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The pathway.

Malaria today afflicts ~500 million people throughout the tropical world.

~1 million, mainly children, die each year

Fig.?1 NMDAR model showing binding sites for agonists and antagonists. The extracellular portions of NR1 and NR2 subunits consist of two domains, the modulatory domain and the agonist binding domain. and D- are agonists for NR1 subu...

Mehdi Ghasemi , Steven C. Schachter

The NMDA receptor complex as a therapeutic target in epilepsy: a review

Epilepsy & Behavior Volume 22, Issue 4 2011 617 - 640 http://dx.doi.org/10.1016/j.yebeh.2011.07.024

Malaria today afflicts ~500 million people throughout the tropical world.

~1 million, mainly children, die each year

KMO inhibition protects mice against cerebral malaria • Plasmodium berghei infected mice die in 7-9 days showing neurological dysfunction • Treat with 200mg/Kg Ro-61-8048 over 12 days (every 1 or 2 days); parasitaemia the same but no neurological dysfunction • Euthanasied at 21 days, pronounced anaemia

Kynurenic Anthranilic Quinolinic Treatment Picolinic acid acid acid acid MIP-1α Control + 309 ± 49 1.3 ± 0.5 2.0 ± 0.3 144 ± 34 0.58 ± 0.06 vehicle Control + Ro- 265 ± 32 10.2 ± 1.5 76.9 ± 23.3 150 ± 32 0.57 ± 0.09 61-8048

P. berghei 1,077 ± 265b 3.5 ± 0.9 2.7 ± 0.6 186 ± 28 4.11 ± 0.18c ANKA + vehicle

P. berghei 300 ± 72 35.6 ± 6.0d 487 ± 96d 170 ± 29 2.26 ± 0.28d ANKA + Ro- 61-8048 Figure 2 Molecular interplay between endothelium and astrocytes, with some functional consequences ? the example of cerebral malaria (CM). In this case the pathogen ( Plasmodium falciparum -infected red blood cell,

Trypanosomes in blood CNS stage model

Trypanosoma brucei brucei GVR35 murine model

Well established & characterised International standard for CNS disease T.b. GVR35 investigations

Monitor parasitem ia in blood 7 14 21 28 35 Early CNS Acute Infection stage Late CNS stage Trypanosome •Parasites proliferate in the haemo- •Parasites established within s identified in lymphatic system and peripheral the CNS the inter- tissues •Stage 1 treatments are no ventricular •Infection can be cured by treatment longer effective foramen with stage 1 drugs Evaluating chemotherapy

T.b. GVR35 Administer Drugs

+ve

Monitor Treatment unsuccessful 21 Up to 180 -ve days Parasites +ve in CNS Treatment successful Up to 180 days Takes too long -ve Develop improved models to assess drugs against Human African Trypanosomiasis

Tryp-2-Viz

In Vivo Imaging System Multi-Photon Laser Scanning Magnetic Resonance Imaging (IVIS) Microscopy (MPLSM) (MRI) Use of IVIS imaging to monitor CNS stage disease Use of IVIS imaging to monitor CNS stage disease

Organs of GVR35-infected mice imaged ex vivo

Day 35 post infection Use of IVIS imaging to monitor CNS stage disease

Brains of GVR35-infected mice imaged ex vivo

Day 13

olfactory bulbs brainstem Suramin

Current Drugs

Pentamidine Melarsoprol

Eflornithine Nifurtimox Use of IVIS imaging to monitor chemotherapy

Melarsoprol treatment at day 21 post infection

Topical application: 3.6 mg x 3 days Use of IVIS imaging to monitor chemotherapy

Diminazene aceturate (Berenil) treatment at day 21 post infection

40mg/kg x 1 injected intraperitoneally Multi-photon Laser Scanning Microscopy

Imaging from the superficial meninges and into the brain parenchyma

Microscope objective

Skull bone (autofluores cent) (Bone removed)

Dura

Arachnoid layer

Blood labelled with Level of tight Collagen dextran-rhodamine, 70 kD junctions jac 120331

Picture by Richard Wheeler, www.richardwheeler.net Multi-photon Laser Scanning Microscopy

Trypanosomes in pial vessels in the brain in vivo

D3 post infection

T. bb 427 + mCherry

Blood vessels labelled with FITC-dextran

Multi-photon Laser Scanning Microscopy

Trypanosomes in the superficial meninges in vivo

1000 Real time 100 (removed)

D13 post in SA space 2 10 infection Thinned skull

N/mm Dura

0 1 0 10 20 30 Arachnoid40 Days since infection GVR35 + Pia mater mCherry 400

300 Coles 2012 GVR35 Blood vessels 200

labelled with in SA space 2 FITC-dextran 100 S427

N/mm 0 5 6 7 8 9 25-30 micron below skull Log(N/mL in blood)

The number of extravascular trypanosomes per unit area of superficial meninges depends on time since infection, not blood parasitaemia.

Roles in inflammation & cancer

The major cellular and molecular targets of . Blockade of N -methyl- D - receptors was the first specific site of action to be identified

Trevor W. Stone , Nicholas Stoy , L. Gail Darlington

An expanding range of targets for kynurenine metabolites of

Trends in Pharmacological Sciences null 2012 null http://dx.doi.org/10.1016/j.tips.2012.09.006 The first enzyme in the , indolamine-2,3- (IDO), plays a key role in regulation of the immune system by virtue of its activation by mediators such as -?. In addition to the effects of kynurenic acid...

Trevor W. Stone , Nicholas Stoy , L. Gail Darlington Kynurenine pathway inhibition as a therapeutic strategy for neuroprotection

FEBS Journal Volume 279, Issue 8, pages 1386-1397, 27 MAR 2012 DOI: 10.1111/j.1742-4658.2012.08487.x http://onlinelibrary.wiley.com/doi/10.1111/j.1742-4658.2012.08487.x/full#f1 FEBS Journal Volume 279, Issue 8, pages 1386-1397, 27 MAR 2012 DOI: 10.1111/j.1742-4658.2012.08487.x http://onlinelibrary.wiley.com/doi/10.1111/j.1742-4658.2012.08487.x/full#f2 Kynurenine pathway inhibition as a therapeutic strategy for neuroprotection

structures are shown of several of the blocking compounds based on the structure of kynurenic acid. Most act at the glycine- B receptor site on the NMDA receptor, the preferred site of action of kynurenic acid. Kynurenine pathway inhibition as a therapeutic strategy for neuroprotection

The structures are shown of the two main inhibitors of the kynurenine pathway that are neuroprotective and prevent excitotoxicity by blocking or KMO. FEBS Journal Volume 279, Issue 8, pages 1386-1397, 27 MAR 2012 DOI: 10.1111/j.1742-4658.2012.08487.x http://onlinelibrary.wiley.com/doi/10.1111/j.1742-4658.2012.08487.x/full#f3

Putative arginase pathway

50000000 5000000 1000000 800000 700000 40000000 4000000 800000 600000 30000000 3000000 600000 500000 400000 20000000 2000000 400000 300000 10000000 1000000 200000 200000 100000 0 0 0 0 24 48 72 0 24 48 72 0 24 48 72 0 0 24 48 72 hours hours hours hours

Not Aminopropyl- Arginase decarboxylase transferase

L- L-Ornithine

1400000 250000 1200000 200000 1000000 800000 150000 600000 100000 400000 200000 50000 N-Acetylputrescine N-Acetylornithine 0 0 0 24 48 72 0 24 48 72 hours hours Putative arginase

Most significant effects (top 10 & bottom 10) Cofactors Cofactors ??? Mass RT Formula Isomers Metabolite Pathway 1 2 135.068 Multiple substrates 4 5.51 C8H9NO 4 2-Phenylacetamide metabolism 5.71 1.22 161.047 6 7.95 C9H7NO2 7 2-Indolecarboxylic acid N/A 5.4 1.71 175.063 11.1 3 0 C10H9NO2 12 3-Indoleglycolaldehyde Tryptophan metabolism 5 2.41 335.148 1 9.42 C16H21N3O5 1 Gly-Pro-Tyr Peptide(tri-) 4.73 1.71 Multiple products 189.042 5 7.34 C10H7NO3 6 Kynurenate Tryptophan metabolism 4.48 0.99 175.063 2 5.71 C10H9NO2 12 Indole-3-acetate Tryptophan metabolism 4.45 1.36 196.063 7 5.40 C12H8N2O 2 2-hydroxyphenazine Secondary Metabolism 4.24 1.62 145.052 8 5.54 C9H7NO 7 3-Methyleneoxindole N/A 3.73 1.3 Cofactors 1 is essential 250.062 (NAD+) 4 9.31 C8H14N2O5S 2 Glu-Cys Peptide(di-) 3.45 1.38 236.079 10.5 6 5 C11H12N2O4 2 L-Formylkynurenine Tryptophan metabolism 3.41 1.51

301.142 8 9.41 C16H19N3O3 3 Trp-Pro Peptide(di-) 0.33 0.74 264.153 18.1 Putative arginase

Most significant effects (top 10 & bottom 10) Cofactors Cofactors Mass RT Formula Isomers Metabolite Pathway 1 2 135.068 Phenylalanine Multiple substrates 4 5.51 C8H9NO 4 2-Phenylacetamide metabolism 5.71 1.22 • Tryptophan 161.047 6 7.95 C9H7NO2 7 2-Indolecarboxylic acid N/A 5.4 1.71 • Trp peptides 175.063 11.1 3 0 C10H9NO2 12 3-Indoleglycolaldehyde Tryptophan metabolism 5 2.41 335.148 1 9.42 C16H21N3O5 1 Gly-Pro-Tyr Peptide(tri-) 4.73 1.71 Multiple products 189.042 5 7.34 C10H7NO3 6 Kynurenate Tryptophan metabolism 4.48 0.99 • Tryptophan pathway 175.063 • Oxidoreductase (or 2 5.71 C10H9NO2 12 Indole-3-acetate Tryptophan metabolism 4.45 1.36 196.063 aminotransferase) 7 5.40 C12H8N2O 2 2-hydroxyphenazine Secondary Metabolism 4.24 1.62 145.052 8 5.54 C9H7NO 7 3-Methyleneoxindole N/A 3.73 1.3 Cofactors 1 is essential 250.062 4 9.31 C8H14N2O5S 2 Glu-Cys Peptide(di-) 3.45 1.38 (NAD+) 236.079 10.5 6 5 C11H12N2O4 2 L-Formylkynurenine Tryptophan metabolism 3.41 1.51

301.142 8 9.41 C16H19N3O3 3 Trp-Pro Peptide(di-) 0.33 0.74 264.153 18.1 Putative arginase

Tryptophan metabolism:

Putative arginase

L-Tryptophan: possible enzyme

Tryptophan 2,3-dioxygenase

O2 L-Tryptophan L-Formylkynurenine

18000000 500000 16000000 450000 400000 14000000 350000 12000000 300000 10000000 250000 8000000 200000 6000000 150000 4000000 100000 2000000 50000 0 0 BYC1 BYC2 BYE1 BYE2 BYC1 BYC2 BYE1 BYE2 Putative arginase

Tryptophan metabolism:

Putative arginase

L-Tryptophan: possible enzyme

Tryptophan 2'-dioxygenase

O2 NH3 CO2 L-Tryptophan 3-Indoleglycolaldehyde

18000000 140000

16000000 120000 14000000 100000 12000000 10000000 80000

8000000 60000 6000000 40000 4000000 2000000 20000 0 0 BYC1 BYC2 BYE1 BYE2 BYC1 BYC2 BYE1 BYE2 Putative arginase

Tryptophan metabolism:

Putative arginase

L-Tryptophan: possible enzyme

Tryptophan dehydrogenase

+ + NAD H2O NH3 H NADH L-Tryptophan Indolepyruvate

18000000 16000000 14000000 12000000 10000000 ? 8000000 6000000 Not 4000000 detected 2000000 0 BYC1 BYC2 BYE1 BYE2 Putative arginase

Tryptophan metabolism:

Putative arginase

Xanthurenate: possible enzyme

Kynurenine aminotransferase

H₂O

3-Hydroxy -L-kynurenine 2-Oxoglutarate L-Glutamate Xanthurenate

350000 90000 300000 80000 250000 70000

200000 60000 50000 150000 40000 100000 30000 20000 50000 10000 0 0 BYC1 BYC2 BYE1 BYE2 BYC1 BYC2 BYE1 BYE2 Putative arginase

Kynurenate: possible enzyme

Kynurenine aminotransferase

H₂O

Glyoxylate L-Kynurenine Glycine Kynurenate

180000 160000 140000 120000 ? 100000 80000 Not 60000 detected 40000 20000 0 BYC1 BYC2 BYE1 BYE2

*50 patients *35 controls *Bloods taken ASAP after presentation then at day *1,2,3,4,7 & 14 *HPLC, fluorescence to measure: tryp, kynurenine KA, AA and 3HAA Altered kynurenine metabolism correlates with infarct volume in stroke

European Journal of Neuroscience Volume 26, Issue 8, pages 2211-2221, 24 SEP 2007 DOI: 10.1111/j.1460-9568.2007.05838.x http://onlinelibrary.wiley.com/doi/10.1111/j.1460-9568.2007.05838.x/full#f1

Kynurenine pathway appears to elevate in these stroke patients, as measured in blood

Stroke Biomarkers

• 40 healthy controls • 40 stroke • 20 TIA • Urine samples Urinary peptides Metabolite profiling distinguishes stroke and TIA

Jesse Dawson Colette Keenan Karl Burgess

STROKE TIA

C10H7NO3 C13H16N204 C5H8O5 C9H13N5O4 C4H6N4O3 C5H7N03 C H NO Kynurenate 10 7 C9H3 9NO3 H3O3P C4H8O5 C H N 0 Formyl-N-acetyl-5-methoxykynurenamine 13 16 C62H410 05 C8H12O4 C6H10NO3 C H NO N-acetyl-anthrinilate 9 9 C53H 7NO4 C11H17NO8 C6H1005 C H NO Dihydroxyquinoline 9 7 C62H 11N04 C8H1404 C11H12N2O4 C H 0 Benzenediol 6 6 2C 9H7NO2 C4H6O4 C5H9N04S2 C H 0 L-Formyl-kynurenineC5H9N04S2 6 10 C5 15H24O3 C6H602 C 3H6O5S C H O C10H19N3O5 C83H166 O5 S C H N O CHC HOOS 7 12 2 6 8 4163

C16H30O2 CH O S 4 3

O

OH serotonin NH2 N tryptophan H

CH3 H N O 2 H2N O The Kynurenine O O O

CH3 OH N-formyl kynurenine NH NH formyl –N-acetyl- pathway 5-methoxy OH O kynurenamine

H2N O O

OH

NH2 kynurenine

NH2 O O

OH OH OH 3-OH kynurenine NH2 benzenediol OH kynurenic acid O N OH OH O O O

OH OH OH

NH NH OH 2 NH OH 2 HO O N-acetyl 3-OH anthranilinic acid anthranilinic acid dihydroxyquinoline anthranilinic acid

N O O H OH OH 2-amino-3- carboxymuconate HO semialdehyde Changed in stroke NH2 O

O

OH Changed in TIA O O N picolinic acid N OH OH

GVR35 kynurenine Stroke Jean Rodgers Jesse Dawson Peter Kennedy Colette Keenan Trevor Stone Karl Burgess

In vivo Imaging Enzyme work Elmarie Myburgh Darren Creek Ryan Ritchie Felicity Lumb Jeremy Mottram Brunda Nijagal Katharina Johnston