A lipid-anchored neurokinin 1 receptor antagonist prolongs pain relief by a three-pronged mechanism of action targeting the receptor at the plasma membrane and in endosomes.

G-protein-coupled receptors (GPCRs) are traditionally known for signaling at the plasma membrane, but they can also signal from endosomes after internalization to control important pathophysiological processes. In spinal neurons, sustained endosomal signaling of the neurokinin 1 receptor (NK1R) mediates nociception, as demonstrated in models of acute and neuropathic pain. An NK1R antagonist, Spantide I (Span), conjugated to cholestanol (Span-Chol), accumulates in endosomes, inhibits endosomal NK1R signaling, and causes prolonged antinociception. However, the extent to which the Chol-anchor influences long-term location and activity is poorly understood. Herein, we used fluorescent correlation spectroscopy and targeted biosensors to characterize Span-Chol over time. The Chol-anchor increased local concentration of probe at the plasma membrane. Over time we observed an increase in NK1R-binding affinity and more potent inhibition of NK1R-mediated calcium signaling. Span-Chol, but not Span, caused a persistent decrease in NK1R recruitment of ß-arrestin and receptor internalization to early endosomes. Using targeted biosensors, we mapped the relative inhibition of NK1R signaling as the receptor moved into the cell. Span selectively inhibited cell surface signaling, whereas Span-Chol partitioned into endosomal membranes and blocked endosomal signaling. In a preclinical model of pain, Span-Chol caused prolonged antinociception (>9 h), which is attributable to a three-pronged mechanism of action: increased local concentration at membranes, a prolonged decrease in NK1R endocytosis, and persistent inhibition of signaling from endosomes. Identifying the mechanisms that contribute to the increased preclinical efficacy of lipid-anchored NK1R antagonists is an important step toward understanding how we can effectively target intracellular GPCRs in disease.

Potential perioperative cardiovascular outcomes in cannabis/cannabinoid users. A call for caution.

Background: Cannabis is one of the most widely used psychoactive substances. Its components act through several pathways, producing a myriad of side effects, of which cardiovascular events are the most life-threatening. However, only a limited number of studies address cannabis's perioperative impact on patients during noncardiac surgery. Methods: Studies were identified by searching the PubMed, Medline, EMBASE, and Google Scholar databases using relevant keyword combinations pertinent to the topic. Results: Current evidence shows that cannabis use may cause several cardiovascular events, including abnormalities in cardiac rhythm, myocardial infarction, heart failure, and cerebrovascular events. Additionally, cannabis interacts with anticoagulants and antiplatelet agents, decreasing their efficacy. Finally, the interplay of cannabis with inhalational and intravenous anesthetic agents may lead to adverse perioperative cardiovascular outcomes. Conclusions: The use of cannabis can trigger cardiovascular events that may depend on factors such as the duration of consumption, the route of administration of the drug, and the dose consumed, which places these patients at risk of drug-drug interactions with anesthetic agents. However, large prospective randomized clinical trials are needed to further elucidate gaps in the body of knowledge regarding which patient population has a greater risk of perioperative complications after cannabis consumption.

New small molecule fluorescent probes for G protein-coupled receptors: valuable tools for drug discovery.

G protein-coupled receptors (GPCRs) are essential signaling proteins and tractable therapeutic targets. To develop new drug candidates, GPCR drug discovery programs require versatile, sensitive pharmacological tools for ligand binding and compound screening. With the availability of new imaging modalities and proximity-based ligand binding technologies, fluorescent ligands offer many advantages and are increasingly being used, yet labeling small molecules remains considerably more challenging relative to peptides. Focusing on recent fluorescent small molecule studies for family A GPCRs, this review addresses some of the key challenges, synthesis approaches and structure-activity relationship considerations, and discusses advantages of using high-resolution GPCR structures to inform conjugation strategies. While no single approach guarantees successful labeling without loss of affinity or selectivity, the choice of fluorophore, linker type and site of attachment have proved to be critical factors that can significantly affect their utility in drug discovery programs, and as discussed, can sometimes lead to very unexpected results.

Neurokinin 1 receptor signaling in endosomes mediates sustained nociception and is a viable therapeutic target for prolonged pain relief.

Typically considered to be cell surface sensors of extracellular signals, heterotrimeric GTP-binding protein (G protein)-coupled receptors (GPCRs) control many pathophysiological processes and are the target of 30% of therapeutic drugs. Activated receptors redistribute to endosomes, but researchers have yet to explore whether endosomal receptors generate signals that control complex processes in vivo and are viable therapeutic targets. We report that the substance P (SP) neurokinin 1 receptor (NK1R) signals from endosomes to induce sustained excitation of spinal neurons and pain transmission and that specific antagonism of the NK1R in endosomes with membrane-anchored drug conjugates provides more effective and sustained pain relief than conventional plasma membrane-targeted antagonists. Pharmacological and genetic disruption of clathrin, dynamin, and ß-arrestin blocked SP-induced NK1R endocytosis and prevented SP-stimulated activation of cytosolic protein kinase C and nuclear extracellular signal-regulated kinase, as well as transcription. Endocytosis inhibitors prevented sustained SP-induced excitation of neurons in spinal cord slices in vitro and attenuated nociception in vivo. When conjugated to cholestanol to promote endosomal targeting, NK1R antagonists selectively inhibited endosomal signaling and sustained neuronal excitation. Cholestanol conjugation amplified and prolonged the antinociceptive actions of NK1R antagonists. These results reveal a critical role for endosomal signaling of the NK1R in the complex pathophysiology of pain and demonstrate the use of endosomally targeted GPCR antagonists.