Ligand efficacy modulates conformational dynamics of the µ-opioid receptor.

The µ-opioid receptor (µOR) is an important target for pain management1 and molecular understanding of drug action on µOR will facilitate the development of better therapeutics. Here we show, using double electron-electron resonance and single-molecule fluorescence resonance energy transfer, how ligand-specific conformational changes of µOR translate into a broad range of intrinsic efficacies at the transducer level. We identify several conformations of the cytoplasmic face of the receptor that interconvert on different timescales, including a pre-activated conformation that is capable of G-protein binding, and a fully activated conformation that markedly reduces GDP affinity within the ternary complex. Interaction of ß-arrestin-1 with the µOR core binding site appears less specific and occurs with much lower affinity than binding of Gi.

Syntheses and Reactions of Pyrroline, Piperidine Nitroxide Phosphonates.

Organophosphorus compounds occupy a significant position among the plethora of organic compounds, but a limited number of paramagnetic phosphorus compounds have been reported, including paramagnetic phosphonates. This paper describes the syntheses and further transformations of pyrroline and piperidine nitroxide phosphonates by well-established methods, such as the Pudovik, Arbuzov and Horner-Wadsworth-Emmons (HWE) reactions. The reaction of paramagnetic a-bromoketone produced a vinylphosphonate in the Perkow reaction. Paramagnetic a-hydroxyphosphonates could be subjected to oxidation, elimination and substitution reactions to produce various paramagnetic phosphonates. The synthesized paramagnetic phosphonates proved to be useful synthetic building blocks for carbon-carbon bond-forming reactions in the Horner-Wadsworth-Emmons olefination reactions. The unsaturated compounds achieved could be transformed into various substituted pyrroline nitroxides, proxyl nitroxides and paramagnetic polyaromatics. The Trolox® equivalent antioxidant capacity (TEAC) of new phosphonates was also screened, and tertiary a-hydroxyphosphonatate nitroxides exhibited remarkable antioxidant activity.

Conformational dynamics of the µ-opioid receptor determine ligand intrinsic efficacy.

The µ-opioid receptor (µOR) is an important target for pain management and the molecular understanding of drug action will facilitate the development of better therapeutics. Here we show, using double electron-electron resonance (DEER) and single-molecule fluorescence resonance energy transfer (smFRET), how ligand-specific conformational changes of the µOR translate into a broad range of intrinsic efficacies at the transducer level. We identify several cytoplasmic receptor conformations interconverting on different timescales, including a pre-activated receptor conformation which is capable of G protein binding, and a fully activated conformation which dramatically lowers GDP affinity within the ternary complex. Interaction of ß-arrestin-1 with the µOR core binding site appears less specific and occurs with much lower affinity than binding of G protein Gi.

Novel Orientation-Sensitive Spin Probes for Graphene Oxide Membranes Study.

Spin probe EPR spectroscopy is currently the only method to quantitatively report on the orientational ordering of graphene oxide membranes. This technique is based on the analysis of EPR spectra of a membrane containing stable radicals sorbed on oxidized graphene planes. The efficiency of the method depends on the spin probe structure; therefore, it is important to find stable paramagnetic substances that are most sensitive to the alignment of graphene oxide membranes. In the present work, three novel stable nitroxide radicals containing aromatic fragments with two nitrogen atoms were tested as spin probes to study graphene oxide membranes. The spin-Hamiltonian parameters of the radicals in graphite oxide powder and orientational order parameters of the probes inside graphene oxide membrane were determined. The sensitivity of one of these radicals to membrane orientational ordering was found to be higher than for any of spin probes used previously. A likely reason for this higher sensitivity is the presence of heteroatoms which can facilitate interaction between paramagnetic molecules and oxygen-containing groups on the inner surface of the membrane. The new high-sensitivity spin probe may significantly increase the potential of EPR spectroscopy for studying the internal structure of graphene oxide membranes.

Proof-of-Concept for the Analgesic Effect and Thermoregulatory Safety of Orally Administered Multi-Target Compound SZV 1287 in Mice: A Novel Drug Candidate for Neuropathic Pain.

SZV 1287 (3-(4,5-diphenyl-1,3-oxazol-2-yl)propanal oxime) is a novel multi-target candidate under preclinical development for neuropathic pain. It inhibits amine oxidase copper containing 3, transient receptor potential ankyrin 1 and vanilloid 1 (TRPV1) receptors. Mainly under acidic conditions, it is transformed to the cyclooxygenase inhibitor oxaprozin, which is ineffective for neuropathy. Therefore, an enterosolvent capsule is suggested for oral formulation, which we investigated for nociception, basic kinetics, and thermoregulatory safety in mice. The antihyperalgesic effect of SZV 1287 (10, 20, 50, and 200 mg/kg, p.o.) was determined in partial sciatic nerve ligation-induced traumatic neuropathy by aesthesiometry, brain and plasma concentrations by HPLC, and deep body temperature by thermometry. Its effect on proton-induced TRPV1 activation involved in thermoregulation was assessed by microfluorimetry in cultured trigeminal neurons. The three higher SZV 1287 doses significantly, but not dose-dependently, reduced neuropathic hyperalgesia by 50% of its maximal effect. It was quickly absorbed; plasma concentration was stable for 2 h, and it entered into the brain. Although SZV 1287 significantly decreased the proton-induced TRPV1-mediated calcium-influx potentially leading to hyperthermia, it did not alter deep body temperature. Oral SZV 1287 inhibited neuropathic hyperalgesia and, despite TRPV1 antagonistic action and brain penetration, it did not influence thermoregulation, which makes it a promising analgesic candidate.

TRPA1 Ion Channel Determines Beneficial and Detrimental Effects of GYY4137 in Murine Serum-Transfer Arthritis.

Modulation of nociception and inflammation by sulfide in rheumatoid arthritis and activation of transient receptor potential ankyrin 1 (TRPA1) ion channels by sulfide compounds are well documented. The present study aims to investigate TRPA1-mediated effects of sulfide donor GYY4137 in K/BxN serum-transfer arthritis, a rodent model of rheumatoid arthritis. TRPA1 and somatostatin sst4 receptor wild-type (WT) and knockout mice underwent K/BxN serum transfer and were treated daily with GYY4137. Functional and biochemical signs of inflammation were recorded, together with histological characterization. These included detection of hind paw mechanical hyperalgesia by dynamic plantar esthesiometry, hind paw volume by plethysmometry, and upside-down hanging time to failure. Hind paw erythema, edema, and passive movement range of tibiotarsal joints were scored. Somatostatin release from sensory nerve endings of TRPA1 wild-type and knockout mice in response to polysulfide was detected by radioimmunoassay. Polysulfide formation from GYY4137 was uncovered by cold cyanolysis. GYY4137 aggravated mechanical hyperalgesia in TRPA1 knockout mice but ameliorated it in wild-type ones. Arthritis score was lowered by GYY4137 in TRPA1 wild-type animals. Increased myeloperoxidase activity, plasma extravasation, and subcutaneous MIP-2 levels of hind paws were detected in TRPA1 knockout mice upon GYY4137 treatment. Genetic lack of sst4 receptors did not alter mechanical hyperalgesia, edema formation, hanging performance, arthritis score, plasma extravasation, or myeloperoxidase activity. TRPA1 WT animals exhibited smaller cartilage destruction upon GYY4137 administration. Sodium polysulfide caused TRPA1-dependent somatostatin release from murine nerve endings. Sulfide released from GYY4137 is readily converted into polysulfide by hypochlorite. Polysulfide potently activates human TRPA1 receptors expressed in Chinese hamster ovary (CHO) cells. According to our data, the protective effect of GYY4137 is mediated by TRPA1, while detrimental actions are independent of the ion channel in the K/BxN serum-transfer arthritis model in mice. At acidic pH in inflamed tissue sulfide is released from GYY4137 and reacts with neutrophil-derived hypochlorite. Resulting polysulfide might be responsible for TRPA1-mediated antinociceptive and anti-inflammatory as well as TRPA1-independent pro-inflammatory effects.

Indanedione spin labelling of Na,K-ATPase.

The indanedione series of vinyl ketone spin-labelling reagents has been extended in two ways: by increasing the length of the rigid spacer between the reactive centre and the nitroxide ring, or by introducing an electrophilic substituent (that could also hinder its rotation) at the bridge head position of the nitroxide ring. Three reagents of this new series have been used to spin label the Class II thiol groups of membranous Na,K-ATPase from Squalus acanthias. With a conjugated diene spacer, the majority of spin labels are strongly held but a minor population is relatively mobile at 37 degrees C. With a conjugated triene spacer, the nitroxide is still strongly held but a portion of the label is non-covalently bound. The 4-bromo-pyrroline derivative (with short vinyl spacer) is tightly held at the attachment site, and the conventional electron paramagnetic resonance (EPR) spectra distinguish between the two enantiomeric structures which differ in their mobility at 37 degrees C. Saturation transfer EPR (ST-EPR) spectra of this label at 4 degrees C have been used to determine the dependence of the protein rotational mobility on ionic strength. Electrostatic repulsion contributes to the lateral interactions between Na,K-ATPase molecules.

Structural determination of spin label immobilization and orientation: a Monte Carlo minimization approach.

Electron paramagnetic resonance (EPR) is often used in the study of the orientation and dynamics of proteins. However, there are two major obstacles in the interpretation of EPR signals: (a) most spin labels are not fully immobilized by the protein, hence it is difficult to distinguish the mobility of the label with respect to the protein from the reorientation of the protein itself; (b) even in cases where the label is fully immobilized its orientation with respect to the protein is not known, which prevents interpretation of probe reorientation in terms of protein reorientation. We have developed a computational strategy for determining whether or not a spin label is immobilized and, if immobilized, predicting its conformation within the protein. The method uses a Monte Carlo minimization algorithm to search the conformational space of labels within known atomic level structures of proteins. To validate the method a series of spin labels of varying size and geometry were docked to sites on the myosin head catalytic and regulatory domains. The predicted immobilization and conformation compared well with the experimentally determined mobility and orientation of the label. Thus, probes can now be targeted to report on various modes of molecular dynamics: immobilized probes to report on protein backbone and domain dynamics or floppy probes to report on the extent of steric restriction experienced by the side chain.