ALX 5407: A Potent, Selective Inhibitor of the hGlyT1 Glycine Transporter

Abstract

High-affinity glycine transport in neurons and glial cells is a primary means of inactivating synaptic glycine. We have synthesized a potent selective inhibitor of glycine transporter 1 (GlyT1), and characterized its activity using a quail fibroblast cell line (QT6). The glycine transporters GlyT1A, GlyT1B, GlyT1C, and GlyT2 were stably expressed in QT6 cells. The transporters expressed in these cells exhibited appropriate characteristics as described previously for these genes: Na(+)/Cl(-) dependence, appropriate K(m) values for glycine uptake, and appropriate pharmacology, as defined in part by the ability of N-methyl glycine (sarcosine) to competitively inhibit glycine transport. Furthermore, the characteristics of the transporters in the cell lines recapitulate the characteristics of glycine transporters observed in tissue preparations. We developed a sarcosine derivative, (R)-(N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl])sarcosine (ALX 5407), and examined its activity against the cloned glycine transporters. ALX 5407 completely inhibited glycine transport in the GlyT1 cells, with an IC(50) value of 3 nM, but had little or no activity at the human GlyT2 transporter, at other binding sites for glycine, or at other neurotransmitter transporters. The inhibition of glycine transport was essentially irreversible. ALX 5407 represents a novel tool in the investigation of N-methyl-D-aspartate-receptor function. This class of drug may lead to novel therapies in the treatment of schizophrenia.

Neurochemical, Electrophysiological and Pharmacological Profiles of the Selective Inhibitor of the Glycine Transporter-1 SSR504734, a Potential New Type of Antipsychotic

“…This effect of SSR504734 was stereoselective since the (R,R) enantiomer SSR506204 was far less potent. SSR504734 inhibited glycine transport at human and rat GlyT1s (IC 50 s ca 20 nM) with a potency in between that reported SSR504734, a reversible GlyT1 inhibitorfor ALX5407 (3 nM, Atkinson et al, 2001;220 nM, Herdon et al, 2001, both for hGlyT1c; 26 nM for hGlyT1b, Smith et al, 2004; 10 nM for rGlyT1a, Kinney et al, 2003) and for ORG 24598 (120 nM for hGlyT1b, Brown et al, 2001), and far above that of sarcosine or glycyldodecylamide, two earlier GlyT1 inhibitors (IC 50 s greater than 10 mM; present results; Javitt and Frusciante, 1997). SSR504734 was similarly potent in mice (IC 50 : 3875 nM, in cortical homogenate, not reported in Materials and methods and Results), a point we verified because of the use of this species in several tests.…”

“…Note: ALX5407 (considered by some authors as producing an essentially irreversible blockade of GlyT1; Atkinson et al, 2001;Aubrey and Vandenberg, 2001) was used as a comparator GlyT1 inhibitor solely in tests susceptible to give an indication of the degree of reversibility of the blockade of glycine uptake.…”

“…By increasing synaptic concentration of glycine in the vicinity of NMDA receptors, blockers of GlyT1 are expected to potentiate glutamatergic transmission, and as such represent promising targets for pharmacological intervention against schizophrenia Millan, 2002;Slassi and Egle, 2004). Several GlyT1 blockers (ALX5407; also known as NFPS, ORG 24461, ORG 24598 and CP 802,079) have been reported to possess the preclinical profile of putative antipsychotics (to cite a few studies, Bergeron et al, 1998;Atkinson et al, 2001;Brown et al, 2001;Harsing et al, 2003, Kinney et al, 2003, Le Pen et al, 2003, Martina et al, 2004, but data for each compound are generally scant and/or scattered across several papers, and none of these compounds seems to have been developed clinically.…”

Glycine transport inhibitors for the treatment of pain

“…β-alanine is an alternate GlyR agonist, but is not a substrate of GlyTs and in contrast to glycine, the time course of β-alanine-evoked currents is insensitive to NAGly, which suggests that NAGly mediates its effects at glycinergic synapses by inhibiting GlyTs. In this study, synaptic currents were also investigated, and although miniature inhibitory post-synaptic currents [47,54,55]. Thus, NAGly enhances inhibitory glycinergic synaptic transmission by inhibiting GlyT2.…”

“…The GlyT1 inhibitors, ORG25935 and ALX5407 (NFPS), and the GlyT1 substrate, sarcosine have also been tested in mouse models of neuropathic pain [38,54,55,59]. Reduction in GlyT1 activity would be expected to increase the activity of both GlyRs and NMDARs and thus influence both inhibitory and excitatory neurotransmission.…”