G protein-coupled receptors are a large family of proteins that regulate many processes in the body and are the target of one third of clinically used drugs. A subset of these receptors plays an important role in pain, including the neurokinin-1 (NK1) receptor, which is activated by a pain-transmitting neuropeptide called substance P. FDA-approved drugs that target the NK1 receptor are used to prevent nausea and vomiting associated with chemotherapy or surgery. Scientists previously hoped that the NK1 receptor would be a promising target for treating pain but drugs targeting the receptor failed to control pain in clinical trials in the past. One reason why drugs targeting the NK1 receptor may not have been effective against pain is that most drugs block receptors at the surface of cells. In a new study published in the peer-reviewed Journal, Proceedings of the National Academy of Sciences (PNAS), illustrates how pain signaling occurs inside cells rather than at the surface, highlighting the need for drugs that can reach receptors within cells. Altering the chemical properties of an anti-nausea drug enables it to enter an interior compartment of the cell and provide long-lasting pain relief, according to a new study led by researchers at NYU College of Dentistry’s Pain Research Center led by Professor Nigel Bunnett, chair of the Department of Molecular Pathobiology. The research team showed that GCPRs signal pain not from the surface of cells, but from compartments inside the cell called endosomes. The researchers aimed to develop effective therapies for pain relief by designing antagonists that can penetrate cells, remain in endosomes (intracellular compartments), and disrupt endosomal signaling. They focused on the neurokinin 1 receptor (NK1R), which is involved in pain signaling. To enhance the membrane penetration and retention of the NK1R antagonist netupitant in acidified endosomes, the researchers synthesized analogs of netupitant with modified lipophilicity (ability to dissolve in lipids) and acidity. These modified analogs, which were lipophilic and acidic, were able to antagonize NK1R signaling from endosomes. Furthermore, in mice expressing the human NK1R, these analogs provided potent, efficacious, and long-lasting pain relief.
Aprepitant and netupitant are both neurokinin 1 receptor (NK1R) antagonists that are used in medical practice for different purposes. Aprepitant is an FDA-approved NK1R antagonist primarily used for the prevention of chemotherapy-induced nausea and vomiting. It is available in oral and intravenous formulations. The mechanism of action of aprepitant involves blocking the binding of substance P, a neuropeptide involved in triggering nausea and vomiting, to the NK1R. Netupitant is another NK1R antagonist that is commonly used in combination with palonosetron, a 5-HT3 receptor antagonist, for the prevention of both acute and delayed chemotherapy-induced nausea and vomiting caused by highly emetogenic chemotherapy (chemotherapy agents with a high likelihood of causing nausea and vomiting). This combination therapy is marketed as NEPA. Netupitant, like aprepitant, exerts its pharmacological effects by binding to and blocking the NK1R. However, netupitant is structurally distinct from aprepitant and exhibits higher receptor affinity and longer receptor occupancy, leading to prolonged blockade of substance P signaling.
The researchers also investigated the role of natural variants of NK1R that exhibit abnormal signaling and endosomal trafficking in maintaining pain. They found that mice expressing a truncated form of human NK1R, corresponding to a naturally occurring variant with aberrant signaling and trafficking, had reduced nociceptive (pain-related) responses to substance P, a molecule that activates NK1R.
Sustained signaling in endosomes is necessary for the hyperexcitability of pain-sensing neurons involved in chronic pain, as a result, treating pain may require the development of drugs that penetrate cells, are retained in endosomes, and disrupt signaling inside the cell. The authors focused on two drugs, aprepitant and netupitant, both NK1 receptor antagonists used to prevent nausea and vomiting. Studying NK1 receptors in the lab has the benefit of clinically available drugs that target the receptor, but also comes with challenges, as there are large differences between the NK1 receptor in mice and humans. To overcome this, the researchers genetically modified mice to express the human NK1 receptor. In a previous study, the authors showed that encapsulating aprepitant in nanoparticles could deliver the drug to endosomes to block pain, but in the new study, aprepitant only briefly disrupted endosomal signaling in cellular studies and stopped pain in mice for short periods. Modifying the second drug, netupitant, held much more promise. The researchers changed the chemical properties of the drug to make it more capable of penetrating a cell’s lipid membrane. They also altered the charge on the molecule within an acidic environment so that once the drug entered the acidic environment of an endosome, it would stay trapped inside and accumulate.
These changes allowed the modified netupitant to readily penetrate cells to reach the endosome and block signaling of the NK1 receptor in endosomes with a much more prolonged effect in cells. The altered netupitant also had a more potent and long-lasting analgesic effect in mice than aprepitant and the regular form of netupitant. The research team studied mice with a different type of NK1 receptor on the outer membrane of the cell, rather than inside. These mice were more resistant to pain than those with human NK1 receptors inside the cell, illustrating the importance of endosomes in signaling pain and the need for treatments that can penetrate cells.
In a nutshell, the authors identified criteria for designing antagonists of endosomal receptors, which can penetrate cells, remain in endosomes, and disrupt endosomal signaling. The findings also provided evidence supporting the idea that endosomal signaling of GPCRs, such as NK1R, contributes to pain and potentially other diseases. The new study offers insights into strategies for targeting intracellular GPCRs for the treatment of various diseases. Future studies will focus on other animal models to develop new therapies for pain that block GCPRs in endosomes.
Hegron A, Peach CJ, Tonello R, Seemann P, Teng S, Latorre R, Huebner H, Weikert D, Rientjes J, Veldhuis NA, Poole DP, Jensen DD, Thomsen ARB, Schmidt BL, Imlach WL, Gmeiner P, Bunnett NW. Therapeutic antagonism of the neurokinin 1 receptor in endosomes provides sustained pain relief. Proc Natl Acad Sci U S A. 2023 ;120(22):e2220979120. doi: 10.1073/pnas.2220979120