Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

The serotonin 5-HT₇ receptors: two decades of research

Like most neurotransmitters, serotonin possesses a simple structure. However, the pharmacological consequences are more complex and diverse. Serotonin is involved in numerous functions in the human body including the control of appetite, sleep, memory and learning, temperature regulation, mood, behavior, cardiovascular function, muscle contraction, endocrine regulation, and depression. Low levels of serotonin may be associated with several disorders, namely increase in aggressive and angry behaviors, clinical depression, Parkinson's disease, obsessive-compulsive disorder, eating disorders, migraine, irritable bowel syndrome, tinnitus, and bipolar disease. These effects are mediated via different serotonin (5-HT) receptors. In this review, we will focus on the last discovered member of this serotonin receptor family, the 5-HT7 receptor. This receptor belongs to the G protein-coupled receptor superfamily and was cloned two decades ago. Later, different splice variants were described but no major functional differences have been described so far. All 5-HT7 receptor variants are coupled to G alpha(s) proteins and stimulate cAMP formation. Recently, several interacting proteins have been reported, which can influence receptor signaling and trafficking

Downregulation of 5-HT7 serotonin receptors by the atypical antipsychotics clozapine and olanzapine: role of motifs in the C-terminal domain and interaction with GASP-1

The human 5-HT7 serotonin receptor, a G-protein-coupled receptor (GPCR), activates adenylyl cyclase constitutively and upon agonist activation. Biased ligands differentially activate 5-HT7 serotonin receptor desensitization, internalization and degradation in addition to G protein activation. We have previously found that the atypical antipsychotics clozapine and olanzapine inhibited G protein Lysosome activation and, surprisingly, induced both internalization and lysosomal degradation of 5-HT7 receptors. Here, we aimed to determine the mechanism of clozapine- and olanzapine-mediated degradation of 5-HT7 receptors. In the C-terminus of the 5-HT7 receptor, we identified two YXX Phi motifs, LR residues, and a palmitoylated cysteine anchor as potential sites involved in receptor trafficking to lysosomes followed by receptor degradation. Mutating either of these sites inhibited clozapine- and olanzapine-mediated degradation of 5-HT7 receptors and also interfered with G protein activation. In addition, we tested whether receptor degradation was mediated by the GPCR-associated sorting protein-1 (GASP-1). We show that GASP-1 binds the 5-HT7 receptor and regulates the clozapine-mediated degradation. Mutations of the identified motifs and residues, located in or close to Helix-VIII of the 5-HT7 receptor, modified antipsychotic-stimulated binding of proteins (such as GASP-1), possibly by altering the flexibility of Helix-VIII, and also interfered with G protein activation. Taken together, our data demonstrate that binding of clozapine or olanzapine to the 5-HT7 receptor leads to antagonist-mediated lysosomal degradation by exposing key residues in the C-terminal tail that interact with GASP-1

Brain penetrant small 18F-GnRH receptor (GnRH-R) antagonists: Synthesis and preliminary positron emission tomography imaging in rats

<h4 id="absSec_2">Introduction</h4>The gonadotropin releasing hormone receptor (GnRH-R) has a well-described neuroendocrine function in the anterior pituitary. However, little is known about its function in the central nervous system (CNS), where it is most abundantly expressed in hippocampus and amygdala. Since peptide ligands based upon the endogenous decapetide GnRH do not pass the blood brain-barrier, we are seeking a high-affinity small molecule GnRH-R ligand suitable for brain imaging by positron emission tomography. We have previously reported the radiosynthesis and <em>in vitro</em> evaluation of two novel [¹⁸F]fluorinated GnRH-R ligands belonging to the furamide class of antagonists, with molecular weight less than 500 Da. We now extend this work using palladium coupling for the synthesis of four novel radioligands, with putatively reduced polar surface area and hydrophilicity relative to the two previously described compounds, and report the uptake of these ¹⁸F-labeled compounds in brain of living rats.</p><h4 id="absSec_2">Methods</h4><p id="sp0085">We synthesized reference standards of the small molecule GnRH-R antagonists as well as mesylate precursors for ¹⁸F-labeling. The antagonists were tested for binding affinity for both human and rat GnRH-R. Serum and blood stability <em>in vitro</em> and <em>in vivo</em> were studied. Biodistribution and PET imaging studies were performed in male rats in order to assess brain penetration <em>in vivo</em>.</p><h4 id="absSec_3">Results</h4><p id="sp0090">A palladium coupling methodology served for the synthesis of four novel fluorinated furamide GnRH receptor antagonists with reduced heteroatomic count. Radioligand binding assays <em>in vitro</em> revealed subnanomolar affinity of the new fluorinated compounds for both human and rat GnRH-R. The ¹⁸F-GnRH antagonists were synthesized from the corresponding mesylate precursors in 5-15% overall radiochemical yield. The radiolabeled compounds demonstrated good <em>in vivo</em> stability. PET imaging with the ¹⁸F-radiotracers in naive rats showed good permeability into brain and rapid washout, but absence of discernible specific binding <em>in vivo</em>.</p><h4 id="absSec_4">Conclusions</h4><p id="sp0095">The novel small molecule ¹⁸F-fluorinated GnRH-R antagonist compounds show high receptor affinity <em>in vitro</em>, and may prove useful for quantitative autoradiographic studies <em>in vitro</em>. The compounds were permeable to the blood-brain barrier, but nonetheless failed to reveal significant specific binding in brain of living rats. Nonetheless, our approach may serve as a foundation for designing PET ligands suitable to image the GnRH-R distribution in brain