Rational Design of Nanobody80 Loop Peptidomimetics: Towards Biased β2 Adrenergic Receptor Ligands

5 years ago

Rational Design of Nanobody80 Loop Peptidomimetics: Towards Biased β2 Adrenergic Receptor Ligands

Jesper Mosolff Mathiesen, Krisztina Fehér, Cecilia Fabris, Els Pardon, Charlotte Martin, Nick Devoogdt, Samuel L. C. Moors, Daniel Sejer Pedersen, May C. Morris, Cecilia Betti, Steven Ballet, Jan Steyaert, Frank De Proft, José C. Martins, Vicky Caveliers, Marion Peyressatre, Mia Danielsen

G protein-coupled receptors (GPCRs) play an important role in many cellular responses; as such, their mechanism of action is of utmost interest. To gain insight into the active conformation of GPCRs, the X-ray crystal structures of nanobody (Nb)-stabilized β2-adrenergic receptor (β2AR) have been reported. Nb80, in particular, is able to bind the intracellular G protein binding site of β2AR and stabilize the receptor in an active conformation. Within Nb80, the complementarity-determining region 3 (CDR3) is responsible for most of the binding interactions. Hence, we hypothesized that peptidomimetics of the CDR3 loop might be sufficient for binding to the receptor, inhibiting the interaction of β2AR with intracellular GPCR interacting proteins (e.g., G proteins). Based on previous crystallographic data, a set of peptidomimetics were synthesized that, similar to the Nb80 CDR3 loop, adopt a β-hairpin conformation. Syntheses, conformational analysis, binding and functional in vitro assays, as well as internalization experiments, were performed. We demonstrate that peptidomimetics can structurally mimic the CDR3 loop of a nanobody and its function by inhibiting G protein coupling as measured by partial inhibition of cAMP production. Looping out: Cyclic peptidomimetics were designed as potential tools to structurally mimic the CDR3 loop of nanobodies (see figure). Herein, the synthesis, conformational analysis, binding, and functional in vitro assays are presented, together demonstrating that a single CDR3 loop mimetic could not functionally mimic the parent nanobody.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1002/chem.201701321