Allosteric interactions between catecholamine receptors and other G protein-coupled receptors: Pharmacological and functional characterization

Abstract

Catecholamines, including dopamine (DA) and norepinephrine (NE), are widely distributed in the body and constitute a class of conventional neurotransmitters and hormones that occupy key positions in the regulation of physiological processes and in the development of neurological, psychiatric, endocrine and cardiovascular diseases. There is a linkage between a variety of genes related to DA (e.g. D4 receptor) and NE (e.g. α2A-adrenoceptor) and the vulnerability for developing attention deficit hyperactivity disorder (ADHD), which is characterized by pervasive symptoms of inattention, impulsivity and/or hyperactivity. In addition, adenosine, acting on adenosine receptors (AR), is a modulator of other receptors such as D1-like and D2-like DA receptors (DR). DA and adenosine receptor complexes are involved in the control of the direct and indirect pathway of motor control in basal ganglia, in which adrenergic receptors are also involved. NE, DA and adenosine receptors belong to the GPCR family, also known as seven transmembrane domain receptors. GPCRs have an enormous biomedical importance. It is estimated that about 35% of approved drugs target GPCRs. Thus, is not surprising that lots of models have been developed in order to explain the pharmacological behavior of these receptors. A large number of GPCRs have been described to form homodimers, heterodimers and higher order oligomers with different pharmacological and functional properties than of its individual components. The aim of this Thesis has been to study and characterize the molecular interactions at pharmacological and functional level of heterodimers between catecholamine receptors and between catecholamine and adenosine receptors involved in several neurological pathologies related to imbalances in attention, impulsivity and motor control. For simplicity, for pharmacologically characterize GPCRs, most of the developed models consider them as monomeric entities. Thus, it is not surprising that, when working with receptor dimers but using monomeric models, some parameters obtained may be erroneous. Then, first of all, we went deeper into the study of allosteric interactions within GPCRs using the dimer receptor model, which considers GPCRs as dimers. Along our study, we have given further evidences that homodimers are the GPCR predominant species, and that allosteric interactions between orthosteric ligands of the different protomers of a GPCR heteromer, have important implications in the field of catecholamine receptor pharmacology. A paradigmatic example of complex allosteric interactions is the A2AR-A2AR-D2R-D2R-Gs-Gi-AC5 heteromer. Moreover, since bivalent ligands are the best example of oligomer selective-ligands that can interact simultaneously with GPCR dimers with high affinity and subtype selectivity, we have developed a precise strategy for developing them. Using computational tools that considered the TM interfaces, distances between orthosteric binding sites and the mode of interaction of the pharmacophore units, we have higher success in affinity results. In particular, the obtained GPCR bivalent ligand had a picomolar binding affinity for the dopamine D2 receptor (D2R) homodimer. However, to obtain oligomer-selective bivalent ligands, the selected pharmacophores must be highly specific. This is not always easy to find. As an example, we have demonstrated that catecholamine receptors constitute a “functional” family of GPCRs. Specifically, in this study we have shown that DA and synthetic DA receptor ligands are able to bind to α2Rs and activate the same signaling pathways as NE. In addition, we have demonstrated the existence of functional D4R-D2SR in vitro and, for the first time, functional D4R-α2AR heteromers in vitro and in rodent brain tissues not only with the D4.4R but also with the D4.7R variant, prevalent in ADHD. Significant different properties of these heteromers were D4R variant-dependent. Finally, given that D2R, D4R and α2AR, are involved in the pathophysiology of ADHD, we suggest that D4R-D2R and α2AR-D4R heteromers could be target for the therapeutic treatment of such neurological disorders.

Les catecolamines dopamina (DA) i norepinefrina (NE) tenen una funció clau en la regulació de processos fisiològics i en el desenvolupament de diverses patologies. Existeix una correlació entre gens relacionats amb la DA (com el receptor D4) i amb la NE (com el receptor α2A) i la vulnerabilitat per desenvolupar el trastorn per dèficit d’atenció i hiperactivitat (TDAH). A més, l’adenosina, actuant sobre els receptors d’adenosina (AR), és un modulador dels receptors de DA tipus D1 i D2, que controlen el moviment als ganglis basals, on també es troben implicats els adrenoreceptors. Els receptors de NE, DA i adenosina pertanyen a la família dels GPCR, que té una gran importància biomèdica, essent diana d’un 35% dels fàrmacs aprovats. És conegut que els GPCRs formen homodimers, heterodimers i oligòmers més complexos amb noves propietats farmacològiques i funcionals. En aquesta Tesi hem caracteritzat les interaccions moleculars entre receptors de catecolamines i entre receptors de catecolamines i d’adenosina, involucrats en patologies neurològiques relacionades amb l’atenció, la impulsivitat i el control motor. Concretament, hem donat evidències que els homodimers de GPCRs són les espècies predominants a l’organisme i que les interaccions alostèriques entre lligands ortostèrics dins un heteròmer tenen importants implicacions farmacològiques. També hem generat un protocol de síntesis de lligands bivalents molt eficient. Aquests lligands permeten actuar sobre un oligòmer concret, minimitzant els efectes secundaris en comparació amb fàrmacs dirigits a monomèrs. Hem demostrat que els receptors de catecolamines constitueixen una mateixa família funcional donada la promiscuïtat entre els seus lligands. Finalment, hem descrit l’existència de complexos entre els receptors D4 i D2S i entre D4 i α2A, trobant diferències funcionals segons la variant del receptor D4 involucrada. Donat que els receptors D2R, D4R i α2AR estan involucrats en el TDAH, aquests heteròmers poden ser una nova diana terapèutica per a aquesta patologia.