Receptor-mediated activation and pharmacological inhibition of heterotrimeric G proteins

Abstract

Heterotrimeric guanine-nucleotide binding proteins (G proteins) are key signal transducers in animal and human cells, where they forward signals transmitted by active G protein-coupled receptors (GPCRs) and trigger intracellular signaling cascades. This thesis presents pharmacological studies on (i) macrocyclic G&alpha;_Ga protein inhibitors and (ii) on the G&alpha; protein activation by the adenosine A_2B receptor (A_2BAR), a promiscuous GPCR. <br /> (i) The structurally related natural products YM-254890 (YM) and FR900359 (FR) are potent and selective inhibitors of the G&alpha;_q/11 protein family. Recently, our group developed tritium-labeled derivatives, which display large differences in residence time, despite having a similar affinity for the G&alpha;_q protein. We identified lipophilic interactions between the inhibitor binding site and an isopropyl group, which is exclusively present in FR and its derivatives, as the molecular basis for the long residence time of the FR-derived radioligand. Furthermore, our data suggest a complex binding mechanism of macrocyclic G&alpha;_q protein inhibitors, involving a conformational selection step. The proposed mechanism was supported by molecular dynamics simulation studies. Next, we established structure-affinity relationships and structure-residence time relationships of a series of YM and FR derivatives. Additionally, YM- and FR-derived radioligands were employed to analyze the G&alpha;_q/11 protein expression in mouse tissues by autoradiography. <br /> (ii) The A_2BAR constitutes a promising drug target for the immunotherapy of cancer. Its physiological functions, however, remain enigmatic. Here, we determine the G&alpha; protein activation fingerprint of the A_2BAR by calcium mobilization assays and novel BRET assay systems. In summary, we found that the A_2BAR is a promiscuous receptor, which activated nearly all G&alpha; protein subunits. However, it did so with varying degrees of efficacy. It couples most efficaciously to G&alpha;_s, G&alpha;₁₅, and G&alpha;₁₂ proteins, but activates all other G&alpha; proteins with submaximal efficacy (&lt; 50% of control receptors). Importantly, each employed agonist displays a different G&alpha; coupling profile. Finally, both the expression levels of the G&alpha; protein and the GPCR are decisive for effective coupling of the A_2BAR to a certain G&alpha; protein. <br /> Altogether, this work provides profound insights into the molecular pharmacology of macrocyclic G&alpha;_q protein inhibitors, focusing on their binding kinetics – a parameter which is gaining recognition in drug design. Furthermore, we created a G&alpha; protein coupling profile of the A_2BAR and identified relevant parameters that influence A_2BAR-mediated G&alpha; activation to gain more insight into A_2BAR signal transmission.