The oxytocin receptor is an emerging therapeutic target for social disorders such as schizophrenia and autism. However, treating these disorders of the central nervous system requires ligands to be able to cross the blood brain barrier and interact with receptors in the brain. The native ligand of this g protein-coupled receptor is oxytocin, a nonapeptide with a short half-life and poor brain penetration. This makes it a poor centrally acting therapeutic. Small molecule agonists may be useful for these indications, however mimicking the activation capability of a peptide with a small molecule can be difficult, and there is currently no solved structure of this protein to aid with structure-based drug design.
My work aims to use a novel mammalian cell directed evolution platform to introduce mutations to the oxytocin receptor that increase its expression levels and stability. This will enable us to express and purify the oxytocin receptor, and solve the structure using cryo-electron microscopy.
Lentivirus was produced by transfecting HEK293T cells with lentiviral plasmids and a mutant oxytocin receptor library created using error prone PCR. Virus was titrated onto HEK293F cells to a multiplicity of infection of 30%, where there is on average an infection rate of 1 receptor per cell. This was determined using an IRES mCherry cell marker. These cells underwent 3 rounds of fluoresence activated cell sorting using a novel fluorescent agonist, enriching the highest binding population. Individual mutants were isolated from this enriched population and were assayed for expression, binding, signalling and stability.
This population will undergo further random mutagenesis and screening before being purified from either insect or mammalian cells. We hope this approach will help overcome the bottlenecks of poor expression and detergent instability that have hindered structure determination of the oxytocin receptor thus far.