Harald Huebner

A new D2 dopamine receptor agonist allosterically modulates A2A adenosine receptor signalling by interacting with the A2A/D2 receptor heteromer

The structural and functional interaction between D₂ dopamine receptor (DR) and A(2A) adenosine receptor (AR) has suggested these two receptors as a pharmacological target in pathologies associated with dopamine dysfunction, such as Parkinsons disease. In transfected cell lines it has been demonstrated the activation of D₂DR induces a significant negative regulation of A(2A)AR-mediated responses, whereas few data are at now available about the regulation of A(2A)AR by D₂DR agonists at receptor recognition site. In this work we confirmed that in A(2A)AR/D₂DR co-transfected cells, these receptors exist as homo- and hetero-dimers. The classical D₂DR agonists were able to negatively modulate both A(2A)AR affinity and functionality. These effects occurred even if any significant changes in A(2A)AR/D₂DR energy transfer interaction could be detected in BRET experiments. Since the development of new molecules able to target A(2A)/D₂ dimers may represent an attractive tool for innovative pharmacological therapy, we also identified a new small molecule, 3-(3,4-dimethylphenyl)-1-(2-piperidin-1-yl)ethyl)piperidine (compound 1), full agonist of D₂DR and modulator of A(2A)-D₂ receptor dimer. This compound was able to negatively modulate A(2A)AR binding properties and functional responsiveness in a manner comparable to classical D₂R agonists. In contrast to classical agonists, compound 1 led to conformational changes in the quaternary structure in D₂DR homomers and heteromers and induced A(2A)AR/D₂DR co-internalization. These results suggest that compound 1 exerts a high control of the function of heteromers and could represent a starting point for the development of new drugs targeting A(2A)AR/D₂ DR heteromers.

Multicomponent Synthesis and Biological Evaluation of a Piperazine-Based Dopamine Receptor Ligand Library.

A series of 1,4-disubstituted piperazine-based compounds were designed, synthesized, and evaluated as dopamine D2/D3 receptor ligands. The synthesis relies on the key multicomponent split-Ugi reaction, assessing its great potential in generating chemical diversity around the piperazine core. With the aim of evaluating the effect of such diversity on the dopamine receptor affinity, a small library of compounds was prepared, applying post-Ugi transformations. Ligand stimulated binding assays indicated that some compounds show a significant affinity, with K i values up to 53 nM for the D2 receptor. Molecular docking studies with the D2 and D3 receptor homology models were also performed on selected compounds. They highlighted key interactions at the indole head and at the piperazine moiety, which resulted in good agreement with the known pharmacophore models, thus helping to explain the observed structure-activity relationship data. Molecular insights from this study could enable a rational improvement of the split-Ugi primary scaffold, toward more selective ligands.

Structure-Based Design and Discovery of New M2 Receptor Agonists

Muscarinic receptor agonists are characterized by apparently strict restraints on their tertiary or quaternary amine and their distance to an ester or related center. On the basis of the active state crystal structure of the muscarinic M2 receptor in complex with iperoxo, we explored potential agonists that lacked the highly conserved functionalities of previously known ligands. Using structure-guided pharmacophore design followed by docking, we found two agonists (compounds 3 and 17), out of 19 docked and synthesized compounds, that fit the receptor well and were predicted to form a hydrogen-bond conserved among known agonists. Structural optimization led to compound 28, which was 4-fold more potent than its parent 3. Fortified by the discovery of this new scaffold, we sought a broader range of chemotypes by docking 2.2 million fragments, which revealed another three micromolar agonists unrelated either to 28 or known muscarinics. Even pockets as tightly defined and as deeply studied as that of the muscarinic reveal opportunities for the structure-based design and the discovery of new chemotypes.

A new small molecule, D2 dopamine receptor agonist, as an allosteric modulator of A2A adenosine receptor signalling

The rodent P2X7k splice variant, which utilizes a different exon 1 and is expressed in both wild type and the Glaxo P2X7−/−mice, has at least a 10-fold greater sensitivity to agonists ATP and BzATP than the full length mouse P2X7a variant, as determined from patch clamp experiments. ThemP2X7k receptor also coupled to the rapid uptake of the cationic dye ethidium, and this process was unaffected by the SNP, P451L, that previously was shown to inhibit mP2X7a receptor-mediated dye uptake. In HEK293 cells, activation of mP2X7k also triggered the uptake of the anionic dye Lucifer yellow, the rate of which was dramatically increased by a 5-min pre-incubation with the pannexin-1 inhibitor, carbenoxolone (CBX) at 37°C. CBX also increased mP2X7k-mediated ethidium uptake but the Ca2+ signal evoked by activation of mP2X7k was unaffected, suggesting that CBX selectively affects ‘large pore’ formation. Themechanismof action of CBX is unclear but the results are not consistentwith pannexin-1 being the dye uptake pathway. P2X7k is expressed in mice spleen T-lymphocytes where NAD is reported to be a physiologically important agonist. In HEK293 cells, 10 uM NAD stimulated mP2X7k-mediated ethidium uptake whereas mP2X7a did not respond to concentrations up to 300 uM NAD. NAD also stimulated a rapid Ca2+ response in HEK293 cells expressing mP2X7k. The coexpression of mP2X7a and mP2X7k in HEK cells reduced the amplitude of the NAD response compared to cells expressing mP2X7k alone. Our results suggest the expression of P2X7k increases the diversity of P2X7 receptor signaling in rodents.