This is an Accepted Article that has been peer-reviewed and approved for publication in the FEBS Journal, but has yet to undergo copy-editing and proof correction. Please cite this article as an “Accepted Article”; doi: 10.1111/j.1742-4658.2012.08487.x

Kynurenine pathway inhibition as a therapeutic strategy for neuroprotection

Trevor W Stone1, Caroline M Forrest1, L Gail Darlington2

1Institute for Neuroscience and Psychology,

College of Medical, Veterinary & Life Sciences,

West Medical Building,

University of Glasgow,

Glasgow G12 8QQ, U.K.

and

2Epsom General Hospital

Epsom

Surrey KT18 7EG

Article type : Minireview Trevor.Stone@glasgow.ac.uk

Running title:-

Kynurenines and neuronal viability

Correspondence:-

Prof. T W Stone, West Medical Building, University of Glasgow, Glasgow G12 8QQ,

UK

Key-words:-

Tryptophan; kynurenine; quinolinic acid; kynurenic acid; neurodegeneration;

neuroprotection;

Abstract

The oxidative pathway for the metabolism of tryptophan along the kynurenine

pathway generates quinolinic acid, an agonist at N-methyl-D-aspartate (NMDA)

receptors, as well as kynurenic acid which is an antagonist at glutamate and nicotinic

receptors. The pathway has become recognised as a key player in the mechanisms

of neuronal damage and neurodegenerative disorders. As a result, manipulation of

the pathway, so that the balance between the levels of components of the pathway

can be modified, has become an attractive target for the development of

pharmacological agents with the potential to treat those disorders. This review

summarises some of the relevant background information on the pathway itself

before identifying some of the chemical strategies for its modification, with examples

of their successful application in animal models of infection, stroke, traumatic brain

damage, cerebral malaria and cerebral trypanosomiasis.

Key-words:-

Tryptophan; kynurenine; quinolinic acid; kynurenic acid; neurodegeneration;

neuroprotection;

Abbreviations

AA: anthranilic acid

AMPA: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid.

CNS : central nervous system

CSF: cerebrospinal fluid

FCE28833A: 3,4-dichlorobenzoylalanine

3HAA: 3-hydroxyanthranilic acid

IDO: indoleamine-2,3-dioxygenase

IL-1β : interleukin-1β

KAT: kynurenine aminotransferase

KMO: kynurenine-3-monoxygenase

L689,560: 2-carboxy-5,7-dichloro-4-[[(N-phenylamino)-carbonyl]amino]-1,2,3,4-

tetrahydroquinoline

L701,252: 4-hydroxy-3-(cyclopropylcarbonyl)-7-chloroquinoline-2(1H)-one

L701,324: 4-hydroxy-7-chloro-3-(3-phenyloxy)phenyl-quinoline-2(1H)-one

MDL 100,748: 4-[(carboxymethyl)amino]-5,7-dichloroquinoline-2-carboxylic acid

MDL29,951: 3-(4,6-dichloro-2-carboxyindole-3-yl)propionic acid

MPP+ : 1-methyl-phenylpyridinium.

NAD: nicotinamide adenine dinucleotide

NMDA: N-methyl-D-aspartate

Ro61-8048: 3,4-dimethoxy-N-[4-(3-nitrophenyl)-thiazol-2-yl]-benzenesulfonamide

TDO: tryptophan-2,3-dioxygenase

Th cells: T helper cells

ZD9379: 7-chloro-4-hydroxy-2-(4-methoxy-2-methylphenyl)-1,2,5,10-

tetrahydropyridazino-[4,5b]quinoline-1,10-dione sodium

Introduction

Although quinolinic acid (2,3-pyridine-dicarboxylic acid) was recognised as a

metabolite of tryptophan for many years, it was thought to be merely an inactive

precursor in the synthesis of nicotinic acid and, thus, nicotinamide and the ubiquitous

enzyme co-factor nicotinamide adenine dinucleotide (NAD). It was known that direct

administration of high concentrations into the brain could induce convulsions [1],

although the mechanism was unknown. This situation changed when quinolinic acid

was shown to activate selectively the population of glutamate receptors that are also

sensitive to N-methyl-D-aspartate (NMDA) [2], receptors which were soon thereafter

to be implicated in synaptic transmission and neuronal plasticity phenomena such as

long-term potentiation and long-term depression. This discovery led to the direct

demonstration that over-activation of NMDA receptors by quinolinic acid could

produce neuronal degeneration in the brain [3], raising the possibility of a role in

several forms of accidental or disease-related brain injury [4-6].

After exploring the potential neuro-activity of other tryptophan metabolites along the

same pathway (kynurenines) it was later found that kynurenic acid was an antagonist

at NMDA receptors, with a lesser ability to block other glutamate receptors

responding to kainate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

(AMPA) [7]. Several families of glutamate receptor blockers for potential therapeutic

use were designed based on the structure of kynurenic acid [8-10], as will be

discussed below.

Excitotoxicity

Although quinolinic acid can produce damage by its activation of NMDA receptors

[3,11] it can also induce, or facilitate the production of, reactive oxygen species such

as hydrogen peroxide, superoxide and hydroxyl radicals [12,13]. Indeed these

molecular species may contribute to, and potentiate, the neurotoxic effects of

quinolinic acid in vivo [14]. The kynurenine pathway and quinolinic acid generation

are activated by pro-inflammatory factors, and this interaction may be relevant to the

notion that inflammation triggered within the central nervous system (CNS) may

contribute to the development of neurodegenerative disorders such as Alzheimer's

disease and Huntington's disease. This possibility is strengthened by data showing a

potentiation of quinolinic acid neurotoxicity by the pro-inflammatory cytokine

interleukin-1β (IL-1β) [15], which has itself been implicated in the damaging effects of

stroke. The cytokine may produce neurodegeneration itself, and increase cell death

produced by quinolinic acid or other NMDA receptor agonists, at least partly by

potentiating amino acid-induced calcium influx [16]. This is, however, a controversial

area, since there is some evidence that IL-1β can inhibit excitotoxic neuronal damage

[17] and a deficiency may increase neuronal injury [18]. Some of these differences

may be attributable to the use of different experimental models and cytokine

concentrations. Interestingly, the toxicity of this combination can be prevented by

antagonists at the A2A receptors for adenosine, compounds which have been shown

to be neuroprotective against injurious stimuli and chemical insults in a variety of

conditions [19]. This might therefore be consistent with a role for quinolinic acid in

neurotoxicity induced by inflammatory stimuli or a range of alternative triggers such

as the dopaminergic toxin 1-methyl-phenylpyridinium (MPP+) and the glutamate

receptor agonist kainic acid.

A commonly voiced concern about the possible role of quinolinic acid in neuronal

damage is that its concentrations in the blood and cerebrospinal fluid (CSF), which

are usually less than 50nM, are much lower than the millimolar levels often required

to induce neuronal damage. However, the levels of compounds in body fluids

represent substantial dilutions from their cellular sites of origin and release, and the

local concentrations of quinolinic acid may be many times greater in the extracellular

space around those activated glial cells and macrophages which produce it. Indeed,

since quinolinic acid is generated in the same types of immune-competent cells that

can generate IL-1β [20,21], their combined presence at higher levels could further

enhance local toxicity. These possibilities are entirely consistent with the increasing

evidence that microglial activation makes a substantial contribution to various form of

brain damage.

In addition, even after its dilution from its sites of generation, quinolinic acid can

attain potentially toxic levels in the blood or cerebrospinal fluid (CSF) following

cerebral insults such as trauma, ischaemia [11, 22, 23] or microbial infections [24].

Finally, there may be subsets of neurons which are particularly sensitive to quinolinic

acid toxicity. Some cells can be killed by maintained exposure to concentrations of

around 100nM quinolinic acid, levels only slightly higher than the resting levels of

approximately 10-50nM.

The relationship between quinolinic acid and neurodegenerative disorders has

attracted a great deal of interest. Special interest has developed into a possible role

in Huntington's disease in the light of molecular studies in yeast models [25] and a

strong association between activity along the kynurenine pathway and the length of

the CAG triplet nucleotide repeat sequence in patients at different stages of the

disorder [26]. This link between neurotoxicity and neurodegeneration is addressed in

other chapters in this issue.

Other kynurenines.

Although the emphasis to date in the understanding of neurodegeneration has

been focussed on quinolinic acid, this compound represents only one component of

the kynurenine pathway of tryptophan oxidation (Figure 1)[10, 11, 27]. The pathway

also generates a glutamate antagonist, kynurenic acid [10, 28] and highly redox-

active compounds such as 3-hydroxykynurenine and 3-hydroxyanthranilic acid.

Kynurenic acid is recognised largely for its ability to block glutamate receptors,

especially the NMDAR at which it blocks the actions of the co-agonist glycine. In

studies of cultured neurons as well as brain slices, kynurenic acid blocked the effects

of exogenously applied acetylcholine or α7-nicotinic receptor-selective agonists [29].

Subsequent work revealed that kynurenic acid could block that component of

excitatory post-synaptic potentials (epsps) mediated by the activation of cholinergic,

nicotinic α7 receptors in hippocampal interneurons [30] - epsps also blocked by

methyl-lycaconitine, an established antagonist at the α7 receptor. Dihydro-β-

erythroidine, which blocks α4β2 receptors, was ineffective, confirming the importance

of the α7 receptors. Kynurenic acid reduced the amplitude of these epsps with an

EC50 of 136 μM and was more potent in blocking the nicotinic epsps than the full,

glutamate-mediated epsps. The ability of kynurenic acid to block nicotinic synaptic

transmission may be important to kynurenic acid pharmacology in the hippocampus,

although it is less potent than when used to block exogenously applied

cholinomimetics in culture. The difference in potency could also be a result of the

different state of differentiation of cells in culture, the relative absence of glial cells or

the different relationships between synaptic terminals and glia with their complement

of enzymes and transporters.

Several clinical studies have examined the levels of kynurenic acid in patient

groups. The levels in blood increased significantly in a sub-population of patients who

died within 21 days of a stroke compared with patients who survived for a longer

period [31]. A possible relationship between the levels of kynurenic acid and patient

mortality has been noted by several groups [32, 33], possibly analogous to reports

that indoleamine-2,3-dioxygenase (IDO; Fig. 1) activity increases with age. One

reason for such a relationship may lie in the fact that, in addition to its formation by

kynurenine aminotransferase (KAT), kynurenic acid can be formed by the non-

enzymic oxidation of kynurenine and tryptophan via indole-3-pyruvic acid [34], a

reaction which is increased by oxidative stress. Increased levels of nitric oxide have

been noted after brain injury, and this can inhibit superoxide dismutase. The resulting

increase in superoxide anions could oxidise indolepyruvate to kynurenic acid,

consistent with reports that nitric oxide donors increase kynurenic acid production

[35].

In fact, KAT exists in two major forms, KAT I being primarily cytosolic in location,

and KAT II being primarily mitochondrial. The latter is identical to α-aminoadipate

aminotransferase. A third form of KAT has been identified, although much less is

known about its selectivity and activity. There is some influence of diet and nutritional

status on this and other kynurenine pathway enzymes, since KAT, kynureninase and

kynurenine-3-monoxygenase are at least partly dependent on the availability of

vitamin B6 (pyridoxine; PLP) for their activity.

3-hydroxyanthranilic acid can generate reactive oxygen species such as hydrogen

peroxide and superoxide when in the presence of transition metal ions but it is also a

highly efficient scavenger of free radicals [36]. Under physiological conditions, 3-

hydroxyanthranilic acid can auto-oxidise to quinoneimines, but the reaction products

can then oxidise other molecules [37, 38]. The balance of this redox cycling

behaviour will depend on local concentrations of iron and copper, the levels and

activities of other free radical generators and anti-oxidants, and pH, which influences

both redox activity and metal ion availability [39].

Some of the kynurenine compounds are of increasing interest in disorders of brain

function. The experimental evidence for a role of kynurenines in stroke injuries is

firmly based on the demonstration that inhibition of the pathway reduces the neuronal

damage which follows cerebral vessel occlusion in rodents [40]. In a recent study on

humans, blood samples were taken from patients as soon as possible after entering

hospital in the immediate aftermath of a stroke and for up to 14 days thereafter [31].

As expected from earlier work by others, the existence of brain damage was

indicated by an early increase in the levels of protein S100B, although this persisted

for several days, perhaps reflecting the progressive development of delayed neuronal

damage. Neopterin levels, a well-established marker of inflammation [41] were also

substantially elevated, together with activation of the kynurenine pathway, since

blood levels of kynurenine and tryptophan revealed an increased kynurenine:

tryptophan ratio consistent with activation of the initial enzymes of the pathway –

[IDO] and tryptophan-2,3-dioxygenase [TDO]. There was also a highly significant

decrease in the ratio of 3-hydroxyanthranilic acid: anthranilic acid which was strongly

correlated with infarct volume indicated in computed tomography brain scans [31].

The reason for the changed ratio is not clear, though the reciprocal changes

suggests a biochemical connection such as the conversion of anthranilic acid (AA)

into 3-hydroxyanthranilic acid (3HAA) [42]. The changed ratio, observed now in

several disorders including osteoporosis, chronic brain injury, Huntington’s disease,

coronary heart disease, thoracic disease, stroke and depression [43] could then

indicate that inflammation generates a decrease in that conversion.

The loss of 3HAA may have important consequences for the immune system. 3-

hydroxyanthranilic acid inhibits the proliferation of CD8+ T cells [44]. It can also

suppress the responses of T cells to allogeneic stimuli [45], acting primarily on Th1

rather than Th2 cells [46], and decreases the ability of dendritic cells to activate T

cells [47]. The overall result of the changed 3HAA:AA ratio, therefore, would seem to

be protective, limiting the inflammatory response, including the activation of microglia

which are thought to contribute to brain damage following stroke.

Anthranilic acid interacts with copper to form an anti-inflammatory complex able to

remove highly injurious reactive oxygen species [48,49]. Several anthranilic acid

derivatives have similar, marked anti-inflammatory activity [50] and it is an intriguing

possibility that the high levels of anthranilic acid in some disorders such as strokes

might be converted to anti-inflammatory compounds as a mechanism to reduce

tissue damage.

The kynurenine pathway as a pharmacological target

Interest in the kynurenine pathway as a potential site of drug action has centred

around the possibility of modifying the balance between the endogenous

concentrations of quinolinic acid and it’s antagonist, kynurenic acid. This interest is

expanding in view of the wide range of clinical disorders in which abnormalities in the

pathway have been proposed, including AIDS-related dementia [51, 52] and stroke

[31], but with probably the greatest interest in the neurodegenerative disorders. One

of the popular hypotheses for the aetiology of disorders such as Alzheimer's disease,

Parkinson's disease and Huntington's disease is that there is an ongoing over-

activation of glutamate receptors, especially NMDA receptors. This hypothesis has

received much support in principle from the discovery that the spinal degenerative

disorder amyotrophic lateral sclerosis, or motoneurone disease, is the result of a

defective glutamate transporter. The resulting accumulation of extracellular glutamate

generates oxidative stress that ultimately leads to the demise of the motoneurones.

By analogy, a loss of transporters or increased presence of receptors for glutamate in

localised regions of the brain such as the nucleus basalis, neostriatum or substantia

nigra could cause or contribute to the neuronal death in Alzheimer's disease,

Huntington's disease and Parkinson's disease respectively. It is probable also that

much of the chronic brain damage occurring after stroke injury is the result of

‘delayed neurodegeneration’ which is caused, not by the initial insult itself, but by

secondary processes entrained by that insult.

These secondary processes may be classified generally as inflammatory, since

there is growing evidence that they are mediated by cytokines and chemokines

produced by glial cells that are immunologically activated by products of the initial

insult. These compounds play a key role in attracting and modulating the activity of

peripheral monocytes and macrophages that invade the CNS and participate in the

removal of damaged tissue and the control of potential infections. One consequence

of this central inflammatory response, however, seems to be the enhancement of

neuronal damage to some extent (as noted above). The activation of immune-

competent cells – peripheral macrophages or central microglia – also includes

induction of the kynurenine pathway, so that quinolinic acid will also be generated

and could contribute to the ‘inflammatory’ response and the later phases of damage

(known as ‘delayed degeneration’). This view is supported by the demonstration that

the excitotoxic response to kainic acid – a response generally attributed almost

exclusively to the direct activation of kainate receptors and the consequent calcium

influx – can be reduced significantly by the co-administration of m-nitrobenzoyl-

alanine, an inhibitor of kynurenine-3-monoxygenase (KMO; Fig. 1) which reduces the

production of quinolinic acid [53]. The implication is that the secondary activation of

microglia includes the generation of increased levels of quinolinic acid or other

kynurenines (see below) that exacerbate neuronal damage.

Infections of the CNS.

By activating IDO, viral components or bacterial lipopolysaccharides increase the

production of several kynurenines. There are substantially elevated levels of

quinolinic acid in the brains of children with bacterial infections of the CNS, changes

which correlate well with markers of immune activation such as neopterin [52].

Septicaemia is similarly associated with increased serum and CSF quinolinic acid

(10-fold in serum and 30-fold in CSF) and kynurenine. Infection of mice by Herpes

simplex virus type 1 raised the levels of quinolinic acid in mice, in parallel with

paralysis.

More recently, attention has focussed on parasitic infections, with reports that the

KMO inhibitor 3,4-dimethoxy-N-[4-(3-nitrophenyl)-thiazol-2-yl]-benzenesulfonamide

(Ro61-8048) described by Roever et al. [54] could prevent death and ataxia in mice

infected with the malaria parasite Plasmodium [55]. This protection was associated

with the predictably raised levels of kynurenic acid, and also of anthranilic acid and

the chemotactic monocyte chemoattractant protein-1. A similar protection has now

been demonstrated in trypanosomiasis (sleeping sickness), with a significant

reduction by Ro61-8048 of the later stages of brain pathology occurring in mice

infected with Trypanosoma brucei parasites [56].

Ischaemic damage.

In addition to the work on stroke introduced above, a delayed increase of quinolinic

acid was noted in gerbils subjected to a period of cerebral ischaemia, with quinolinic

acid levels rising to 50-fold their basal value after 7 days [57]. There was an

accompanying increase in the activity of several of the kynurenine pathway enzymes

in those brain regions experiencing an interrupted blood supply. Intracisternally

applied tryptophan was converted to quinolinic acid in damaged but not normal areas

of brain, consistent with a local production at the sites of injury. There was no change

of kynurenine aminotransferase activity and, as a result, there was an increased ratio

of quinolinic acid: kynurenic acid which would tend to exacerbate the degree of

neuronal injury. Central microglia and macrophages may contribute to delayed

neuronal death after cerebral ischaemia [58] since they possess and secrete

quinolinic acid as noted above and as reflected in the existence of quinolinic acid-

positive microglia in the brain following transient global ischaemia [59].

Huntington's disease

The injection or infusion of quinolinic acid into the rodent striatum has become a

widely accepted paradigm for generating electrophysiological, neuropathological and

behavioural changes closely resembling those seen in patients with Huntington's

disease [60], particularly symptoms which develop in the early stages of the disorder

[61]. Even in non-human primates quinolinic acid is able to induce motor disabilities

very similar to the involuntary movements of Huntington’s disease [62, 63].

When considered together with the clinical evidence for parallel changes in the

CAG triplet repeat sequence, symptoms, and kynurenine metabolism [26], there is a

real possibility that interference with the pathway could slow or prevent the

development of symptoms or the progression of the disorder. Inhibition of KMO, with

the resulting increase of kynurenic acid levels and possible lowering of quinolinic acid

production, represents a promising avenue for this therapeutic approach.

A therapeutic strategy

Kynurenic acid analogues

A major effort to develop antagonists acting directly at the glutamate binding sites

resulted in a large number of compounds with therapeutic promise, although some

exhibited neurotoxic and psychological side effects, including neuronal vacuolisation

or disturbing psychotomimetic effects. These problems contributed to a shift in

emphasis towards the glycine-B co-agonist site on the NMDA receptor, with the

beneficial corollary that kynurenic acid analogues generally cross the blood–brain

barrier more easily than many of the glutamate site quinoxaline ligands.

The most obvious way to base a therapeutic strategy for neuroprotection on the

kynurenine pathway is to mimic the glutamate blocking activity of kynurenic acid,

since over-activation of the various glutamate receptors may be a key characteristic

of brain damage in stroke or neurodegeneration. To this end, several approaches

have been reported in which the kynurenic acid molecule itself is modified by the

addition of halogen atoms. This can generate potent antagonistic analogues such as

5,7-dichlorokynurenic acid with an IC50 of only 80 nM as an antagonist at the

glycine-B site on the NMDA receptor. The potency of these compounds is increased

further if the 4-hydroxy group of kynurenic acid is substituted by acetic acid or similar

moieties. Such additions have led to amido- and thio-substituted compounds such as

MDL 100,748 (4-[(carboxymethyl)amino]-5,7-dichloroquinoline-2-carboxylic acid) [64,

65] (Fig. 2). Some of these compounds have shown clear therapeutic potential as

neuroprotectants or anticonvulsants, while L689,560 (2-carboxy-5,7-dichloro-4-[[(N-

phenylamino)-carbonyl]amino]-1,2,3,4-tetrahydroquinoline) (Fig. 2) has been used

extensively to displace compounds at the strychnine-resistant (NMDA-linked) glycine

binding site. A valuable discovery was made when it was found that 3-phenyl

substituents retained potent activity at the NMDA/glycine site but they were also

more lipophilic than earlier compounds and, presumably partly as a result of this

property, were bioavailable after oral administration [66]. The presence of a 3-keto

grouping was retained in quinones such as L701,252 (4-hydroxy-3-

(cyclopropylcarbonyl)-7-chloroquinoline-2(1H)-one) (Fig. 2) which showed nanomolar

potency at displacing L689,560 binding, and was an effective anticonvulsant in mice.

Kynurenic acid has also been converted into 2-quinolone sulphonamide analogues

with good antagonistic potency.

A breakthrough in kynurenic acid pharmacology arrived with the demonstration that

the quinoline nucleus could be replaced by the indole nucleus with a retention of

glutamate antagonism. Of many subsequent analogues of this structure MDL29,951

(3-(4,6-dichloro-2-carboxyindole-3-yl)propionic acid) [67, 68] (Fig. 2)proved to be

highly effective, although oral bioavailability was reduced. Lipid solubility and blood–

brain barrier penetration are increased if the 3-position of the kynurenate nucleus is

occupied by highly lipophilic substituents, as in L701,324 (4-hydroxy-7-chloro-3-(3-

phenyloxy)phenyl-quinoline-2(1H)-one (Fig. 2) [69]. Both this compound and a

sulphur-containing analogue have good antagonistic activity at the glycine-B co-

agonist site and acceptable systemic and oral bioavailability. The B ring of the

kynurenate nucleus is also amenable to modification by a range of substituents.

Neuronal damage produced by focal cerebral ischaemia can be reduced by a

number of compounds in rats, even when administered several hours after the insult.

A long half-life, leading to greater persistence of compounds in the brain may

account for the efficacy of the Zeneca compound ZD9379 (7-chloro-4-hydroxy-2-(4-

methoxy-2-methylphenyl)-1,2,5,10-tetrahydropyridazino-[4,5b]quinoline-1,10-dione

sodium) (Fig. 2) which, with a half-life of 34 hours in rats, was able to protect the

brain up to at least 24 hours after middle cerebral artery occlusion [70].

Replacement of the nitrogenous ring of kynurenate by a seven-membered ring to

yield benzazepinedione compounds allows retention of antagonism at NMDA

receptors, protection against cerebral ischaemia and the ability to displace

strychnine-resistant glycine binding in vitro or ex vivo [71].

Pro-drugs

The use of pro-drugs to deliver kynurenic acid or its analogues directly into the

brain provides an alternative approach to overcoming the limitations of the blood-

brain barrier. Esterified analogues of kynurenic acid penetrate into the CNS

significantly more rapidly than kynurenic acid itself. Once within the brain

parenchyma, such compounds can be converted to kynurenic acid itself. Similarly,

esterified 4-amino analogues are converted into kynurenic acid in the brain, with

retention or increase in their functional activity, while precursor analogues such as L-

4-chloro-kynurenine and 4,6-dichlorokynurenine are metabolised to the highly potent

kynurenic acid derivatives 7-chlorokynurenic acid and 5,7-dichlorokynurenic acid [72,

73].

Enzyme inhibitors

A different approach is to interfere with the enzymes of the kynurenine pathway so

as to modify the ratio between quinolinic acid and kynurenic acid levels, or to alter

the relative concentrations of other components of the pathway. The ratio of

quinolinic acid: kynurenic acid is largely determined by KMO (Fig. 1) and a number of

investigators have attempted to define the molecular features of molecules required

to achieve inhibition of this enzyme without having non-specific effects on related

flavine mono-oxygenases. A similar shift of balance should be attainable by inhibiting

kynureninase.

The feasibility of this approach was first demonstrated by the development of

nicotinylalanine as an inhibitor of kynureninase and KMO [74-76] (Fig. 1). The

Inhibition of KMO reduced the levels of endogenous quinolinic acid but increased the

conversion of kynurenine to kynurenic acid. These neurochemical changes were

substantially greater when nicotinylalanine was administered together with

kynurenine and the acidic transport inhibitor probenecid, which limits the efflux of

kynurenic acid formed within the brain. The change in the ratio of quinolinic acid:

kynurenic acid was assumed to underlie the anticonvulsant and neuroprotective

properties of nicotinylalanine.

Related compounds developed since this initial proof of concept work generated

meta-nitrobenzoylalanine which preferentially inhibits KMO, while ortho-

methoxybenzoylalanine preferentially inhibits kynureninase [ 77, 78]. The former

produces higher levels of kynurenine and kynurenic acid in the brain and peripheral

tissues, but both have been shown to increase the amount of kynurenic acid in the

hippocampus in vivo. This probably accounts for the decrease of locomotion and

suppression of seizures in sensitive strains of mice [79]. The inhibition of KMO

produced the expected fall in 3-hydroxykynurenine levels together with the increase

of kynurenic acid.

FCE28833A (3,4-dichlorobenzoylalanine) (Fig. 3) is active after systemic

administration and inhibits KMO more effectively than meta-nitrobenzoylalanine

leading to an increase of kynurenine and kynurenic acid in the brain. Concentrations

of both metabolites were increased substantially in the rat hippocampus after a single

systemic injection and, interestingly from a therapeutic viewpoint, the levels of

kynurenic acid remained high for 24 h after the injection [80].

The compound used almost exclusively at the present time to inhibit KMO is one of

a series of N-(4-phenylthiazol-2-yl) benzenesulphonamides. This compound, Ro61-

8048 (Fig. 3) inhibits KMO with an IC50 of only 37 nM. It also increased the levels of

kynurenic acid in the extracellular fluid of gerbil brain after oral administration [54].

A second approach to preventing the synthesis of quinolinic acid is to inhibit 3-

hydroxyanthranilic acid 3,4-dioxygenase. Good inhibition is produced by a series of

4-halo-3-hydroxyanthranilic acids which produce a corresponding reduction in the

formation of quinolinic acid [81, 82].

Conclusion

In summary, the discovery that tryptophan metabolites have direct agonist or

antagonist activity at glutamate and nicotinic receptors in the CNS has resulted in an

enormous amount of work to generate more effective or more brain-penetrant

analogues of those metabolites, or to develop approaches which modify their

endogenous concentrations. The proof of principle that interference with the

kynurenine pathway is indeed neuroprotective is now well established in models for

conditions as different as epileptic seizures, cerebral malaria, stroke and

trypanosomiasis. The over-riding advantage of persevering to manipulate the

kynurenine pathway as a key site of action for new therapeutic agents lies in the fact

that the pathway is activated primarily, if not exclusively, in those areas which have

been subjected to damage or are undergoing degeneration. In this important respect,

modulators of the kynurenine pathway will have inestimable advantages over

compounds which block non-specifically all glutamate or nicotinic receptors

throughout the brain and, indeed, throughout peripheral tissues where they are also

physiologically important.

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Figure 1

Diagrammatic summary of the major component compounds and enzymes of the

kynurenine pathway for the oxidation of tryptophan.

Sequence of steps in the pathway from tryptophan to nicotinic acid and NAD,

including the compounds quinolinic acid and kynurenic acid which are of primary

relevance to the text.

Figure 2

Chemical structures of compounds discussed

The 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.

Figure 3

Chemical structures of compounds discussed.

The structures are shown of the two main inhibitors of the kynurenine pathway that

are neuroprotective and prevent excitoxicity by blocking kynureninase or kynurenine-

3-monoxygenase (KMO).