The Kynurenine

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. Glycine and D-serine 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

Treatment Picolinic acid Kynurenic

acid Anthranilic

acid Quinolinic

acid MIP-1α

Control + vehicle

309 ± 49 1.3 ± 0.5 2.0 ± 0.3 144 ± 34 0.58 ± 0.06

Control + Ro-61-8048

265 ± 32 10.2 ± 1.5 76.9 ± 23.3 150 ± 32 0.57 ± 0.09

P. berghei ANKA + vehicle

1,077 ± 265b 3.5 ± 0.9 2.7 ± 0.6 186 ± 28 4.11 ± 0.18c

P. berghei ANKA + Ro-61-8048

300 ± 72 35.6 ± 6.0d 487 ± 96d 170 ± 29 2.26 ± 0.28d

Figure 2 Molecular interplay between endothelium and astrocytes, with some functional consequences ? the example of cerebral malaria (CM). In this case the pathogen (<ce:italic> Plasmodium falciparum</ce:italic> -infected red blood cell, <ce:ital...

Trypanosomes in blood

Trypanosoma brucei brucei GVR35 murine model

T.b. GVR35

CNS stage model

Well established & characterised International standard for CNS disease

investigations

Acute Infection Early CNS

stage

•Parasites proliferate in the haemo-lymphatic system and peripheral tissues •Infection can be cured by treatment with stage 1 drugs

•Parasites established within the CNS •Stage 1 treatments are no longer effective

Late CNS stage

7 14 21 28 35

Monitor parasitemia in blood

Trypanosomes identified in

the inter-ventricular

foramen

Evaluating chemotherapy

T.b. GVR35

21

Administer Drugs

Monitor

Up to 180 days

-ve

+ve

Treatment unsuccessful

-ve

Up to 180 days

Treatment successful

+ve Parasites

in CNS

Takes too long

Develop improved models to assess drugs against Human African Trypanosomiasis

Magnetic Resonance Imaging (MRI)

In Vivo Imaging System (IVIS)

Multi-Photon Laser Scanning Microscopy (MPLSM)

Tryp-2-Viz

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

olfactory bulbs brainstem

Day 13

Current Drugs

Suramin

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

Microscope objective

Blood labelled with dextran-rhodamine, 70 kD

Skull bone (autofluores

cent)

Imaging from the superficial meninges and into the brain parenchyma

Multi-photon Laser Scanning Microscopy

Dura

Arachnoid layer

Level of tight junctions

Collagen

(Bone removed)

jac 120331

Picture by Richard Wheeler, www.richardwheeler.net

D3 post infection T. bb 427 + mCherry Blood vessels labelled with FITC-dextran

Trypanosomes in pial vessels in the brain in vivo

Multi-photon Laser Scanning Microscopy

Trypanosomes in the superficial meninges in vivo

D13 post infection GVR35 + mCherry Blood vessels labelled with FITC-dextran

Real time

Multi-photon Laser Scanning Microscopy

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

GVR35

S427

6 7 8 9

0

100

200

300

400

Log(N/mL in blood)N

/mm

2in

SA

sp

ace

5

0 10 20 30 401

10

100

1000

Days since infection

N/m

m2

in S

A s

pace

0

(removed)

Dura

Thinned skull

Arachnoid

Pia mater

Coles 2012

25-30 micron below skull

The major cellular and molecular targets of kynurenic acid. Blockade of <ce:italic> N</ce:italic> -methyl-<ce:small-caps> D</ce:small-caps> -aspartic acid receptors was the first specific site of action to be identified <ce:cross-ref ref...

Roles in inflammation & cancer

Figure 3 Potential sites at which kynurenine, long regarded as biologically inactive, may act to regulate the balance of cells produced in the immune system.

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

An expanding range of targets for kynurenine metabolites of tryptophan

Trends in Pharmacological Sciences null 2012 null

http://dx.doi.org/10.1016/j.tips.2012.09.006

The first enzyme in the kynurenine pathway, indolamine-2,3-dioxygenase (IDO), plays a key role in regulation of the immune system by virtue of its activation by mediators such as interferon-?. 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

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#f2

structures are shown of several of the glutamate receptor 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

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

The structures are shown of the two main inhibitors of the kynurenine pathway that are neuroprotective and prevent excitotoxicity by blocking kynureninase or KMO.

Ornithine decarboxylase

Putative arginase Polyamine pathway

0

1000000

2000000

3000000

4000000

5000000

0 24 48 72

hours

0

200000

400000

600000

800000

1000000

0 24 48 72

hours

L-Ornithine Putrescine Spermidine

N-Acetylornithine N-Acetylputrescine 0

200000

400000

600000

800000

1000000

1200000

1400000

0 24 48 72

hours

0

50000

100000

150000

200000

250000

0 24 48 72

hours

0

100000

200000

300000

400000

500000

600000

700000

800000

0 24 48 72 hours

Aminopropyl- transferase

Not Arginase

0

10000000

20000000

30000000

40000000

50000000

0 24 48 72

hours

L-Arginine

Putative arginase

Most significant effects (top 10 & bottom 10)

Mass RT Formula Isomers Metabolite Pathway Cofactors

1 Cofactors

2

135.0684 5.51 C8H9NO 4 2-Phenylacetamide

Phenylalanine metabolism 5.71 1.22

161.0476 7.95 C9H7NO2 7 2-Indolecarboxylic acid N/A 5.4 1.71

175.0633

11.10 C10H9NO2 12 3-Indoleglycolaldehyde Tryptophan metabolism 5 2.41

335.1481 9.42 C16H21N3O5 1 Gly-Pro-Tyr Peptide(tri-) 4.73 1.71

189.0425 7.34 C10H7NO3 6 Kynurenate Tryptophan metabolism 4.48 0.99

175.0632 5.71 C10H9NO2 12 Indole-3-acetate Tryptophan metabolism 4.45 1.36

196.0637 5.40 C12H8N2O 2 2-hydroxyphenazine Secondary Metabolism 4.24 1.62

145.0528 5.54 C9H7NO 7 3-Methyleneoxindole N/A 3.73 1.3

250.0624 9.31 C8H14N2O5S 2 Glu-Cys Peptide(di-) 3.45 1.38

236.0796

10.55 C11H12N2O4 2 L-Formylkynurenine Tryptophan metabolism 3.41 1.51

301.1428 9.41 C16H19N3O3 3 Trp-Pro Peptide(di-) 0.33 0.74

264.153 18.1

???

Multiple substrates

Multiple products

Cofactors 1 is essential (NAD+)

Putative arginase

Most significant effects (top 10 & bottom 10)

Mass RT Formula Isomers Metabolite Pathway Cofactors

1 Cofactors

2

135.0684 5.51 C8H9NO 4 2-Phenylacetamide

Phenylalanine metabolism 5.71 1.22

161.0476 7.95 C9H7NO2 7 2-Indolecarboxylic acid N/A 5.4 1.71

175.0633

11.10 C10H9NO2 12 3-Indoleglycolaldehyde Tryptophan metabolism 5 2.41

335.1481 9.42 C16H21N3O5 1 Gly-Pro-Tyr Peptide(tri-) 4.73 1.71

189.0425 7.34 C10H7NO3 6 Kynurenate Tryptophan metabolism 4.48 0.99

175.0632 5.71 C10H9NO2 12 Indole-3-acetate Tryptophan metabolism 4.45 1.36

196.0637 5.40 C12H8N2O 2 2-hydroxyphenazine Secondary Metabolism 4.24 1.62

145.0528 5.54 C9H7NO 7 3-Methyleneoxindole N/A 3.73 1.3

250.0624 9.31 C8H14N2O5S 2 Glu-Cys Peptide(di-) 3.45 1.38

236.0796

10.55 C11H12N2O4 2 L-Formylkynurenine Tryptophan metabolism 3.41 1.51

301.1428 9.41 C16H19N3O3 3 Trp-Pro Peptide(di-) 0.33 0.74

264.153 18.1

Multiple substrates

• Tryptophan

• Trp peptides

Multiple products

• Tryptophan pathway

• Oxidoreductase (or aminotransferase)

Cofactors 1 is essential (NAD+)

Putative arginase

Tryptophan

metabolism:

Putative arginase

L-Tryptophan: possible enzyme

Tryptophan 2,3-dioxygenase

L-Tryptophan L-Formylkynurenine

0

2000000

4000000

6000000

8000000

10000000

12000000

14000000

16000000

18000000

BYC1 BYC2 BYE1 BYE2

0

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

BYC1 BYC2 BYE1 BYE2

O2

Putative arginase

Tryptophan

metabolism:

Putative arginase

L-Tryptophan: possible enzyme

Tryptophan 2'-dioxygenase

L-Tryptophan 3-Indoleglycolaldehyde

0

2000000

4000000

6000000

8000000

10000000

12000000

14000000

16000000

18000000

BYC1 BYC2 BYE1 BYE2 0

20000

40000

60000

80000

100000

120000

140000

BYC1 BYC2 BYE1 BYE2

O2 NH3 CO2

Putative arginase

Tryptophan

metabolism:

Putative arginase

L-Tryptophan: possible enzyme

Tryptophan dehydrogenase

L-Tryptophan Indolepyruvate

0

2000000

4000000

6000000

8000000

10000000

12000000

14000000

16000000

18000000

BYC1 BYC2 BYE1 BYE2

H2O NH3 H+

? Not detected

NAD+ NADH

Putative arginase

Tryptophan

metabolism:

L-Glutamate

Putative arginase

Xanthurenate: possible enzyme

Kynurenine aminotransferase

Xanthurenate 3-Hydroxy -L-kynurenine

0

50000

100000

150000

200000

250000

300000

350000

BYC1 BYC2 BYE1 BYE2 0

10000

20000

30000

40000

50000

60000

70000

80000

90000

BYC1 BYC2 BYE1 BYE2

2-Oxoglutarate

H₂O

Putative arginase

Kynurenate: possible enzyme

Kynurenate

?

Glycine Glyoxylate L-Kynurenine

0

20000

40000

60000

80000

100000

120000

140000

160000

180000

BYC1 BYC2 BYE1 BYE2

Not detected

Kynurenine aminotransferase

H₂O

*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

C10H7NO3

C13H16N204

C9H13N5O4 C9H9NO3 C6H1005

C5H7NO4 C6H11N04 C9H7NO2

C15H24O3 C10H19N3O5

C7H12N2O6

C16H30O2

C4H6N4O3

H3O3P

C8H12O4 C11H17NO8 C8H1404

C4H6O4 C6H602

C5H8O5 C5H7N03

C4H8O5 C6H10NO3 C6H1005

C11H12N2O4 C5H9N04S2 C3H6O5S

C8H16O CH4O3S

STROKE TIA

C10H7NO3

C13H16N204

C9H13N5O4 C9H9NO3 C6H1005

C5H7NO4 C6H11N04 C9H7NO2

C15H24O3 C10H19N3O5

C7H12N2O6

C16H30O2

C4H6N4O3

H3O3P

C8H12O4 C11H17NO8 C8H1404

C4H6O4 C6H602

C5H8O5 C5H7N03

C4H8O5 C6H10NO3 C6H1005

C11H12N2O4

C5H9N04S2

C3H6O5S C8H16O

CH4O3S

STROKE TIA

C10H7NO3

C13H16N204

C9H13N5O4 C9H9NO3 C6H1005

C5H7NO4 C6H11N04 C9H7NO2

C15H24O3 C10H19N3O5

C7H12N2O6

C16H30O2

C4H6N4O3

H3O3P

C8H12O4 C11H17NO8 C8H1404

C4H6O4 C6H602

C5H8O5 C5H7N03

C4H8O5 C6H10NO3 C6H1005

C11H12N2O4 C5H9N04S2 C3H6O5S

C8H16O CH4O3S

STROKE TIA

C10H7NO3 Kynurenate C13H16N204 Formyl-N-acetyl-5-methoxykynurenamine C9H9NO3 N-acetyl-anthrinilate C9H7NO2 Dihydroxyquinoline

C6H602 Benzenediol

C6H1005 L-Formyl-kynurenine

NH

NH2

O

OH

N

OH

O

OH

NH2

OH

O

OH

NH2

O

OH

O

OH

OH

NO

OH

O

OH

NO

OH

tryptophan

kynurenine

3-OH kynurenine

3-OH anthranilinic acid

2-amino-3- carboxymuconate semialdehyde

picolinic acid quinolinic acid

kynurenic acid NH2

O

OH

OH

ONH2

NH2

O

OH

ONH2

N-formyl kynurenine

NH

O

OH

ONH2

OH

NH

OONH2

O

CH3

O

CH3

serotonin

formyl –N-acetyl-5-methoxy kynurenamine

N

OH

OH

dihydroxyquinoline

NH

O

OH

OOH

NH2

O

OH

anthranilinic acid N-acetyl anthranilinic acid

benzenediol

Changed in stroke

Changed in TIA

OH

OH

The Kynurenine pathway

GVR35 kynurenine Jean Rodgers Peter Kennedy Trevor Stone In vivo Imaging Elmarie Myburgh Ryan Ritchie Jeremy Mottram

Stroke Jesse Dawson Colette Keenan Karl Burgess Enzyme work Darren Creek Felicity Lumb Brunda Nijagal Katharina Johnston