Archives

  • 2018-07
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • br Results and discussion br Conclusion In this

    2022-07-01


    Results and discussion
    Conclusion In this study the first binding assays based on a nonlabelled reporter ligand addressing GlyT1 are described. Following the concept of MS Binding Assays recently introduced in our group, binding of the well-known GlyT1 inhibitor Org24598 employed as reporter ligand towards GlyT1 could be monitored using LC-MS for quantification. For this purpose, a highly sensitive LC-ESI-MS/MS method was developed that enables reliable quantification of Org24598 in binding experiments in a range from 5 pM to 1 nM using the five times deuterated racemic species of the marker, [2H5]Org24461, as internal standard. The use of a deuterated internal standard might give rise to the assumption that the established GlyT1 MS Binding assays employing Org24598 as nonlabelled marker do not really represent a label-free assay technique and furthermore, that the access to this internal standard may be also somehow restricted. However, in this context it should be mentioned that deuterated compounds can be employed without any safety or environmental concern or restriction in every lab and that the two step synthesis of [2H5]Org24461 described in this study is based on commercially available compounds and does not require skills beyond basic synthetic chemistry. The most important argument in the discussion of this issue is, however, that the use of a deuterated internal standard is not essential for marker quantification by means of LC-ESI-MS/MS in MS Binding Assays as demonstrated in former studies (Massink et al., 2015; Neiens et al., 2015; Schuller et al., 2017). In this project during method development, it has been found, for example, that also another commercially available GlyT1 inhibitor, namely ALX5407, can be used for this purpose. After validation of the developed LC-ESI-MS/MS method for quantification of Org24598 it was used as readout for filtration based MS Binding Assays performed in analogy to known GlyT1 radioligand binding assays and MS Binding Assays recently established for other neurotransmitter transporters. In saturation experiments, the affinity of Org24598 at GlyT1 could be characterized with a Kd of 16.8 nM. The developed MS Binding Assays were also used to determine the affinities of known GlyT ligands at GlyT1 in competition experiments. A comparison of the affinities obtained in MS Binding Assays with results from literature showed an almost perfect match thus demonstrating the reliability of the established GlyT1 MS Binding Assays.
    Material and methods
    Conflicts of interest
    Acknowledgments Special thanks go to AbbVie Germany and in particular to Dr. Willi Amberg for providing the CHO-K1 cell line which expresses GlyT1. We are also thankful to Lars Allmendinger (LMU München, Department of Pharmacy, Center of Drug Research, Munich, Germany) for analysis of the internal standard [2H5]Org24461 and for help in preparation of the manuscript regarding this subject.
    Introduction Glycine is a neurotransmitter that serves an inhibitory function in the spinal cord and 17 aag synthesis stem, but also is an obligatory co-agonist at excitatory glutamate receptors of the NMDA type (Kemp and Leeson, 1993). While it was initially believed that the concentration of glycine in the synaptic cleft would be high enough to saturate the glycine site on the NMDA receptor, later pharmacological and electrophysiological studies indicate that this might be not the case due to the action of the glycine transporter GLYT1 (Zafra and Gimenez, 2008). Similarly to glycine, GLYT1 also seems to accomplish a dual role in neurotransmission. First, it is highly expressed in glycinergic areas of the nervous system where it localize predominantly in glial cells (Zafra et al., 1995a), and mice lacking GLYT1 showed an impaired glycinergic neurotransmission that was attributed to an increase in the extracellular glycine close to strychnine sensitive glycine receptors (Gomeza et al., 2003). Second, GLYT1 also has been identified in neuronal elements throughout all the brain closely associated to glutamatergic pathways. GLYT1 is enriched in presynaptic boutons, where it largely colocalize with the vesicular glutamate transporter vGLUT1, and it is also present in the postsynaptic densities of asymmetric synapses (Cubelos et al., 2005). Immunoprecipitation assays showed the existence of immunoprecipitable complexes containing both NMDA and GLYT1 (Cubelos et al., 2005). These anatomical and biochemical evidence support a role of GLYT1 in controlling the glycine concentration in the microenvironment of the NMDA receptor and are consistent with functional studies showing that N[3-(4’-fluorophenyl)-3-(4’-phenylphenoxy)propyl]sarcosine (NFPS), a specific inhibitor of GLYT1, potentiates the NMDA-mediated responses both in vitro and in vivo (Bergeron et al., 1998, Chen et al., 2003, Kinney et al., 2003). The possible role of GLYT1 in glutamatergic neurotransmission was also confirmed in heterozygous Glyt1 +/− animals that expressed only 50% of GLYT1 and had an enhanced hippocampal NMDA receptor function and better memory retention (Tsai et al., 2004). While this evidence indicate that the glycine concentration in the microenvironment of NMDA receptors is regulated by reuptake through GLYT1, it is unclear how the levels of glycine in forebrain glutamatergic synapses oscillate in response to neuronal activity. Glycine release from hippocampal synaptosomes (Galli et al., 1993, Luccini et al., 2008) and cultured neurons (Fatima-Shad and Barry, 1998) has been reported. Additionally, a recent study showed the existence of a vesicular release of glycine in glutamatergic terminals of the hippocampus in response to neuronal depolarization, and this might modulate NMDA receptor function (Muller et al., 2013). However, the source of this glycine must be other than classical glycinergic terminals since boutons containing both the plasma membrane transporter GLYT2 and the vesicular glycine transporter (VIAAT) are absent in these forebrain regions (Dumoulin et al., 1999). Thus, changes in the extracellular glycine levels might occur either through modifications of the forward or reverse operation of the glycine transport systems, or by an unusual vesicular release, or by a combination of these mechanisms.