Structure-Activity Relationships of 7-Substituted Dimethyltyrosine-Tetrahydroisoquinoline Opioid Peptidomimetics.

The opioid receptors modulate a variety of biological functions, including pain, mood, and reward. As a result, opioid ligands are being explored as potential therapeutics for a variety of indications. Multifunctional opioid ligands, which act simultaneously at more than one type of opioid receptor, show promise for use in the treatment of addiction, pain, and other conditions. Previously, we reported the creation of bifunctional kappa opioid receptor (KOR) agonist/mu opioid receptor (MOR) partial agonist ligands from the classically delta opioid receptor (DOR) antagonist selective dimethyltyrosine-tetrahydroisoquinoline (Dmt-Tiq) scaffold through the addition of a 7-benzyl pendant on the tetrahydroisoquinoline ring. This study further explores the structure-activity relationships surrounding 7-position pendants on the Dmt-Tiq scaffold. Some analogues maintain a KOR agonist/MOR partial agonist profile, which is being explored in the development of a treatment for cocaine addiction. Others display a MOR agonist/DOR antagonist profile, which has potential to be used in the creation of a less addictive pain medication. Ultimately, we report the synthesis and in vitro evaluation of novel opioid ligands with a variety of multifunctional profiles.

Use of Functional Polymorphisms To Elucidate the Peptide Binding Site of TAP Complexes.

TAP1/TAP2 complexes translocate peptides from the cytosol to the endoplasmic reticulum lumen to enable immune surveillance by CD8(+) T cells. Peptide transport is preceded by peptide binding to a cytosol-accessible surface of TAP1/TAP2 complexes, but the location of the TAP peptide-binding pocket remains unknown. Guided by the known contributions of polymorphic TAP variants to peptide selection, we combined homology modeling of TAP with experimental measurements to identify several TAP residues that interact with peptides. Models for peptide-TAP complexes were generated, which indicate bent conformation for peptides. The peptide binding site of TAP is located at the hydrophobic boundary of the cytosolic membrane leaflet, with striking parallels to the glutathione binding site of NaAtm1, a transporter that functions in bacterial heavy metal detoxification. These studies illustrate the conservation of the ligand recognition modes of bacterial and mammalians transporters involved in peptide-guided cellular surveillance.

Structural adaptations of proteins to different biological membranes.

To gain insight into adaptations of proteins to their membranes, intrinsic hydrophobic thicknesses, distributions of different chemical groups and profiles of hydrogen-bonding capacities (α and ß) and the dipolarity/polarizability parameter (π*) were calculated for lipid-facing surfaces of 460 integral α-helical, ß-barrel and peripheral proteins from eight types of biomembranes. For comparison, polarity profiles were also calculated for ten artificial lipid bilayers that have been previously studied by neutron and X-ray scattering. Estimated hydrophobic thicknesses are 30-31Å for proteins from endoplasmic reticulum, thylakoid, and various bacterial plasma membranes, but differ for proteins from outer bacterial, inner mitochondrial and eukaryotic plasma membranes (23.9, 28.6 and 33.5Å, respectively). Protein and lipid polarity parameters abruptly change in the lipid carbonyl zone that matches the calculated hydrophobic boundaries. Maxima of positively charged protein groups correspond to the location of lipid phosphates at 20-22Å distances from the membrane center. Locations of Tyr atoms coincide with hydrophobic boundaries, while distributions maxima of Trp rings are shifted by 3-4Å toward the membrane center. Distributions of Trp atoms indicate the presence of two 5-8Å-wide midpolar regions with intermediate π* values within the hydrocarbon core, whose size and symmetry depend on the lipid composition of membrane leaflets. Midpolar regions are especially asymmetric in outer bacterial membranes and cell membranes of mesophilic but not hyperthermophilic archaebacteria, indicating the larger width of the central nonpolar region in the later case. In artificial lipid bilayers, midpolar regions are observed up to the level of acyl chain double bonds.

Translation of structure-activity relationships from cyclic mixed efficacy opioid peptides to linear analogues.

Most opioid analgesics used in the treatment of pain are mu opioid receptor (MOR) agonists. While effective, there are significant drawbacks to opioid use, including the development of tolerance and dependence. However, the coadministration of a MOR agonist with a delta opioid receptor (DOR) antagonist slows the development of MOR-related side effects, while maintaining analgesia. We have previously reported a series of cyclic mixed efficacy MOR agonist/DOR antagonist ligands. Here we describe the transfer of key features from these cyclic analogs to linear sequences. Using the linear MOR/DOR agonist, Tyr-DThr-Gly-Phe-Leu-Ser-NH2 (DTLES), as a lead scaffold, we replaced Phe(4) with bulkier and/or constrained aromatic residues shown to confer DOR antagonism in our cyclic ligands. These replacements failed to confer DOR antagonism in the DTLES analogs, presumably because the more flexible linear ligands can adopt binding poses that will fit in the narrow binding pocket of the active conformations of both MOR and DOR. Nonetheless, the pharmacological profile observed in this series, high affinity and efficacy for MOR and DOR with selectivity relative to KOR, has also been shown to reduce the development of unwanted side effects. We further modified our lead MOR/DOR agonist with a C-terminal glucoserine to improve bioavailability. The resulting ligand displayed high efficacy and potency at both MOR and DOR and no efficacy at KOR.

Unveiling the membrane-binding properties of N-terminal and C-terminal regions of G protein-coupled receptor kinase 5 by combined optical spectroscopies.

G protein-coupled receptor kinase 5 (GRK5) is thought to associate with membranes in part via N- and C-terminal segments that are typically disordered in available high-resolution crystal structures. Herein we investigate the interactions of these regions with model cell membrane using combined sum frequency generation (SFG) vibrational spectroscopy and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. It was found that both regions associate with POPC lipid bilayers but adopt different structures when doing so: GRK5 residues 2-31 (GRK5(2-31)) was in random coil whereas GRK5(546-565) was partially helical. When the subphase for the GRK5(2-31) peptide was changed to 40% TFE/60% 10 mM phosphate pH 7.4 buffer, a large change in the SFG amide I signal indicated that GRK5(2-31) became partially helical. By inspecting the membrane behavior of two different segments of GRK5(2-31), namely, GRK5(2-24) and GRK5(25-31), we found that residues 25-31 are responsible for membrane binding, whereas the helical character is imparted by residues 2-24. With SFG, we deduced that the orientation angle of the helical segment of GRK5(2-31) is 46 ± 1° relative to the surface normal in 40% TFE/60% 10 mM phosphate pH = 7.4 buffer but increases to 78 ± 11° with higher ionic strength. We also investigated the effect of PIP2 in the model membrane and concluded that the POPC:PIP2 (9:1) lipid bilayer did not change the behavior of either peptide compared to a pure POPC lipid bilayer. With ATR-FTIR, we also found that Ca(2+)·calmodulin is able to extract both peptides from the POPC lipid bilayer, consistent with the role of this protein in disrupting GRK5 interactions with the plasma membrane in cells.

Synthesis and evaluation of 4-substituted piperidines and piperazines as balanced affinity µ opioid receptor (MOR) agonist/δ opioid receptor (DOR) antagonist ligands.

In this letter, we describe a series of 4-substituted piperidine and piperazine compounds based on tetrahydroquinoline 1, a compound that shows balanced, low nanomolar binding affinity for the mu opioid receptor (MOR) and the delta opioid receptor (DOR). We have shown that by changing the length and flexibility profile of the side chain in this position, binding affinity is improved at both receptors by a significant degree. Furthermore, several of the compounds described herein display good efficacy at MOR, while simultaneously displaying DOR antagonism. The MOR agonist/DOR antagonist has shown promise in the reduction of negative side effects displayed by selective MOR agonists, namely the development of dependence and tolerance.

OPM database and PPM web server: resources for positioning of proteins in membranes.

The Orientations of Proteins in Membranes (OPM) database is a curated web resource that provides spatial positions of membrane-bound peptides and proteins of known three-dimensional structure in the lipid bilayer, together with their structural classification, topology and intracellular localization. OPM currently contains more than 1200 transmembrane and peripheral proteins and peptides from approximately 350 organisms that represent approximately 3800 Protein Data Bank entries. Proteins are classified into classes, superfamilies and families and assigned to 21 distinct membrane types. Spatial positions of proteins with respect to the lipid bilayer are optimized by the PPM 2.0 method that accounts for the hydrophobic, hydrogen bonding and electrostatic interactions of the proteins with the anisotropic water-lipid environment described by the dielectric constant and hydrogen-bonding profiles. The OPM database is freely accessible at http://opm.phar.umich.edu. Data can be sorted, searched or retrieved using the hierarchical classification, source organism, localization in different types of membranes. The database offers downloadable coordinates of proteins and peptides with membrane boundaries. A gallery of protein images and several visualization tools are provided. The database is supplemented by the PPM server (http://opm.phar.umich.edu/server.php) which can be used for calculating spatial positions in membranes of newly determined proteins structures or theoretical models.

Salt bridges overlapping the gonadotropin-releasing hormone receptor agonist binding site reveal a coincidence detector for G protein-coupled receptor activation.

G protein-coupled receptors (GPCRs) play central roles in most physiological functions, and mutations in them cause heritable diseases. Whereas crystal structures provide details about the structure of GPCRs, there is little information that identifies structural features that permit receptors to pass the cellular quality control system or are involved in transition from the ground state to the ligand-activated state. The gonadotropin-releasing hormone receptor (GnRHR), because of its small size among GPCRs, is amenable to molecular biological approaches and to computer modeling. These techniques and interspecies comparisons are used to identify structural features that are important for both intracellular trafficking and GnRHR activation yet distinguish between these processes. Our model features two salt (Arg(38)-Asp(98) and Glu(90)-Lys(121)) and two disulfide (Cys(14)-Cys(200) and Cys(114)-Cys(196)) bridges, all of which are required for the human GnRHR to traffic to the plasma membrane. This study reveals that both constitutive and ligand-induced activation are associated with a "coincidence detector" that occurs when an agonist binds. The observed constitutive activation of receptors lacking Glu(90)-Lys(121), but not Arg(38)-Asp(98) ionic bridge, suggests that the role of the former connection is holding the receptor in the inactive conformation. Both the aromatic ring and hydroxyl group of Tyr(284) and the hydrogen bonding of Ser(217) are important for efficient receptor activation. Our modeling results, supported by the observed influence of Lys(191) from extracellular loop 2 (EL2) and a four-residue motif surrounding this loop on ligand binding and receptor activation, suggest that the positioning of EL2 within the seven-α-helical bundle regulates receptor stability, proper trafficking, and function.

Anisotropic solvent model of the lipid bilayer. 2. Energetics of insertion of small molecules, peptides, and proteins in membranes.

A new computational approach to calculating binding energies and spatial positions of small molecules, peptides, and proteins in the lipid bilayer has been developed. The method combines an anisotropic solvent representation of the lipid bilayer and universal solvation model, which predicts transfer energies of molecules from water to an arbitrary medium with defined polarity properties. The universal solvation model accounts for hydrophobic, van der Waals, hydrogen-bonding, and electrostatic solute-solvent interactions. The lipid bilayer is represented as a fluid anisotropic environment described by profiles of dielectric constant (ε), solvatochromic dipolarity parameter (π*), and hydrogen bonding acidity and basicity parameters (α and ß). The polarity profiles were calculated using published distributions of quasi-molecular segments of lipids determined by neutron and X-ray scattering for DOPC bilayer and spin-labeling data that define concentration of water in the lipid acyl chain region. The model also accounts for the preferential solvation of charges and polar groups by water and includes the effect of the hydrophobic mismatch for transmembrane proteins. The method was tested on calculations of binding energies and preferential positions in membranes for small-molecules, peptides and peripheral membrane proteins that have been experimentally studied. The new theoretical approach was implemented in a new version (2.0) of our PPM program and applied for the large-scale calculations of spatial positions in membranes of more than 1000 peripheral and integral proteins. The results of calculations are deposited in the updated OPM database ( http://opm.phar.umich.edu ).

Anisotropic solvent model of the lipid bilayer. 1. Parameterization of long-range electrostatics and first solvation shell effects.

A new implicit solvation model was developed for calculating free energies of transfer of molecules from water to any solvent with defined bulk properties. The transfer energy was calculated as a sum of the first solvation shell energy and the long-range electrostatic contribution. The first term was proportional to solvent accessible surface area and solvation parameters (σ(i)) for different atom types. The electrostatic term was computed as a product of group dipole moments and dipolar solvation parameter (η) for neutral molecules or using a modified Born equation for ions. The regression coefficients in linear dependencies of solvation parameters σ(i) and η on dielectric constant, solvatochromic polarizability parameter π*, and hydrogen-bonding donor and acceptor capacities of solvents were optimized using 1269 experimental transfer energies from 19 organic solvents to water. The root-mean-square errors for neutral compounds and ions were 0.82 and 1.61 kcal/mol, respectively. Quantification of energy components demonstrates the dominant roles of hydrophobic effect for nonpolar atoms and of hydrogen-bonding for polar atoms. The estimated first solvation shell energy outweighs the long-range electrostatics for most compounds including ions. The simplicity and computational efficiency of the model allows its application for modeling of macromolecules in anisotropic environments, such as biological membranes.

Conformationally restricted analogs of the direct thrombin inhibitor FM 19.

The serine protease thrombin plays several key roles in the clotting cascade within the hemostatic system, such as in fibrin formation and platelet activation. Thus, development of an inhibitor that binds to the enzyme's active site (a direct thrombin inhibitor) offers an approach for the treatment of thrombus-associated diseases. Previous structure-activity relationship studies originally based on the bradykinin breakdown product Arg-Pro-Pro-Gly-Phe (RPPGF) led to the development of lead compound FM 19 (d-Arg-Oic-Pro-d-Ala-Phe(p-Me)-NH(2)). The recently determined X-ray structure of FM 19 in the active site of thrombin has revealed sites of modification to potentially improve inhibition. In this study, we report the synthesis and biological characterization of nine peptides that replace only the d-Arg residue of the FM 19 sequence, investigating ways to add conformational restriction, modification of the basic moiety at the end of the side chain, and removal of the charge from the N-terminus. Two of these peptides, 6 and 7 (IC(50) values of 0.51 and 0.45 µM, respectively), show similar potency to the best compounds in the FM 19 series reported thus far.

SAR Matrices Enable Discovery of Mixed Efficacy µ-Opioid Receptor Agonist Peptidomimetics with Simplified Structures through an Aromatic-Amine Pharmacophore.

We previously described the development of potent µ-opioid receptor (MOR)-agonist/δ-opioid receptor (DOR)-antagonist peptidomimetic ligands as an approach toward effective analgesics with reduced side effects. In this series, a tetrahydroquinoline (THQ) or substituted phenyl is employed to link two key pharmacophore elements, a dimethyltyrosine amino acid and typically an aromatic pendant. Using new and previously reported analogues, we constructed a structure-activity relationship (SAR) matrix that probes the utility of previously reported amine pendants. This matrix reveals that the MOR-agonist/DOR-antagonist properties of these ligands do not change when a tetrahydroisoquinoline (THIQ) pendant is used, despite removal of substituents on the core phenyl ring. Based on this observation, we retained the THIQ pendant and replaced the phenyl core with simpler aliphatic chain structures. These simpler analogues proved to be potent MOR-agonists with high variability in their effects at the DOR and the κ-opioid receptor (KOR). These data show that the amine of the THIQ pendant may be a novel pharmacophore element that favors high MOR-efficacy, whereas the aromatic ring of the THIQ pendant may produce high MOR-potency. Combined, the two pharmacophores within the THIQ pendant may be a structurally efficient means of converting opioid peptides and peptidomimetics into potent and efficacious MOR-agonists.

Purification and functional reconstitution of monomeric mu-opioid receptors: allosteric modulation of agonist binding by Gi2.

Despite extensive characterization of the mu-opioid receptor (MOR), the biochemical properties of the isolated receptor remain unclear. In light of recent reports, we proposed that the monomeric form of MOR can activate G proteins and be subject to allosteric regulation. A mu-opioid receptor fused to yellow fluorescent protein (YMOR) was constructed and expressed in insect cells. YMOR binds ligands with high affinity, displays agonist-stimulated [(35)S]guanosine 5'-(gamma-thio)triphosphate binding to Galpha(i), and is allosterically regulated by coupled G(i) protein heterotrimer both in insect cell membranes and as purified protein reconstituted into a phospholipid bilayer in the form of high density lipoprotein particles. Single-particle imaging of fluorescently labeled receptor indicates that the reconstituted YMOR is monomeric. Moreover, single-molecule imaging of a Cy3-labeled agonist, [Lys(7), Cys(8)]dermorphin, illustrates a novel method for studying G protein-coupled receptor-ligand binding and suggests that one molecule of agonist binds per monomeric YMOR. Together these data support the notion that oligomerization of the mu-opioid receptor is not required for agonist and antagonist binding and that the monomeric receptor is the minimal functional unit in regard to G protein activation and strong allosteric regulation of agonist binding by G proteins.

Pharmacological chaperones restore function to MC4R mutants responsible for severe early-onset obesity.

Heterozygous null mutations in the melanocortin-4 receptor (MC4R) cause early-onset obesity in humans, indicating that metabolic homeostasis is sensitive to quantitative variation in MC4R function. Most of the obesity-causing MC4R mutations functionally characterized so far lead to intracellular retention of receptors by the cell's quality control system. Thus, recovering cell surface expression of mutant MC4Rs could have a beneficial therapeutic value. We tested a pharmacological chaperone approach to restore cell surface expression and function of 10 different mutant forms of human melanocortin-4 receptor found in obese patients. Five cell-permeant MC4R-selective ligands were tested and displayed pharmacological chaperone activities, restoring cell surface targeting and function of the receptors with distinct efficacy profiles for the different mutations. Such mutation-specific efficacies suggested a structure-activity relationship between compounds and mutant receptor conformations that may open a path toward personalized therapy. In addition, one of the five pharmacological chaperones restored function to most of the mutant receptors tested. Combined with its ability to reach the central nervous system and its selectivity for the MC4R, this pharmacological chaperone may represent a candidate for the development of a targeted therapy suitable for a large subset of patients with MC4R-deficient obesity.

Incorporation of Agouti-Related Protein (AgRP) Human Single Nucleotide Polymorphisms (SNPs) in the AgRP-Derived Macrocyclic Scaffold c[Pro-Arg-Phe-Phe-Asn-Ala-Phe-dPro] Decreases Melanocortin-4 Receptor Antagonist Potency and Results in the Discovery of Melanocortin-5 Receptor Antagonists.

While the melanocortin receptors (MCRs) are known to be involved in numerous biological pathways, the potential roles of the MC5R have not been clearly elucidated in humans. Agouti-related protein (AgRP), an MC3R/MC4R antagonist and MC4R inverse agonist, contains an exposed ß-hairpin loop composed of six residues (Arg-Phe-Phe-Asn-Ala-Phe) that is imperative for binding and function. Within this active loop of AgRP, four human missense polymorphisms were deposited into the NIH Variation Viewer database. These polymorphisms, Arg111Cys, Arg111His, Phe112Tyr, and Ala115Val (AgRP full-length numbering), were incorporated into the peptide macrocycles c[Pro1-Arg2-Phe3-Phe4-Xaa5-Ala6-Phe7-dPro8], where Xaa was Dap5 or Asn5, to explore the functional effects of these naturally occurring substitutions in a simplified AgRP scaffold. All peptides lowered potency at least 10-fold in a cAMP accumulation assay compared to the parent sequences at the MC4Rs. Compounds MDE 6-82-3c, ZMK 2-82, MDE 6-82-1c, ZMK 2-85, and ZMK 2-112 are also the first AgRP-based chemotypes that antagonize the MC5R.

Aromatic-Amine Pendants Produce Highly Potent and Efficacious Mixed Efficacy µ-Opioid Receptor (MOR)/δ-Opioid Receptor (DOR) Peptidomimetics with Enhanced Metabolic Stability.

We previously reported a novel SAR campaign that converted a metabolically unstable series of µ-opioid receptor (MOR) agonist/δ-opioid receptor (DOR) antagonist bicyclic core peptidomimetics with promising analgesic activity and reduced abuse liabilities into a more stable series of benzylic core analogues. Herein, we expanded the SAR of that campaign and determined that the incorporation of amines into the benzylic pendant produces enhanced MOR-efficacy in this series, whereas the reincorporation of an aromatic ring into the pendant enhanced MOR-potency. Two compounds, which contain a piperidine (14) or an isoindoline (17) pendant, retained the desired opioid profile in vitro, possessed metabolic half-lives of greater than 1 h in mouse liver microsomes (MLMs), and were active antinociceptive agents in the acetic acid stretch assay (AASA) at subcutaneous doses of 1 mg/kg.

Functional characterization and structural modeling of obesity associated mutations in the melanocortin 4 receptor.

Mutations in the melanocortin 4 receptor (MC4R) gene are the most common known cause of monogenic human obesity. The MC4R gene was sequenced in 2000 subjects with severe early-onset obesity. We detected seven different nonsense and 19 nonsynonymous mutations in a total of 94 probands, some of which have been reported previously by others. We functionally characterized the 11 novel obesity associated missense mutations. Seven of these mutants (L54P, E61K, I69T, S136P, M161T, T162I, and I269N) showed impaired cell surface trafficking, reduced level of maximal binding of the radioligand [125I]NDP-MSH, and reduced ability to generate cAMP in response to ligand. Four mutant MC4Rs (G55V, G55D, S136F, and A303T) displayed cell surface expression and agonist binding similar to the wild-type receptor but showed impaired cAMP production, suggesting that these residues are likely to be critical for conformational rearrangement essential for receptor activation. Homology modeling of these mutants using a model of MC4R based on the crystal structure of the beta2-adrenoreceptor was used to provide insights into the possible structural basis for receptor dysfunction. Transmembrane (TM) domains 1, 3, 6, 7, and peripheral helix 8 appear to participate in the agonist-induced conformational rearrangement necessary for coupling of ligand binding to signaling. We conclude that G55V, G55D, S136F, and A303T mutations are likely to strengthen helix-helix interactions between TM1 and TM2, TM3 and TM6, and TM7 and helix 8, respectively, preventing relative movement of these helices during receptor activation. The combination of functional studies and structural modeling of naturally occurring pathogenic mutations in MC4R can provide valuable information regarding the molecular mechanism of MC4R activation and its dysfunction in human disease.

A covalent peptide inhibitor of RGS4 identified in a focused one-bead, one compound library screen.

BACKGROUND: Regulators of G protein signaling (RGSs) accelerate GTP hydrolysis by Galpha subunits and profoundly inhibit signaling by G protein-coupled receptors (GPCRs). The distinct expression patterns and pathophysiologic regulation of RGS proteins suggest that inhibitors may have therapeutic potential. We recently described a focused one-bead, one-compound (OBOC) library screen to identify peptide inhibitors of RGS4. Here we extend our observations to include another peptide with a different mechanism of action. RESULTS: Peptide 5nd (Tyr-Trp-c [Cys-Lys-Gly-Leu-Cys]-Lys-NH2, S-S) blocks the RGS4-Galphao interaction with an IC50 of 28 microM. It forms a covalent, dithiothreitol (DTT) sensitive adduct with a mass consistent with the incorporation of one peptide per RGS. Peptide 5nd activity is abolished by either changing its disulfide bridge to a methylene dithioether bridge, which cannot form disulfide bridges to the RGS, or by removing all cysteines from the RGS protein. However, no single cysteine in RGS4 is completely necessary or sufficient for 5nd activity. CONCLUSION: Though it has some RGS selectivity, 5nd appears to be a partially random cysteine modifier. These data suggest that it inhibits RGS4 by forming disulfide bridges with the protein.

Structural Simplification of a Tetrahydroquinoline-Core Peptidomimetic µ-Opioid Receptor (MOR) Agonist/δ-Opioid Receptor (DOR) Antagonist Produces Improved Metabolic Stability.

We have previously reported a series of µ-opioid receptor (MOR) agonist/δ-opioid receptor (DOR) antagonist ligands to serve as potential nonaddictive opioid analgesics. These ligands have been shown to be active in vivo, do not manifest withdrawal syndromes or reward behavior in conditioned-place preference assays in mice, and do not produce dependence. Although these attributes are promising, these analogues exhibit poor metabolic stability in mouse liver microsomes, likely due to the central tetrahydroquinoline scaffold in this series. As such, a structure-activity relationship (SAR) campaign was pursued to improve their metabolic stability. This resulted in a shift from our original bicyclic tetrahydroquinoline core to a monocyclic benzylic-core system. By eliminating one of the rings in this scaffold and exploring the SAR of this new core, two promising analogues were discovered. These analogues (5l and 5m) had potency and efficacy values at MOR better or comparable to morphine, retained their DOR-antagonist properties, and showed a 10-fold improvement in metabolic stability.

Dual Pharmacophores Explored via Structure-Activity Relationship (SAR) Matrix: Insights into Potent, Bifunctional Opioid Ligand Design.

Short-acting µ-opioid receptor (MOR) agonists have long been used for the treatment of severe, breakthrough pain. However, selective MOR agonists including fentanyl and morphine derivatives are limited clinically due to high risks of dependence, tolerance, and respiratory depression. We recently reported the development of a long-acting, bifunctional MOR agonist/δ-opioid receptor (DOR) antagonist analgesic devoid of tolerance or dependence in mice (AAH8, henceforth referred to as 2B). To address the need for short-acting treatments for breakthrough pain, we present a series of novel, short-acting, high-potency MOR agonist/DOR antagonist ligands with antinociceptive activity in vivo. In this study, we utilized a two-dimensional structure-activity relationship matrix to identify pharmacological trends attributable to combinations of two key pharmacophore elements within the chemotype. This work enhances our ability to modulate efficacy at MOR and DOR, accessing a variety of bifunctional profiles while maintaining high affinity and potency at both receptors.