Site-directed mutagenesis to reveal essential residues for 5-HT7 receptor binding and activation

Serotonin (5-hydroxytryptamine, 5-HT) is a key neurotransmitter implicated in neuropsychiatric disturbances such as depression, anxiety and psychosis. Recent studies on knockout animals and using selective 5-HT7 receptor antagonists have provided strong s upport that the G-protein-coupled 5-HT7 receptor plays a role in the onset of depression. Hence, the development and investigation of novel 5-HT7 receptor ligands may lead to a new class of novel antidepressant agents. To better understand the process of ligand binding to and activation of the 5-HT7 receptor and which essential residues are involved, we will use structural modelling of 5-HT7 receptors and site-directed mutagenesis combined with biological assays to identify which amino acids are essential for ligand binding and receptor activation. Determination of which amino acids are to be mutated is based upon the fact that the 5-HT1A and 5-HT7 receptors, which share a similar affinity for many ligands, have a high sequence homology in their putative binding sites. Putatively important residues have so far been identified in the 7th transmembrane domain (TM7) and in the second intracellular loop of the h5-HT7(a) receptor and will be tested first. Using in silico methods for drug discovery requires the careful integration of several computational tools into a single streamlined process. In this case, the first step is the homology modelling and its refinement of the target GPCR. The validated model will then be used to help identify putative ligands fi rst by 3D screening (docking) of large virtual compound libraries, followed by a scoring and selection process that leads to a small number of virtual hits. The virtual hits will subsequently be purchased and tested in experimental assays for ligand bindi ng and receptor activation to verify their activity.