In vitro and in vivo pharmacological activities of 14-o-phenylpropyloxymorphone, a potent mixed mu/delta/kappa-opioid receptor agonist with reduced constipation in mice

Pain, particularly chronic pain, is still an unsolved medical condition. Central goals in pain control are to provide analgesia of adequate efficacy and to reduce complications associated with the currently available drugs. Opioids are the mainstay for the treatment of moderate to severe pain. However, opioid pain medications also cause detrimental side effects, thus highlighting the need of innovative and safer analgesics. Opioids mediate their actions via the activation of opioid receptors, with the mu-opioid receptor as the primary target for analgesia, but also for side effects. One long-standing focus of drug discovery is the pursuit for new opioids exhibiting a favorable dissociation between analgesia and adverse effects. In this study, we describe the in vitro and in vivo pharmacological profiles of the 14-O-phenylpropyl substituted analog of the mu-opioid agonist 14-O-methyloxymorphone (14-OMO). The consequence of the substitution of the 14-O-methyl in 14-OMO with a 14-O-phenylpropyl group on in vitro binding and functional activity, and in vivo behavioral properties (nociception and gastrointestinal motility) was investigated. In binding studies, 14-O-phenylpropyloxymorphone (POMO) displayed very high affinity at mu-, delta-, and kappa-opioid receptors (Ki values in nM, mu:delta:kappa = 0.073:0.13:0.30) in rodent brain membranes, with complete loss of mu-receptor selectivity compared to 14-OMO. In guinea-pig ileum and mouse vas deferens bioassays, POMO was a highly efficacious and full agonist, being more potent than 14-OMO. In the [35S]GTPγS binding assays with membranes from CHO cells expressing human opioid receptors, POMO was a potent mu/delta-receptor full agonist and a kappa-receptor partial agonist. In vivo, POMO was highly effective in acute thermal nociception (hot-plate test, AD50= 0.7 nmol/kg) in mice after subcutaneous administration, with over 70- and 9000-fold increased potency than 14-OMO and morphine, respectively. POMO-induced antinociception is mediated through the activation of the mu-opioid receptor, and it does not involve delta- and kappa-opioid receptors. In the charcoal test, POMO produced fourfold less inhibition of the gastrointestinal transit than 14-OMO and morphine. In summary, POMO emerges as a new potent mixed mu/delta/kappa-opioid receptor agonist with reduced liability to cause constipation at antinociceptive doses

Phylogenetic Analysis of the Thylakoid ATP/ADP Carrier Reveals New Insights into Its Function Restricted to Green Plants

ATP is the common energy currency of cellular metabolism in all living organisms. Most of them synthesize ATP in the cytosol or on the mitochondrial inner membrane, whereas land plants, algae, and cyanobacteria also produce it on the thylakoid membrane during the light-dependent reactions of photosynthesis. From the site of synthesis, ATP is transported to the site of utilization via intracellular membrane transporters. One major type of ATP transporters is represented by the mitochondrial ADP/ATP carrier family. Here we review a recently characterized member, namely the thylakoid ATP/ADP carrier from Arabidopsis thaliana (AtTAAC). Thus far, no orthologs of this carrier have been characterized in other organisms, although similar sequences can be recognized in many sequenced genomes. Protein Sequence database searches and phylogenetic analyses indicate the absence of TAAC in cyanobacteria and its appearance early in the evolution of photosynthetic eukaryotes. The TAAC clade is composed of carriers found in land plants and some green algae, but no proteins from other photosynthetic taxa, such as red algae, brown algae, and diatoms. This implies that TAAC-like sequences arose only once before the divergence of green algae and land plants. Based on these findings, it is proposed that TAAC may have evolved in response to the need of a new activity in higher photosynthetic eukaryotes. This activity may provide the energy to drive reactions during biogenesis and turnover of photosynthetic complexes, which are heterogeneously distributed in a thylakoid membrane system composed of appressed and non-appressed regions

Function and evolution of channels and transporters in photosynthetic membranes

Chloroplasts from land plants and algae originated from an endosymbiotic event, most likely involving an ancestral photoautotrophic prokaryote related to cyanobacteria. Both chloroplasts and cyanobacteria have thylakoid membranes, harboring pigment-protein complexes that perform the light-dependent reactions of oxygenic photosynthesis. The composition, function and regulation of these complexes have thus far been the major topics in thylakoid membrane research. For many decades, we have also accumulated biochemical and electrophysiological evidence for the existence of solute transthylakoid transport activities that affect photosynthesis. However, research dedicated to molecular identification of the responsible proteins has only recently emerged with the explosion of genomic information. Here we review the current knowledge about channels and transporters from the thylakoid membrane of Arabidopsis thaliana and of the cyanobacterium Synechocystis sp. PCC 6803. No homologues of these proteins have been characterized in algae, although similar sequences could be recognized in many of the available sequenced genomes. Based on phylogenetic analyses, we hypothesize a host origin for most of the so far identified Arabidopsis thylakoid channels and transporters. Additionally, the shift from a non-thylakoid to a thylakoid location appears to have occurred at different times for different transport proteins. We propose that closer control of and provision for the thylakoid by products of the host genome has been an ongoing process, rather than a one-step event. Some of the proteins recruited to serve in the thylakoid may have been the result of the increased specialization of its pigment-protein composition and organization in green plants. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00018-013-1412-3) contains supplementary material, which is available to authorized users

Mechanistic Characterization of the Pharmacological Profile of HS-731, a Peripherally Acting Opioid Analgesic, at the µ-, δ-, κ-Opioid and Nociceptin Receptors

Accumulated preclinical and clinical data show that peripheral restricted opioids provide pain relief with reduced side effects. The peripherally acting opioid analgesic HS-731 is a potent dual μ-/δ-opioid receptor (MOR/DOR) full agonist, and a weak, partial agonist at the κ-opioid receptor (KOR). However, its binding mode at the opioid receptors remains elusive. Here, we present a comprehensive in silico evaluation of HS-731 binding at all opioid receptors. We provide insights into dynamic interaction patterns explaining the different binding and activity of HS-731 on the opioid receptors. For this purpose, we conducted docking, performed molecular dynamics (MD) simulations and generated dynamic pharmacophores (dynophores). Our results highlight two residues important for HS-731 recognition at the classical opioid receptors (MOR, DOR and KOR), particular the conserved residue 5.39 (K) and the non-conserved residue 6.58 (MOR: K, DOR: W and KOR: E). Furthermore, we assume a salt bridge between the transmembrane helices (TM) 5 and 6 via K2275.39 and E2976.58 to be responsible for the partial agonism of HS-731 at the KOR. Additionally, we experimentally demonstrated the absence of affinity of HS-731 to the nociceptin/orphanin FQ peptide (NOP) receptor. We consider the morphinan phenol Y1303.33 responsible for this affinity lack. Y1303.33 points deep into the NOP receptor binding pocket preventing HS-731 binding to the orthosteric binding pocket. These findings provide significant structural insights into HS-731 interaction pattern with the opioid receptors that are important for understanding the pharmacology of this peripheral opioid analgesic

Mechanistic Understanding of Peptide Analogues, DALDA, [Dmt1]DALDA, and KGOP01, Binding to the Mu Opioid Receptor

The mu opioid receptor (MOR) is the primary target for analgesia of endogenous opioid peptides, alkaloids, synthetic small molecules with diverse scaffolds, and peptidomimetics. Peptide-based opioids are viewed as potential analgesics with reduced side effects and have received constant scientific interest over the years. This study focuses on three potent peptide and peptidomimetic MOR agonists, DALDA, [Dmt1]DALDA, and KGOP01, and the prototypical peptide MOR agonist DAMGO. We present the first molecular modeling study and structure–activity relationships aided by in vitro assays and molecular docking of the opioid peptide analogues, in order to gain insight into their mode of binding to the MOR. In vitro binding and functional assays revealed the same rank order with KGOP01 > [Dmt1]DALDA > DAMGO > DALDA for both binding and MOR activation. Using molecular docking at the MOR and three-dimensional interaction pattern analysis, we have rationalized the experimental outcomes and highlighted key amino acid residues responsible for agonist binding to the MOR. The Dmt (2′,6′-dimethyl-L-Tyr) moiety of [Dmt1]DALDA and KGOP01 was found to represent the driving force for their high potency and agonist activity at the MOR. These findings contribute to a deeper understanding of MOR function and flexible peptide ligand–MOR interactions, that are of significant relevance for the future design of opioid peptide-based analgesics