Cecilia Betti

Structural basis for bifunctional peptide recognition at human δ-opioid receptor

Bifunctional μ- and δ-opioid receptor (OR) ligands are potential therapeutic alternatives, with diminished side effects, to alkaloid opiate analgesics. We solved the structure of human δ-OR bound to the bifunctional δ-OR antagonist and μ-OR agonist tetrapeptide H-Dmt-Tic-Phe-Phe-NH2 (DIPP-NH2) by serial femtosecond crystallography, revealing a cis-peptide bond between H-Dmt and Tic. The observed receptor-peptide interactions are critical for understanding of the pharmacological profiles of opioid peptides and for development of improved analgesics.

Variation of the net charge, lipophilicity, and side chain flexibility in Dmt(1)-DALDA: Effect on Opioid Activity and Biodistribution.

The influence of the side chain charges of the second and fourth amino acid residues in the peptidic μ opioid lead agonist Dmt-d-Arg-Phe-Lys-NH(2) ([Dmt(1)]-DALDA) was examined. Additionally, to increase the overall lipophilicity of [Dmt(1)]-DALDA and to investigate the Phe(3) side chain flexibility, the final amide bond was N-methylated and Phe(3) was replaced by a constrained aminobenzazepine analogue. The in vitro receptor binding and activity of the peptides, as well as their in vivo transport (brain in- and efflux and tissue biodistribution) and antinociceptive properties after peripheral administration (ip and sc) in mice were determined. The structural modifications result in significant shifts of receptor binding, activity, and transport properties. Strikingly, while [Dmt(1)]-DALDA and its N-methyl analogue, Dmt-d-Arg-Phe-NMeLys-NH(2), showed a long-lasting antinociceptive effect (>7 h), the peptides with d-Cit(2) generate potent antinociception more rapidly (maximal effect at 1h postinjection) but also lose their analgesic activity faster when compared to [Dmt(1)]-DALDA and [Dmt(1),NMeLys(4)]-DALDA.

Stabilisation of a short α-helical VIP fragment by side chain to side chain cyclisation: a comparison of common cyclisation motifs by circular dichroism.

A model octapeptide segment derived from vasoactive intestinal peptide (VIP) was utilised to investigate the effect of several conventional cyclisation methods on the α‐helical conformation in short peptide fragments. Three of the classical macrocyclisation techniques (i.e. lactamisation, ring‐closing metathesis and Huisgen cycloaddition) were applied, and the conformations of the resulting cyclic peptides, as well as their linear precursors, were compared by CD analysis. The visibly higher folding propensity of the triazole‐tethered peptide after azide‐alkyne CuAAC macrocyclisation illustrates that the secondary structure of a short peptide fragment can differ significantly depending on the chemical strategy used to covalently cross‐link side chain residues in a ‘helical’ fragment. Copyright

Antimicrobial peptides in frog poisons constitute a molecular toxin delivery system against predators

Animals using toxic peptides and proteins for predation or defense typically depend on specialized morphological structures, like fangs, spines, or a stinger, for effective intoxication. Here we show that amphibian poisons instead incorporate their own molecular system for toxin delivery to attacking predators. Skin-secreted peptides, generally considered part of the amphibian immune system, permeabilize oral epithelial tissue and enable fast access of cosecreted toxins to the predator’s bloodstream and organs. This absorption-enhancing system exists in at least three distantly related frog lineages and is likely to be a widespread adaptation, determining the outcome of predator–prey encounters in hundreds of species.To avoid being eaten, poisonous prey animals must rely on fast passage of toxins across a predator’s oral tissue, a major barrier to large molecules. Here, Raaymakers et al. show that antimicrobial peptides co secreted with frog toxins enhance intoxication of a snake predator by permeabilizing oral cell layers.

Dual Alleviation of Acute and Neuropathic Pain by Fused Opioid Agonist-Neurokinin 1 Antagonist Peptidomimetics.

Herein, the synthesis and biological evaluation of dual opioid agonists-neurokinin 1 receptor (NK1R) antagonists is described. In these multitarget ligands, the two pharmacophores do not overlap, and this allowed maintaining high NK1R affinity and antagonist potency in compounds 12 and 13. Although the fusion of the two ligands resulted in slightly diminished opioid agonism at the μ- and δ-opioid receptors (MOR and DOR, respectively), as compared to the opioid parent peptide, balanced MOR/DOR activities were obtained. Compared to morphine, compounds 12 and 13 produced more potent antinociceptive effects in both acute (tail-flick) and neuropathic pain models (von Frey and cold plate). Similarly to morphine, analgesic tolerance developed after repetitive administration of these compounds. To our delight, compound 12 did not produce cross-tolerance with morphine and high antihyperalgesic and antiallodynic effects could be reinstated after chronic administration of each of the two compounds.

One-Pot Isomerization–Cross Metathesis–Reduction (ICMR) Synthesis of Lipophilic Tetrapeptides

An efficient, versatile and rapid method toward homologue series of lipophilic tetrapeptide derivatives (herein, the opioid peptides H-TIPP-OH and H-DIPP-OH) is reported. High atom economy and a minimal number of synthetic steps resulted from a one-pot tandem isomerization-cross metathesis-reduction sequence (ICMR), applicable both in solution and solid phase methodology. The broadly applicable synthesis proceeds with short reaction times and simple work-up, as illustrated in this work for alkylated opioid tetrapeptides.

Rational design of Nanobody80 loop peptidomimetics : towards biased β2 adrenergic receptor ligands

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 (β2 AR) have been reported. Nb80, in particular, is able to bind the intracellular G protein binding site of β2 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 β2 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.

Nanobody‐Enabled Reverse Pharmacology on G‐Protein‐Coupled Receptors

The conformational complexity of transmembrane signaling of G-protein-coupled receptors (GPCRs) is a central hurdle for the design of screens for receptor agonists. In their basal states, GPCRs have lower affinities for agonists compared to their G-protein-bound active state conformations. Moreover, different agonists can stabilize distinct active receptor conformations and do not uniformly activate all cellular signaling pathways linked to a given receptor (agonist bias). Comparative fragment screens were performed on a β2 -adrenoreceptor-nanobody fusion locked in its active-state conformation by a G-protein-mimicking nanobody, and the same receptor in its basal-state conformation. This simple biophysical assay allowed the identification and ranking of multiple novel agonists and permitted classification of the efficacy of each hit in agonist, antagonist, or inverse agonist categories, thereby opening doors to nanobody-enabled reverse pharmacology.

χ-Space Screening of Dermorphin-Based Tetrapeptides through Use of Constrained Arylazepinone and Quinolinone Scaffolds

Herein, the synthesis of novel conformationally constrained amino acids, 4-amino-8-bromo-2-benzazepin-3-one (8-Br-Aba), 3-amino-3,4-dihydroquinolin-2-one, and regioisomeric 4-amino-naphthoazepinones (1- and 2-Ana), is described. Introduction of these constricted scaffolds into the N-terminal tetrapeptide of dermorphin (i.e., H-Tyr-d-Ala-Phe-Gly-NH2) induced significant shifts in binding affinity, selectivity, and in vitro activity at the μ- and δ-opioid receptors (MOP and DOP, respectively). A reported constrained μ-/δ-opioid lead tetrapeptide H-Dmt-d-Arg-Aba-Gly-NH2 was modified through application of various constrained building blocks to identify optimal spatial orientations in view of activity at the opioid receptors. Interestingly, when the aromatic moieties were turned toward the C-terminus of the peptide sequences, (partial) (ant)agonism at MOP and weak (ant)agonism at DOP were noticed, whereas the incorporation of the 1-Ana residue led toward balanced low nanomolar MOP/DOP binding and in vitro agonism.