An Immunocytochemical-Derived Correlate for Evaluating the Bridging of Heteromeric Mu-Delta Opioid Protomers by Bivalent Ligands

Bivalent ligands that contain two pharmacophores linked by a spacer are promising tools to investigate the pharmacology of opioid receptor heteromers. Evidence for occupation of neighboring protomers by two phamacophores of a single bivalent ligand (bridging) has relied mainly on pharmacological data. In the present study, we have employed an immunocytochemical correlate to support <i>in vivo</i> biological studies that are consistent with bridging. We show that a bivalent mu agonist/delta antagonist (MDAN-21) that is devoid of tolerance due to possible bridging of mu and delta protomers prevents endocytosis of the heteromeric receptors in HEK-293 cells. Conversely, a bivalent ligand (MDAN-16) with a short spacer or monovalent mu agonist give rise to robust internalization. The data suggest that the immobilization of proximal mu and delta protomers is due to bridging by MDAN-21. The finding that MDAN-21 and its shorter spacer homologue MDAN-16 possess equivalent activity in HEK-293 cells, but produce dramatically divergent internalization of mu-delta heteromer, is relevant to the role of internalization and tolerance

Clinically Employed Opioid Analgesics Produce Antinociception via μ‑δ Opioid Receptor Heteromers in Rhesus Monkeys

Morphine and related drugs are widely employed as analgesics despite the side effects associated with their use. Although morphine is thought to mediate analgesia through mu opioid receptors, delta opioid receptors have been implicated in mediating some side effects such as tolerance and dependence. Here we present evidence in rhesus monkeys that morphine, fentanyl, and possibly methadone selectively activate mu-delta heteromers to produce antinociception that is potently antagonized by the delta opioid receptor antagonist, naltrindole (NTI). Studies with HEK293 cells expressing mu-delta heteromeric opioid receptors exhibit a similar antagonism profile of receptor activation in the presence of NTI. In mice, morphine was potently inhibited by naltrindole when administered intrathecally, but not intracerebroventricularly, suggesting the possible involvement of mu-delta heteromers in the spinal cord of rodents. Taken together, these results strongly suggest that, in primates, mu-delta heteromers are allosterically coupled and mediate the antinociceptive effects of three clinically employed opioid analgesics that have been traditionally viewed as mu-selective. Given the known involvement of delta receptors in morphine tolerance and dependence, our results implicate mu-delta heteromers in mediating both antinociception and these side effects in primates. These results open the door for further investigation in humans

Optimizing Drug Design by Merging Generative AI With Active Learning Frameworks

Traditional drug discovery programs are being transformed by the advent of machine learning methods. Among these, Generative AI methods (GM) have gained attention due to their ability to design new molecules and enhance specific properties of existing ones. However, current GM methods have limitations, such as low affinity towards the target, unknown ADME/PK properties, or the lack of synthetic tractability. To improve the applicability domain of GM methods, we have developed a workflow based on a variational autoencoder coupled with active learning steps. The designed GM workflow iteratively learns from molecular metrics, including drug likeliness, synthesizability, similarity, and docking scores. In addition, we also included a hierarchical set of criteria based on advanced molecular modeling simulations during a final selection step. We tested our GM workflow on two model systems, CDK2 and KRAS. In both cases, our model generated chemically viable molecules with a high predicted affinity toward the targets. Particularly, the proportion of high-affinity molecules inferred by our GM workflow was significantly greater than that in the training data. Notably, we also uncovered novel scaffolds significantly dissimilar to those known for each target. These results highlight the potential of our GM workflow to explore novel chemical space for specific targets, thereby opening up new possibilities for drug discovery endeavors

The δ Opioid Receptor Agonist SNC80 Selectively Activates Heteromeric μ–δ Opioid Receptors

Coexpressed and colocalized μ- and δ-opioid receptors have been established to exist as heteromers in cultured cells and <i>in vivo</i>. However the biological significance of opioid receptor heteromer activation is less clear. To explore this significance, the efficacy of selective activation of opioid receptors by SNC80 was assessed <i>in vitro</i> in cells singly and coexpressing opioid receptors using a chimeric G-protein-mediated calcium fluorescence assay, SNC80 produced a substantially more robust response in cells expressing μ–δ heteromers than in all other cell lines. Intrathecal SNC80 administration in μ- and δ-opioid receptor knockout mice produced diminished antinociceptive activity compared with wild type. The combined <i>in vivo</i> and <i>in vitro</i> results suggest that SNC80 selectively activates μ–δ heteromers to produce maximal antinociception. These data contrast with the current view that SNC80 selectively activates δ-opioid receptor homomers to produce antinociception. Thus, the data suggest that heteromeric μ–δ receptors should be considered as a target when SNC80 is employed as a pharmacological tool <i>in vivo</i>

Bortezomib-induced neuropathy is in part mediated by the sensitization of TRPV1 channels

Abstract TRPV1 is an ion channel that transduces noxious heat and chemical stimuli and is expressed in small fiber primary sensory neurons that represent almost half of skin nerve terminals. Tissue injury and inflammation result in the sensitization of TRPV1 and sustained activation of TRPV1 can lead to cellular toxicity though calcium influx. To identify signals that trigger TRPV1 sensitization after a 24-h exposure, we developed a phenotypic assay in mouse primary sensory neurons and performed an unbiased screen with a compound library of 480 diverse bioactive compounds. Chemotherapeutic agents, calcium ion deregulators and protein synthesis inhibitors were long-acting TRPV1 sensitizers. Amongst the strongest TRPV1 sensitizers were proteasome inhibitors, a class that includes bortezomib, a chemotherapeutic agent that causes small fiber neuropathy in 30–50% of patients. Prolonged exposure of bortezomib produced a TRPV1 sensitization that lasted several days and neurite retraction in vitro and histological and behavioral changes in male mice in vivo. TRPV1 knockout mice were protected from epidermal nerve fiber loss and a loss of sensory discrimination after bortezomib treatment. We conclude that long-term TRPV1 sensitization contributes to the development of bortezomib-induced neuropathy and the consequent loss of sensation, major deficits experienced by patients under this chemotherapeutic agent