The concept of ligand bias at G protein-coupled receptors broadens the possibilities for agonist activities and provides the opportunity to develop safer, more selective therapeutics. Morphine pharmacology in b-arrestin-2 knockout mice suggested that a ligand that promotes coupling of the m-opioid receptor (MOR) to G proteins, but not b-arrestins, would result in higher analgesic efficacy, less gastrointestinal dysfunction, and less respiratory suppression than morphine. Here we report the discovery of TRV130 ([(3-methoxythiophen-2-yl)methyl]({2-[(9R)-9-(pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]ethyl})amine), a novel MOR G protein-biased ligand. In cell-based assays, TRV130 elicits robust G protein signaling, with potency and efficacy similar to morphine, but with far less b-arrestin recruitment and receptor internalization. In mice and rats, TRV130 is potently analgesic while causing less gastrointestinal dysfunction and respiratory suppression than morphine at equianalgesic doses. TRV130 successfully translates evidence that analgesic and adverse MOR signaling pathways are distinct into a biased ligand with differentiated pharmacology. These preclinical data suggest that TRV130 may be a safer and more tolerable therapeutic for treating severe pain.

Endoplasmic reticulum (ER) stress signaling is an adaptive cellular response to the loss of ER Ca 2؉homeostasis and/or the accumulation of misfolded, unassembled, or aggregated proteins in the ER lumen. ER stress-activated signaling pathways regulate protein synthesis initiation and can also trigger apoptosis through the ER-associated caspase 12. Viruses that utilize the host cell ER as an integral part of their life cycle would be predicted to cause some level of ER stress. Bovine viral diarrhea virus (BVDV) is a positive-stranded RNA virus of the Flaviviridae family. BVDV and related flaviviruses use the host ER as the primary site of envelope glycoprotein biogenesis, genomic replication, and particle assembly. We are using a cytopathic strain of BVDV (cpBVDV) that causes cellular apoptosis as a model system to determine how virus-induced ER stress contributes to pathogenesis. We show that, in a natural infection of MDBK cells, cpBVDV activates the ER transmembrane kinase PERK (PKR-like ER kinase) and causes hyperphosphorylation of the translation initiation factor eIF2␣, consistent with the induction of an ER stress response. Additionally, we show that initiation of cellular apoptosis correlates with downregulation of the antiapoptotic Bcl-2 protein, induced expression of caspase 12, and a decrease in intracellular glutathione levels. Defining the molecular stress pathways leading to cpBVDV-induced apoptosis provides the basis to study how other ER-tropic viruses, such as hepatitis C and B viruses, modulate the host cell ER stress response during the course of persistent infection.

The hepatitis C virus envelope protein, E2, is an endoplasmic reticulum (ER)-bound protein that contains a region of sequence homology with the double-stranded RNA-activated protein kinase PKR and its substrate, the eukaryotic translation initiation factor 2 (eIF2). We previously reported that E2 modulates global translation through inhibition of the interferon-induced antiviral protein PKR through its PKR-eIF2␣ phosphorylation site homology domain (PePHD). Here we show that the PKR-like ER-resident kinase (PERK) binds to and is also inhibited by E2. At low expression levels, E2 induced ER stress, but at high expression levels, and in vitro, E2 inhibited PERK kinase activity. Mammalian cells that stably express E2 were refractory to the translation-inhibitory effects of ER stress inducers, and E2 relieved general translation inhibition induced by PERK. The PePHD of E2 was required for the rescue of translation that was inhibited by activated PERK, similar to our previous findings with PKR. Here we report the inhibition of a second eIF2␣ kinase by E2, and these results are consistent with a pseudosubstrate mechanism of inhibition of eIF2␣ kinases. These findings may also explain how the virus promotes persistent infection by overcoming the cellular ER stress response.Hepatitis C virus (HCV) is a major public health problem, with at least 2.7 million persons infected in the United States and more than 170 million infected worldwide. Most HCV infections become persistent, which often leads to chronic liver disease, including cirrhosis and hepatocellular carcinoma. Viral persistence may be partially attributable to the ability of the virus to evade the host immune responses. Viral variants that escape innate or acquired immunity are likely to also be important in allowing the virus to establish persistence. For instance, many strains of HCV are resistant to alpha interferon (IFN) therapy and may also be resistant to endogenous IFN. Several mechanisms for IFN resistance have been proposed, including interactions of both the E2 envelope protein and NS5A with the IFN-induced double-stranded RNA-activated protein kinase PKR (7, 23). HCV E2 protein is one of two envelope glycoproteins and contains a region of sequence homology with a region of PKR that contains autophosphorylation sites that affect the activation of the kinase (23, 24). Adjacent to this region of PKR homology lies a sequence identical to the phosphorylation site in the alpha subunit of the eukaryotic translation initiation factor 2 (eIF2␣) targeted by PKR for translational inhibition during virus infection. This region in E2, termed the PKR-eIF2␣ phosphorylation site homology domain (PePHD), is required for inhibition of PKR by E2 (23). HCV E2 containing PePHD sequences from IFN-resistant strains of virus inhibits PKR, confirming that these sequences are important for the interaction of E2 with PKR.

Selective delta opioid receptor agonists are promising potential therapeutic agents for the treatment of various types of pain conditions. A spirocyclic derivative was identified as a promising hit through screening. Subsequent lead optimization identified compound 20 (ADL5859) as a potent, selective, and orally bioavailable delta agonist. Compound 20 was selected as a clinical candidate for the treatment of pain.

Selective, nonpeptidic delta opioid receptor agonists have been the subject of great interest as potential novel analgesic agents. The discoveries of BW373U86 (1) and SNC80 (2) contributed to the rapid expansion of research in this field. However, poor drug-like properties and low therapeutic indices have prevented clinical evaluation of these agents. Doses of 1 and 2 similar to those required for analgesic activity produce convulsions in rodents and nonhuman primates. Recently, we described a novel series of potent, selective, and orally bioavailable delta opioid receptor agonists. The lead derivative, ADL5859 (4), is currently in phase II proof-of-concept studies for the management of pain. Further structure activity relationship exploration has led to the discovery of ADL5747 (36), which is approximately 50-fold more potent than 4 in an animal model of inflammatory pain. On the basis of its favorable efficacy, safety, and pharmacokinetic profile, 36 was selected as a clinical candidate for the treatment of pain.