Maura Marinozzi

Type 2 metabotropic glutamate (mGlu) receptors tonically inhibit transmitter release in rat caudate nucleus: in vivo studies with (2S,1'S,2'S,3'R)-2-(2'-carboxy-3'-phenylcyclopropyl)glycine, a new potent and selective antagonist.

Anatomical, biochemical and electrophysiological studies have previously shown that cortico‐striatal terminals contain abundant presynaptic group 2 metabotropic glutamate (mGlu) receptors. Using brain slices we have previously shown that these receptors inhibit depolarization‐induced transmitter release. Using microdialysis in freely moving rats, we now report the effects of group 2 mGlu receptor agonists and antagonists on glutamate concentration in the caudate extracellular fluid. A mild decrease (20–30%) in glutamate concentration in caudate dialysates was observed when 1S,3R‐1‐aminocyclopentane‐1,3‐dicarboxylic acid or (2S,3S,4S)‐α‐carboxycyclopropyl‐glycine (L‐CCG‐1), mGlu receptor agonists, was locally administered. On the contrary, α‐methyl‐4‐carboxyphenylglycine, an antagonist of type 1 and type 2 mGlu receptors, increased the glutamate concentration in dialysates by up to 3.5‐fold, and its effects were prevented by the simultaneous administration of L‐CCG‐1, a preferential type 2 mGlu receptor agonist. A significant increase of glutamate output in striatal dialysate was also found after local administration of (2S,1′S,2′S,3′R)‐2‐(2′‐carboxy‐3′‐phenylcyclopropyl)glycine, another structurally unrelated, relatively selective and potent type 2 mGlu receptor antagonist. The results suggest that type 2 mGlu receptors tonically inhibit transmitter release from corticostriatal terminals. Since the cortico‐striatal pathway profoundly affects the function of a large percentage of caudate neurons, it is reasonable to predict that the use of selective type 2 mGlu receptor agents will be helpful for scientific and therapeutic studies on the physiopathology of basal ganglion disorders.

Specific inhibition of kynurenate synthesis enhances extracellular dopamine levels in the rodent striatum

Fluctuations in the endogenous levels of kynurenic acid (KYNA), a potent alpha7 nicotinic and NMDA receptor antagonist, affect extracellular dopamine (DA) concentrations in the rat brain. Moreover, reductions in KYNA levels increase the vulnerability of striatal neurons to NMDA receptor-mediated excitotoxic insults. We now assessed the role of a key KYNA-synthesizing enzyme, kynurenine aminotransferase II (KAT II), in these processes in the rodent striatum, using KAT II KO mice-which have reduced KYNA levels-and the selective KAT II inhibitor (S)-4-(ethylsulfonyl)benzoylalanine (S-ESBA) as tools. S-ESBA (applied by reverse dialysis) raised extracellular DA levels in the striatum of KYNA-deficient mice threefold and caused a much larger, 15-fold increase in wild-type mice. In the rat striatum, S-ESBA produced a 35% reduction in extracellular KYNA, which was accompanied by a 270% increase in extracellular DA. The latter effect was abolished by co-infusion of 100 nM KYNA. Intrastriatal S-ESBA pre-treatment augmented the size of a striatal quinolinate lesion by 370%, and this potentiation was prevented by co-infusion of KYNA. In separate animals, acute inhibition of KAT II reduced the de novo synthesis of KYNA during an early excitotoxic insult without enhancing the formation of the related neurotoxic metabolites 3-hydroxykynurenine and quinolinate. Taken together, these results provide further support for the concept that KAT II is a critical determinant of functionally relevant KYNA fluctuations in the rodent striatum.

Modulators of the kynurenine pathway of tryptophan metabolism: synthesis and preliminary biological evaluation of (S)-4-(ethylsulfonyl)benzoylalanine, a potent and selective kynurenine aminotransferase II (KAT II) inhibitor.

In mammals, most non-proteinogenic l-tryptophan is metabolized by a complex enzymatic cascade known as the kynurenine pathway (KP, Scheme 1), which ultimately leads to the production of NAD . The KP has received considerable interest over the last decade since several of its components are endowed with neuroactive properties. Thus, in a branch of the pathway initiated by the rate-limiting enzyme kynurenine 3-hydroxylase, the central metabolite kynurenine (KYN, 1) is transformed into the pro-excitotoxic free-radical generator 3-hydroxykynurenine (3-HK, 2) and then further into the excitotoxin quinolinic acid (QUIN, 3). A second branch of the KP leads from KYN to kynurenic acid (KYNA, 4), a competitive antagonist of the GlyB site of the NMDA receptor complex and a noncompetitive antagonist of the a7* nicotinic acetylcholine receptor. The transamination of KYN to KYNA is irreversible and catalyzed by kynurenine aminotransferases (KATs). At least two isoforms (KAT I and KAT II) are present in the mammalian brain and have been characterized by biochemical and genetic methods. The KP provides several targets for drugs that could be of use in the treatment of CNS diseases and disorders. For example, the inhibition of kynurenine 3-hydroxylase decreases 3HK and QUIN production and causes an increase in KYNA formation, leading to protection against excitotoxic insults. This concept has been verified using in vitro and in vivo models of brain ischemia, although it has not yet been successfully translated into clinically deliverable drugs. In contrast, the inhibition of KAT, and especially of KAT II, results in decreased KYNA production. In the brain, this causes diminished inhibition of a7* nicotinic acetylcholine receptor function and enhanced glutamate release. 12] KAT inhibitors could therefore be useful in disorders related to glutamatergic and cholinergic hypofunction, such as learning and memory deficits. So far, however, only a few potent KAT inhibitors have been described. During the past years, we and others have shown that the benzoylalanine nucleus is particularly amenable to

D-3,4-‘cyclopropylglutamate’ isomers as nmda receptor ligands: Synthesis and enantioselective activity.

Abstract Dirhodium(II) tetraacetate catalyzed decomposition of ethyl diazoacetate in the presence of D-Cbz-vinylglycine methyl ester (11) afforded a mixture of the cyclopropyl esters D-CGA A-D (13) from which the corresponding 2R-acids 7-10 were obtained and their absolute configurations assigned. The (2R,3S,4R) α-(carboxycyclopropyl)glycine (D-CGA C, 9 resulted to be the most potent and selective among the NMDA receptor ligands yet reported.

Determination of bile salt critical micellization concentration on the road to drug discovery.

With the discovery of the bile acid (BA)-activated nuclear and membrane receptors, the role of BAs as signalling molecules in important paracrine and endocrine networks has been fully documented in the last decade. Besides regulating their own synthesis and transport, BAs have been demonstrated being involved in triggering the adaptive response to cholestasis and other insults to liver. More to the point, their recognized ability to control the general energy-related metabolism and inflammation processes has contributed to justify the renewed interest towards this class of amphiphilic steroidal compounds. All these evidences feed a continuing interest in the BA research aimed at designing and synthesizing new side chain- and body-modified derivatives endowed with improved biological and physico-chemical profiles, as well as with proper ADMET behaviour. In this context, the micellar aggregation of BAs, and the respective critical micellization concentration (CMC) value (determined on the BA sodium salt, BS), is considered a key parameter that needs to be determined in the preliminary phase of compound characterization, being implicated in cytotoxicity issues. An extraordinary variety of different analytical techniques and methods have been proposed along the years with the aim of better identifying the start of the self-aggregation process of BS monomers. The unicity of the physico-chemical nature of such class of compounds can be invoked to explain this unusual interest. Accordingly, a number of both invasive and non-invasive approaches have been developed along with a limited number of indirect chromatographic-based estimation strategies. Worth to be mentioned among the non-invasive determination methods are those based on potentiometry, freezing point depression, surface tension, nuclear magnetic resonance, viscosimetry, turbidimetry, microcalorimetry, refractometry, conductimetry, spectrophotometry, cholesterol solubilization, and monoglucuronide solubilization. Dye solubilization- and fluorescence-based methods deserve instead credit among the invasive methodological approaches. Indirect chromatographic methods based on capillary electrophoresis and high performance liquid chromatography analysis also demonstrated to be profitably exploited for the CMC estimation, especially when a small amount of sample is available. The collection of literature data reveals that the CMC value of a given BS is markedly related to the method selected for determining it as well as to the experimental conditions applied during the analysis.

The effect of mobile phase composition in the enantioseparation of pharmaceutically relevant compounds with polysaccharide-based stationary phases

Mobile phase variables have a deep influence on the chromatographic behavior with polysaccharide-based chiral stationary phases. Basic additives are generally used to minimize peak broadening arising from unwanted interactions between polar solutes and underivatized silanols. However, basic additives can improve enantioselectivity through disruption of hydrogen bonds and modification of the polymer morphology. Acidic additives are incorporated into the mobile phase during the analysis of acidic compounds as efficiency enhancers. Acidic additives can also improve enantioselectivity by minimizing within the chiral recognition site nonenantioselective retention. Peak shape without acidic additive in the eluent could be severely distorted during the analysis of salified compounds. Concentration and type of alcohol modifier can have an effect on the morphology of the polymer. The different winding of the chiral selector, caused by alcohol modifiers of different size/shape, ultimately results in different stereo environment of the chiral cavities in the polymer chain. Trace amounts of water in normal-phase eluents can affect retention time, tailing, and resolution. Deliberate addition of water to the eluent can improve peak resolution and save analysis time and solvent needs. Immobilized-type polysaccharide-derived chiral stationary phases offer new selectivity profiles and often improved enantioselectivity.

Modulation of glutamate receptor pathways in the search for new neuroprotective agents

Excessive stimulation of excitatory amino acid (EAA) receptors is responsible for a wide variety of acute and chronic neurological impairments. A separate line of investigation has focused on oxidative stress as one of the main reasons for several of these degenerative disorders. Current evidence has confirmed that activation of both ionotropic and metabotropic glutamate receptors can also result in either neuroprotection or neurodegeneration according to the role played by oxidative stress mechanisms. An outline of this research, together with our recent results aimed at the discovery of new subtype selective modulators of the central nervous system pathways as well as new classes of free radical scavengers, is presented.

Beyond Bile Acids: Targeting Farnesoid X Receptor (FXR) with Natural and Synthetic Ligands

The modulation of FXR receptor remains an attractive area in drug discovery to develop novel therapeutic opportunities for liver and metabolic disorders. Despite the large variety of FXR ligands reported so far, only a very restricted number of agonists have entered in clinical settings. In this review article we provide the reader with an overview on the different classes of natural and synthetic ligands that have been developed by academic groups and pharmaceutical companies to target FXR. We discuss their structure-activity relationships, analyzing the binding modes that some of these compounds adopt to interact with the receptor.

Synthesis, molecular modeling and preliminary biological evaluation of 1-amino-3-phosphono-3-cyclopentene-1-carboxylic acid and 1-amino-3-phosphono-2-cyclopentene-1-carboxylic acid, two novel agonists of metabotropic glutamate receptors of group III

On the basis of a pharmacophore definition of mGlu4 agonists, the two novel semi-rigid derivatives 12 and 13 were designed and synthesized. The preliminary biological evaluation demonstrated that both compounds interact with hmGlu4a, while ineffective at group II receptor subtypes. In particular, derivative 13 is a full hmGlu4a agonist with an EC50 = 17 microM.

Pyrazole[3,4-e][1,4]thiazepin-7-one derivatives as a novel class of Farnesoid X Receptor (FXR) agonists

A virtual screening procedure was applied to the discovery of structurally diverse non-steroidal Farnesoid X Receptor (FXR) agonists. From 117 compounds selected by virtual screening, a total of 47 compounds were found to be FXR agonists, with 34 of them showing activity below a concentration of 20 μM. 1H-Pyrazole[3,4-e][1,4]thiazepin-7-one-based hit compound 7 was chosen for hit-to-lead optimization. A large number of 1H-pyrazole[3,4-e][1,4]thiazepin-7-one derivatives was designed, synthesized, and evaluated by a cell-based luciferase transactivation assay for their agonistic activity against FXR. Most of them exhibited low micromolar range of potency and very high efficacy.