autoradiography-as-simple-powerful-method-for-visualization

Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The ...

University of Copenhagen

The αβδ subtype of the γ-aminobutyric acid (GABA) type A receptors (GABARs) has been shown to be implicated in high-affinity binding of the neuromodulator γ-hydroxybutyric acid (GHB), but may not be the only GHB high-affinity binding sites. Monastrol has been identified as a modulator of GHB high-affinity binding and is furthermore reported as an allosteric modulator selective for the αβδ GABARs. Therefore, structural determinants for selectivity at the two targets were investigated. 39 structural diverse monastrol analogues were synthesized by employing the Biginelli cyclocondensation and examined for modulation of GHB high-affinity binding using the GHB-specific ligand [H]NCS-382 [(E,RS)-6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene)acetic acid] in rat brain homogenate. Only limited modifications were allowed on the monastrol scaffold in order to maintain modulation of GHB high-affinity binding. However, three analogues of monastrol (11, 12 and 24) enhanced the maximal binding of [H]NCS-382 to a higher maximal level than seen for monastrol itself. Selected compounds were further characterized as modulators at αβδ, αβγ and αβ GABARs. Most of these modulators were shown to have δ-specific GABA-potentiating effects. The dual effect shown for monastrol to modulate the GHB high-affinity binding and αβδ GABAR activity was also shown for the compounds 11, 18 and 24. Compound 29 displayed minimal modulatory effect on GABARs and therefore appears to be a GHB high-affinity binding preferring modulator. However, compounds 34 and 37 were shown to be αβδ GABAR selective modulators, without modulatory effects on GHB high-affinity binding. Thus, our study shows that minor modifications in the structure of monastrol affects the selectivity profile for the two targets under study enabling separation of the dual activity.

Monastrol, a 3,4-dihydropyrimidin-2(1H)-thione, as structural scaffold for the development of modulators for GHB high-affinity binding sites and αβδ GABA receptors.

Virtual screening offers an efficient alternative to high-throughput screening in the identification of pharmacological tools and lead compounds. Virtual screening is typically based on the matching of target structures or ligand pharmacophores to commercial or in-house compound catalogues. This study provides the first proof-of-concept for our recently reported method where pharmacophores are instead constructed based on the inference of residue-ligand fragments from crystal structures. We demonstrate its unique utility for G protein-coupled receptors, which represent the largest families of human membrane proteins and drug targets. We identified five neutral antagonists and one inverse agonist for the histamine H receptor with potencies of 0.7-8.5 μM in a recombinant receptor cell-based inositol phosphate accumulation assay and validated their activity using a radioligand competition binding assay. H receptor antagonism is of large therapeutic value and our ligands could serve as starting points for further lead optimisation. The six ligands exhibit four chemical scaffolds, whereof three have high novelty in comparison to the known H receptor ligands in the ChEMBL database. The complete pharmacophore fragment library is freely available through the GPCR database, GPCRdb, allowing the successful application herein to be repeated for most of the 285 class A GPCR targets. The method could also easily be adapted to other protein families.

Identification of Histamine H Receptor Ligands Using a New Crystal Structure Fragment-based Method.

Imbalances in GABA-mediated tonic inhibition are involved in several pathophysiological conditions. A classical way of controlling tonic inhibition is through pharmacological intervention with extrasynaptic GABA receptors that sense ambient GABA and mediate a persistent GABAergic conductance. An increase in tonic inhibition may, however, also be obtained indirectly by inhibiting glial GABA transporters (GATs). These are sodium-coupled membrane transport proteins that normally act to terminate GABA neurotransmitter action by taking up GABA into surrounding astrocytes. The aim of the review is to provide an overview of glial GATs in regulating tonic inhibition, especially in epilepsy and stroke. This entails a comprehensive summary of changes known to occur in GAT expression levels and signalling following epileptic and ischemic insults. Further, we discuss the accumulating pharmacological evidence for targeting GATs in these diseases.

Article	Year
Monastrol, a 3,4-dihydropyrimidin-2(1H)-thione, as structural scaffold for the development of modulators for GHB high-affinity binding sites and αβδ GABA receptors. European journal of medicinal chemistry\| PubMed ID: 28683403	2017
Identification of Histamine H Receptor Ligands Using a New Crystal Structure Fragment-based Method. Scientific reports\| PubMed ID: 28684785	2017
Glial GABA Transporters as Modulators of Inhibitory Signalling in Epilepsy and Stroke. Advances in neurobiology\| PubMed ID: 28828609	2017

Petrine Wellendorph

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