Screening for New Inhibitors of Glycine Transporter 1 and 2 by Means of MS Binding Assays.

A straightforward screening of a compound library comprising 2439 substances for the identification of new inhibitors for the neurotransmitter transporters GlyT1 and GlyT2 is described. Screening and full-scale competition experiments were performed using recently developed GlyT1 and GlyT2 MS Binding Assays. That way for both targets, GlyT1 and GlyT2, ligands were identified, which exhibited affinities (pKi values) in the low micromolar to sub-micromolar range. The majority of these binders exhibit new chemical scaffolds in the class of GlyT1 and GlyT2 inhibitors, which could be of interest for the development of new ligands with improved affinities for the target proteins. Additionally, compounds with excellent fluorescent properties were found for GlyT2, which renders them promising compounds for future fluorescence-based techniques. All in all, this study demonstrates that MS Binding Assays represent a powerful technology platform also well suited for the screening of compound libraries in a highly reliable and effective manner.

MS Binding Assays for Glycine Transporter 2 (GlyT2) Employing Org25543 as Reporter Ligand.

This study describes the first binding assay for glycine transporter 2 (GlyT2) following the concept of MS Binding Assays. The selective GlyT2 inhibitor Org25543 was employed as a reporter ligand and it was quantified with a highly sensitive and rapid LC-ESI-MS/MS method. Binding of Org25543 at GlyT2 was characterized in kinetic and saturation experiments with an off-rate of 7.07×10-3 s-1 , an on-rate of 1.01×106  M-1 s-1 , and an equilibrium dissociation constant of 7.45 nM. Furthermore, the inhibitory constants of 19 GlyT ligands were determined in competition experiments. The validity of the GlyT2 affinities determined with the binding assay was examined by a comparison with published inhibitory potencies from various functional assays. With the capability for affinity determination towards GlyT2 the developed MS Binding Assays provide the first tool for affinity profiling of potential ligands and it represents a valuable new alternative to functional assays addressing GlyT2.

MS binding assays for GlyT1 based on Org24598 as nonlabelled reporter ligand.

In this study an alternative to radioligand binding assays addressing the glycine transporter 1 (GlyT1) based on quantification of a nonlabelled reporter ligand by means of mass spectrometry (MS) is presented. The established MS Binding Assays employ the GlyT1 inhibitor Org24598 as reporter ligand for which a highly sensitive LC-ESI-MS/MS (liquid chromatography electrospray ionization tandem mass spectrometry) method was developed. A validation of this LC-ESI-MS/MS method with respect to selectivity, linearity, accuracy and precision according to the FDA guidance demonstrated its reliability for quantification of Org24598 in binding experiments. For the implementation of GlyT1 binding experiments conditions in accordance to known GlyT1 radioligand binding assays and already known filtration based MS Binding Assays were chosen. In saturation experiments the affinity of Org24598 towards GlyT1 could be characterized with an equilibrium dissociation constant (Kd) of 16.8 ±â€¯2.2 nM that is well in agreement with the affinity determined in radioligand binding assays. Finally, several known GlyT ligands were studied in competition experiments and the determined inhibition constants (Ki) compared with results from radioligand binding and uptake assays. The almost perfect correlation of the affinities obtained in the MS based binding experiments with results from literature clearly indicates that the established GlyT1 MS Binding Assays are a powerful substitute for the GlyT1 radioligand binding assays so far used for affinity profiling and screening. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.

Osteoblast-derived paracrine factors regulate angiogenesis in response to mechanical stimulation.

Angiogenesis is a process by which new blood vessels emerge from existing vessels through endothelial cell sprouting, migration, proliferation, and tubule formation. Angiogenesis during skeletal growth, homeostasis and repair is a complex and incompletely understood process. As the skeleton adapts to mechanical loading, we hypothesized that mechanical stimulation regulates "osteo-angio" crosstalk in the context of angiogenesis. We showed that conditioned media (CM) from osteoblasts exposed to fluid shear stress enhanced endothelial cell proliferation and migration, but not tubule formation, relative to CM from static cultures. Endothelial cell sprouting was studied using a dual-channel collagen gel-based microfluidic device that mimics vessel geometry. Static CM enhanced endothelial cell sprouting frequency, whereas loaded CM significantly enhanced both frequency and length. Both sprouting frequency and length were significantly enhanced in response to factors released from osteoblasts exposed to fluid shear stress in an adjacent channel. Osteoblasts released angiogenic factors, of which osteopontin, PDGF-AA, IGBP-2, MCP-1, and Pentraxin-3 were upregulated in response to mechanical loading. These data suggest that in vivo mechanical forces regulate angiogenesis in bone by modulating "osteo-angio" crosstalk through release of paracrine factors, which we term "osteokines".