Our exploration of Domain focused on the
Our exploration of Domain 1 focused on the role of the carbonyl linker in (). We found that replacement of this group by a methylene unit as in or direct attachment of the thiophene ring to the central aromatic core as in did not significantly impact potency. The thiophene ring in could be replaced by phenyl () and cyclohexyl (). In the case of phenyl analogues only a methylene linker was found to be equivalent in activity to the carbonyl group. A biphenyl system as in led to a decrease in activity. Cyclohexyl analogues and without a carbonyl group linker were also significantly less active.
The effect of substituents in directly attached phenyl groups is detailed in .
-Substitution as in and was found to improve potency. Introduction of a fluorine Dipraglurant led to some loss of activity (), a methoxy group () reduced activity even further. A limited exploration of heteroaromatic and fused systems as in – did not yield any compounds with greater activity.
details the effect of combining preferred substituents from Domains 1 and 2. This approach led to the discovery of several compounds with significantly improved in vitro activity over the initial hit . Incorporation of alkoxy and alkyl substituents into five or six-membered aliphatic rings as in – and – was found to be particularly beneficial for in vitro activity.
In order to assess the ability of our compounds to selectively increase cortical glycine levels in an animal model, we measured their effect on glycine levels in rat cerebrospinal fluid (CSF) after subcutaneous administration () at a dose of 30mg/kg. Animals were euthanized and 50–100ml of CSF was sampled immediately after death from the cisterna magna., , The concentration of glycine and test compounds in the CSF was determined using an LC/MS method in positive electrospray mode ().
Our initial hit molecule showed no significant effect on CSF glycine levels. 2-Chloro-phenyl substituted analogue had a weak effect on CSF glycine levels, but 2-thiophene substituted proved to have a similar effect on glycine levels as ALX-5407 . Corresponding to the increase in glycine levels compound levels of measured in CSF were also significantly higher than those measured for , , and .
Introduction Glycine is a major inhibitory neurotransmitter in the CNS and controls motor and sensory signal transduction. GlyT1 and GlyT2 affect glycinergic neurotransmission by regulating the extracellular glycine concentration . The expression of GlyT2 is confined to glycinergic neurons in caudal regions of the central nervous system (CNS), while GlyT1 is expressed in most regions of the CNS, mainly in glial cells and to a lesser extent in a subpopulation of glutamatergic neurons , . It is unknown whether GlyTs are also expressed in the peripheral nervous system (PNS). Although inhibitory glycine receptors are absent in the PNS , GlyTs might contribute here to the regulation of ionotropic glutamate receptors of the N-methyl-d-aspartate (NMDA) subtype . Here, glycine, just as d-serine, acts as an essential coagonist by binding at the NR1 subunit of the receptor . Interestingly, it has been described that the glycine binding NR1 subunit of the NMDA receptor is upregulated at least in the spinal cord in the context of neuropathic pain . Since changes in glycine-dependent neurotransmission have been shown to play a major role in chronic pain, the role of GlyTs has gained increasing attention recently. The pain-relieving effects of GlyT inhibitors are well documented in a variety of experimental pain models . Yet, whether the development of chronic pain is associated with changes in GlyT activity in the nervous system is still unknown.
Western blot analysis Protein samples were prepared by homogenizing and solubilizing the tissue in phosphate buffered saline (PBS) including sodium dodecyl sulfate (SDS 10mg/ml) and 1mM phenylmethanesulfonylfluoride (PMSF). After centrifugation (50,000×g, 15min, 4°C) the content was measured using the Lowry method . Equal amounts of protein (20μg) were separated by SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes in a semidry electroblotting system. Nonspecific protein binding was blocked by incubating the membranes with 5% nonfat milk protein in PBS-Tween. Incubation with primary antibodies (rabbit polyclonal anti-GlyT1 No. 176 (1:1,000) or rabbit polyclonal anti-GlyT2 No. 218 (1:1,000), respectively , rabbit polyclonal anti-N-methyl-d-aspartate (NMDA) receptor subunit NR1 antibody (1:1000, Cell Signaling, Cambridge, UK) followed. Blots were incubated with horseradish peroxidase-linked anti-rabbit IgG. Equal loading of protein was verified by probing the membrane with anti-tubulin antibody (mouse monoclonal; 1:50,000; Sigma–Aldrich, St. Louis, MO, USA) or anti-GAPDH antibody (mouse monoclonal; 1:40,000), Sigma–Aldrich). Immunoreactive bands were detected using an enhanced chemiluminescence system (Santa Cruz, Dallas, TX, USA). The blots were quantified using GelScan 6.0 software (Decon Science Tec, Frankfurt, Germany). The results are expressed as Differential Integrated Density (DID) and presented as normalized density ratios of the protein to the housekeeper protein. Each experiment was run in duplicates.