2-Aminonicotinic acid 1-oxides are chemically stable inhibitors of quinolinic acid synthesis in the mammalian brain: a step toward new antiexcitotoxic agents.

3-Hydroxyanthranilic acid 3,4-dioxygenase (3-HAO) is the enzyme responsible for the production of the neurotoxic tryptophan metabolite quinolinic acid (QUIN). Elevated brain levels of QUIN are observed in several neurodegenerative diseases, but pharmacological investigation on its role in the pathogenesis of these conditions is difficult because only one class of substrate-analogue 3-HAO inhibitors, with poor chemical stability, has been reported so far. Here we describe the design, synthesis, and biological evaluation of a novel class of chemically stable inhibitors based on the 2-aminonicotinic acid 1-oxide nucleus. After the preliminary in vitro evaluation of newly synthesized compounds using brain tissue homogenate, we selected the most active inhibitor and showed its ability to acutely reduce the production of QUIN in the rat brain in vivo. These findings provide a novel pharmacological tool for the study of the mechanisms underlying the onset and propagation of neurodegenerative diseases.

Computational Insights into the Mechanism of Inhibition of OASS-A by a Small Molecule Inhibitor.

O-Acetylserine sulfhydrylase (isoform A, OASS-A) is a PLP-dependent enzyme involved in the last step of cysteine biosynthesis in many pathogens. Many microorganisms use cysteine as the main building block for sulfur-containing antioxidants, and cysteine depletion in several pathogens resulted in a reduced antibiotic resistance, thus leading to the identification of OASS as novel suitable molecular targets to overcome antimicrobial resistances. The precise molecular mechanism of OASS-A inhibition by small peptides or by small molecule inhibitors is still unclear. To shed more lights on the structural basis underlying the inhibition mechanism for OASS, we engaged ourselves in studying the dynamic properties of this enzyme. In this paper, we describe a computational study involving unbiased MD simulations of OASS-A from Haemophilus influenzae (HiOASS) in its inhibitor free, PLP-bound form, and in complex with a pentapeptide inhibitor and with UPAR40, a small molecule which we have recently reported as a potent OASS-A inhibitors. We proposed that UPAR40 inhibits HiOASS-A through the stabilization of a closed conformation. Moreover, preliminary docking studies and sequence analysis allow us to speculate about the non-specificity of UPAR40 toward a particular OASS enzyme species or isoforms.

Inhibition of NaV1.6 sodium channel currents by a novel series of 1,4-disubstituted-triazole derivatives obtained via copper-catalyzed click chemistry.

We have synthesized and evaluated a series of 1,4-disubstituted-triazole derivatives for inhibition of the rat Na(V)1.6 sodium channel isoform, an isoform thought to play an important role in controlling neuronal firing. Starting from a series of 2,4(1H)-diarylimidazoles previously published, we decided to extend the SAR study by replacing the imidazole with a different heterocyclic scaffold and by varying the aryl substituents on the central aromatic ring. The 1,4-disubstituted 1,2,3-triazoles were prepared employing the copper-catalyzed azide-alkyne cycloaddition (CuAAC). Many of the new molecules were able to block the rNa(v)1.6 currents at 10 µM by over 20%, displaying IC(50) values ranging in the low micromolar, thus indicating that triazole can efficiently replace the central heterocyclic core. Moreover, the introduction of a long chain at C4 of the central triazole seems beneficial for increased rNa(v)1.6 current block, whereas the length of N1 substituent seems less crucial for inhibition, as long as a phenyl ring is not direcly connected to the triazole. These results provide additional information on the structural features necessary for block of the voltage-gated sodium channels. These new data will be exploited in the preparation of new compounds and could result in potentially useful AEDs.

Enzymatic transamination of D-kynurenine generates kynurenic acid in rat and human brain.

In the mammalian brain, the α7 nicotinic and NMDA receptor antagonist kynurenic acid is synthesized by irreversible enzymatic transamination of the tryptophan metabolite l-kynurenine. d-kynurenine, too, serves as a bioprecursor of kynurenic acid in several organs including the brain, but the conversion is reportedly catalyzed through oxidative deamination by d-amino acid oxidase. Using brain and liver tissue homogenates from rats and humans, and conventional incubation conditions for kynurenine aminotransferases, we show here that kynurenic acid production from d-kynurenine, like the more efficient kynurenic acid synthesis from l-kynurenine, is blocked by the aminotransferase inhibitor amino-oxyacetic acid. In vivo, focal application of 100 µM d-kynurenine by reverse microdialysis led to a steady rise in extracellular kynurenic acid in the rat striatum, causing a 4-fold elevation after 2 h. Attesting to functional significance, this increase was accompanied by a 36% reduction in extracellular dopamine. Both of these effects were duplicated by perfusion of 2 µM l-kynurenine. Co-infusion of amino-oxyacetic acid (2 mM) significantly attenuated the in vivo effects of d-kynurenine and essentially eliminated the effects of l-kynurenine. Thus, enzymatic transamination accounts in part for kynurenic acid synthesis from d-kynurenine in the brain. These results are discussed with regard to implications for brain physiology and pathology.

Addressing the Conformational Flexibility of Serine Racemase by Combining Targeted Molecular Dynamics, Conformational Sampling and Docking Studies.

Serine racemase (SR) is a PLP-dependent enzyme catalyzing the racemization of L-Ser into D-Ser, which is now recognized as an endogenous co-agonist at the NMDA receptor complex. As indicated by available X-ray structures, SR undergoes significant conformational changes during ligand recognition, and it is conceivable that, in addition to the reported X-ray structures, other intermediate conformational states may have relevance in drug discovery studies. Targeted molecular dynamics (TMD) simulations are an effective tools to follow the conformational transition of a protein under the constrain of reaching a known target states. In this study we use TMD to simulate the transition between the open and closed form of SR in the presence of the endogenous substrate or an orthosteric inhibitor. The trajectories thus obtained are then clustered to collect an ensemble of intermediate conformational states. Docking of a small library of known SR inhibitors or closely related inactive analogs demonstrates that ensemble docking performs better than docking on available crystal structures, thus indicating that inclusion of conformational flexibility into ligand design strategies will be crucial for the development of new classes of SR inhibitors.

Dysfunctional kynurenine pathway metabolism in the R6/2 mouse model of Huntington's disease.

Elevated concentrations of neurotoxic metabolites of the kynurenine pathway (KP) of tryptophan degradation may play a causative role in Huntington's disease (HD). The brain levels of one of these compounds, 3-hydroxykynurenine (3-HK), are increased in both HD and several mouse models of the disease. In the present study, we examined this impairment in greater detail using the R6/2 mouse, a well-established animal model of HD. Initially, mutant and age-matched wild-type mice received an intrastriatal injection of (3)H-tryptophan to assess the acute, local de novo production of kynurenine, the immediate bioprecursor of 3-HK, in vivo. No effect of genotype was observed between 4 and 12 weeks of age. In contrast, intrastriatally applied (3)H-kynurenine resulted in significantly increased neosynthesis of (3)H-3-HK, but not other tritiated KP metabolites, in the R6/2 striatum. Subsequent ex vivo studies in striatal, cortical and cerebellar tissue revealed substantial increases in the activity of the biosynthetic enzyme of 3-HK, kynurenine 3-monooxygenase and significant reductions in the activity of its degradative enzyme, kynureninase, in HD mice starting at 4 weeks of age. Decreased kynureninase activity was most evident in the cortex and preceded the increase in kynurenine 3-monooxygenase activity. The activity of other KP enzymes showed no consistent brain abnormalities in the mutant mice. These findings suggest that impairments in its immediate metabolic enzymes jointly account for the abnormally high brain levels of 3-HK in the R6/2 model of HD.

On the relationship between the two branches of the kynurenine pathway in the rat brain in vivo.

In the mammalian brain, kynurenine aminotransferase II (KAT II) and kynurenine 3-monooxygenase (KMO), key enzymes of the kynurenine pathway (KP) of tryptophan degradation, form the neuroactive metabolites kynurenic acid (KYNA) and 3-hydroxykynurenine (3-HK), respectively. Although physically segregated, both enzymes use the pivotal KP metabolite l-kynurenine as a substrate. We studied the functional consequences of this cellular compartmentalization in vivo using two specific tools, the KAT II inhibitor BFF 122 and the KMO inhibitor UPF 648. The acute effects of selective KAT II or KMO inhibition were studied using a radiotracing method in which the de novo synthesis of KYNA, and of 3-HK and its downstream metabolite quinolinic acid (QUIN), is monitored following an intrastriatal injection of (3)H-kynurenine. In naïve rats, intrastriatal BFF 122 decreased newly formed KYNA by 66%, without influencing 3-HK or QUIN production. Conversely, UPF 648 reduced 3-HK synthesis (by 64%) without affecting KYNA formation. Similar, selective effects of KAT II and KMO inhibition were observed when the inhibitors were applied acutely together with the excitotoxin QUIN, which impairs local KP metabolism. Somewhat different effects of KMO (but not KAT II) inhibition were obtained in rats that had received an intrastriatal QUIN injection 7 days earlier. In these neuron-depleted striata, UPF 648 not only decreased both 3-HK and QUIN production (by 77% and 66%, respectively) but also moderately raised KYNA synthesis (by 27%). These results indicate a remarkable functional segregation of the two pathway branches in the brain, boding well for the development of selective KAT II or KMO inhibitors for cognitive enhancement and neuroprotection, respectively.

Mitochondrial aspartate aminotransferase: a third kynurenate-producing enzyme in the mammalian brain.

The tryptophan metabolite kynurenic acid (KYNA), which is produced enzymatically by the irreversible transamination of l-kynurenine, is an antagonist of alpha7 nicotinic and NMDA receptors and may thus modulate cholinergic and glutamatergic neurotransmission. Two kynurenine aminotransferases (KAT I and II) are currently considered the major biosynthetic enzymes of KYNA in the brain. In this study, we report the existence of a third enzyme displaying KAT activity in the mammalian brain. The novel KAT had a pH optimum of 8.0 and a low capacity to transaminate glutamine or alpha-aminoadipate (the classic substrates of KAT I and KAT II, respectively). The enzyme was inhibited by aspartate, glutamate, and quisqualate but was insensitive to blockade by glutamine or anti-KAT II antibodies. After purification to homogeneity, the protein was sequenced and the enzyme was identified as mitochondrial aspartate aminotransferase (mitAAT). Finally, the relative contributions of KAT I, KAT II, and mitAAT to total KAT activity were determined in mouse, rat, and human brain at physiological pH using anti-mitAAT antibodies. KAT II was most abundant in rat and human brain, while mitAAT played the major role in mouse brain. It remains to be seen if mitAAT participates in cerebral KYNA synthesis under physiological and/or pathological conditions in vivo.

Synthesis and preliminary pharmacological evaluation of the four stereoisomers of (2S)-2-(2'-phosphono-3'-phenylcyclopropyl)glycine, the first class of 3'-substituted trans C1'-2'-2-(2'-phosphonocyclopropyl)glycines.

Four stereoisomers of (2S)-2-(2'-phosphono-3'-phenylcyclopropyl)glycine were synthesized by a stereocontrolled synthetic procedure and evaluated as mGluRs ligands. The (2S,1'R,2'S,3'R)-isomer (PPCG-2) showed to be a group III mGluRs selective ligand endowed with a moderate potency as mGluR4/mGluR6 agonist.

Perinatal kynurenine 3-hydroxylase inhibition in rodents: pathophysiological implications.

The kynurenine pathway (KP) of tryptophan degradation contains three neuroactive metabolites: the neuroinhibitory agent kynurenic acid (KYNA) and, in a competing branch, the free radical generator 3-hydroxykynurenine (3-HK) and the excitotoxin quinolinic acid (QUIN). These three "kynurenines" derive from a common precursor, L-kynurenine, and are recognized for their role in brain physiology and pathophysiology. Inhibition of kynurenine 3-hydroxylase, the enzyme responsible for 3-HK formation, shifts KP metabolism in the mature brain toward enhanced KYNA formation. We now tested the cerebral effects of kynurenine 3-hydroxylase inhibition in immature rodents. Rat pups treated with the kynurenine 3-hydroxylase inhibitor UPF 648 (30 mg/kg, i.p.) 10 min after birth showed substantial increases in cerebral and liver kynurenine and KYNA levels up to 24 hr later, whereas 3-HK and QUIN levels were simultaneously decreased. Administered to pregnant rats or mice on the last day of gestation, UPF 648 (50 mg/kg, i.p.) produced qualitatively similar changes (i.e., large increases in kynurenine and KYNA and reductions in 3-HK and QUIN) in the brain and liver of the offspring. Rat pups delivered by UPF 648-treated mothers and immediately exposed to neonatal asphyxia showed further enhanced brain KYNA levels. These studies demonstrate that acute kynurenine 3-hydroxylase inhibition effectively shifts cerebral KP metabolism in neonatal rodents toward increased KYNA formation. Selective inhibitors of this enzyme may therefore provide neuroprotection in newborns and will also be useful for the experimental evaluation of the long-term effects of perinatal KP impairment.

Synthesis and preliminary biological evaluation of (2S,1'R,2'S)- and (2S,1'S,2'R)-2-(2'-phosphonocyclopropyl)glycines, two novel conformationally constrained l-AP4 analogues.

Two novel constrained l-AP4 analogues, (2S,1'R,2'S)- and (2S,1'S,2'R)-2-(2'-phosphonocyclopropyl)glycines (7) and (8), were synthesized and evaluated as mGluR ligands. Compound 7 showed to be a group III mGluRs agonist with micromolar activity.

Modulation of the kynurine pathway of tryptophan metabolism in search for neuroprotective agents. Focus on kynurenine-3-hydroxylase.

A novel potent and selective kynurenine-3-hydroxylase inhibitor is descibed along a preliminary evaluation in a in vivo gerbil model of its ability to increase the kynurenine and kynurenic acid concentration in both plasma and brain. These data support the notion that kynurenine-3-hydroxylase inhibitors may have a sustained therapeutic potential in those diseases characterized by unbalance in the QUIN/KYNA branches of the kynurenine pathway.