to be close to the ribosome

(depicted in gray, Rb) that can be found associated with the endoplasmic reticulum (orange) or free in the cytosol. mRNA (shown in gray) leaving the

nucleus (blue) is translated to produce an amino acid peptide chain (depicted as yellow beads) that will fold into an active protein. The role of the

nuclear PfA-M1 is currently unknown. The remaining PfMAPs are thought to be involved in Hb digestion. Hb is transported into the parasite from the

red blood cell (RBC) through vesicles. The Hb molecule is delivered to a specialized food vacuole or digestive vacuole (DV). Once internalized, a

proteolytic cascade occurs incorporating numerous proteases of various mechanistic classes. Once the Hb is broken down to smaller peptides

(typically 2-8 amino acids in length, depicted as red ‘beads on a string’), the PfMAPs take over the final digestion to produce free amino acids. PfA-M1

is functional in both the DV and the cytosol although evidence would suggest that its primary role is inside the DV. PfA-M17 and PfM18AAP (colored by

chain) are cytosolic and thought to contribute to Hb digestion, in addition to putative other roles in the Pf lifecycle. The specificity of each PfMAP is

shown by single letter amino acid codes (shown inside black circles). The preference for each amino acid is shown in descending order.

Current Opinion in Structural Biology 2013, 23:828–835 www.sciencedirect.com

The Plasmodium falciparum metalloaminopeptidases McGowan 833

cellular localization and role in growth and development References and recommended reading

Papers of particular interest, published within the period of review,

suggest that, like PfA-M17, PfM18AAP may also have

have been highlighted as:

important functions in addition to Hb digestion [10,47].

of special interest

of outstanding interest

The biological assembly of the PfM18AAP is a dodeca-

mer that forms a tetrahedron shape (Figure 3) in a similar

1. Schechter I, Berger A: On the size of the active site in proteases.

arrangement to the M42 glutamyl aminopeptidases [48]. I. Papain. Biochem Biophys Res Commun 1967, 27:157-162.

PfM18AAP shares only 34% identity with the human 2. Rawlings ND, Tolle DP, Barrett AJ: Evolutionary families of

peptidase inhibitors. Biochem J 2004, 378:705-716.

enzyme and contains a large insertion of 65 amino in

the regulatory domain that is unique to the Pf enzyme (it 3. Enserink M: Malaria’s drug miracle in danger. Science 2010,

328:844-846.

is not present in other Plasmodium M18 enzymes that

share > 75% identity). The role of this unique insertion 4. Miller LH, Ackerman HC, Su XZ, Wellems TE: Malaria biology and

disease pathogenesis: insights for new treatments. Nat Med

remains unknown [10,46 ,47]. The location of this loop

2013, 19:156-167.

indicates that it may act as a sentinel, guarding the pore

5. D‘Alessandro U: Existing antimalarial agents and malaria-

entry to the inner active site cavity (Figure 3). PfM18AAP

treatment strategies. Expert Opin Pharmacother 2009,

thus resembles a proteasomal-like machine with multiple 10:1291-1306.

active sites able to degrade peptide substrates that enter 6. Gardner MJ, Hall N, Fung E, White O, Berriman M, Hyman RW,

Carlton JM, Pain A, Nelson KE, Bowman S et al.: Genome

the central lumen [46 ]. Each active site contains two

sequence of the human malaria parasite Plasmodium

well-coordinated zinc ions [46 ]. The active site of each

falciparum. Nature 2002, 419:498-511.

monomer is a small closed cavity that has a small entrance

˚ 7. Dalal S, Klemba M: Roles for two aminopeptidases in vacuolar

(diameter 5.6 A) [46 ]. Studies using chelation agents hemoglobin catabolism in Plasmodium falciparum. J Biol

Chem 2007, 282:35978-35987.

have shown that altering the type of metal ion can

increase catalytic efficiency but does not affect binding 8. Naughton JA, Nasizadeh S, Bell A: Downstream effects of

haemoglobinase inhibition in Plasmodium falciparum-

of the substrate [47]. To date, no potent inhibitors of

infected erythrocytes. Mol Biochem Parasitol 2010, 173:81-87.

PfM18AAP have been published; however, a high-

9. Harbut MB, Velmourougane G, Dalal S, Reiss G, Whisstock JC,

throughput screening approach has been undertaken

Onder O, Brisson D, McGowan S, Klemba M, Greenbaum DC:

and results of this bioassay are publically available from Bestatin-based chemical biology strategy reveals distinct

roles for malaria M1- and M17-family aminopeptidases. Proc

the PubChem BioAssay.

Natl Acad Sci U S A 2011, 108:E526-E534.

This study reports a multidiscilinary effort to functionally analyze the two

genetically essential neutral aminopeptidases, PfA-M1 and PfA-M17. It

Conclusions and outlook combines activity-based profiling, biochemical and peptidomic

approaches with structural biology to identify PfA-M1 as an essential

The artemisinin-based combination therapies (ACTs) are

hemoglobinase in P. falciparum. Inhibition of PfA-M1 prevents proteolysis

the current regime for malaria and have been deployed of hemoglobin peptides. It also identifies that inhibition of PfA-M17 is

lethal to parasites early in the life cycle, suggesting an essential role

globally with tremendous success for over a decade.

outside of hemoglobin digestion.

Recent reports, however, of the emergence of resistance

10. Lauterbach SB, Coetzer TL: The M18 aspartyl aminopeptidase

to the artemisinins in South-East Asia is alarming. Should

of Plasmodium falciparum binds to human erythrocyte

the ACTs fail, no suitable first-line treatments are avail- spectrin in vitro. Malar J 2008, 7:161.

able for Pf malaria [49]. The PfMAPs have essential

11. Skinner-Adams TS, Stack CM, Trenholme KR, Brown CL,

biological functions in Pf (Figure 4) and represent attrac- Grembecka J, Lowther J, Mucha A, Drag M, Kafarski P,

McGowan S et al.: Plasmodium falciparum neutral

tive targets for the development of novel antimalarial

aminopeptidases: new targets for anti-malarials. Trends

agents. Similarities between different classes of enzyme Biochem Sci 2010, 35:53-61.

raise the possibility of designing combination inhibi-

12. Trenholme KR, Brown CL, Skinner-Adams TS, Stack C, Lowther J,

tors — one compound to target one or more PfMAPs, To J, Robinson MW, Donnelly SM, Dalton JP, Gardiner DL:

Aminopeptidases of malaria parasites: new targets for

thus slowing the emergence of drug resistant parasites.

chemotherapy. Infect Disord Drug Targets 2010, 10:217-225.

The PfA-M1 and PfA-M17 enzymes show promise in this

13. Drag M, Salvesen GS: Emerging principles in protease-based

regard. To overcome the many challenges of producing

drug discovery. Nat Rev Drug Discov 2010, 9:690-701.

drugs against the PfMAPs, a simultaneous dual approach

14. Lowther WT, Matthews BW: Structure and function of the

will be necessary. Our understanding of the biological role methionine aminopeptidases. Biochim Biophys Acta 2000,

1477:157-167.

that the enzymes fulfill within the parasite lifecycle is

absolutely pivotal to success as are the initial compound 15. Mauriz JL, Martin-Renedo J, Garcia-Palomo A, Tunon MJ,

Gonzalez-Gallego J: Methionine aminopeptidases as potential

discovery campaigns to identify, test, optimize and

targets for treatment of gastrointestinal cancers and other

develop potent inhibitors that can be used in downstream tumours. Curr Drug Targets 2010, 11:1439-1457.

drug development.

16. Chen X, Xie S, Bhat S, Kumar N, Shapiro TA, Liu JO: Fumagillin

and fumarranol interact with P. falciparum methionine

aminopeptidase 2 and inhibit malaria parasite growth in vitro

Acknowledgements and in vivo. Chem Biol 2009, 16:193-202.

SM is an Australian Research Council Future Fellow (FT100100690) and

17. Chen X, Chong CR, Shi L, Yoshimoto T, Sullivan DJ Jr, Liu JO:

she thanks both the Australian Research Council and National Health and

Inhibitors of Plasmodium falciparum methionine

Medical Research for funding support.

www.sciencedirect.com Current Opinion in Structural Biology 2013, 23:828–835

834 Catalysis and regulation

aminopeptidase 1b possess antimalarial activity. Proc Natl 31. Florent I, Derhy Z, Allary M, Monsigny M, Mayer R, Schrevel J: A

Acad Sci U S A 2006, 103:14548-14553. Plasmodium falciparum aminopeptidase gene belonging to

the M1 family of zinc-metallopeptidases is expressed in

18. Wu Y, Wang X, Liu X, Wang Y: Data-mining approaches reveal

erythrocytic stages. Mol Biochem Parasitol 1998,

hidden families of proteases in the genome of malaria 97:149-160.

parasite. Genome Res 2003, 13:601-616.

32. Cunningham E, Drag M, Kafarski P, Bell A: Chemical target

19. Krissinel E, Henrick K: Secondary-structure matching (SSM), a

validation studies of aminopeptidase in malaria parasites

new tool for fast protein structure alignment in three

using alpha-aminoalkylphosphonate and phosphonopeptide

dimensions. Acta Crystallogr D Biol Crystallogr 2004,

inhibitors. Antimicrob Agents Chemother 2008, 52:3221-3228.

60:2256-2268.

33. Skinner-Adams TS, Lowther J, Teuscher F, Stack CM,

20. Goldberg DE, Slater AF, Cerami A, Henderson GB: Hemoglobin

Grembecka J, Mucha A, Kafarski P, Trenholme KR, Dalton JP,

degradation in the malaria parasite Plasmodium falciparum:

Gardiner DL: Identification of phosphinate dipeptide analog

an ordered process in a unique organelle. Proc Natl Acad Sci U

inhibitors directed against the Plasmodium falciparum M17

S A 1990, 87:2931-2935.

leucine aminopeptidase as lead antimalarial compounds.

J Med Chem 2007, 50:6024-6031.

21. Kolakovich KA, Gluzman IY, Duffin KL, Goldberg DE:

Generation of hemoglobin peptides in the acidic digestive

34. Nankya-Kitaka MF, Curley GP, Gavigan CS, Bell A, Dalton JP:

vacuole of Plasmodium falciparum implicates peptide

Plasmodium chabaudi chabaudi and P. falciparum: inhibition

transport in amino acid production. Mol Biochem Parasitol

of aminopeptidase and parasite growth by bestatin and

1997, 87:123-135.

nitrobestatin. Parasitol Res 1998, 84:552-558.

22. Rosenthal PJ: Hydrolysis of erythrocyte proteins by proteases

35. Harbut MB, Velmourougane G, Reiss G, Chandramohanadas R,

of malaria parasites. Curr Opin Hematol 2002, 9:140-145.

Greenbaum DC: Development of bestatin-based activity-based

probes for metallo-aminopeptidases. Bioorg Med Chem Lett

23. Allary M, Schrevel J, Florent I: Properties, stage-dependent

2008, 18:5932-5936.

expression and localization of Plasmodium falciparum M1

family zinc-aminopeptidase. Parasitology 2002, 125:1-10.

36. Velmourougane G, Harbut MB, Dalal S, McGowan S, Oellig CA,

Meinhardt N, Whisstock JC, Klemba M, Greenbaum DC:

24. McGowan S, Porter CJ, Lowther J, Stack CM, Golding SJ,

Synthesis of new (S)-bestatin-based inhibitor libraries reveals

Skinner-Adams TS, Trenholme KR, Teuscher F, Donnelly SM,

a novel binding mode in the S1 pocket of the essential malaria

Grembecka J et al.: Structural basis for the inhibition of the

M1 metalloaminopeptidase. J Med Chem 2011, 54:1655-1666.

essential Plasmodium falciparum M1 neutral aminopeptidase.

The paper identified a plasticity in the PfA-M1 S1 pocket that showed,

Proc Natl Acad Sci U S A 2009, 106:2537-2542.

despite a buried active site, that the enzyme was able to alter its shape

and size to accommodate inhibitory compounds. The plasticity of the S1

25. Poreba M, McGowan S, Skinner-Adams T, Trenholme KR,

pocket has implications for rational design of more potent and selective

Gardiner DL, Whisstock JC, To J, Salvesen GS, Drag M, Dalton JP:

inhibitors.

Fingerprinting the substrate specificity of M1 and M17 neutral

aminopeptidases of human malaria, Plasmodium falciparum.

37. Flipo M, Beghyn T, Leroux V, Florent I, Deprez BP, Deprez-

PLoS ONE 2012, 2:e31938.

Poulain RF: Novel selective inhibitors of the zinc plasmodial

This paper defines the substrate specificity of PfA-M1 and PfA-M17 by

aminopeptidase PfA-M1 as potential antimalarial agents.

screening in vitro proteolytic activity against a library of 61 fluorogenic

J Med Chem 2007, 50:1322-1334.

substrates derived from natural and non-natural amino acids. A detailed

substrate fingerprint for each protease is detailed. 38. Deprez-Poulain R, Flipo M, Piveteau C, Leroux F, Dassonneville S,

Florent I, Maes L, Cos P, Deprez B: Structure–activity

0

26. Dalal S, Ragheb DR, Klemba M: Engagement of the S1, S1 and

relationships and blood distribution of antiplasmodial

0

S2 subsites drives efficient catalysis of peptide bond

aminopeptidase-1 inhibitors. J Med Chem 2012,

hydrolysis by the M1-family aminopeptidase from 55:10909-10917.

Plasmodium falciparum. Mol Biochem Parasitol 2012, 183:70-

The paper is concerned with the apparent gap between the reported in

77.

vitro and in vivo results for inhibitors of PfA-M1. Potent in vitro inhibitors

This study employs a small library of X-Ala and Ala-X dipeptide substrates

often display poor antiplasmodial activity. They report the design of a

0

to probe the specificities of the PfA-M1 S1 and S1 subsites. Both

recent inhibitor (BDM14471) which takes into account potency, physi-

subsites show a preference for basic and hydrophobic sidechains over

cochemical parameters and metabolic stability. The report that the

polar and acidic residues. The analysis of subsite specificity starts to

inhibitor can enter the red blood cell, however, beyond that it was difficult

investigate how this enzyme is able to efficiently generate amino acids

to determine if it had penetrated the parasite.

from highly sequence diverse hemoglobin peptides.

39. Kannan Sivaraman K, Paiardini A, Sienczyk M, Ruggeri C,

27. Azimzadeh O, Sow C, Geze M, Nyalwidhe J, Florent I:

Oellig CA, Dalton JP, Scammells PJ, Drag M, McGowan S:

Plasmodium falciparum PfA-M1 aminopeptidase is trafficked

Synthesis and structure–activity relationships of phosphonic

via the parasitophorous vacuole and marginally delivered to

mimetics as inhibitors of the M1 and M17

the food vacuole. Malar J 2010, 9:189.

aminopeptidases from Plasmodium falciparum. J Med Chem

2013, 56:5213-5217.

28. Klemba M: On the location of the aminopeptidase N homolog

PfA-M1 in Plasmodium falciparum. Proc Natl Acad Sci U S A

40. Skinner-Adams TS, Peatey CL, Anderson K, Trenholme KR,

2009, 106:E55.

Krige D, Brown CL, Stack C, Nsangou DM, Mathews RT,

Thivierge K et al.: The aminopeptidase inhibitor CHR-2863 is an

29. Whisstock JC, McGowan S, Trenholme KR, Gardiner DL,

orally bioavailable inhibitor of murine malaria. Antimicrob

Dalton JP: Reply to Klemba: intracellular processing of the

Agents Chemother 2012, 56:3244-3249.

membrane-bound PfA-M1 neutral aminopeptidase, a target

This paper shows that it is possible to treat malaria by oral administration

for new antimalarials. Proc Natl Acad Sci U S A 2009, 106:E56.

of aminopeptidase inhibitors. The compound tested, CHR-2863, is clo-

sely related to Tosedostat, a compound in Phase I/II trials for AML and

30. Ragheb D, Dalal S, Bompiani KM, Ray WK, Klemba M:

myelodysplasia patients.

Distribution and biochemical properties of an M1-family

aminopeptidase in Plasmodium falciparum indicate a role in

41. Stack CM, Lowther J, Cunningham E, Donnelly S, Gardiner DL,

vacuolar hemoglobin catabolism. J Biol Chem 2011,

Trenholme KR, Skinner-Adams TS, Teuscher F, Grembecka J,

286:27255-27265.

Mucha A et al.: Characterization of the Plasmodium falciparum

This paper further probes the location of the PfA-M1 enzyme and the

M17 leucyl aminopeptidase. A protease involved in amino acid

relevance that cellular location has to proteolytic function. It states that

regulation with potential for antimalarial drug development.

the N-terminal extension of PfA-M1 (that is removed before proteolytic

J Biol Chem 2007, 282:2069-2080.

activity is noted) acts to dual target the enzyme by providing a signal

peptide and a putative alternate translation initiation site. The study 42. Lee JY, Song SM, Seok JW, Jha BK, Han ET, Song HO, Yu HS,

provides strong evidence that the reported p68 isoform of PfA-M1 is Hong Y, Kong HH, Chung DI: M17 leucine aminopeptidase of the

not a true species of PfA-M1 and provides justification for the activity of human malaria parasite Plasmodium vivax. Mol Biochem

the enzyme in an acid pH. Parasitol 2010, 170:45-48.

Current Opinion in Structural Biology 2013, 23:828–835 www.sciencedirect.com

The Plasmodium falciparum metalloaminopeptidases McGowan 835

43. Martin RE, Kirk K: Transport of the essential nutrient isoleucine the X-ray crystal structure showed that the enzyme forms a dodeca-

in human erythrocytes infected with the malaria parasite meric structure. Comparison to the human M18 homolog is described.

Plasmodium falciparum. Blood 2007, 109:2217-2224.

47. Teuscher F, Lowther J, Skinner-Adams TS, Spielmann T, Dixon MW,

44. McGowan S, Oellig CA, Birru WA, Caradoc-Davies TT, Stack CM, Stack CM, Donnelly S, Mucha A, Kafarski P, Vassiliou S et al.:

Lowther J, Skinner-Adams T, Mucha A, Kafarski P, Grembecka J The M18 aspartyl aminopeptidase of the human malaria

et al.: Structure of the Plasmodium falciparum M17 parasite Plasmodium falciparum. J Biol Chem 2007,

aminopeptidase and significance for the design of drugs 282:30817-30826.

targeting the neutral . Proc Natl Acad Sci U S A

2010, 107:2449-2454. 48. Dura MA, Rosenbaum E, Larabi A, Gabel F, Vellieux FM,

This paper described the X-ray crystal structure of PfA-M17 and com- Franzetti B: The structural and biochemical characterizations

pares the bound and unbound active sites of PfA-M1 with that of PfA- of a novel TET peptidase complex from Pyrococcus horikoshii

M17. Implications for the design of combination inhibitors, targeting both reveal an integrated peptide degradation system in

enzymes, are discussed. hyperthermophilic Archaea. Mol Microbiol 2009,

72:26-40.

45. Maric S, Donnelly SM, Robinson MW, Skinner-Adams T,

Trenholme KR, Gardiner DL, Dalton JP, Stack CM, Lowther J: The 49. O‘Brien C, Henrich PP, Passi N, Fidock DA: Recent clinical and

M17 leucine aminopeptidase of the malaria parasite molecular insights into emerging artemisinin resistance in

Plasmodium falciparum: importance of active site metal ions Plasmodium falciparum. Curr Opin Infect Dis 2011,

in the binding of substrates and inhibitors. Biochemistry 2009, 24:570-577.

48:5435-5439.

50. Jones PM, Robinson MW, Dalton JP, George AM: The

46. Sivaraman KK, Oellig CA, Huynh K, Atkinson SC, Poreba M, Plasmodium falciparum malaria M1 alanyl aminopeptidase

Perugini MA, Trenholme KR, Gardiner DL, Salvesen G, Drag M (PfA-M1): insights of catalytic mechanism and function from

et al.: X-ray crystal structure and specificity of the Plasmodium MD simulations. PLoS ONE 2011, 6:e28589.

falciparum malaria aminopeptidase PfM18AAP. J Mol Biol

2012, 422:495-507. 51. Burley SK, David PR, Taylor A, Lipscomb WN: Molecular

˚

This paper describes the X-ray crystal structure of PfM18AAP and a structure of leucine aminopeptidase at 2.7-A resolution.

detailed analysis of substrate specificity. Unlike previous publications, Proc Natl Acad Sci U S A 1990, 87:6878-6882.

www.sciencedirect.com Current Opinion in Structural Biology 2013, 23:828–835