New Chemo- and Immunotherapeutic

Approaches in Chagas’ Disease

Rosa A. Maldonado MedinaFederal University of São Paulo

(UNIFESP)

.

• Chagas’ disease affects 16-18 million people in

Latin America, from Mexico to Argentina.

• The ethiological agent is the protozoan parasite

Trypanosoma cruzi

• Current treatment (benznidazole, Rochagan) is

partially effective (~60% cure).

Recently, drug-resistant strains have been found.

Chagas’ Disease(American trypanosomiasis)

Chemotherapeutic Target Properties

a) The molecule must perform essential functions in the parasite.

Life or death

b) It can be selectively inhibited:

Present in T. cruzi, but not in mammals

Carbohydrates Lipids Proteins

Basic Metabolism

Oleate (-12) desaturase (ODTc ):

A New Chemotherapeutic Target

in Chagas’ Disease

• OD is absent in humans, then it can be selectively inhibited.

• Linoleic acid is a major component of GPI anchor of T. cruzi trypomastigote

mucins and phospholipids. So, the product of OD seems to perform essential

functions for the parasite.

OD fulfills all the requirements as a potential chemotherapeutic target.

9-

12- desaturase (OD)

(C18:0)---X* (C18:1)---X

NAD(P)H

(C18:2)---X

NAD(P)

9- desaturase

12-

Stearoyl (C18:0)--- Oleoyl (C18:1)---X

NAD(P)H

Linoleoyl (C18:2)---X

NAD(P)

15- desaturaseNAD(P)H

(C18:3)---X

NAD(P) -

NAD(P)H

Linolenoyl(C18: )---X

NAD(P)

9-

12- desaturase (OD)

(C18:0)---X* (C18:1)---X

NAD(P)H

(C18:2)---X

NAD(P)

9- desaturase

12-

Stearoyl (C18:0)--- Oleoyl (C18:1)---X

NAD(P)H

Linoleoyl (C18:2)---X

NAD(P)

15- desaturaseNAD(P)H

(C18:3)---X

NAD(P) -

NAD(P)H

Linolenoyl(C18: )---X

NAD(P)X* = CoA or ACP (acyl carrier protein) or phospholipid.

Cloning of the T. cruzi Oleate Desaturase (ODTc)

P1, P2= degenerated primersgDNA

P1

P2PCR

High similarity with 12-desaturase from plants

(1003 bp)

Size selection library

Sac I Genomic clone Sac I 3 kb

Mannosyl Transferase

5´-ODTc 3´- missing

Sph

I

Sac I

Kpn I

Hind

II

3.0

Kb

3´-RACE PCR

Sac I Sac I Sac I

Mannosyl Transferase

ODTcI ODTc II

3 Kb 2.7 Kb

~8 Kb

1.2 Kb

Sac I Sac I Sac I

3 Kb 2.7 Kb

Sph I Sph I

1.2 Kb

Chromosomal localization

Chrom. Mb Dm28c Sylvio CL Y CL

Band X10/7 Brener

IX 1.03

VII 0.95

VI 0.85

I 0.45

Strain

Phylogenetic groups: I (sylvatic cycle): Dm28c, Sylvio X10/7II (domestic cycle): Y, CLHybrid: CL Brener

CL-Brener

0.95

0.85

0.45

Mb

Y CL

1.03

0.85

Mb

0.45

Dm28c S.X10/7

Mb

Alignment of Trypanosomatid ODs vs. Plant OD

T.cruziL.major

T.bruceiB.officinalis

T.cruziL.major

T.bruceiB.officinalis

T.cruziL.major

T.bruceiB.officinalis

T.cruziL.major

T.bruceiB.officinalis

T.cruziL.major

T.bruceiB.officinalis

T.cruziL.major

T.bruceiB.officinalis

T.cruziL.major

T.bruceiB.officinalis

Putative transmembrane motifs

His-rich motifs: possibly participating in catalysis

PSORT II - k-nearest neighbor (k-NN) algorithm: 44.4 %: endoplasmic reticulum

1 54 .......... ...MSRVDNL TVAPGSPDVM KAVLKSGRNK .ENNVIVPS. .SALTIREIQ EKIPVKYFER .......... .......... .........M KSVLKAGLKK .DVDVRVPS. .AELTIKQIQ DLIPAKYFER MLPKQQMGGS VCNASIETVN TEATDPSEAK KIVLNAGRSE .KVNVYVPP. .STLMVRDIQ EQIPAEYFQR .......... .......... ...MGGGGRM PVPTKGKKSK SDVFQRVPSE KPPFTVGDLK KVIPPHCFQR 55 124 NTTRSVMFLL RDLAQVALTY AIMYAVALPL ATSMEVSAAR TVADGGALSL MAGTAMTTAA WLLKGVLWAV SAVWSMFYVF RDICQLLVVY CIMYRAVMPA LAGLES...R MYEAAGAT.. WASWSLVSA. ..VKLAVWNV SMWRSFSYLS RDMFQLFLTF VIMYNFVLPM LDSSLLN... .......... ....AVPPVA WLSRAAAWMI SVLHSFSYVV YDLVIAALFF YTASRYIHLQ PHPLSY.... .......... .......... .....VAWPL 125 194 FWFVQGLNGT ALWVLAHECG HQAFSPMKGL NDAVGMILHS ALLVPYHSWR ITHGGHHKHT NHLTKDLVFV FWFVQGLNGT ALWVLAHECG HQAFSPYRSL NNAVGLLLHS SVLVPYHSWR ITHGNHHKHT NHLTKDTVFV YWFIQGLNGT ALWVLAHECG HQAFCNSRRV NNAVGMILHS ALLVPYHSWR LTHGTHHKHT NHLTKDLVFV YWFCQGSVLT GVWVIAHECG HHAFSDYQWL DDTVGLLLHS ALLVPYFSWK YSHRRHHSNT GSLERDEVFV 195 262 PEKRNSVVEL VEDA..PLVS LIQTLIIFLF GWPAHLLFNA SGQEFGRLAC HFDPGAPFFR SEDRHDVVLS PRKESKVIDL LEET..PLVM VSNMLIMFIF GWPGYLLFNV ASQDYGRRTN HFEPSSPLFR KEDAHDIVLS PVQRSAVGEA VEEA..PIVM LWNMALMFLF GWPMHLLVNV GGQKFDRFTS HFDPNAPFFR RADYNNVMVS PKKRSGISWS SEYLNNPPGR VLVLLVQLTL GWPLYLMFNV SGRPYDRFAC HFDPKSPIYN DRERLQIYIS 263 332 NVGIVSALFV IFSSVYRFGF TNVFYWYIVP YLWVNFWLVY ITYLQHTDIR IPHYTHEHWT FVRGALAAVD DIGIFAALTV LGLCTYQYGA YNVLCWYVVP YMWVNFWLLY ITYLQHTDIR IPHYNHEHWT FVRGALAAVD NMGVLLTLSI LGACSWSFGF AVVVRWYLIP YLWVNFWLVY ITYMQHSDVR LPHYTHDHWT YVRGAVAAVD DAGIVAVMYG LYRLVAAKGV AWVVCYYGVP LLVVNGFLVL ITYLQHTQPS LPHYDSSEWD WLKGALATVD 333 402 RDYGFVLNTW LHHIKIPTPS HQDSHVVHHL FSKMPHYNAI QVTRKYIREI LGATYITDER SLWKMLWEQR RDYGFLINNW LHHI...... .HDSHVVHHL FSQMPFYHAI EVTRHHIKAI LGDNYVEDRR PLIDALCTSW RDFGPLLNSW LHHIN..... ..DSHVVHHL FSQMPHYNAI EVTRKHIRDI LGDLYVTDAK PLLKSLVHTW RDYG.FLNKV LHNIT..... ..DTHVAHHL FSTMPHYHAM EAT.KAIKPI LGDYYQCDRT PVFKAMYREV 403 419 KECRYVVPAE G...VCVFHG ...... KECAYVVPSE G...VSVFYG FNKKRA RECRYVVPSE G...ICIYRS ...... KECIYVEADE GDNKKGVFWY KNKL..

Homologous expression of ODTc fused to GFP

gDNA5`-Xba I

Xba I -5`

Xba I Xba IODTc

PCR

Neor

AMPr

GFPODTc

Xba IXba I Eco RV

pTEX

Electroporation

LIT

G418 (100 g/mL)

1% blood

Selection

Transfected parasites

50 g plasmid

1 x 106 epimastigotes/ 200 L

Co-localization of ODtc in the Endoplasmic Reticulum

ER-TrackerBlue-White DPX

OD-GFP

Phase Merge

Molecular characterization of OD in other trypanosomatids

HmBc

La LsCf

2.5

1.5

1.0

KbCf Hm Ls Tf Tr Tl

10.05.03.02.5

1.51.0

Kb

Southern - gDNA Southern - cDNA

Chromosomal localization

Tc Es Ls M Tc Es Ls

0.450.610.820.95

1.1

Mb

Tc Cf Hm Tcon Tf M Tc Cf Hm TconTf

Mb

0.380.450.610.680.750.820.95

Biochemical studies of the native OD

activity in different trypanosomatids

Freeze-dried

1010 parasites

+ 5 Ci [14C]-oleic acid

16 h, at 28 or 37ºC

Metabolic labeling and lipid analysis

Washed

Lipid extraction (C:M, 2:1; C:M:W, 1:2:0.8)

TAG

C18:1

PI

PCLPC

PE/PME

IPC

ErgDAG

T. cruziEpimastigote

PA/MAG

LPC

LPI

TAG

C18:1

PE/PMEPI

PC

Erg DAG

T. cruziTrypomastigote

TAG

C18:1Erg

DAG

PI/IPC

PE/PME

PA/MAG

LPC

Cf La Hm Ls

TAG

PC

PE/PME

Cf La Hm Ls

Total lipids

HPTLC

Autoradiography

Fatty acid hydrogenation of Trypanosomatid phospholipids

Hydrogenation + - + - + - + - + - + -

C18:1

C18:0

Tc epi Tc tryp Cf La Hm Ls

C18:2

C18:3

Phospholipids (PC, PE, PI)

Alkaline hydrolysis

+/-Hydrogenation

RP-HPTLC

Autoradiography

Methylation

C18:1

C18:0

C18:2

C18:3

• ODTc gene has 2 copies in tandem array and is transcribed in

all T. cruzi stages.

• The enzyme is localized in the endoplasmic reticulum.

• The enzymes -12 and -15 desaturases are active in

trypanosomatids.

• OD seems to be promising chemotherapeutic target in

Chagas’ disease, leishmaniasis and African trypanosomiasis,

as well as in fungal infections.

CONCLUSIONS

The Spider Antimicrobial Peptide Gomesin

as a New Adjuvant

in the Immunotherapy against T. cruzi

• Involved in innate immunity; found from bacteria to mammals

• Cationic molecules composed of 12 to 50 amino acids

• Frequently rich in cysteine residues

• Positively charged in physiological pH, and with amphypathic structure

in -helix, -sheet or both

• Primary action mechanism is the permeabilization of the microbial

membrane

Antimicrobial peptides (AMP)

Gomesin

• Isolated from hemocytes of the Brazilian

tarantula spider Acanthoscurria

gomesiana (Silva Jr. et al, JBC, 2000)

• Mass of 2270 Da, 18 amino acids

(ZCRRLCYKQRCVTYCRGRa), two

disulfide bonds (Cys6-11 e Cys2-15)

• Antibacterial (Gram-negative and Gram-

positive) and antifungal activity

Gomesin kills T. cruzi epimastigotes in vitro

Concentration (M)

Su

rviv

al (

%) DL50 = 6.5 M

0 1 2 3 4 5 6 7 8 9 10

1.5×107 Control

Liposomal Gomesin

Free Gomesin

Empty Liposome

Time (days after infection)

Par

asite

mia

(N

o. o

f pa

rasi

tes/

mL)

In vivo activity of gomesin in the experimental infection of mice BALB/c by T. cruzi

1.0×107

0.5×107

0

In vivo activity of gomesin in the experimental infection of mice BALB/c by T. cruzi

Sur

viva

l (%

)

0 10 20 30 40 50 60 700

20

40

100

0 10 20 30 40 50 60 70

Control

Liposomal Gomesin

Free Gomesin

Empty Liposome

Non Infected +Liposomal Gomesin

Control

Non Infected + Free Gomesin

Time (days after infection)

60

80

How gomesin was protecting the animals if it was not by directly killing the parasites?

• One possibility was that gomesin like defensins (Biragyn et al., Science,

2002) was acting on the mouse innate immune system, inducing pro-

inflammatory cytokines, through the activation of Toll-like receptors.

• TLRs are primary sensors of the innate immunity. They recognize

Pathogen-Associated Molecular Patterns (PAMPs)(e.g. LPS, GPIs, etc),

initiating signaling pathways that stimulate the host defense against

microorganisms.

METHODOLOGY

• Transfected CHO/CD14 cells: CHO/CD14/TLR2

CHO/CD14/TLR4

CHO/CD14/mMD2

CHO/CD14/mTLR4

• Gomesin stock solution was cleaned-up of endotoxins using an affinity column of polimyxin B (depletion of LPS)

• The stimulation of the TLR was measured by the expression of CD25 on the surface of the cells by FACS

mutants not able tosignal via TLR4

Activation of TLR4 by Gomesin

CD

25 e

xp

ress

ion

Fo

ld i

nc

rea

se

None Gom S.a. LPS0.0

2.5

5.0

7.5

10.0

CHO/CD14/mMD2

CHO/CD14/mTLR4

CHO/CD14/TLR2

CHO/CD14/TLR4

Stimulus

12 0.12 0.001 1.2x10-6

CD

25 e

xp

ress

ion

Fo

ld i

nc

rea

se

Gomesin concentration (nM)

Gomesin activates TLR4 at low

nanomolar to picomolar range

Gomesin activates Toll-like receptor 4

Cytoplasm

TLR-4

Plasmamembrane

Signaling cascade

Nucleus

NF-B

Activationof

immune responsegenes

Synthesis ofPro-inflammatory

Cytokinesand

Chemokines

MyD88

IRAK

IB P

CD14

?

2

GOMESIN3

4

56

MD-2

CONCLUSIONS

• Gomesin stimulates TLR4, so it is a new PAMP.

• Gomesin could be used as an adjuvant of the innate

immune response against Chagas’ infection and, probably,

other infectious diseases.

University of São Paulo (USP)

ICB• Lab. Parasite Glycobiology

Igor C. Almeida

Renata K. Kuniyoshi

• Lab. of Biochem. and Immunol. of Arthropodes

Sirlei Daffre

Marcello R. Burgierman

IQ

Maria Terêsa Miranda

Iolanda Cuccovia

Federal University of São Paulo (UNIFESP)

Antônio Miranda

Marcos Fazio

University of DundeeAlan Fairlamb

Financial support

FAPESP/ CNPq WHO/TDRThe Wellcome Trust-UK

ACKNOWLEDGMENTS