Review Article Serine Proteases of Malaria Parasite ...downloads.hindawi.com/journals/ipid/2014/453186.pdflow a nity interaction between the host and parasite and erythrocyte invasion

Review ArticleSerine Proteases of Malaria Parasite Plasmodium falciparumPotential as Antimalarial Drug Targets

Asrar Alam

Department of Biological Sciences Tata Institute of Fundamental Research Homi Bhabha Road Colaba Mumbai-400005 India

Correspondence should be addressed to Asrar Alam asraralam22yahoocoin

Received 25 October 2013 Revised 2 January 2014 Accepted 7 January 2014 Published 11 March 2014

Academic Editor Mary E Marquart

Copyright copy 2014 Asrar Alam This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Malaria is a major global parasitic disease and a cause of enormous mortality and morbidity Widespread drug resistance againstcurrently available antimalarials warrants the identification of novel drug targets and development of new drugs Malarial proteasesare a group of molecules that serve as potential drug targets because of their essentiality for parasite life cycle stages and feasibilityof designing specific inhibitors against them Proteases belonging to various mechanistic classes are found in P falciparum ofwhich serine proteases are of particular interest due to their involvement in parasite-specific processes of egress and invasion In Pfalciparum a number of serine proteases belonging to chymotrypsin subtilisin and rhomboid clans are foundThis review focuseson the potential of P falciparum serine proteases as antimalarial drug targets

1 Global Malaria Burden and Need forDevelopment of Novel Antimalarials

Malaria caused by protozoan parasite Plasmodium is a majorglobal parasitic disease [1] Malaria in humans is caused byfive Plasmodium species namely P falciparum P vivax Povale P malariae and P knowlesi Of these P falciparum isthe causative agent of severe malaria and the major cause ofmalaria-related fatality

According to World Malaria Report of 2013 there werean estimated 207 million clinical cases of malaria in 2012and an estimated 627000 deaths with about 90 of deathsoccurring in sub-Saharan Africa International efforts tocontrol malaria have resulted in significant reduction ofmalaria-related deaths Between 2000 and 2012 malaria-related deaths reduced by 29 globally and 31 in theWHO African Region [2] Methods used to prevent thespread of the disease or to protect individuals in areas wheremalaria is endemic include therapeutic and prophylacticdrugs mosquito eradication and prevention of mosquitobites by using insecticide-treated nets (ITNs) indoor residualspray and larval control [2]

Early antimalarial agentswere isolated fromnatural prod-ucts Bark of the cinchona tree and extracts of the wormwood

plant were among the first effective antimalarials Quinolinecompound chloroquine has been the most widely used druguntil recently Resistance to chloroquine started in Africa inthe 1980s causing tremendous resurgence of malaria burden[3 4] Chloroquine resistance prompted many countries toadopt sulfadoxine-pyrimethamine (SP) as the first-line anti-malarial but resistant P falciparum populations were selectedquickly in Africa Southeast Asia and South America It wasabandoned after only 5 years of use in Southeast Asia [5 6]Due to widespread resistance to the available antimalarialsartemisinin-based combination therapies (ACTs) were intro-duced in Asia Africa and South America The artemisininsare potent and rapidly acting antimalarials derived from theChinese sweet wormwood plant Artemisia annua [7 8]Due to their short duration of action artemisinins cannotbe administered alone which results in recrudescent para-sitemia [9] however they can be administered as ACTs overthree days in the combinations with longer-acting antimalar-ials in the forms of artemether-lumefantrine amodiaquine-artesunate andmefloquine-artesunate [10] Despite the effec-tiveness of ACTs use of artemisinin monotherapy resultedin emergence of drug-resistant P falciparum parasites inCambodia-Thailand border region [11 12] According toWHO till now drug resistance has been reported in three

Hindawi Publishing CorporationInterdisciplinary Perspectives on Infectious DiseasesVolume 2014 Article ID 453186 7 pageshttpdxdoiorg1011552014453186

2 Interdisciplinary Perspectives on Infectious Diseases

Plasmodium species P falciparum P vivax and P malariae[13]

Currently treatment ofmalaria is effectedmainly throughthe administration of chloroquine SP and ACTs Prophy-lactic drugs include chloroquine primaquine mefloquinedoxycycline and malarone (atovaquone and proguanil) [14]Despite the availability of antimalarials for both treatmentand prophylaxis the spread of resistance and paucity of moreantimalarials warrants the need for identification of newdrug targets and development of novel drugs

2 Proteases as Antimalarial Drug Targets

Proteases constitute a ubiquitous and highly abundant groupof catalytic and regulatory molecules having widespreadroles in living systemsThey are primarily involved in proteinturnover to their constituent amino acids to generate thebuilding blocks for new proteins and digestion of dietaryproteins in higher organisms Besides protein activationby limited proteolysis is a common means of regulation ofmany physiological processes [15] Proteases constitute themajor virulence factors in various parasitic diseases such asschistosomiasis malaria leishmaniasis Chagas disease andAfrican sleeping sickness Some well-characterized examplesof the roles of proteases in parasite pathogenesis includetheir involvement in the invasion of host cells degradationof hemoglobin and other blood proteins immune evasionand activation of inflammation [16] In this context they arecrucial for the pathogenic organisms both for their survivaland the diseases they cause Their potential as drug targets isunderscored by the feasibility of designing specific inhibitorsagainst them

Proteases recognize an optimum peptide sequence andcatalyze its cleavage at the active site Selective inhibitorstargeting the active sites can be developed Besides the activesites exosites and allosteric sites also participate in substraterecognition Hence selective inhibitors targeting these sitescan also be developed [17]

Protease inhibitors have been successfully used as drugsagainst human immunodeficiency virus (HIV) [18] andhepatitis C virus (HCV) [19] and in treatment of hypertension[20] and coagulopathies [21] The active sites of proteaseshave been successfully targeted against viruses HIV andHCV and angiotensin-converting enzyme in hypertension[22 23] Targeting the active site is not always feasible dueto homology with the host enzymes For example in manycancers development of protease inhibitor-based drugs hasbeen challenging due to the difficulty in selectively targetingthe active sites In such cases allosteric sites could betargeted to achieve the goals [17] Malaria parasite is the mostimportant member of the parasites of phylum Apicomplexawhich invade the host cell and reside in intracellular nichethat is protected from host defenses and provides a richsource of nutrient Asexual erythrocytic life cycle of malariaparasite is responsible for the clinical symptoms of malariaIt starts with the invasion of erythrocytes by the merozoitesreleased from liver The intraerythrocytic parasite feeds onhost hemoglobin and develops from small ring stage form

to a relatively large and metabolically active trophozoitestage parasite which then transforms to multinucleatedschizont Inhibitor-based studies have shown that cysteineaspartic metallo and serine protease activities are crucial forcompletion of this cycle [24] Several previous studies haveimplicated a functional role of serine proteases in egress andinvasion at blood stages [25ndash27]

3 P falciparum Serine Proteases

Serine proteases are widely dispersed in organisms throughevolution and have diverse functions They have beengrouped into thirteen clans [28] Chymotrypsintrypsin-likeand subtilisin-like serine proteases are two major clans ofserine proteases which have highly similar arrangement ofcatalytic triad Asp His and Ser residues and radically differ-ent protein scaffolds that is 120573120573 for chymotrypsin and 120572120573for subtilisin [29] A number of serine proteases belongingto chymotrypsin subtilisin and rhomboid protease clansare found in P falciparum genome A list of P falciparumserine proteases along with their orthologs and putativefunctions is presented in Table S1 (Supplementary Materialsavailable online at httpdxdoiorg1011552014453186)These pro-teases are expressed in a temporally regulatedmanner at the asexual and sexual stages of the parasite lifecycle [30ndash32] Table S2 presents the microarray and massspectrometry based expression profiles of P falciparumserine proteases Some of these proteases are known to beessential for parasite development at the erythrocytic andexoerythrocytic stages suggesting their potential as targetsfor therapeutic intervention

31 P falciparum Chymotrypsin-Like Proteases Two genesencoding for serine proteases of chymotrypsin-like clan(PlasmoDB IDs PF3D7 0807700 and PF3D7 0812200) wereidentified inP falciparum genome PF3D7 0807700 is homol-ogous to DegP heat shock protein family [33] Since DegPacts as a chaperone at low temperature and protease atelevated temperature its role in extracellular process relatedto invasion is unlikely The second putative chymotrypsin-like serine protease PF3D7 0812200 possesses PDZ2 domainbesides the trypsin domainThis domain is found in prokary-otic viral and eukaryotic signaling proteins having GTPaseactivity [34] known to anchor transmembrane proteinsto cytoskeleton and assembly of signaling complexes Thisprotease is also unlikely to be directly involved in invasion

32 P falciparum Subtilisin-Like Proteases Three genesencoding for proteases of another major clan subtilisin-like proteases or subtilases (clan SB) [35] are found in Pfalciparum genome known as PfSUB1 2 and 3 All of themare highly expressed at late asexual blood stages [31] Ofthese PfSUB1 and 2 have been extensively characterizedand implicated in egress and invasion during asexual bloodstage life cycle of the parasite [26 27 36] PfSUB3 is theleast characterized member and preliminary reports haveconfirmed the in vitro serine protease activity of PfSUB3

Interdisciplinary Perspectives on Infectious Diseases 3

and also identified a multifunctional parasite protein pro-filin as its interacting partner [37 38]

321 P falciparum Subtilisin-Like Protease 1 (PfSUB1)PfSUB1 (PlasmoDB ID PF3D7 050700 and MEROPS iden-tification number S08012) is the first identified member ofP falciparum subtilases The primary structure of PfSUB1classifies it in a small group of bacterial-like eukaryoticsubtilases [29 35] PfSUB1 undergoes two major intracellularprocessing steps during maturation The first one takes placeinside the lumen of endoplasmic reticulum and converts theearliest detectable 82 kDa form into a 54 kDa form (p54)[39] The second brefeldin A-sensitive processing step is theconversion of p54 to intracellular 47 kDa terminal processingproduct (p47) both p54 and p47 contain the predictedcatalytic domain [40]

Expression of codon-optimized PfSUB1 gene inrecombinant baculovirus-infected insect cells resulted in thesecretion of the processed form (p54) [39] N-terminal radio-sequencing of the in vitro translated protein showed thecleavage betweenAsp219 andAsn220 within the sequence Leu-Val-Ser-Ala-Asp-Asn-Ile-Asp-Ile-Ser This highly restrictedsubstrate specificity of PfSUB1 is suggestive of a veryspecialized and nondegradative biological function in theparasite [39 41] A substantial fraction of insect cell-secretedp54 was found bound to its 31 kDa propeptide (rp31) whichwas a highly selective high-affinity inhibitor of the proteasewith dissociation constant in nanomolar range (Kisim125 nM)[39] Truncation of 11 residues from the C-terminal of rp31substantially reduced inhibition of PfSUB1 activity [42] Sincesubtilase propeptides are specific inhibitors of their cognateproteases [43ndash47] the inhibitory peptides from the proregionwill facilitate designing of specific inhibitors of the protease

Many Plasmodium proteins possess structural insertionsnot found in their homologs from other genera [48 49]These insertions may provide targets for highly selectivetherapies against P falciparum Comparison of PfSUB1primary structure with its orthologs and related bacterialsubtilisins revealed the presence of both high and lowcomplexity insertions that are predicted to form surfacestrand or loop structures Site-directed mutagenesis deletionof the whole loop insertions or strategic replacementsrevealed that the majority of the loop insertions are criticalfor the activity of the protease [50]

PfSUB1 gene was found to be refractory to deletionin blood stages It was stored in apical organelles distinctfrom those involved in erythrocyte invasion and termed asldquoexonemesrdquo [26] During the final stage of schizont matu-ration it was discharged into the parasitophorous vacuole(PV) and triggered a series of proteolytic events resulting inmerozoite egress [27] In an attempt to identify the mediatorsof egress Arastu-Kapur et al tested the effect of a library of1200 focused serine and cysteine protease inhibitors on bloodstage malaria parasite growth Using the hits from libraryscreening they identified PfSUB1 and dipeptidyl aminopep-tidase 3 (DPAP3) as the primary regulators of egressInhibition of PfSUB1 and DPAP3 caused a block in schizontrupture [27] whereas at a relatively lower concentration

of the inhibitor defective merozoites were released [51]DPAP3 caused maturation of PfSUB1 [27] The maturePfSUB1 caused processing of parasitophorous vacuolarproteins SERA5 [27] and SERA6 [52] which are implicatedin merozoite egress

Merozoite surface protein 1 (MSP1) complex is a largeglycosylphosphatidylinositol (GPI)-anchored protein com-plex which is comprised of MSP1 and its associated partnerproteins MSP6 and MSP7 During erythrocyte invasionthe initial low affinity interaction with the host cell takesplace through this complex [53ndash55] Proteolytic processing(primary processing) of this complex is necessary for initiallow affinity interaction between the host and parasite anderythrocyte invasion At a later stage another processingof this complex is required for movement of the merozoiteinside the host cell (secondary processing) [36] PfSUB1carries out the primary processing of MSP1 complex inthe parasitophorous vacuole in a spatiotemporally regulatedmanner [56] Besides PfSUB1 also cleaves a number of mero-zoite and parasitophorous vacuolar proteins [57] Besidesblood stages SUB1 is also essential for liver developmentalstages Conditional knockout of Plasmodium berghei SUB1(PbSUB1) revealed that SUB1 although not essential for earlyliver stage development was essential for development ofliver stage schizonts and production of merozoites [58] Con-ditional mutagenesis studies showed that PbSUB1-deficientmerozoites were unable to egress from the hepatocytes [59]

Recently attempts have been made to identify PfSUB1inhibitors Maslinic acid (MA) a low toxic natural penta-cyclic triterpene was found to inhibit the P falciparum bloodstage transition from ring to schizont stage by amultitargetedmechanism MA was found to inhibit the proteolytic pro-cessing of the MSP1 complex probably by targeting PfSUB1[60] Characterization of PfSUB1 orthologs from P vivax Pknowlesi and P berghei revealed that although there are anumber of unusual features of the SUB1 substrate bindingcleft cleavage sites in parasite substrates in these proteasesare conserved Two peptidyl alpha-ketoamide inhibitors ofPfSUB1 inhibited all its orthologs suggesting that smallmolecule inhibitors can be developed against this protease[61] A molecular dynamics simulation study of binding ofknown PfSUB1 substrate peptides based on its prodomainrevealed that the prime and nonprime sides of the scissilebondmake themajor contribution to the binding free energyIt comprises the peptide residues P4 to P21015840making this regionof potential interest for designing peptidomimetic inhibitorsagainst PfSUB1 [62] Given its essentiality for parasite bloodand liver stages proteolytic activity on multiple parasiteproteins and role in egress and invasion PfSUB1 qualifies asan attractive antimalarial drug target

322 P falciparum Subtilisin-Like Protease 2 P falciparumsubtilisin-like protease 2 (PfSUB2) (PlasmoDB ID 248PF3D7 1136900 and MEROPS identification numberS08013) is a type I 249 transmembrane protein and ex-pressed at late asexual blood stages Attempts to disrupt Pberghei ortholog of PfSUB2 (PbSUB2) by double-crossoverintegration have been unsuccessful suggesting the potential


of PfSUB2 as a drug target [63] It is secreted into merozoiteapical organelles ldquomicronemesrdquo and plays a critical role inmerozoite invasion of red blood cells (RBCs) [36]

PfSUB2 causes shedding of merozoite adhesins MSP1and apical membrane antigen 1 (AMA1) at a juxtamembranesite during invasion [64] Cleavage of MSP1 by PfSUB2takes place distal to an epidermal growth factor- (EGF-)like domain at its C-terminal called MSP1

19[53] MSP1

19

remains bound to the merozoite surface and it is the onlypart of MSP1 which enters into the host cell Cleavage ofAMA1 takes places 29 residues away from the transmem-brane domain releasing the bulk of the ectodomain Inthis way the juxtamembrane ldquostubrdquo along with its cognatetransmembrane domain (TMD) and cytoplasmic domainenters into the host cell [65] Shedding of these proteinsis essential for productive invasion [65ndash67] Since PfSUB2causes the shedding of both MSP1 and AMA1 at the mov-ing junction during erythrocyte invasion it is termed asldquomerozoite surface sheddaserdquo (MESH) This protein hasnot been expressed in recombinant proteolytically activeform but shows the MESH activity in purified mero-zoites It translocates from the anterior to the posteriorend of the merozoite in an actin-dependent movementas the merozoite enters into the host erythrocyte [36]

Like PfSUB1 PfSUB2 also undergoes proteolytic process-ings in the parasite which could be probable maturationevents The open reading frame encoding for PfSUB2 was invitro translated which revealed that the 1548 kDa primarytranslated product (SUB2p) underwent rapid conversion to74 kDa intermediate species (SUB2I) which was quantita-tively converted to terminal 72 kDa species (SUB2T) [68]The prodomain of PfSUB2 has been found to be a selectiveinhibitor of its ldquosheddaserdquo activity [36] Nuclear magneticresonance (NMR) structure of PfSUB2 prodomain identifieda likely catalytic domain-binding interface region in it whichcould be exploited to design peptidomimetic inhibitor againstthe protease [69] Essentiality of the protease for parasitesurvival involvement in RBC invasion and the initial find-ings suggesting the feasibility of designing inhibitors againstthe protease make PfSUB2 a promising drug target againstmalaria

323 P falciparum Subtilisin-Like Protease 3 P falciparumsubtilisin-like protease 3 (PfSUB3) (PlasmoDB ID PF3D70507200 and MEROPS identification number S08122) isthe third P falciparum subtilase PfSUB3 is the least studiedmember of P falciparum subtilases It is also highly expressedat late asexual blood stages [31] The full-length PfSUB3 geneencodes an 88 kDa protein the 25 kDa C-terminal regionof which has been shown to possess serine protease activity[37] Yeast two-hybrid screening has revealed parasite profilin(PfPRF) a cytoskeletal and proinflammatory molecule as aninteracting partner of PfSUB3 PfPRF was found to inducethe secretion of proinflammatory cytokines IL-12 and TNF-120572 from mouse bone marrow-derived dendritic cells PfSUB3showed proteolytic activity on PfPRF in in vitro assaysand caused cleavage of PfPRF into multiple fragments of

smaller sizes which were hydrolyzed by increasing concen-tration of PfSUB3 [38] It is still not clear if this proteolyticactivity causes maturation of PfPRF or degradation underphysiological conditions Given the serine protease activityof PfSUB3 and multiple physiological functions of PfPRFnamely motility egress and induction of proinflammatorycytokines its role in the related processes needs to be explored[38]

33 P falciparum Rhomboid Proteases Rhomboid proteinsare intramembranous serine proteases with their catalytictriad embedded within the membrane bilayer surroundedby a hydrophilic cavity formed by a protein ring [70] Ninerhomboid protease genes are found in P falciparum genomeP falciparum rhomboids are largely uncharacterized till date

Two characterized members of P falciparum rhomboidsare PfROM1 and PfPROM4 PfROM1 localizes to a thread-like apical organelle of blood stage merozoites termed asldquomononemerdquo [71] and on the surface of sporozoites aftersalivary gland invasion [72] Plasmodium yoelii ROM1 defi-cient parasites were attenuated during erythrocytic and hep-atic stages and defective in parasitophorous vacuole (PV)development [73] PfROM1 andPfROM4helped inmerozoiteinvasion by catalyzing the intramembrane cleavage of themerozoite adhesin AMA1 [65 74] and erythrocyte bindingantigen 175 (EBA-175) respectively [75] PfROM1 andor 4were able to cleave a variety of adhesins involved in hostparasite interaction within the transmembrane domains [74]Although initial reports on these proteases are suggestiveof their importance for parasite development they stillremain to be extensively characterized and assessed for theirtherapeutic value

4 Conclusion

P falciparum serine proteases are of particular interest aspotential antimalarial drug targets due to their role in theprocesses of egress and invasion at erythrocytic and preery-throcytic stages two critical checkpoints where the parasitedevelopment can be blocked Involvement of parasite serineproteases in processing of parasite molecules involved inmolecular interactions during parasite invasion and cleavageof the transmembrane adhesins for the invasion make themattractive drug targets Liver stages although clinically silentare potential targets of drug and vaccine intervention dueto their low abundance and distinct metabolism Study ofproteases expressed at liver stages is an exciting area ofresearch A schematic diagram of role of P falciparum serineproteases at asexual blood stages and liver stages is shown inFigure 1

Extensive biochemical and structural characterization ofthese molecules and high throughput screening for smallmolecules inhibitors will lead to the way of developmentof novel drugs directed against these proteases Besides thepeptidomimetic inhibitors based on the inhibitory region ofthe prodomains can also be developed


Merozoites SubtilisinsRing

Trophozoite Schizont

RBC

Subtilisins andrhomboid proteases

(a)

Sporozoite

Liver

Infected hepatocyteLiver stage

schizont

Release of liver stagemerozoites

(SUB1activity)

(SUB1activity)

(Rhomboid proteaseactivity)

(b)

Figure 1 Role of serine proteases at asexual blood stages (a) andliver stages of Plasmodium falciparum (b) Subtilisin-like proteasesare essential for merozoite invasion and egress in blood stages liverstage schizont development and subsequent liver stage merozoiteegress Rhomboid protease activities are supposed to be involved ininvasion of RBC and liver

Conflict of Interests

The author declares that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Data sources for PlasmoDB Version 10 are gratefullyacknowledged The author is thankful to Professor AtharChishti Tufts University USA for proofreading the paper

References

[1] J G Breman ldquoThe ears of the hippopotamus manifestationsdeterminants and estimates of the malaria burdenrdquoThe Amer-ican Journal of Tropical Medicine and Hygiene vol 64 no 1-2pp 1ndash11 2001

[2] WHO ldquoWorld malaria reportrdquo 2013[3] J F Trape F Legros P Ndiaye et al ldquoChloroquine-resistant

Plasmodium falciparummalaria in Senegalrdquo Transactions of theRoyal Society of Tropical Medicine and Hygiene vol 83 no 6 p761 1989

[4] J R Zucker T K Ruebush II C Obonyo J Otieno and C CCampbell ldquoThemortality consequences of the continued use ofchloroquine in Africa experience in SiayaWestern KenyardquoTheAmerican Journal of Tropical Medicine and Hygiene vol 68 no4 pp 386ndash390 2003

[5] C H Sibley J E Hyde P F G Sims et al ldquoPyrimethamine-sulfadoxine resistance in Plasmodium falciparum what nextrdquoTrends in Parasitology vol 17 no 12 pp 582ndash588 2001

[6] C Wongsrichanalai A L Pickard W H Wernsdorfer and SR Meshnick ldquoEpidemiology of drug-resistant malariardquo LancetInfectious Diseases vol 2 no 4 pp 209ndash218 2002

[7] F Ter Kuile N J White P Holloway G Pasvol and S KrishnaldquoPlasmodium falciparum in vitro studies of the pharmacody-namic properties of drugs used for the treatment of severemalariardquo Experimental Parasitology vol 76 no 1 pp 85ndash951993

[8] N J White ldquoQinghaosu (artemisinin) the price of successrdquoScience vol 320 no 5874 pp 330ndash334 2008

[9] W Ittarat A L Pickard P Rattanasinganchan et al ldquoRecrude-scence in artesunate-treated patients with falciparum malariais dependent on parasite burden not on parasite factorsrdquo TheAmerican Journal of Tropical Medicine and Hygiene vol 68 no2 pp 147ndash152 2003

[10] M A Travassos and M K Laufer ldquoResistance to antimalarialdrugs molecular pharmacologic and clinical considerationsrdquoPediatric Research vol 65 no 5 pp 64Rndash70R 2009

[11] A M Dondorp F Nosten P Yi et al ldquoArtemisinin resistancein Plasmodium falciparum malariardquo New England Journal ofMedicine vol 361 no 5 pp 455ndash467 2009

[12] T J C Anderson S Nair S Nkhorna et al ldquoHigh heritabilityof malaria parasite clearance rate indicates a genetic basisfor artemisinin resistance in western Cambodiardquo Journal ofInfectious Diseases vol 201 no 9 pp 1326ndash1330 2010

[13] WHO ldquoGlobal report on antimalarial drug efficacy and drugresistance 2000ndash2010rdquo 2010

[14] WHO Guidelines for the Treatment of Malaria 2nd edition2010

[15] B Turk ldquoTargeting proteases successes failures and futureprospectsrdquoNature ReviewsDrugDiscovery vol 5 no 9 pp 785ndash799 2006

[16] J H McKerrow C Caffrey B Kelly P Loke and M SajidldquoProteases in parasitic diseasesrdquo Annual Review of Pathologyvol 1 pp 497ndash536 2006

[17] M Drag and G S Salvesen ldquoEmerging principles in protease-based drug discoveryrdquo Nature Reviews Drug Discovery vol 9no 9 pp 690ndash701 2010

[18] C Flexner G Bate and P Kirkpatrick ldquoTipranavirrdquo NatureReviews Drug Discovery vol 4 no 12 pp 955ndash956 2005

[19] I Melnikova ldquoHepatitis C therapiesrdquo Nature Reviews DrugDiscovery vol 7 no 10 pp 799ndash800 2008

[20] C G Smith and J R Vane ldquoThe discovery of captoprilrdquo FASEBJournal vol 17 no 8 pp 788ndash789 2003

[21] I Melnikova ldquoThe anticoagulants marketrdquo Nature ReviewsDrug Discovery vol 8 no 5 pp 353ndash354 2009


[22] N Alkhouri and N N Zein ldquoProtease inhibitors silver bulletsfor chronic hepatitis C infectionrdquo Cleveland Clinic Journal ofMedicine vol 79 no 3 pp 213ndash222 2012

[23] A J P Docherty T Crabbe J P OrsquoConnell and C R GroomldquoProteases as drug targetsrdquo Biochemical Society Symposium no70 pp 147ndash161 2003

[24] P J Rosenthal ldquoHydrolysis of erythrocyte proteins by proteasesof malaria parasitesrdquo Current Opinion in Hematology vol 9 no2 pp 140ndash145 2002

[25] M J Blackman ldquoProteases involved in erythrocyte invasion bythe malaria parasite function and potential as chemotherapeu-tic targetsrdquo Current Drug Targets vol 1 no 1 pp 59ndash83 2000

[26] S Yeoh R A OrsquoDonnell K Koussis et al ldquoSubcellular dis-charge of a serine protease mediates release of invasive malariaparasites from host erythrocytesrdquo Cell vol 131 no 6 pp 1072ndash1083 2007

[27] S Arastu-Kapur E L Ponder U P Fonovic et al ldquoIdentificationof proteases that regulate erythrocyte rupture by the malariaparasite Plasmodium falciparumrdquoNature Chemical Biology vol4 no 3 pp 203ndash213 2008

[28] E Di Cera ldquoSerine proteasesrdquo IUBMB Life vol 61 no 5 pp510ndash515 2009

[29] R J Siezen and J A M Leunissen ldquoSubtilases the superfamilyof subtilisin-like serine proteasesrdquo Protein Science vol 6 no 3pp 501ndash523 1997

[30] Z Bozdech M Llinas B L Pulliam E D Wong J Zhuand J L DeRisi ldquoThe transcriptome of the intraerythrocyticdevelopmental cycle of Plasmodium falciparumrdquo PLoS Biologyvol 1 no 1 article E5 2003

[31] K G le Roch Y Zhou P L Blair et al ldquoDiscovery of genefunction by expression profiling of the malaria parasite lifecyclerdquo Science vol 301 no 5639 pp 1503ndash1508 2003

[32] M Llinas Z Bozdech E D Wong A T Adai and J L DeRisildquoComparative whole genome transcriptome analysis of threePlasmodium falciparum strainsrdquoNucleic Acids Research vol 34no 4 pp 1166ndash1173 2006

[33] M J Pallen and B W Wren ldquoThe HtrA family of serineproteasesrdquo Molecular Microbiology vol 26 no 2 pp 209ndash2211997

[34] C P Ponting ldquoEvidence for PDZdomains in bacteria yeast andplantsrdquo Protein Science vol 6 no 2 pp 464ndash468 1997

[35] N D Rawlings E OrsquoBrien and A J Barrett ldquoMEROPS theprotease databaserdquo Nucleic Acids Research vol 30 no 1 pp343ndash346 2002

[36] P K Harris S Yeoh A R Dluzewski et al ldquoMolecularidentification of a malaria merozoite surface sheddaserdquo PLoSPathogens vol 1 no 3 article e29 pp 0241ndash0251 2005

[37] A Alam R K Bhatnagar and V S Chauhan ldquoExpression andcharacterization of catalytic domain of Plasmodium falciparumsubtilisin-like protease 3rdquoMolecular and Biochemical Parasitol-ogy vol 183 no 1 pp 84ndash89 2012

[38] A Alam R K Bhatnagar U Relan P Mukherjee and VS Chauhan ldquoProteolytic activity of Plasmodium falciparumsubtilisin-like protease 3 on parasite profilin a multifunctionalproteinrdquoMolecular and Biochemical Parasitology vol 191 no 2pp 58ndash62 2013

[39] M Sajid C Withers-Martinez and M J Blackman ldquoMatu-ration and specificity of Plasmodium falciparum subtilisin-likeprotease-1 a malaria merozoite subtilisin-like serine proteaserdquoJournal of Biological Chemistry vol 275 no 1 pp 631ndash641 2000

[40] M J Blackman H FujiokaWH L Stafford et al ldquoA subtilisin-like protein in secretory organelles of Plasmodium falciparummerozoitesrdquo Journal of Biological Chemistry vol 273 no 36 pp23398ndash23409 1998

[41] C Withers-Martinez J W Saldanha B Ely F Hackett TOrsquoConnor and M J Blackman ldquoExpression of recombinantPlasmodium falciparum subtilisin-like protease-1 in insect cellsCharacterization comparison with the parasite protease andhomology modelingrdquo Journal of Biological Chemistry vol 277no 33 pp 29698ndash29709 2002

[42] L Jean F Hackett S R Martin and M J Blackman ldquoFunc-tional characterization of the propeptide of Plasmodium falci-parum subtilisin-like protease-1rdquo Journal of Biological Chem-istry vol 278 no 31 pp 28572ndash28579 2003

[43] Y Li ZHu F Jordan andM Inouye ldquoFunctional analysis of thepropeptide of subtilisin E as an intramolecular chaperone forprotein folding Refolding and inhibitory abilities of propeptidemutantsrdquo Journal of Biological Chemistry vol 270 no 42 pp25127ndash25132 1995

[44] H-W Huang W-C Chen C-Y Wu et al ldquoKinetic studiesof the inhibitory effects of propeptides subtilisin BPNrsquo andcarlsberg to bacterial serine proteasesrdquo Protein Engineering vol10 no 10 pp 1227ndash1233 1997

[45] A Boudreault D Gauthier and C Lazure ldquoProprotein con-vertase PC13-related peptides are potent slow tight-bindinginhibitors of murine PC13 and Hfurinrdquo Journal of BiologicalChemistry vol 273 no 47 pp 31574ndash31580 1998

[46] Y Yabuta H Takagi M Inouye and U Shinde ldquoFoldingpathway mediated by an intramolecular chaperone propeptidereleasemodulates activation precision of pro-subtilisinrdquo Journalof Biological Chemistry vol 276 no 48 pp 44427ndash44434 2001

[47] M Fugere P C Limperis V Beaulieu-Audy et al ldquoInhibitorypotency and specificity of subtilase-like pro-protein convertase(SPC) prodomainsrdquo Journal of Biological Chemistry vol 277 no10 pp 7648ndash7656 2002

[48] M J Gardner H Tettelin D J Carucci et al ldquoChromosome2 sequence of the human malaria parasite Plasmodium falci-parumrdquo Science vol 282 no 5391 pp 1126ndash1132 1998

[49] E Pizzi and C Frontali ldquoDivergence of noncoding sequencesand of insertions encoding nonglobular domains at a genomicregion well conserved in plasmodiardquo Journal of MolecularEvolution vol 50 no 5 pp 474ndash480 2000

[50] L Jean C Withers-Martinez F Hackett and M J BlackmanldquoUnique insertions within Plasmodium falciparum subtilisin-like protease-1 are crucial for enzyme maturation and activityrdquoMolecular and Biochemical Parasitology vol 144 no 2 pp 187ndash197 2005

[51] S Gemma S Giovani M Brindisi et al ldquoQuinolylhydrazonesas novel inhibitors of Plasmodium falciparum serine proteasePfSUB1rdquo Bioorganic amp Medicinal Chemistry Letters vol 22 no16 pp 5317ndash5321 2012

[52] A Ruecker M Shea F Hackett C Suarez E M A Hirstand K Milutinovic ldquoProteolytic activation of the essentialparasitophorous vacuole cysteine protease SERA6 accompaniesmalaria parasite egress from its host erythrocyterdquoThe Journal ofBiological Chemistry vol 287 no 45 pp 37949ndash37963 2012

[53] M J Blackman H-G Heidrich S Donachie J S McBrideand A A Holder ldquoA single fragment of a malaria merozoitesurface protein remains on the parasite during red cell invasionand is the target of invasion-inhibiting antibodiesrdquo Journal ofExperimental Medicine vol 172 no 1 pp 379ndash382 1990


[54] V K Goel X Li H Chen S-C Liu A H Chishti and S SOh ldquoBand 3 is a host receptor bindingmerozoite surface protein1 during the Plasmodium falciparum invasion of erythrocytesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 100 no 9 pp 5164ndash5169 2003

[55] X Li H Chen T H Oo et al ldquoA co-ligand complex anchorsPlasmodium falciparummerozoites to the erythrocyte invasionreceptor band 3rdquo Journal of Biological Chemistry vol 279 no 7pp 5765ndash5771 2004

[56] M A Child C Epp H Bujard andM J Blackman ldquoRegulatedmaturation of malaria merozoite surface protein-1 is essentialfor parasite growthrdquo Molecular Microbiology vol 78 no 1 pp187ndash202 2010

[57] N C S de Monerri H R Flynn M G Campos et al ldquoGlobalidentification of multiple substrates for Plasmodium falciparumSUB1 an essential malarial processing proteaserdquo Infection andImmunity vol 79 no 3 pp 1086ndash1097 2011

[58] C Suarez K Volkmann A R Gomes O Billker and M JBlackman ldquoThemalarial serine protease SUB1 plays an essentialrole in parasite liver stage developmentrdquo PLoS Pathogens vol 9Article ID e1003811 2013

[59] L Tawk C Lacroix P Gueirard et al ldquoA key role for Plasmod-ium subtilisin-like SUB1 in egress of malaria parasites from hosthepatocytesrdquo The Journal of Biological Chemistry vol 288 no46 pp 33336ndash33346 2013

[60] C Moneriz J Mestres J M Bautista A Diez and A PuyetldquoMulti-targeted activity of maslinic acid as an antimalarialnatural compoundrdquo FEBS Journal vol 278 no 16 pp 2951ndash2961 2011

[61] C Withers-Martinez C Suarez S Fulle et al ldquoPlasmodiumsubtilisin-like protease 1 (SUB1) insights into the active-sitestructure specificity and function of a pan-malaria drug targetrdquoInternational Journal for Parasitology vol 42 no 6 pp 597ndash6122012

[62] S Fulle C Withers-Martinez M J Blackman G M Morrisand P W Finn ldquoMolecular determinants of binding to thePlasmodium subtilisin-like protease 1rdquo Journal of ChemicalInformation and Modeling vol 53 no 3 pp 573ndash583 2013

[63] P Uzureau J-C Barale C J Janse A P Waters and C BBreton ldquoGene targeting demonstrates that the Plasmodiumberghei subtilisin PbSUB2 is essential for red cell invasion andreveals spontaneous genetic recombination eventsrdquo CellularMicrobiology vol 6 no 1 pp 65ndash78 2004

[64] J-C Barale T Blisnick H Fujioka et al ldquoPlasmodium falci-parum subtilisin-like protease 2 a merozoite candidate for themerozoite surface protein 1-42 maturaserdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 11 pp 6445ndash6450 1999

[65] S A Howell F Hackett A M Jongco et al ldquoDistinct mecha-nisms govern proteolytic shedding of a key invasion protein inapicomplexan pathogensrdquo Molecular Microbiology vol 57 no5 pp 1342ndash1356 2005

[66] S L Fleck B Birdsall J Babon et al ldquoSuramin and suraminanalogues inhibit merozoite surface protein-1 secondary pro-cessing and erythrocyte invasion by the malaria parasite Plas-modium falciparumrdquo Journal of Biological Chemistry vol 278no 48 pp 47670ndash47677 2003

[67] S Dutta J D Haynes A Barbosa et al ldquoMode of action ofinvasion-inhibitory antibodies directed against apical mem-brane antigen 1 of Plasmodium falciparumrdquo Infection andImmunity vol 73 no 4 pp 2116ndash2122 2005

[68] F Hackett M Sajid C Withers-Martinez M Grainger andM J Blackman ldquoPfSUB-2 a second subtilisin-like protein inPlasmodium falciparummerozoitesrdquoMolecular andBiochemicalParasitology vol 103 no 2 pp 183ndash195 1999

[69] Y He Y Chen N Oganesyan et al ldquoSolution NMR structureof a sheddase inhibitor prodomain from the malarial parasitePlasmodium falciparumrdquo Proteins vol 80 no 12 pp 2810ndash28172012

[70] S Urban ldquoRhomboid proteins conserved membrane proteaseswith divergent biological functionsrdquo Genes and Developmentvol 20 no 22 pp 3054ndash3068 2006

[71] S SinghM Plassmeyer D Gaur and L HMiller ldquoMononemea new secretory organelle in Plasmodium falciparummerozoitesidentified by localization of rhomboid-1 proteaserdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 104 no 50 pp 20043ndash20048 2007

[72] P Srinivasan I Coppens andM Jacobs-Lorena ldquoDistinct rolesofPlasmodium rhomboid 1 in parasite development andmalariapathogenesisrdquo PLoS Pathogens vol 5 no 1 Article ID e10002622009

[73] I M Vera W L Beatty P Sinnis and K Kim ldquoPlasmodiumprotease rom1 is important for proper formation of the para-sitophorous vacuolerdquo PLoS Pathogens vol 7 no 9 Article IDe1002197 2011

[74] R P Baker R Wijetilaka and S Urban ldquoTwo Plasmodiumrhomboid proteases preferentially cleave different adhesinsimplicated in all invasive stages ofmalariardquo PLoS Pathogens vol2 no 10 p e113 2006

[75] R A OrsquoDonnell F Hackett S A Howell et al ldquoIntramembraneproteolysis mediates shedding of a key adhesin during erythro-cyte invasion by the malaria parasiterdquo Journal of Cell Biologyvol 174 no 7 pp 1023ndash1033 2006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Plasmodium species P falciparum P vivax and P malariae[13]

Currently treatment ofmalaria is effectedmainly throughthe administration of chloroquine SP and ACTs Prophy-lactic drugs include chloroquine primaquine mefloquinedoxycycline and malarone (atovaquone and proguanil) [14]Despite the availability of antimalarials for both treatmentand prophylaxis the spread of resistance and paucity of moreantimalarials warrants the need for identification of newdrug targets and development of novel drugs

2 Proteases as Antimalarial Drug Targets

Proteases constitute a ubiquitous and highly abundant groupof catalytic and regulatory molecules having widespreadroles in living systemsThey are primarily involved in proteinturnover to their constituent amino acids to generate thebuilding blocks for new proteins and digestion of dietaryproteins in higher organisms Besides protein activationby limited proteolysis is a common means of regulation ofmany physiological processes [15] Proteases constitute themajor virulence factors in various parasitic diseases such asschistosomiasis malaria leishmaniasis Chagas disease andAfrican sleeping sickness Some well-characterized examplesof the roles of proteases in parasite pathogenesis includetheir involvement in the invasion of host cells degradationof hemoglobin and other blood proteins immune evasionand activation of inflammation [16] In this context they arecrucial for the pathogenic organisms both for their survivaland the diseases they cause Their potential as drug targets isunderscored by the feasibility of designing specific inhibitorsagainst them

Proteases recognize an optimum peptide sequence andcatalyze its cleavage at the active site Selective inhibitorstargeting the active sites can be developed Besides the activesites exosites and allosteric sites also participate in substraterecognition Hence selective inhibitors targeting these sitescan also be developed [17]

Protease inhibitors have been successfully used as drugsagainst human immunodeficiency virus (HIV) [18] andhepatitis C virus (HCV) [19] and in treatment of hypertension[20] and coagulopathies [21] The active sites of proteaseshave been successfully targeted against viruses HIV andHCV and angiotensin-converting enzyme in hypertension[22 23] Targeting the active site is not always feasible dueto homology with the host enzymes For example in manycancers development of protease inhibitor-based drugs hasbeen challenging due to the difficulty in selectively targetingthe active sites In such cases allosteric sites could betargeted to achieve the goals [17] Malaria parasite is the mostimportant member of the parasites of phylum Apicomplexawhich invade the host cell and reside in intracellular nichethat is protected from host defenses and provides a richsource of nutrient Asexual erythrocytic life cycle of malariaparasite is responsible for the clinical symptoms of malariaIt starts with the invasion of erythrocytes by the merozoitesreleased from liver The intraerythrocytic parasite feeds onhost hemoglobin and develops from small ring stage form

to a relatively large and metabolically active trophozoitestage parasite which then transforms to multinucleatedschizont Inhibitor-based studies have shown that cysteineaspartic metallo and serine protease activities are crucial forcompletion of this cycle [24] Several previous studies haveimplicated a functional role of serine proteases in egress andinvasion at blood stages [25ndash27]

3 P falciparum Serine Proteases

Serine proteases are widely dispersed in organisms throughevolution and have diverse functions They have beengrouped into thirteen clans [28] Chymotrypsintrypsin-likeand subtilisin-like serine proteases are two major clans ofserine proteases which have highly similar arrangement ofcatalytic triad Asp His and Ser residues and radically differ-ent protein scaffolds that is 120573120573 for chymotrypsin and 120572120573for subtilisin [29] A number of serine proteases belongingto chymotrypsin subtilisin and rhomboid protease clansare found in P falciparum genome A list of P falciparumserine proteases along with their orthologs and putativefunctions is presented in Table S1 (Supplementary Materialsavailable online at httpdxdoiorg1011552014453186)These pro-teases are expressed in a temporally regulatedmanner at the asexual and sexual stages of the parasite lifecycle [30ndash32] Table S2 presents the microarray and massspectrometry based expression profiles of P falciparumserine proteases Some of these proteases are known to beessential for parasite development at the erythrocytic andexoerythrocytic stages suggesting their potential as targetsfor therapeutic intervention

31 P falciparum Chymotrypsin-Like Proteases Two genesencoding for serine proteases of chymotrypsin-like clan(PlasmoDB IDs PF3D7 0807700 and PF3D7 0812200) wereidentified inP falciparum genome PF3D7 0807700 is homol-ogous to DegP heat shock protein family [33] Since DegPacts as a chaperone at low temperature and protease atelevated temperature its role in extracellular process relatedto invasion is unlikely The second putative chymotrypsin-like serine protease PF3D7 0812200 possesses PDZ2 domainbesides the trypsin domainThis domain is found in prokary-otic viral and eukaryotic signaling proteins having GTPaseactivity [34] known to anchor transmembrane proteinsto cytoskeleton and assembly of signaling complexes Thisprotease is also unlikely to be directly involved in invasion

32 P falciparum Subtilisin-Like Proteases Three genesencoding for proteases of another major clan subtilisin-like proteases or subtilases (clan SB) [35] are found in Pfalciparum genome known as PfSUB1 2 and 3 All of themare highly expressed at late asexual blood stages [31] Ofthese PfSUB1 and 2 have been extensively characterizedand implicated in egress and invasion during asexual bloodstage life cycle of the parasite [26 27 36] PfSUB3 is theleast characterized member and preliminary reports haveconfirmed the in vitro serine protease activity of PfSUB3














19[53] MSP1

19







4 Conclusion






RBC


(a)

Sporozoite

Liver


schizont


(SUB1activity)

(SUB1activity)


(b)




Acknowledgments


References



















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























19[53] MSP1

19







4 Conclusion






RBC


(a)

Sporozoite

Liver


schizont


(SUB1activity)

(SUB1activity)


(b)




Acknowledgments


References



















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















19[53] MSP1

19







4 Conclusion






RBC


(a)

Sporozoite

Liver


schizont


(SUB1activity)

(SUB1activity)


(b)




Acknowledgments


References



















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















RBC


(a)

Sporozoite

Liver


schizont


(SUB1activity)

(SUB1activity)


(b)




Acknowledgments


References



















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Review Article Serine Proteases of Malaria Parasite ...downloads.hindawi.com/journals/ipid/2014/453186.pdflow a nity interaction between the host and parasite and erythrocyte invasion