Engineering a GPCR-ligand pair that simulates the activation of D(2L) by Dopamine.

In the past decade, engineered G-protein-coupled receptors activated solely by synthetic ligands (RASSLs) have been implemented as a new means to study neurotransmission, which is controlled by G-protein-coupled receptors in vitro and in vivo. In this study, we report an engineered dopamine receptor D(2L) F390(6.52)W, which is the first identified RASSL for the dopamine receptor family. The mutant receptor is characterized by a disrupted ligand binding and complete loss of efficacy for the endogenous ligand, dopamine, which is putatively due to a sterically induced perturbation of H-bonding with conserved serine residues in TM5. Based on this model, we rationally developed an aminoindane-derived set of agonists. Because these agonists forgo analogous H-bonding functionalities, their binding energy does not depend on the respective interactions. Binding affinity and potency were optimized by ligand modifications bearing molecular appendages that obviously interact with a secondary recognition site provided by four hydrophobic residues in TM2 and TM3. Thus, the ferrocenyl carboxamide 5b (FAUC 185) was identified as a synthetic agonist that is able to stimulate the mutant receptor in a manner similar to that by which endogenous dopamine activates the D(2L) wild-type receptor. The engineered dopamine receptor D(2L) F390(6.52)W in combination with FAUC 185 (5b) provides a new tool to probe GPCR functions selectively in specific cell populations in vitro and in vivo.

Novel D3 selective dopaminergics incorporating enyne units as nonaromatic catechol bioisosteres: synthesis, bioactivity, and mutagenesis studies.

Enynes of type 4 and 5 as long chain derivatives of the nonaromatic dopamine D 3 receptor agonist 3 (FAUC 73) were prepared by exploiting chemoselective functionalization of the azido-substituted vinyl triflate 9. Radioligand binding studies indicated excellent D 3 affinity and selectivity over related GPCRs for the terminal alkynes 4c (FAUC 460) and 5c. Biphasic displacement curves gave picomolar K i values for the high affinity binding site of D 3. According to mitogenesis experiments and bioluminescence based cAMP assays, the biphenylcarboxamide 4c and its click chemistry derived triazole analogue 5c behaved as strong partial agonists but relative ligand efficacy significantly depended on the type of functional assay. Site directed mutagenesis involving the mutants D 3 D3.32E, and D 3 F6.51W implied that ligand interactions with D3.32 and F6.51 are highly crucial, giving rise to analogous binding modes for dopamine, classical and enyne type agonists.