Approach to the specificity and selectivity between D2 and D3 receptors by mutagenesis and binding experiments part I: Expression and characterization of D2 and D3 receptor mutants

Abstract

D3/D2 sub-specificity is a complex problem to solve. Indeed, in the absence of easy structural biology of the G-protein coupled receptors, and despite key progresses in this area, the systematic knowledge of the ligand/receptor relationship is difficult to obtain. Due to these structural biology limitations concerning membrane proteins, we favored the use of directed mutagenesis to document a rational towards the discovery of markedly specific D3 ligands over D2 ligands together with basic binding experiments. Using our methodology of stable expression of receptors in HEK cells, we constructed the gene encoding for 24 mutants and 4 chimeras of either D2 or D3 receptors and expressed them stably. Those cell lines, expressing a single copy of one receptor mutant each, were stably constructed, selected, amplified and the membranes from them were prepared. Binding data at those receptors were obtained using standard binding conditions for D2 and D3 dopamine receptors. We generated 26 new molecules derived from D2 or D3 ligands. Using 8 reference compounds and those 26 molecules, we characterized their binding at those mutants and chimeras, exemplifying an approach to better understand the difference at the molecular level of the D2 and D3 receptors. Although all the individual results are presented and could be used for minute analyses, the present report does not discuss the differences between D2 and D3 data. It simply shows the feasibility of the approach and its potential.

Third-Generation Antipsychotics and Lurasidone in the Treatment of Substance-Induced Psychoses: A Narrative Review

“…This high D 3 affinity is significant, considering that most antipsychotics have a lower affinity for this receptor, leading to inadequate D 3 receptor occupancy in the brain. This unique affinity is crucial for cariprazine's clinical efficacy, as indicated by PET studies on schizophrenic patients, demonstrating a stronger effect on the D 3 receptor compared to the D 2 receptor [41][42][43][44][45][46][47] (see Table S6).…”

Biased Agonism or “Biaism” for Dummies: A Commentary

“…For the expression of a transgene, the use of the integrase system leads to a perfectly mastered system in which the transgene is expressed at a control level, similarly to different genes, once inserted in a given region of the cell genome that was described for its neutrality [36]. Furthermore, from one clone to the next, the stable expression of the transgene is of a comparative level, leading to populations of cells expressing similar amounts of the transgene, a very useful tool whenever one wants to compare a mutant to a wild type, or mutant receptors to each other [37]. Finally, as pointed out above, further cautions should be applied in interpreting data on biaism obtained from overexpressing systems [35].…”