Dopamine D₂, D₃, and D₄ Selective Phenylpiperazines as Molecular Probes To Explore the Origins of Subtype Specific Receptor Binding

Abstract

Assembling phenylpiperazines with 7a-azaindole via different spacer elements, we developed subtype selective dopamine receptor ligands of types 1a,c, 2a, and 3a preferentially interacting with D4, D2, and D3, respectively. To complete this set, the methylthio analogues 2b and 3b exceeding the affinity of 2a and 3a by one order of magnitude and the structural intermediate 1b were synthesized. These chemically similar but biologically divergent target compounds served as molecular probes for radioligand displacement experiments, mutagenesis, and docking studies on homology models based on the recent crystal structure of the beta2-adrenergic receptor. Specific interactions with the highly conserved amino acids Asp3.32 and His6.55 and less conserved residues at positions 2.61, 2.64, 3.28, and 3.29 were identified. Inclusion of a carefully modeled extracellular loop 2 displayed two nonconserved residues in EL2 that differently contribute to ligand binding. Obviously, subtype selectivity is caused by nonconserved but frequently mediated by conserved amino acids.

Histidine 6.55 Is a Major Determinant of Ligand-Biased Signaling in Dopamine D2LReceptor

“…The affinities of other compounds remained unchanged or mildly decreased (up to 3.5-fold as for CPD1). These findings are in accordance with those of our previous study (Ehrlich et al, 2009), which demonstrated the D 2L H393…”

“…The cDNA of the human dopamine D2 long (D 2L ) receptor was purchased from the Missouri University of Science and Technology cDNA Resource Center (Rolla, MO). The site-directed mutagenesis was performed as described previously (Ehrlich et al, 2009). The D 2L wild-type, D 2L H393 6.55 A, and D 2L H393 6.55 F receptor cDNAs were subcloned into a pcDNA5/FRT vector (Invitrogen) using NheI/XhoI restriction sites.…”

“…Data were analyzed by nonlinear regression and were best-fit to one-site (monophasic) or two-site (biphasic) competition curves. pK i values were calculated according to Cheng and Prusoff (1973 (Ehrlich et al, 2009), the homologs CPD1 (Hackling et al, 2003) and FAUC346 (Bettinetti et al, 2002) display butylene spacers between the basic center and the two-atom carboxamide moiety. Aripiprazole combines a butlyene linker with a one-atom heteroaromatic ether group (Oshiro et al, 1998).…”

“…We demonstrated that the mutation of His393 6.55 to alanine results in the increased affinity of 1,4-disubstituted phenylpiperazines at the D 2L receptor (Ehrlich et al, 2009). We hypothesized that the additional space created by the H393 6.55 A mutation leads to an increase in conformational freedom of the phenylalanine residues Phe389 6.51 and Phe390 6.52 .…”

A Single Glycine in Extracellular Loop 1 Is the Critical Determinant for Pharmacological Specificity of Dopamine D2 and D3 Receptors

“…We have previously reported that the 3-carbon linked analog of the prototypic D3R selective antagonist, NGB 2904 (N-[4-[4-(2,3-dichlorophenyl)-1-piperazinyl]butyl]-9H-fluorene-2-carboxamide), showed an approximately 100-fold decrease in D3R affinity, but remained D3R selective, as did another 3-carbon linked analog with the 2,3-diCl-substituted 4-phenylpiperazine (Robarge et al, 2001). In contrast, a 2-OCH 3 -substituted 4-phenylpiperazine derivative with a 3-carbon linker has previously been reported to be approximately 14-fold selective for D2R over D3R (Ehrlich et al, 2009). The decrease in D3R affinity in this case was also accompanied by an increase in D2R affinity and could be due to the 2-OCH 3 substitution of the 4-phenylpiperazine bound in the OBS and/or the azaindole as the SP.…”