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Generation and screening of oxime libraries by competitive MS Binding Assays represents a powerful tool for the identification of new compounds, with affinity to mGAT1, the most ...abundant plasma membrane bound GABA transporter in the CNS. By screening a guvacine derived oxime library, new potent inhibitors of mGAT1 had been revealed. In the present study, oxime libraries generated by reaction of a large excess of a rac-nipecotic acid derivative displaying a hydroxylamine functionality in which various aldehydes under suitable conditions, were examined for new potent inhibitors of mGAT1. The pKi values obtained of the best hits were compared with those of related compounds displaying a guvacine instead of a nipecotic acid subunit as hydrophilic moiety. Amongst the new compounds one of the most affine ligands of mGAT1 known so far (pKi = 8.55 ± 0.04) was found.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The relative reactivities of several secondary amines serving as hydride donors in propargylic amines undergoing a 1,5-hydride transfer reaction to yield the respective terminal and 1,3-disubstituted ...allenes were studied. For this study, a two-step procedure was employed. At first, the synthesis of propargylic amines via the CuI-catalyzed aldehyde-alkyne-amine reactions (A3 coupling) was accomplished. The obtained propargylic amines were subsequently transformed to the desired allenes under CdI2 or ZnI2 catalysis. As a result, among the various secondary amines employed, differing in steric bulk, electronic nature, and conformational properties, allyl(tert-butyl)amine was found to be the best hydride donor for the synthesis of terminal allenes. For the synthesis of 1,3-disubstituted allenes, the propyne derivatives containing either a allyl(tert-butyl)amine or a 1,2,3,6-tetrahydropyridine unit in propargylic position performed best. Finally, with the developed procedure, nipecotic acid derivatives containing an N-allenyl substituent were synthesized with good yields using either ZnI2 as catalyst for the preparation of 1-substituted or CdI2 for the synthesis of 1,3-disubstitued allenes.
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IJS, KILJ, NUK, PNG, UL, UM
In this study we present MS Binding Assays for the PCP ion channel binding site of Torpedo californica nicotinic acetylcholine receptor (nAChR) as an alternative to radioligand binding assays. As MS ...Marker Benocyclidine (BTCP) was employed, found to be more affine (Kd of 84.2 nM) than the radioligands, e. g. 3HPCP, used so far in respective binding assays. Based on a highly sensitive and fast LC‐ESI‐MS/MS method for quantification of BTCP samples, BTCP MS Binding Assays for the PCP ion channel binding site of Torpedo nAChR could be established comprising saturation, kinetic and competition experiments. The affinities obtained in competitive BTCP MS Binding Assays for ligands addressing the PCP ion channel binding site of Torpedo nAChR were in excellent accord with those reported from radioligand experiments. Thus, the new BTCP MS Binding Assays represent a potent and reliable alternative to radioligand binding assays used so far for the characterization of ligand binding to the PCP ion channel binding site of the nAChR.
We present the first MS Binding Assays addressing the PCP ion channel binding site of the nicotinic acetylcholine receptor as a substitute for radioligand binding assays utilizing radioligands such as 3HPCP or 3HTCP. Because BTCP has distinctly higher affinity for the binding site than the aforementioned radioligands, it was used as reporter ligand for these MS Binding Assays, comprising kinetic, saturation and competition experiments. Results from competitive MS Binding Assays matched very well with those from radioligand binding experiments reported in the literature, demonstrating the high validity of the new approach.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The inhibitory neurotransmitter γ-aminobutyric acid (GABA) is cleared from the synaptic cleft by the sodium- and chloride-coupled GABA transporter GAT1. Inhibition of GAT1 prolongs the GABAergic ...signaling at the synapse and is a strategy to treat certain forms of epilepsy. In this study, we present the cryo-electron microscopy structure of Rattus norvegicus GABA transporter 1 (rGAT1) at a resolution of 3.1 Å. The structure elucidation was facilitated by epitope transfer of a fragment-antigen binding (Fab) interaction site from the Drosophila dopamine transporter (dDAT) to rGAT1. The structure reveals rGAT1 in a cytosol-facing conformation, with a linear density in the primary binding site that accommodates a molecule of GABA, a displaced ion density proximal to Na site 1 and a bound chloride ion. A unique insertion in TM10 aids the formation of a compact, closed extracellular gate. Besides yielding mechanistic insights into ion and substrate recognition, our study will enable the rational design of specific antiepileptics.
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GEOZS, IJS, IMTLJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
•Human α7 nAChRs activated and desensitized by agonists are potentiated by PNU-120596.•Activity of human α7 nAChRs is resensitized by substituted bispyridinium analogues.•Structure-activity relations ...of substituted bispyridinium analogues were identified.
Organophosphorus compounds, including nerve agents and pesticides, exert their toxicity through irreversible inhibition of acetylcholinesterase (AChE) resulting in an accumulation of acetylcholine and functional impairment of muscarinic and nicotinic acetylcholine receptors. Current therapy comprises oximes to reactivate AChE and atropine to antagonize effects induced by muscarinic acetylcholine receptors. Nicotinic malfunction leading to depression of the central and peripheral respiratory system is not directly treated calling for alternative therapeutic interventions. In the present study, we investigated the electrophysiological properties of the human nAChR subtype α7 (hα7-nAChR) and the functional effect of the 4-tert-butyl bispyridinium (BP) compound MB327 and of a series of novel substituted bispyridinium compounds on the receptors by an automated patch clamp technique. Activation of hα7-nAChRs was induced by nicotine and acetylcholine demonstrating rapid cationic influx up to 100μM. Agonist-induced currents decayed within a few milliseconds revealing fast desensitization of the receptors. Application of higher agonist concentrations led to a decline of current amplitudes which seemed to be due to increasing receptor desensitization. When 100μM of agonist was coapplied with low concentrations of the well characterized α7-specific positive allosteric modulator PNU-120596 (1μM–10μM), the maximum response and duration of nAChR activation were markedly augmented indicating an elongated mean open-time of receptors and prevention of receptor desensitization. However, co-application of increasing PNU-120596 concentrations (>10μM) with agonist induced a decline of potentiated current responses. Although less pronounced than PNU-120596, six of the twenty tested substituted BP compounds, in particular those with a substituent at 3-position and 4-position at the pyridinium moieties, were found to potentiate current responses of hα7-nAChRs, most pronounced MB327.This effect was clearly depended on the presence of the agonist indicating a positive allosteric mechanism of these compounds. Besides potentiation at low concentrations, these compounds seem to interact at different binding sites on hα7-nAChRs since enhancement decreased at high concentrations.
The residual fourteen BP compounds, possessing either an isopropyl-group or more than one group at the pyridinium moiety, antagonized nicotinic currents exhibiting IC50 of low up to high micromolar concentrations (∼1μM–300μM).
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
γ-Amino butyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system (CNS). A malfunction of the GABAergic neurotransmission is connected to several neuronal ...disorders like epilepsy, Alzheimer’s disease, neuropathic pain, and depression. One possibility to enhance GABA levels in the synaptic cleft is to inhibit mGAT1, one of the four known plasma membrane bound GABA transporters, which is considered the most important GABA transporter subtype, being in charge of the removal of GABA from the synaptic cleft after a neuronal impulse. Lipophilic derivatives of nipecotic acid like Tiagabine (Gabitril®), an approved drug used in add-on therapy of epilepsy, are known to inhibit uptake of mGAT1 with high subtype selectivity and affinity. We synthesized new N-substituted nipecotic acid derivatives with a vinyl ether spacer and an unsymmetrical bis-aromatic residue, which carries fluorine substituents at various positions of the aromatic ring-system. The new compounds were characterized with respect to their potency and subtype selectivity as mGAT1 inhibitors.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
This study presents an efficient screening approach based on combination of mass spectrometry (MS) based binding assays (MS Binding Assays) and affinity selection mass spectrometry (ASMS) customized ...for screening of structurally homogeneous libraries sharing a common mass spectrometric fragmentation pattern. After reaction of a nipecotic acid derivative possessing a hydroxylamine functionality with aldehydes, the resulting oxime library was screened accordingly toward the GABA transporter subtype 1 (GAT1), a drug target for several neurological disorders. After assessing sublibraries’ activities for inhibition of reporter ligand binding, hits in active ones were directly identified. This could be achieved by recording mass transitions for the reporter ligand as well as those predicted for the library components in a single LC-MS/MS run with a triple quadrupole mass spectrometer in the multiple reaction monitoring mode. Identification of hits with a predefined affinity could be reliably accomplished by calculation of IC50-values from specific binding concentrations of library constituents and reporter ligand. Application of this strategy revealed six hits, from which two of them were resynthesized for further biological evaluation. Thereby, the best one displayed a pKi of 7.38 in MS Binding Assays and a pIC50 of 6.82 in 3HGABA uptake assays for GAT1.
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•Combination of MS Binding Assays and affinity selection mass spectrometry.•Concept for MS based screening of homogenous compound libraries.•MS detection in the MRM mode based on predicted mass transitions.•Identification of six hits with an IC50-value of ≤100 nM.•Characterization of compounds 9cn and 9cf as potent and selective mGAT1 inhibitors.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
This study describes the screening of dynamic combinatorial libraries based on nipecotic acid as core structure with substituents attached to the 5- instead of the common 1-position for the search of ...novel inhibitors of the GABA transporter GAT1. The generated pseudostatic hydrazone libraries included a total of nearly 900 compounds and were screened for their binding affinities toward GAT1 in competitive mass spectrometry (MS) based Binding Assays. Characterization of the hydrazones with the highest affinities (with cis-configured rac-16gf bearing a 5-(1-naphthyl)furan-2-yl residue and a four atom spacer being the most potent) in binding and uptake experiments revealed an allosteric interaction at GAT1, which was not reported for any other nipecotic acid derivative up to now. Therefore, the herein introduced 5-substituted nipecotic acid derivatives could serve as valuable tools for investigations of allosterically modulated GABA transport mediated by GAT1 and furthermore as starting point for a new class of GAT1 inhibitors.
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 ± 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’.
•GlyT1 binding assays with nonlabelled Org24598 as reporter ligand were developed.•Org24598 binding at GlyT1 could be monitored by means of LC-ESI-MS/MS.•Binding of Org24598 at GlyT1 was characterized in saturation experiments.•Determined affinities for known GlyT ligands are in agreement with literature.•Established GlyT1 MS Binding Assays can substitute radioligand binding assays.
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GEOZS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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.
Reliable and effective: GlyT1 and GlyT2 MS Binding Assays were used for a library screening investigating 2439 compounds. For GlyT1 and GlyT2 several inhibitors were identified, of which GlyT2 Inhibitors exhibiting excellent fluorescent properties were of great interest. The outcome of this study demonstrated that MS Binding Assays are powerful tools for the examination of compound libraries of reasonable size in a straightforward manner.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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