Biochemistry of opioid (morphine) receptors : binding, structure and molecular modelling

Morphine is the most widely used compound among narcotic analgesics and remains the gold standard when the effects of other analgetic drugs are compared. The most characteristic effect of morphine is the modulation of pain perception resulting in an increase in the threshold of noxious stimuli. Antinociception induced by morphine is mediated via opioid receptors, namely the μ-type opioid receptor. Apart from the μ-opioid receptor, two other classical opioid receptors κ- and δ- and one non-classical opioid receptor, the nociceptin receptor was discovered and cloned so far. At the same time endogenous opioids were also discovered, such as enkephalins, endorphins, and dynorphins. The opioid receptors together with the endogenous opioids form the so called endogenous opioid system, which is highly distributed throughout the body and apart from analgesia it has several other important physiological functions. In this article we will review the historical milestones of opioid research − in detail with morphine. The review will also cover the upmost knowledge in the molecular structure and physiological effects of opioid receptors and endogenous opioids and we will discuss opioid receptor modelling − a rapidly evolving field in opioid receptor research

What have we learned from two-pore potassium channels? Their molecular configuration and function in the human heart

Two-pore domain potassium channels (K2P) control excitability, stabilize the resting membrane potential below firing threshold, and accelerate repolarisation in different cells. Until now, fifteen different genes for the six K2P channel subfamily were cloned. The pore-forming part is translated from two genes and they are built up from a dimer of two two-unit transmembrane domains functioning with a wide spectrum of physiological profiles. K2P ion channels were discovered in the last two decades and gave novel opportunity to recognize the complex molecular mechanism of the potassium ion flux, and may lead to the design of individual drug targeting in the future. In this review, we summarise the structure, function, channelopathies and pharmacological silhouette of the two-pore potassium channels in the human tissues. In addition, we present the computer model of the partially reconstructed wild type K2P1/TWIK1 lacking the intracellular C and N terminal loop

Functional characterization of a novel opioid, PZM21, and its influence on behavioural responses to morphine

The concept of opioid ligands biased toward the G protein pathway with minimal recruitment of β-arrestin-2 has become a promising approach for the development of novel, efficient and potentially nonaddictive opioid therapeutics. A recently discovered biased μ-opioid receptor agonist, PZM21, was reported to be analgesic and possess reduced side effects. Here, we aimed to further investigate the behavioural and biochemical properties of PZM21.We evaluated antinociceptive effects of systemic and intrathecal PZM21 administration. Its addiction-like properties were determined using several behavioural approaches: conditioned place preference, locomotor sensitization, precipitated withdrawal and self-administration. Further, we assessed the influence of PZM21 on morphine-induced antinociception, tolerance and reward. Effects of PZM21 on striatal release of monoamines were evaluated using brain microdialysis.PZM21 caused long-lasting dose-dependent antinociception. It did not induce reward- and reinforcement-related behaviour, however, its repeated administration led to antinociceptive tolerance and naloxone-precipitated withdrawal symptoms. Pretreatment with PZM21 enhanced morphine-induced antinociception and attenuated the expression of morphine reward. In comparison to morphine, PZM21 administration led to moderate release of dopamine and robust release of serotonin in the striatum.PZM21 presents antinociceptive efficacy and does not possess rewarding or reinforcing properties. However, its clinical application may be restricted, as it induces tolerance and withdrawal symptoms. Notably, its ability to diminish morphine reward implicates that PZM21 may be useful in opioid use disorder therapy

Discovery of Novel mu-Opioid Receptor Inverse Agonist from a Combinatorial Library of Tetrapeptides through Structure-Based Virtual Screening

Morphine, oxycodone, fentanyl, and other mu-opioid receptors (MOR) agonists have been used for decades in antinociceptive therapies. However, these drugs are associated with numerous side effects, such as euphoria, addiction, respiratory depression, and adverse gastrointestinal reactions, thus, circumventing these drawbacks is of extensive importance. With the aim of identifying novel peptide ligands endowed with MOR inhibitory activity, we developed a virtual screening protocol, including receptor-based pharmacophore screening, docking studies, and molecular dynamics simulations, which was used to filter an in-house built virtual library of tetrapeptide ligands. The three top-scored compounds were synthesized and subjected to biological evaluation, revealing the identity of a hit compound (peptide 1) endowed with appreciable MOR inverse agonist effect and selectivity over delta-opioid receptors. These results confirmed the reliability of our computational approach and provided a promising starting point for the development of new potent MOR modulators

In vitro binding and functional studies of Ac-RYYRIK-ol and its derivatives, novel partial agonists of the nociceptin/orphanin F/Q receptor.

Following the discovery of nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) and its endogenous ligand, an extensive search has started to find selective agonists and antagonists targeting this novel receptor-ligand system due to their therapeutic potentials. By the help of the combinatorial chemistry a series of hexapeptides with a general formula of Ac-RYY-R/K-W/I-R/K-NH(2) having high NOP receptor affinity and selectivity were identified. On the basis of this information we developed a number of novel compounds. The detailed structure-activity studies on the partial agonist Ac-RYYRIK-NH(2) are reported in this communication. Besides the modifications on N- and C-terminal, Arg-Cit exchange was performed on the template structure. The novel hexapeptides were analyzed in radioligand binding, functional biochemical [(35)S]GTPgammaS binding assays by using membranes from rat brains and Chinese hamster ovary cells expressing human NOP receptor. The agonist/antagonist properties were also tested on in the mouse vas deferens bioassay. C-terminal modification yielded a high affinity, selective and potent NOP ligand (Ac-RYYRIK-ol) with a partial agonist property. Several analogs of this compound were synthesized. The presence of the positively charged arginine residue at the first position turned out to be crucial for the biological activity of the hexapeptide. The N-terminal modifications with various acyl groups (ClAc, pivaloyl, formyl, benzoyl, mesyl) decreased the affinity of the ligand towards the receptor and the intrinsic activity for stimulating the G-protein activation was also decreased. The structure-activity studies on the hexapeptide derivatives provided some basic information on the structural requirements for receptor binding and activation

The effects of estrogen on the α2-adrenergic receptor subtypes in rat uterine function in late pregnancy in vitro

Aim To assess the effect of 17β-estradiol pretreatment on the function and expression of α2- adrenergic receptors (ARs) subtypes in late pregnancy in rats. Methods Sprague-Dawley rats (n = 37) were treated with 17β-estradiol for 4 days starting from the 18th day of pregnancy. The myometrial expression of the α2-AR subtypes was determined by real time polymerase chain reaction and Western blot analysis. In vitro contractions were stimulated with (-)-noradrenaline, and its effect was modified with the selective antagonists BRL 44408 (α2A), ARC 239 (α2B/C), and spiroxatrine (α2A). The cyclic adenosine monophosphate (cAMP) accumulation was also measured. The activated G-protein level was investigated by guanosine 5’-O-[gamma-thio]triphosphate (GTPγS) binding assay. Results 17β-estradiol pretreatment decreased the contractile effect of (-)-noradrenaline via the α2-ARs, and abolished the contractile effect via the α2B-ARs. All the α2-AR subtypes’ mRNA was significantly decreased. 17β-estradiol pretreatment significantly increased the myometrial cAMP level in the presence of BRL 44408 (P = 0.001), ARC 239 (P = 0.007), and spiroxatrine (P = 0.045), but did not modify it in the presence of spiroxatrine + BRL 44408 combination (P = 0.073). It also inhibited the G-protein-activating effect of (-)-noradrenaline by 25% in the presence of BRL 44408 + spiroxatrine combination. Conclusions The expression of the α2-AR subtypes is sensitive to 17β-estradiol, which decreases the contractile response of (-)-noradrenaline via the α2B-AR subtype, and might cause changes in G-protein signaling pathway. Estrogen dysregulation may be responsible for preterm labor or uterine inertia via the α2-AR

Food-inspired peptides from spinach Rubisco endowed with antioxidant, antinociceptive and anti-inflammatory properties

Rubiscolin-6 (amino acid sequence: YPLDLF) is a selective & delta;-opioid receptor peptide isolated from spinach Rubisco. Its synthetic analogue, peptide YPMDIV is the most potent described so far for its increased opioid activity, thus in this work it was considered as lead compound for the design of twelve new analogues e.g. LMAS112. Firstly all the novel compounds have been tested for their antinociceptive and anti-inflammatory capacity in vitro and in vivo in order to evaluate their ability to maintain or loss the original activity. Among them peptides LMAS5-8 gave the best results, thus their antioxidant properties have been investigated along with their enzymatic inhibitory ability. Peptide LMAS6 shows a strong antioxidant (154.25 mg TE/g CUPRAC) and inhibitor activity on tyrosinase (84.49 mg KAE/g), indicating a potential role in food industry as anti-browning agent, while peptides LMAS5 and LMAS7 possess a modest cholinesterase inhibitory activity suggesting a conceivable use for nutraceuticals production