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

Abstract

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 β -adrenergic receptor (β AR) have been reported. Nb80, in particular, is able to bind the intracellular G protein binding site of β AR 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 β AR 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.

Computational design and characterization of nanobody-derived peptides that stabilize the active conformation of the β2-adrenergic receptor (β2-AR)

“…Second, an antibody uses only a single loop, its CDR3, to interact directly with the antigen 28 . Because CDR3 within Nb80 is responsible for most of the binding interactions 16 and because the peptidomimetics of the CDR3 loop were likely sufficient for binding to the receptor and inhibiting the interaction of β 2 -AR with its intracellular GPCR-interacting proteins, such as Gαs and β-arrestins, the peptidomimetics of CDR3 structurally mimicked the CDR3 loop of an Nb 29 .

Figure 2Mapping of the putative interaction sites of β 2 -AR and Nb71. ( a ) Position of the domain in the primary structure of Nb71 (residues 88–104).

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Therapeutic Potential of Targeting ß-Arrestin

“…Interestingly, Martin et al. synthesized a set of peptidomimetics which are structurally similar to the complementarity-determining region 3 (CDR3) of the nanobody Nb80, and inhibit ß 2 AR-G protein coupling (Martin et al., 2017).…”

Trifluoromethylated Proline Surrogates as Part of “Pro–Pro” Turn‐Inducing Templates

“…Considering the general synthetic accessibility of building block 2 a , b , which is synthesized as a racemic mixture in two steps, compound 15 was selected over 6 and 14 for incorporation into tripeptide sequences PG‐( R )‐Pro–( R )‐TfmOxa–( S )‐Val‐OH, in which PG is an acetyl group for conformational studies and a fluorenylmethyloxycarbonyl (Fmoc) group for subsequent solid‐phase peptide synthesis (SPPS) incorporation. Valine was selected as the TfmOxa‐flanking residue because it directly correlated with the CDR3 sequence of previously reported nanobody Nb80 mimetic peptide IV (Figure ) . This mimetic was designed by grafting the CDR3 loop on a ( R )‐Pro–( S )‐Pro template.…”

“…B) Overlay of the best fitting molecular dynamics (MD) conformation (Cα root‐mean‐square deviation (RMSD)=1.4 Å) of mimetic IV (orange) and the secondary structure of the CDR3 loop region in Nb80. C) Detailed structure of IV …”