| Summary: | The 5-HT2AR is a target in neuropsychiatric conditions such as psychosis, which is commonly managed with atypical antipsychotics. These have associated side effects due to off-target interactions. Consequently, there is a demand to develop therapeutics that exhibit no affinity for these off-target sites. Moreover, in recent 5-HT2AR X-ray crystal structures, a unique hydrophobic pocket or passage between transmembrane helices 4 and 5 sets it apart from other 5-HT2 receptor subtypes. This structural feature is implicated in the selectivity demonstrated by therapeutic pimavanserin towards the 5-HT2AR, owing to its binding conformation. Thus, presenting it as a target to interrogate for enhancing 5-HT2AR binding.
A structure-based approach was used to design a pimavanserin analogue series targeting transmembrane helices 4 and 5. In silico studies involving docking and MM-GBSA calculations using a 5-HT2AR homology model (RMSD 0.09 Å) predicted several analogues to demonstrate improved binding compared to pimavanserin. Synthesis of the series yielded low to moderate yields. To evaluate the binding of the series experimentally a cell membrane-based fluorescent time-resolved Förster resonance energy transfer (TR-FRET) methodology was chosen. This led to the development of a high-affinity 5-HT2AR fluorescent probe, and determination of kinetic rates, (Kd 1.74 ± 0.46 nM, Kon 1.96 ± 0.13 x 108 M−1 min−1, and Koff 0.42 ± 0.02 min−1) characterised at SNAP-tagged human 5-HT2AR expressed in Chinese hamster ovary membranes.
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