We first investigated the influence of the acyl group
We first investigated the influence of the -acyl group of the glycine amide side chain and found that shorter linkers lead to better activities (–); simple -benzoyl substituents were always superior to the 3-arylpropanoyl group present in the high-throughput hit (). Electron withdrawing substituents on Ar(–) further improved the activity. The aromatic group Arcould also be replaced by small 5- or 6-membered heterocycles (–), whereas alkyl- or cycloalkyl derivatives were not tolerated (–). Most of the derivatives showed good to excellent selectivity against the Glyt2 isoform. Some characteristic examples are shown in .
Introduction Amino pitavastatin glycine acts as a brain neurotransmitter, influencing both inhibitory and excitatory brain activity (Betz, 1992, Johnson and Ascher, 1987). Two glycine transporters GlyT1 and GlyT2, members of the family of sodium dependent neurotransmitter transporters play major role in the regulation of brain neurotransmitter glycine pools (Nelson, 1998). They control local glycine concentration in various brain regions, where their localization has been described using immunohistochemical and pharmacological methods (Borowsky et al., 1993, Jursky and Nelson, 1995, Jursky and Nelson, 1996, Zafra et al., 1995, Cubelos et al., 2005a, Luccini et al., 2008). Further complexity and compartmentalization is reflected by the existence of species specific GlyT1a, b, c as well as GlyT2a, b, c transporter subtypes (Kim et al., 1994, Ponce et al., 1998, Jursky and Baliova, 2002, Ebihara et al., 2004). Similarly as in the case of other transporter family members, acute regulation of glycine concentration during brain activity is achieved by coupling the glycine transporters cycle to sodium/chloride gradient across the membrane and cycling of the transporters molecules between membrane and intracellular compartments (Nelson, 1998, Masson et al., 1999, Robinson, 2002). Cell surface localization is influenced by the transporter N-terminal interactions with Syntaxin 1A and its C-terminal interactions with trafficking and clustering proteins (Geerlings et al., 2001, Cubelos et al., 2005a, Cubelos et al., 2005b). To study the glycine transporter function in vivo, genes for GlyT1 and GlyT2 has been deleted in mouse (Gomeza et al., 2003a, Gomeza et al., 2003b). Despite that both knock-outs die soon after they are born, several important facts about the function of both transporters have been learned from these models. While mice deficient in GlyT1 resemble symptoms of non-ketonic hyperglycinemia, GlyT2 absence leads to hyperplexia (Gomeza et al., 2003a, Gomeza et al., 2003b, Rees et al., 2006, Eulenburg et al., 2006, Harvey et al., 2008). Experiments further suggest that GlyT1 transporter is responsible for termination of inhibitory glycinergic transmission on strychnine sensitive glycine receptor and GlyT2 rather serves for refilling the vesicles in glycinergic presynaptic terminals. NMDA receptor is colocalized with GlyT1 (Cubelos et al., 2005a) and contains glycine/d-serine co-agonist site. It was generally assumed that glycine acts as major co-agonist at this site in vivo. Recent studies however indicate that glial and neuronal-derived d-serine rather than glycine acts as endogenous ligand of NMDA receptor (Mothet et al., 2000, Panatier et al., 2006, Wolosker, 2007). Glycine concentration in cerebrospinal fluid is low micromolar and it should saturate glycine site on NMDA receptor. GlyT1 inhibitors however potentiate NMDA responses, which indicate that transporters lower the glycine concentration under saturating level. Recent discovery of specific glycine transporters inhibitors allowed modification of the local glycine concentration in vivo. Such strategy seems to be very promising for treatment of schizophrenia and other psychoses caused by hypothetical hypofunction of NMDA (Sur and Kinney, 2007, Boulay et al., 2010, Javitt, 2009). Heterozygous GlyT1 knock-out mice are viable and have doubled extracellular glycine concentration. It was however recently reported, that they exhibit abnormal redistribution of hippocampal synaptic NMDA receptors into extra synaptic sites (Imamura et al., 2008). Thus despite mice seems to be phenotypically normal and even some increase of memory retention is observed, elevated brain glycine concentration may introduce unexpected pathological side-effects.