Introduction.The amino acid glutamate is present in high concentrations in the mammalian brain, and it acts as the major excitatory neurotransmitter in the CNS. Through its actions on both ionotropic and metabotropic receptors, glutamate plays an important role in a variety of physiological functions including learning, memory, and developmental plasticity. Excessive activation of glutamate receptors or disturbances in the cellular mechanisms that protect against the adverse consequences of physiological glutamate receptor activation have been implicated in the pathogenesis of a host of neurological disorders. Although several drugs designed to attenuate the pathological consequences of excessive glutamate activation have been shown to reduce injury in experimental models of cerebral ischemia, so far none of these compounds has proven to be effective in the clinical treatment of stroke. 1 N-Acetyl-L-aspartyl-L-glutamate (NAAG) is a peptide neurotransmitter that is widely distributed in the mammalian nervous system. 2 NAAG is both an agonist at metabotropic glutamate receptors (mGluR3) 3 and a mixed agonist/antagonist at the N-methyl-D-aspartate (NMDA) receptor. 4 NAAG is hydrolyzed by the neuropeptidase glutamate carboxypeptidase II (GCPII; also known as N-acetylated R-linked acidic dipeptidase, NAALADase, or NAAG peptidase) to liberate N-acetylaspartate and glutamate both in vitro and in vivo. 5 The role of this metalloprotease GCPII is thus thought to be twofold: (1) to terminate the neurotransmitter activity of NAAG and (2) to liberate glutamate which is then able to act at the various glutamate receptor subtypes. Alterations in the levels of GCPII and NAAG have been observed in disorders that are linked to abnormalities in glutamatergic neurotransmission. 6 As a consequence of these findings, it has been hypothesized that the inhibition of GCPII might provide an effective strategy for achieving neuroprotection in cases of cerebral ischemia by increasing the levels of

Amino acids are among the most important molecules in nature since they play central roles both as building blocks of proteins and as intermediates in metabolism. The amino acid sequence dictates protein folding, the native three dimensional structure, and protein stability. Furthermore, the peculiar chemical properties of the amino acids forming the active site and their interplay determine protein function and regulation. All amino acids found in proteins, except glycine, possess a stereogenic center at the a-carbon atom. Millions of years of evolution have resulted in the virtually complete homochirality of such a stereogenic center, i.e. the L-enantiomer, in mammals. 1 This selection of the L-amino acids by nature is generally considered to be a result of chance. 2 Since the cornerstone of the protein-ligand recognition is the multi-point attachment theory, it turns out that the configuration of the a-carbon atom of amino acids strongly affects the protein-ligand interaction. Nevertheless, during the last half of the twentieth century, various studies evidenced the presence of Damino acids in some plants and bacteria. 3,4 These compounds were either found in a free state or in peptides and proteins. Most bacteria produce significant amounts of D-alanine (D-Ala) and D-glutamate (D-Glu), which are incorporated into peptidoglycan. 5 Peptidoglycan is a strong and elastic polymer of the bacterial wall, which is capable to counteract the osmotic pressure of the cell, maintaining cell shape and anchoring components of the cell envelope. 6 The number of D-amino acids present in the structure of peptidoglycan seems to constitute a measure of protection against peptidase and protease attacks. So far, no peptidase capable of hydrolyzing a peptide bond characterized by the sequence D-D or D-L amino acids has been isolated in mammals. In addition, several antibiotics produced by prokaryotes (e.g. bacitracin, actinomycin D) contain D-amino acids (Figure 1). It has been recently demonstrated that bacteria synthesize a pool of different D-amino acids, including D-methionine (D-Met) and D-leucine (D-Leu) in Vibrio cholerae and D-tyrosine (D-Tyr) and D-phenylalanine (D-Phe) in Bacillus subtilis. By selectively incorporating them in the peptidoglycan cell wall, bacteria cope to different environmental stresses. 7

Glutamate is the major excitatory neurotransmitter of the central nervous system (CNS) and may induce cytotoxicity through persistent activation of glutamate receptors and oxidative stress. Its extracellular concentration is maintained at physiological concentrations by high affinity glutamate transporters of the solute carrier 1 family (SLC1). Glutamate is also present in islet of Langerhans where it is secreted by the ␣-cells and acts as a signaling molecule to modulate hormone secretion. Whether glutamate plays a role in islet cell viability is presently unknown. We demonstrate that chronic exposure to glutamate exerts a cytotoxic effect in clonal ␤-cell lines and human islet ␤-cells but not in ␣-cells. In human islets, glutamate-induced ␤-cell cytotoxicity was associated with increased oxidative stress and led to apoptosis and autophagy. We also provide evidence that the key regulator of extracellular islet glutamate concentration is the glial glutamate transporter 1 (GLT1). GLT1 localizes to the plasma membrane of ␤-cells, modulates hormone secretion, and prevents glutamate-induced cytotoxicity as shown by the fact that its down-regulation induced ␤-cell death, whereas GLT1 up-regulation promoted ␤-cell survival. In conclusion, the present study identifies GLT1 as a new player in glutamate homeostasis and signaling in the islet of Langerhans and demonstrates that ␤-cells critically depend on its activity to control extracellular glutamate levels and cellular integrity.

A series of Δ(2)-isoxazoline constrained analogues of procaine/procainamide (7a-k and 8a-k) were prepared and their inhibitory activity against DNA methyltransferase 1 (DNMT1) was tested. Among them, derivative 7b is far more potent in vitro (IC(50) = 150 μM) than other non-nucleoside inhibitors and also exhibits a strong and dose-dependent antiproliferative effect against HCT116 human colon carcinoma cells. The binding mode of 7b with the enzyme was also investigated by means of a simple competition assay as well as of docking simulations conducted using the recently published crystallographic structure of human DNMT1. On the basis of the findings, we assessed that the mode of inhibition of 7b is consistent with a competition with the cofactor and propose it as a novel lead compound for the development of non-nucleoside DNMT inhibitors.

We developed a new class of covalent inhibitors of Plasmodium falciparum glyceraldehyde-3-phosphate dehydrogenase, a validated target for the treatment of malaria, by screening a small library of 3-bromo-isoxazoline derivatives that inactivate the enzyme through a covalent, selective bond to the catalytic cysteine, as demonstrated by mass spectrometry. Substituents on the isoxazolinic ring modulated the potency up to 20-fold, predominantly due to an electrostatic effect, as assessed by computational analysis.