molecules Article The Meta-Position of Phe4 in Leu-Enkephalin Regulates Potency, Selectivity, Functional Activity, and Signaling Bias at the Delta and Mu Opioid Receptors 1, 2, 1 3 Robert J. Cassell y, Krishna K. Sharma y , Hongyu Su , Benjamin R. Cummins , Haoyue Cui 4, Kendall L. Mores 1, Arryn T. Blaine 1, Ryan A. Altman 2,* and Richard M. van Rijn 1,5,6,* 1 Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue University, West Lafayette, IN 47907, USA; [email protected] (R.J.C.); [email protected] (H.S.); [email protected] (K.L.M.); [email protected] (A.T.B.) 2 Department of Medicinal Chemistry, The University of Kansas, Lawrence, KS 66045, USA; [email protected] 3 Department of Chemistry, Purdue University, West Lafayette, IN 47907, USA; [email protected] 4 College of Wuya, Shenyang Pharmaceutical University, Shenyang 110016, China; [email protected] 5 Purdue Institute for Drug Discovery, Purdue University, West Lafayette, IN 47907, USA 6 Purdue Institute for Integrative Neuroscience, Purdue University, West Lafayette, IN 47907, USA * Correspondence: [email protected] (R.A.A.); [email protected] (R.M.v.R.) These authors contributed equally to this work. y Academic Editors: Mariana Spetea and Helmut Schmidhammer Received: 20 November 2019; Accepted: 10 December 2019; Published: 12 December 2019 Abstract: 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. Keywords: Leu-enkephalin; beta-arrestin; mu opioid receptor; delta opioid receptor; biased signaling; DADLE; ischemia; plasma stability Molecules 2019, 24, 4542; doi:10.3390/molecules24244542 www.mdpi.com/journal/molecules Molecules 2019, 24, 4542 2 of 15 1.Molecules Introduction 2019, 24, x FOR PEER REVIEW 2 of 15 5 1 2 3 4 5 Leu 5--enkephalinenkephalin (Tyr 1-Gly-Gly2--GlyGly3--PhePhe4--LeuLeu5, ,Figure Figure 1)1) is an endogenous opioid peptidepeptide producedproduced in vertebratevertebrate speciesspecies includingincluding rodents, rodents, primates primates and and humans humans [1 [1–4–]4] that that results results from from the the metabolism metabolism of 5 proenkephalinof proenkephalin or dynorphinor dynorphin [5]. Pharmacologically,[5]. Pharmacologically, Leu -enkephalin Leu5-enkephalin agonizes agonizes the δ opioid the δ receptor opioid (receptorδOR) with (δOR) moderate with moderate selectivity selectivity over the overµ opioid the µ receptor opioid receptor (µOR), but (µOR), does but not does significantly not significantl interacty 5 withinteract the withκ opioid the κ receptor opioid [receptor6]. As a neurotransmitter[6]. As a neurotransmitter in pain circuits, in pain Leu circuits,-enkephalin Leu5-enkephalin possesses antinociceptivepossesses antinociceptive properties [7 ],properties whereas peripherally, [7], whereas the peptide peripherally, demonstrates the cardiovascularpeptide demonstrates effects [8]. Overcardiovascular the last twoeffects decades, [8]. Over improved the last two pharmacological decades, improved characterization pharmacological of opioid characterization pathways has of revealedopioid pathways that activation has revealed of an opioid that activation receptor canof an trigger opioid two receptor distinct can pathways, trigger twoβ-arrestin-dependent distinct pathways, orβ-arrestinβ-arrestin-independent-dependent or β- (i.e.,arrestin G protein-mediated)-independent (i.e. and, G protein that these-mediated) pathways and di ffthaterentially these pathways modulate antinociceptiondifferentially modulate and side antinociception effect profiles [and9]. Despiteside effect the profiles increasing [9]. Despite number the of studiesincreasing that number implicate of δstudiesOR mediated that implicateβ-arrestin δOR recruitment mediated β with-arrestin various recruitment (patho)physiological with various e (patho)physiologicalffects, such as tolerance effects, [10], alcoholsuch as intaketolerance [11, 12[10]] and, alcoholδOR agonist-induced intake [11,12] and seizures δOR [agonist10], the- roleinduced of β-arrestin seizures recruitment [10], the role towards of β- δarrestinOR-induced recruitment cardioprotection towards δOR remains-induced unclear. cardioprotection remains unclear. OH OH Leu5-Enkephalin (Leu-Enk) iPr O O H H N N O H3N N N N H H H N OH O O O 3 O O HN O OH iPr O O iPr H H NH N N O – H3N N N O2C H H Rubiscolin-6 O O Me O O HN N O H3N DADLE OH iPr O O NH HN O – O i iPr O Pr O O O NH H H – N N N O2C H3N N N O O H H HN O O O Aza-β-homoleucine-enkephalin Rubiscolin-5 i O– Pr Figure 1.1. Overview of unbiased (Leu5--enkephalinenkephalin and and DADLE) and biased (Aza-(Aza-β-Homoleucine--Homoleucine- Enkephalin, Rubiscolin-5Rubiscolin-5 and -6)-6) δδOROR peptides.peptides. From a translationaltranslational perspective, peptide-basedpeptide-based probes provide an ideal tool for studying the cardioprotective eeffectsffects of of the theδ OR,δOR, given given their their low low brain brain penetration. penetration. While While numerous numerous enkephalin-like enkephalin- peptideslike peptides have have been been synthesized synthesized that that interact interact with with excellent excellent potency potency and and selectivity selectivityfor for δδORsORs and µµORsORs [13] [13],, a a majority majority of of studies studies [14] [14 investigating] investigating δORδOR involvement involvement in ischemia in ischemia have have utilized utilized the 2 5 thesynthetic synthetic peptide peptide D-Alad2,-Ala D-Leu, 5d-enkephalin-Leu -enkephalin (DADLE (DADLE, Figure, 1) Figure [15,16]1)[, because15,16], DADLE because possessesDADLE possessesimproved improvedproteolytic proteolytic stability and stability improved and improvedselectivity selectivityfor δORs foroverδORs µORs over relativeµORs to relative Leu5- toenkephalin Leu5-enkephalin [6,17]. However, [6,17]. However,DADLE’s DADLEdiscovery’s in discovery 1977 [18] in, predated 1977 [18 identification], predated identification of β-arrestin ofas βa -arrestinmodulator as aof modulator opioid signaling of opioid [14,19 signaling–21], With [14,19 the–21 ],use With of contemporary the use of contemporary cellular assays cellular it is assays now itapparent is now apparentthat DADLE that pharmacologicallyDADLE pharmacologically signals similarly signals to similarly Leu5-enkephalin, to Leu5-enkephalin, though it recruits though β it- recruitsarrestin β2 -arrestin(arrestin 23) (arrestin slightly 3)more slightly efficaciously. more effi caciously.Given the Givensimilarities the similarities between DADLE between andDADLE Leu5- andenkephalin, Leu5-enkephalin, it is unclear it isto unclearwhat degree to what β-arrestin degree recruitmentβ-arrestin recruitment contributes contributes to or detracts to orfrom detracts these frompeptides’ these in peptides’ vivo cardioprotectivein vivo cardioprotective efficacy, and effi cacy,new analogs and new with analogs distinct with phar distinctmacolo pharmacoloogicalogical profiles profilesare necessary are necessary to probe to these probe contributions. these contributions. To better investigate investigate the the role role of of δORδOR-mediated-mediated β-arrestinβ-arrestin signaling signaling in ischemic in ischemic protection, protection, the thedevelopment development of δOR of δOR selective selective agonists agonists that that have have either either low, low, intermediate intermediate or or high high β--arrestinarrestin is desired; however, reports of δOR selective peptide-based biased ligands remain limited. Recently, the naturally occurring peptides rubiscolin-5 and -6 (Figure 1) were classified as G protein-biased (β- arrestin 2 efficacy = 15% and 20%, respectively; δOR bias factor = 2.0) δOR selective peptides, albeit with only micromolar potencies [22]. While a number of synthetic peptides display nanomolar Molecules 2019, 24, 4542 3 of 15 desired; however, reports of δOR selective peptide-based biased ligands remain limited. Recently, the naturally occurring peptides rubiscolin-5 and -6 (Figure1) were classified as G protein-biased (β-arrestin 2 efficacy = 15% and 20%, respectively; δOR bias factor = 2.0) δOR selective peptides, albeit with only micromolar potencies [22]. While a number of synthetic peptides display nanomolar potencies