Global control of RNA polymerase II.

RNA polymerase II (Pol II) is the multi-protein complex responsible for transcribing all protein-coding messenger RNA (mRNA). Most research on gene regulation is focused on the mechanisms controlling which genes are transcribed when, or on the mechanics of transcription. How global Pol II activity is determined receives comparatively less attention. Here, we follow the life of a Pol II molecule from 'assembly of the complex' to nuclear import, enzymatic activity, and degradation. We focus on how Pol II spends its time in the nucleus, and on the two-way relationship between Pol II abundance and activity in the context of homeostasis and global transcriptional changes.

Peripheral Administration of Selective Glycine Transporter-2 Inhibitor, Oleoyl-D-Lysine, Reverses Chronic Neuropathic Pain but Not Acute or Inflammatory Pain in Male Mice.

Aberrations in spinal glycinergic signaling are a feature of pain chronification. Normalizing these changes by inhibiting glycine transporter (GlyT)-2 is a promising treatment strategy. However, existing GlyT2 inhibitors (e.g., ORG25543) are limited by narrow therapeutic windows and severe dose-limiting side effects, such as convulsions, and are therefore poor candidates for clinical development. Here, intraperitoneally administered oleoyl-D-lysine, a lipid-based GlyT2 inhibitor, was characterized in mouse models of acute (hot plate), inflammatory (complete Freund's adjuvant), and chronic neuropathic (chronic constriction injury) pain. Side effects were also assessed on a numerical rating score, convulsions score, for motor incoordination (rotarod), and for respiratory depression (whole body plethysmography). Oleoyl-D-lysine produced near complete antiallodynia for chronic neuropathic pain, but no antiallodynia/analgesia in inflammatory or acute pain. No side effects were seen at the peak analgesic dose, 30 mg/kg. Mild side effects were observed at the highest dose, 100 mg/kg, on the numerical rating score, but no convulsions. These results contrasted markedly with ORG25543, which reached less than 50% reduction in allodynia score only at the lethal/near-lethal dose of 50 mg/kg. At this dose, ORG25543 caused maximal side effects on the numerical rating score and severe convulsions. Oleoyl-D-lysine (30 mg/kg) did not cause any respiratory depression, a problematic side effect of opiates. These results show the safe and effective reversal of neuropathic pain in mice by oleoyl-D-lysine and provide evidence for a distinct role of glycine in chronic pain over acute or short-term pain conditions. SIGNIFICANCE STATEMENT: Partially inhibiting glycine transporter (GlyT)-2 can alleviate chronic pain by restoring lost glycinergic function. Novel lipid-based GlyT2 inhibitor ol-D-lys is safe and effective in alleviating neuropathic pain, but not inflammatory or acute pain. Clinical application of GlyT2 inhibitors may be better suited to chronic neuropathic pain over other pain aetiologies.

Critical Assessment of G Protein-Biased Agonism at the µ-Opioid Receptor.

G protein-biased agonists of the µ-opioid receptor (MOPr) have been proposed as an improved class of opioid analgesics. Recent studies have been unable to reproduce the original experiments in the ß-arrestin2-knockout mouse that led to this proposal, and alternative genetic models do not support the G protein-biased MOPr agonist hypothesis. Furthermore, assessment of putatively biased ligands has been confounded by several factors, including assay amplification. As such, the extent to which current lead compounds represent mechanistically novel, extremely G protein-biased agonists is in question, as is the underlying assumption that ß-arrestin2 mediates deleterious opioid effects. Addressing these current challenges represents a pressing issue to successfully advance drug development at this receptor and improve upon current opioid analgesics.

Intrinsic Efficacy of Opioid Ligands and Its Importance for Apparent Bias, Operational Analysis, and Therapeutic Window.

Evidence from several novel opioid agonists and knockout animals suggests that improved opioid therapeutic window, notably for analgesia versus respiratory depression, is a result of ligand bias downstream of activation of the µ-opioid receptor (MOR) toward G protein signaling and away from other pathways, such as arrestin recruitment. Here, we argue that published claims of opioid bias based on application of the operational model of agonism are frequently confounded by failure to consider the assumptions of the model. These include failure to account for intrinsic efficacy and ceiling effects in different pathways, distortions introduced by analysis of amplified (G protein) versus linear (arrestin) signaling mechanisms, and nonequilibrium effects in a dynamic signaling cascade. We show on both theoretical and experimental grounds that reduced intrinsic efficacy that is unbiased across different downstream pathways, when analyzed without due considerations, does produce apparent but erroneous MOR ligand bias toward G protein signaling, and the weaker the G protein partial agonism is the greater the apparent bias. Experimentally, such apparently G protein-biased opioids have been shown to exhibit low intrinsic efficacy for G protein signaling when ceiling effects are properly accounted for. Nevertheless, such agonists do display an improved therapeutic window for analgesia versus respiratory depression. Reduced intrinsic efficacy for G proteins rather than any supposed G protein bias provides a more plausible, sufficient explanation for the improved safety. Moreover, genetic models of G protein-biased opioid receptors and replication of previous knockout experiments suggest that reduced or abolished arrestin recruitment does not improve therapeutic window for MOR-induced analgesia versus respiratory depression. SIGNIFICANCE STATEMENT: Efforts to improve safety of µ-opioid analgesics have focused on agonists that show signaling bias for the G protein pathway versus other signaling pathways. This review provides theoretical and experimental evidence showing that failure to consider the assumptions of the operational model can lead to large distortions and overestimation of actual bias. We show that low intrinsic efficacy is a major determinant of these distortions, and pursuit of appropriately reduced intrinsic efficacy should guide development of safer opioids.

Low intrinsic efficacy for G protein activation can explain the improved side effect profiles of new opioid agonists.

Biased agonism at G protein-coupled receptors describes the phenomenon whereby some drugs can activate some downstream signaling activities to the relative exclusion of others. Descriptions of biased agonism focusing on the differential engagement of G proteins versus ß-arrestins are commonly limited by the small response windows obtained in pathways that are not amplified or are less effectively coupled to receptor engagement, such as ß-arrestin recruitment. At the µ-opioid receptor (MOR), G protein-biased ligands have been proposed to induce less constipation and respiratory depressant side effects than opioids commonly used to treat pain. However, it is unclear whether these improved safety profiles are due to a reduction in ß-arrestin-mediated signaling or, alternatively, to their low intrinsic efficacy in all signaling pathways. Here, we systematically evaluated the most recent and promising MOR-biased ligands and assessed their pharmacological profile against existing opioid analgesics in assays not confounded by limited signal windows. We found that oliceridine, PZM21, and SR-17018 had low intrinsic efficacy. We also demonstrated a strong correlation between measures of efficacy for receptor activation, G protein coupling, and ß-arrestin recruitment for all tested ligands. By measuring the antinociceptive and respiratory depressant effects of these ligands, we showed that the low intrinsic efficacy of opioid ligands can explain an improved side effect profile. Our results suggest a possible alternative mechanism underlying the improved therapeutic windows described for new opioid ligands, which should be taken into account for future descriptions of ligand action at this important therapeutic target.

Morphine-induced respiratory depression is independent of ß-arrestin2 signalling.

BACKGROUND AND PURPOSE: GPCRs can signal through both G proteins and ß-arrestin2. For the µ-opioid receptor, early experimental evidence from a single study suggested that G protein signalling mediates analgesia, whereas ß-arrestin2 signalling mediates respiratory depression and constipation. Consequently, for more than a decade, much research effort has been focused on developing biased µ-opioid agonists that preferentially target G protein signalling over ß-arrestin signalling, as it was believed that such drugs would be analgesics devoid of respiratory depressant activity. However, the prototypical compounds that have been developed based on this concept have so far failed in clinical and preclinical development. EXPERIMENTAL APPROACH: The present study was set up to re-examine opioid-induced respiratory depression in ß-arrestin2 knockout mice. To this end, a consortium was formed consisting of three different laboratories located in different countries to evaluate independently opioid-induced respiratory depression. KEY RESULTS: Our consensus results unequivocally demonstrate that the prototypical µ-opioid agonist morphine (3.75-100 mg·kg-1 s.c. or 3-30 mg·kg-1 i.p.) as well as the potent opioid fentanyl (0.05-0.35 mg·kg-1 s.c.) do indeed induce respiratory depression and constipation in ß-arrestin2 knockout mice in a dose-dependent manner indistinguishable from that observed in wild-type mice. CONCLUSION AND IMPLICATIONS: Our findings do not support the original suggestion that ß-arrestin2 signalling plays a key role in opioid-induced respiratory depression and call into question the concept of developing G protein-biased µ-opioid receptor agonists as a strategy for the development of safer opioid analgesic drugs.

A tetrapeptide class of biased analgesics from an Australian fungus targets the µ-opioid receptor.

An Australian estuarine isolate of Penicillium sp. MST-MF667 yielded 3 tetrapeptides named the bilaids with an unusual alternating LDLD chirality. Given their resemblance to known short peptide opioid agonists, we elucidated that they were weak (Ki low micromolar) µ-opioid agonists, which led to the design of bilorphin, a potent and selective µ-opioid receptor (MOPr) agonist (Ki 1.1 nM). In sharp contrast to all-natural product opioid peptides that efficaciously recruit ß-arrestin, bilorphin is G protein biased, weakly phosphorylating the MOPr and marginally recruiting ß-arrestin, with no receptor internalization. Importantly, bilorphin exhibits a similar G protein bias to oliceridine, a small nonpeptide with improved overdose safety. Molecular dynamics simulations of bilorphin and the strongly arrestin-biased endomorphin-2 with the MOPr indicate distinct receptor interactions and receptor conformations that could underlie their large differences in bias. Whereas bilorphin is systemically inactive, a glycosylated analog, bilactorphin, is orally active with similar in vivo potency to morphine. Bilorphin is both a unique molecular tool that enhances understanding of MOPr biased signaling and a promising lead in the development of next generation analgesics.