Optimized Proteomic Mass Spectrometry Characterization of Recombinant Human μ‑Opioid Receptor Functionally Expressed in <i>Pichia pastoris</i> Cell Lines

Human μ-opioid receptor (hMOR) is a class-A G-protein-coupled receptor (GPCR), a prime therapeutic target for the management of moderate and severe pain. A chimeric form of the receptor has been cocrystallized with an opioid antagonist and resolved by X-ray diffraction; however, further direct structural analysis is still required to identify the active form of the receptor to facilitate the rational design of hMOR-selective agonist and antagonists with therapeutic potential. Toward this goal and in spite of the intrinsic difficulties posed by the highly hydrophobic transmembrane motives of hMOR, we have comprehensively characterized by mass spectrometry (MS) analysis the primary sequence of the functional hMOR. Recombinant hMOR was overexpressed as a C-terminal c-myc and 6-his tagged protein using an optimized expression procedure in <i>Pichia pastoris</i> cells. After membrane solubilization and metal-affinity chromato­graphy purification, a procedure was devised to enhance the concentration of the receptor. Subsequent combinations of in-solution and in-gel digestions using either trypsin, chymotrypsin, or proteinase K, followed by matrix-assisted laser desorption ionization time-of-flight MS or nanoliquid chromatography coupled with tandem MS analyses afforded an overall sequence coverage of up to >80%, a level of description first attained for an opioid receptor and one of the six such high-coverage MS-based analyses of any GPCR

Proposed Bioactive Conformations of Opiorphin, an Endogenous Dual APN/NEP Inhibitor

The conformational profiles for the endogenous peptide Opiorphin and a set of seven analogues exhibiting different inhibitory activities toward human aminopeptidase N (hAPN) and human neprilysin (hNEP) were independently computed to deduce a bioactive conformation that Opiorphin may adopt when binding these two enzymes. The conformational space was thoroughly sampled using an iterative simulated annealing protocol, and a library of low-energy conformers was generated for each peptide. Bioactive Opiorphin conformations fitting our experimental structure–activity relationship data were identified for hAPN and hNEP using computational pairwise comparisons between each of the unique low-energy conformations of Opiorphin and its analogues. The obtained results provide a structural explanation for the dual hAPN and hNEP inhibitory activity of Opiorphin and show that the inborn flexibility of Opiorphin is essential for its analgesic activity

Tuning Transthyretin Amyloidosis Inhibition Properties of Iododiflunisal by Combinatorial Engineering of the Nonsalicylic Ring Substitutions

Two series of iododiflunisal and diflunisal analogues have been obtained by using a two step sequential reaction solution-phase parallel synthesis. The synthesis combined an aqueous Suzuki-Miyaura cross-coupling and a mild electrophilic aromatic iodination step using a new polymer-supported iodonium version of Barluenga’s reagent. From a selected set of 77 noniodinated and 77 iodinated diflunisal analogues, a subset of good transthyretin amyloid inhibitors has been obtained with improved turbidimetry inhibition constants, high binding affinity to transthyretin, and good selectivity for TTR compared to other thyroxine binding proteins

Structure–Activity Relationship Study of Opiorphin, a Human Dual Ectopeptidase Inhibitor with Antinociceptive Properties

Toward developing new potential analgesics, this first structure–activity relationship study of opiorphin (H-Gln-Arg-Phe-Ser-Arg-OH), a human peptide inhibiting enkephalin degradation, was performed. A systematic Ala scanning proved that Phe³ is a key residue for neprilysin and aminopeptidase N (AP-N) ectoenkephalinase inhibition. A series of Phe³-halogenated analogues revealed that halogen bonding based optimization strategies are not applicable to this residue. Additional substituted Phe³ derivatives showed that replacing l-Phe³ for d-Phe³ increased the AP-N inhibition potency by 1 order of magnitude. NMR studies and molecular mechanics calculations indicated that the improved potency may be due to CH−π stacking interactions between the aromatic ring of d-Phe³ and the Hγ protons of Arg². This structural motif is not possible for the native opiorphin and may be useful for the design of further potent and metabolically stable analogues

Modulation of the Fibrillogenesis Inhibition Properties of Two Transthyretin Ligands by Halogenation

The amyloidogenic protein transthyretin (TTR) is thought to aggregate into amyloid fibrils by tetramer dissociation which can be inhibited by a number of small molecule compounds. Our analysis of a series of crystallographic protein-inhibitor complexes has shown no clear correlation between the observed molecular interactions and the in vitro activity of the inhibitors. From this analysis, it emerged that halogen bonding (XB) could be mediating some key interactions. Analysis of the halogenated derivatives of two well-known TTR inhibitors has shown that while flufenamic acid affinity for TTR was unchanged by halogenation, diflunisal gradually improves binding up to 1 order of magnitude after iodination through interactions that can be interpreted as a suboptimal XB (carbonyl Thr106: I...O distance 3.96–4.05 Å; CI...O angle 152–156°) or as rather optimized van der Waals contacts or as a mixture of both. These results illustrate the potential of halogenation strategies in designing and optimizing TTR fibrillogenesis inhibitors

Insights on the Interaction between Transthyretin and Aβ in Solution. A Saturation Transfer Difference (STD) NMR Analysis of the Role of Iododiflunisal

Several strategies against Alzheimer disease (AD) are directed to target Aβ-peptides. The ability of transthyretin (TTR) to bind Aβ-peptides and the positive effect exerted by some TTR stabilizers for modulating the TTR–Aβ interaction have been previously studied. Herein, key structural features of the interaction between TTR and the Aβ(12–28) peptide (<b>3</b>), the essential recognition element of Aβ, have been unravelled by STD-NMR spectroscopy methods in solution. Molecular aspects related to the role of the TTR stabilizer iododiflunisal (IDIF, <b>5</b>) on the TTR–Aβ complex have been also examined. The NMR results, assisted by molecular modeling protocols, have provided a structural model for the TTR–Aβ interaction, as well as for the ternary complex formed in the presence of IDIF. This basic structural information could be relevant for providing light on the mechanisms involved in the ameliorating effects of AD symptoms observed in AD/TTR^± animal models after IDIF treatment and eventually for designing new molecules toward AD therapeutic drugs

Modulation of the Interaction between a Peptide Ligand and a G Protein-Coupled Receptor by Halogen Atoms

Systematic halogenation of two native opioid peptides has shown that halogen atoms can modulate peptide–receptor interactions in different manners. First, halogens may produce a steric hindrance that reduces the binding of the peptide to the receptor. Second, chlorine, bromine, or iodine may improve peptide binding if their positive σ-hole forms a halogen bond interaction with negatively charged atoms of the protein. Lastly, the negative electrostatic potential of fluorine can interact with positively charged atoms of the protein to improve peptide binding