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  • s6 kinase Due to the therapeutic potential of GlyT

    2021-09-23

    Due to the therapeutic potential of GlyT1 inhibitors, numerous drug screening campaigns have been started - and are still going on - which aimed at the identification of compounds with biological activity at or affinity for this drug target. For characterization of biological activity at GlyT1, transport assays based on radiolabelled glycine as substrate are employed, in case a considerable throughput is required (Brown et al., 2001; Kopec et al., 2009; Santora et al., 2018). When only single compounds are to be characterized regarding their inhibitory activity at GlyT1 electrophysiological studies may be suitable as well (Mezler et al., 2008). For identification of GlyT1 ligands or for determination of their affinity radioligand binding assays are used (Amberg et al., 2018; Mezler et al., 2008; Wang et al., 2018). Both techniques, i.e. transport and binding assays, are exclusively based on the use of radioisotopes such as 3H and 14C. As the use of radioisotopes is, however, inherently coupled to several serious drawbacks, such as safety issues, restrictions set by authorities and problems with remaining waste, there is a strong need for label-free assay techniques addressing GlyT1. For GlyT1 transport assays based on radiolabelled glycine already a fluorescence based alternative has been reported, recording changes in the membrane potential as a consequence of the net inward flux of a single positive charge for every glycine molecule transported by the transporter (Allan et al., 2006). With respect to binding assays addressing GlyT1, however, to the best of our knowledge, up to now no alternative to radioligand binding assays has been described. Furthermore, all of the radioligands used for this purpose so far are not commercially available, as they have been synthesized in-house by pharma companies for their own purposes. Due to this unsatisfactory situation we decided to establish an alternative to GlyT1 radioligand binding assays by applying the concept of MS Binding Assays recently introduced by our group (Grimm et al., 2015a, 2015b; Hess et al., 2011a, 2011b; Neiens et al., 2015, 2018a, 2018b; Schuller et al., 2017; Zepperitz et al., 2006, 2008). MS Binding Assays are based on the use of a nonlabelled reporter ligand instead of a ligand labelled with a radioisotope, the former of which is therefore termed “native marker”, “MS Marker” or simply “marker” that is quantified by means of mass spectrometry (MS). Binding experiments following this strategy can be performed as simple as radioligand binding experiments by incubation of the target with the marker (together with test compounds if necessary). MS Binding Assays require – like radioligand binding assays – for termination of the binding experiment a s6 kinase of the formed target-marker-complexes from nonbound marker which is typically achieved by filtration. In MS Binding Assays, subsequently to this separation step, the formerly bound marker is finally quantified by LC-MS after its liberation and elution from the target-marker-complexes remaining on the filter with an organic solvent, whereas in radioligand binding experiments in contrast the bound marker remaining on the filter employed for separation is quantified by LSC (liquid scintillation counting). For the establishment of GlyT1 MS Binding Assays, at first, an appropriate marker has to be identified. As several tritium labelled GlyT1 inhibitors have already proved their suitability as reporter ligands in filtration based binding experiments (Alberati et al., 2012; Herdon et al., 2010; Mallorga et al., 2003; Mezler et al., 2008; Zhang et al., 2011), we decided to start with a nonlabelled GlyT1 inhibitor already employed as radioligand. Therefore, the commercially available GlyT1 selective inhibitors ALX5407 (Fig. 1; (R)-2) and Org24598 (Fig. 1; (R)-3) were selected for this purpose. For none of both compounds a method for a highly sensitive quantification has been described so far. Hence, at first such a method using liquid chromatography electrospray ionization with tandem mass spectrometry (LC-ESI-MS/MS), a technique assumed to be best suited for this task, had to be established (Höfner and Wanner, 2015). The analytical range required for marker quantification can be estimated from the equilibrium dissociation constant (Kd) of the marker towards GlyT1 taking into account the commonly accepted directives for radioligand binding assays suggesting examination of nominal marker concentrations in saturation experiments between 0.1 Kd and 10 Kd while the target concentration should not exceed 0.1 Kd to avoid marker depletion (Bylund et al., 2004; Hulme, 1992). Hence, as a rule of thumb, at least a marker concentration of about 0.01 Kd should be quantifiable (Grimm et al., 2015b). Taking into account the Kd values described for ALX5407 and Org24598 ranging from 4 to 21 nM (Alberati et al., 2012; Mallorga et al., 2003; Zhang et al., 2011) it was aspired to reach a lower limit of quantification (LLOQ) in the lower pM range. After the final selection of the marker, i.e. whether ALX5407 or Org24598 should be used, based on its suitability for LC-MS quantification, the developed LC-ESI-MS/MS method should be validated according to the FDA guidance for bioanalytical method validation (FDA, 2018) to guarantee its reliability for marker quantification in binding experiments. Subsequently, binding experiments should be performed in analogy to known GlyT1 radioligand binding assays and MS Binding Assays established for other neurotransmitter transporters of the SLC6 family (Alberati et al., 2012; Grimm et al., 2015a, 2015b; Herdon et al., 2010; Hess et al., 2011a, 2011b; Mallorga et al., 2003; Mezler et al., 2008; Neiens et al., 2018a, 2018b; Zepperitz et al., 2006, 2008; Zhang et al., 2011). Thereby, the affinities of the marker and known GlyT ligands (see Fig. 1) towards GlyT1 should be characterized in saturation and competition experiments, respectively. Finally, the results obtained with the established GlyT1 MS Binding Assays should be compared with those from literature to prove their reliability.