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

As tool compounds to study cardiac ischemia, the endogenous δ-opioid receptors (δOR) agonist Leu5-enkephalin and the more metabolically stable synthetic peptide (d-Ala2, d-Leu5)-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 Phe4 position of Leu5-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 Phe4 of Leu5-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 Phe4 may be combined with other modifications to Leu5-enkephalin to deliver improved agonists with finely tuned potency, selectivity, bias and drug-like properties.

From a translational perspective, peptide-based probes provide an ideal tool for studying the cardioprotective effects of the δOR, given their low brain penetration. While numerous enkephalin-like peptides have been synthesized that interact with excellent potency and selectivity for δORs and µORs [13], a majority of studies [14] investigating δOR involvement in ischemia have utilized the synthetic peptide D-Ala 2 ,D-Leu 5 -enkephalin (DADLE, Figure   1) [15,16], because DADLE possesses improved proteolytic stability and improved selectivity for δORs over µORs relative to Leu 5 -enkephalin [6,17]. However, DADLE's discovery in 1977 [18], predated identification of β-arrestin as a modulator of opioid signaling [14,[19][20][21], With the use of contemporary cellular assays it is now apparent that DADLE pharmacologically signals similarly to Leu 5 -enkephalin, though it recruits β-arrestin 2 (arrestin 3) slightly more efficaciously. Given the similarities between DADLE and Leu 5 -enkephalin, it is unclear to what degree β-arrestin recruitment contributes to or detracts from these peptides' in vivo cardioprotective efficacy, and new analogs with distinct pharmacolgocial profiles are necessary to probe these contributions.
To better investigate the role of δOR-mediated β-arrestin signaling in ischemic protection, the development of δOR selective agonists that have either low, intermediate or high β-arrestin is desired; however, reports of δOR selective peptide-based biased ligands remain limited.

Results and Discussion
Design Considerations: To probe the meta position of Phe 4 , we initially considered known structure-activity-relationship trends at the ortho and para positions of this residue. Considering that halogenated substituents at these positions perturbed binding affinity, δOR selectivity, and stability properties of the Leu 5 -enkephalin [28], we initially hypothesized that meta-halogenated analogs might similarly perturb the parent scaffold ( Figure 3A, 1a-1d). An additional set of analogs bearing electron-donating (1e-1f) and -withdrawing groups (1g-1i) would further probe interactions at this site, including the electronic character of the Phe 4 ring. Finally, pyridine analogs (1j-1l) would present H-bond accepting contacts about the ring, as well as provide analogs that present dipoles at similar vectors as to previously successful halogenated substituents.
Solid Phase Synthesis of Peptides: All peptides were synthesized using a rapid solid phase peptide synthesis protocol on an automated peptide synthesizer using an Fmoc protection strategy [34,35] and N,N′-diisopropylcarbodiimide and oxyma as the coupling reagents ( Figure   3B). Fmoc-Leu-Wang resin was utilized as a starting template for this synthetic protocol. All coupling steps and Fmoc-deprotection steps were carried out at 70 ºC under an atmosphere of N 2 .
Though the improved δOR affinity at the meta position is significantly greater than the perturbations imparted by Cl, Br, or I at the ortho position (up to 6-to 17-fold, based on δOR construct) [28]. Notably, the meta-chlorinated and -brominated analogs (1b, and 1c) are 500-900fold more potent in cAMP assays at δOR than at µOR, despite exhibiting little differences in binding affinity at δOR relative to µOR ( Table 1). Interestingly, the meta-Cl analog (1b) had stronger bias towards G protein-signaling at δOR ( Table 3, bias factor 1.6), but towards βarrestin 2 recruitment at µOR relative to Leu 5 -enkephalin ( Table 3, bias factor 0.004) as well as DAMGO ( Table 3, bias factor 0.3), which provides a unique pharmacological profile for future uses. In contrast, meta-F and -CN and -NO 2 -substitutions (1a,h,i) improved δOR functional selectivity ( Table 3), but lost β-arrestin 2 potency at δOR relative to Leu 5 -enkephalin.
Additionally, meta-OMe and -NO 2 substitution (1f, 1i) provided both potent and biased analogs, with G protein coupling activities comparable to Leu 5 -enkephalin ( Table 2, IC 50 = 0.14-0.47 nM vs. 1.02 nM), but with improved bias factors relative to Leu 5 -enkephalin at both δOR ( Table 3, bias factor 6.9-7.5) and µOR ( Table 3, bias factor 8.4-11.1). Of these two analogs, the -NO 2substituted analog 1i exhibited higher δOR functional G protein selectivity ( Table 3, 104-fold δOR selectivity) relative to the -OMe analog 1f ( Table 3, 58-fold δOR selectivity). Though pyridyl-substituted analogs (1j-l) showed poor potency and efficacy for the δOR and µOR relative to Leu 5 -enkephalin ( Table 2), the 3-and 4-pyridyl analogs (1k-l) showed strong bias at µOR ( Table 3, bias factor 17-331), when compared to the full agonist DAMGO. However, if instead of DAMGO, Leu 5 -enkephalin was used as the reference compound, most analogs, with the exception of 1k and 1l lost G protein bias ( Table 3, bias factor <1), because the analogs generally were more potent and efficacious than Leu 5 -enkephalin in recruiting β-arrestin 2 at µOR ( Table 2), with the exception of 1k ( Table 2, 36% recruitment efficacy). Given that exogenous Leu 5 -enkephalin analogs in vivo would compete with endogenous Leu 5 -enkephalin and not DAMGO, our results highlight the limitations of interpreting bias factors (particularly using an unnatural compound, such as DAMGO, as a reference) and the associated risk of using bias factor as a major driver of lead optimization. Compared to DADLE, the Leu 5 -enkephalin analogs 1b and 1c displayed a similar signaling bias profile, but were more potent and selective for δOR, and thus may provide a better tool compound to target δORs for the treatment of cardiac ischemia. However, compounds 1b and 1c are also more potent than DADLE at recruiting β-arrestin 2 to µOR, resulting in a lower bias factor (Figure 4). In contrast, analog 1i has a favorable pharmacology relative to DADLE, with similar selectivity as 1c, but improved G-protein bias at δOR and µOR relative to DADLE.
Given the potential adverse effects associated with strong µOR-mediated β-arrestin 2 recruitment, analog 1k is potentially useful, as it exhibited weak β-arrestin 2 recruitment (36% efficacy) and concomitant strong bias factor for G-protein signaling. However, cAMP potency for 1k at δOR was more than one log unit weaker than DADLE, and thus it may be necessary further optimize the ligand by increasing δOR potency in the cAMP assay, while retaining the low µOR β-arrestin 2 recruitment efficacy (Figure 4).

Stability:
The stability of all compounds to Sprague Dawley rat plasma was assessed to study the influence of the meta-and picoline/pyridine-substitutions at Phe 4 relative to Leu 5 -enkephalin (Table 4). For this parent compound, the predominant known routes of metabolism and clearance occur through cleavage of Tyr 1 -Gly 2 by aminopeptidase N [36,37], and of Gly 3 -Phe 4 by angiotensin converting enzyme [38], and combined, the plasma metabolism occurs with a half-life (t 1/2 ) of < 10 min. In general, meta substituted Phe 4 analogs exhibited improved plasma stability compared with Leu 5 -enkephalin with half-lives typically >20 min. The 3-fluoro derivative (1a) was the most stable analog with a half-life of 82.3 min. From UPLC-mass spectrometry analysis of degradation fragments, meta-substitution did not greatly impede the proteolysis at the Tyr 1 -Gly 2 site, but instead slowed digestion at the Gly 3 -Phe 4 site ( Table 4).
Thus, the improved stability of our Phe 4 -substituted analogs presumably derived from perturbation/deceleration of angiotensin-converting enzyme activity. Picoline peptides also displayed improved stability though interestingly, UPLC-mass spectrometry analysis indicated that degradation of all pyridyl-substituted analogs (1j-l) predominantly occurred through Tyr 1 -Gly 2 as opposed to meta-substituted analogs 1a-i that degraded through cleavage of Gly 3 -Phe 4 .

Table 4. Rat Plasma Stability of Leu 5 -enkephalin and Its Analogs
Compound

Half-life (min) 95% CI Degradation products (first appearance)
Leu 5 -enkephalin 9. opioid. The generated pharmacological data herein may aid computational modeling efforts to reveal ligand-receptor interactions at δOR and µOR that will guide the development of novel peptides with tuned selectivity and signaling profiles. The novel Leu 5 -enkephalin analogs have superior δOR selectivity over µOR relative to DADLE, the gold-standard peptide for studying the role of δOR in cardiac ischemia. Additionally, the analogs generally have lower β-arrestin recruitment efficacy at µOR, which could further reduce potential adverse in vivo effects.
Finally, relative to DADLE, the meta-substituents tune the bias profile at δOR (either more or less biased towards G-protein signaling), and the resulting tool compounds should be useful for investigating the importance of δOR mediated β-arrestin signaling in the peptides cardioprotective effects.

Synthetic Chemistry -General Considerations.
Unless specified, all chemicals were purchased from commercial sources and used without further purification. All solvents used for synthesis were of analytical grade and used without further purification. Proton nuclear magnetic resonance ( 1 H NMR) spectra, and carbon nuclear magnetic resonance ( 13 C NMR) spectra were recorded on a Bruker AVIII 500 AVANCE spectrometer with a CPDUL cryoprobe (500 and 126 MHz, respectively) or a Bruker DRX 500 spectrometer (500 and 126 MHz, respectively). Peptides were synthesized using an Aapptec Focus XC automated peptide synthesizer coupled with a heating system using a solid phase peptide synthesis protocol using Fmoc chemistry.
Preparative RP-HPLC was performed using an appropriate column and solvent system (described below) and final purity of peptides was determined by UV area % from UPLC analysis. Peptides were purified by Teledyne ISCO EZ Prep system on RediSep® C18 Prep column (30x250 mm, 100 Å). Purity analysis of final peptides was carried out using a Waters

Synthesis of Peptides.
Peptides were synthesized using a solid phase peptide synthesis protocol using an Aapptec Focus XC automated peptide synthesizer coupled with a heating system using the Fmoc chemistry and Wang resin as solid support [34]. To prepare the resin for synthesis, a Cell culture and biased signaling assays. cAMP inhibition and β-arrestin 2 recruitment assays were performed as previously described [12]. In brief, for cAMP inhibition assays HEK 293 (Life Technologies, Grand Island, NY, USA) cells were transiently transfected in a 1:3 ratio with  (Table S2) as previously described [12]. Subsequently, bias factors were calculated using Leu 5 -enkephalin as reference compound for δOR and using either DAMGO or Leu 5 -enkephalin as reference compound for µOR, respectively. Leu 5 -enkephalin and DAMGO were more potent in the cAMP (G protein) assay than in the β-Arrestin 2 recruitment assay, and thus were not unbiased, but rather G protein-biased to begin with. A bias factor >1 meant that the agonist was more G protein-biased than the reference compound; A bias factor <1 meant that the agonist was less G protein-biased than the reference compound.
Data and Statistical analysis. All data are presented as means ± standard error of the mean, and analysis was performed using GraphPad Prism 8 software (GraphPad Software, La Jolla, CA).
For in vitro assays, nonlinear regression was conducted to determine pIC 50 (cAMP) or pEC 50 (βarrestin 2 recruitment). Technical replicates were used to ensure the reliability of single values, specifically each data point for binding and β-arrestin recruitment was run in duplicate, and for the cAMP assay in triplicate. The averages of each independent run were counted as a single experiment and combined to provide a composite curve in favor of providing a 'representative' curve. In each experimental run, a positive control/reference compound was utilized to allow the data to be normalized and to calculate the log bias value.

Supplementary Material
Supplementary materials are available online. Specifically, for compounds 1a-l: Additional pharmacological characterization, stability data, NMR spectra and characterization data for peptides, UPLC traces for determining purity.