The Meta-Position of Phe4 in Leu-Enkephalin Regulates Potency, Selectivity, Functional Activity, and Signaling Bias at the Delta and Mu Opioid Receptors

Abstract

As tool compounds to study cardiac ischemia, the endogenous δ-opioid receptors (δOR) agonist Leu 5 -enkephalin and the more metabolically stable synthetic peptide (d-Ala 2 , d-Leu 5 )-enkephalin are frequently employed. However, both peptides have similar pharmacological profiles that restrict detailed investigation of the cellular mechanism of the δOR's protective role during ischemic events. Thus, a need remains for δOR peptides with improved selectivity and unique signaling properties for investigating the specific roles for δOR signaling in cardiac ischemia. To this end, we explored substitution at the Phe 4 position of Leu 5 -enkephalin for its ability to modulate receptor function and selectivity. Peptides were assessed for their affinity to bind to δORs and µ-opioid receptors (µORs) and potency to inhibit cAMP signaling and to recruit β-arrestin 2. Additionally, peptide stability was measured in rat plasma. Substitution of the meta-position of Phe 4 of Leu 5 -enkephalin provided high-affinity ligands with varying levels of selectivity and bias at both the δOR and µOR and improved peptide stability, while substitution with picoline derivatives produced lower-affinity ligands with G protein biases at both receptors. Overall, these favorable substitutions at the meta-position of Phe 4 may be combined with other modifications to Leu 5 -enkephalin to deliver improved agonists with finely tuned potency, selectivity, bias and drug-like properties.

Modulating β Arrestin-2 Recruitment at the δ- and μ-Opioid Receptors

“…In general, the potency for the peptides to recruit b-Arr 2 at dOR was 10-fold lower than for the peptides to inhibit cAMP at dOR (Table 2), which matches previous findings. 28 Despite their similar binding profiles (Table 1) and potencies inhibiting cAMP ( Figure 3A,C Figure 4A), and we speculate that perturbation of these helices causes intracellular distortion that in turn affects β-Arr 2 recruitment.…”

Identification of a Novel Delta Opioid Receptor Agonist Chemotype with Potential Negative Allosteric Modulator Capabilities

“…Next, we performed a structure activity relationship (SAR) by catalog using 14 analogs of compound 1 ( Figure 4 , Table 2 ) to investigate how compound 1 may bind to δOR and to possibly identify compounds with improved pharmacology. In our experience, potency for δOR agonism in the PathHunter β-arrestin assay is generally lower than for the cAMP assay [ 21 ]. Therefore, to assess if analogs of compound 1 displayed improved δOR potency we first characterized the compounds in the cAMP assay.…”

Opioids and Their Receptors: Present and Emerging Concepts in Opioid Drug Discovery

“…Endogenous and naturally occurring opioid peptides have continuously served as important leads for the design of peptide analogues, with a repertoire of structural modifications that can be targeted when exploring SARs or focusing on the improvement of their pharmacodynamics and the pharmacokinetics of peptide active compounds. Four research articles [5][6][7][8] focused on this subject.…”

“…Cassell et al [7] explored SAR trends, at the meta-position of Phe 4 , of the endogenous DOR peptide Leu 5 -enkephalin, demonstrating that substitution at this position variously regulated DOR and MOR affinity and G protein activity, enabled the fine-tuning of β-arrestin2 recruitment to both receptors, and increased the plasma stability of the derived peptides. The resulting peptide analogues should be useful tools for studying the role of DOR in cardiac ischemia and the importance of DOR mediated β-arrestin2 signaling in the peptides cardioprotective effects.…”