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
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100345
ISSN1083-351X
AutoresN. Quynh, Priyank A. Shenoy, Tim Quach, Jeffri S. Retamal, Arisbel B. Gondin, Holly R. Yeatman, Luigi Aurelio, Joshua Conner, Daniel P. Poole, Meritxell Canals, Cameron J. Nowell, Bim Graham, Thomas P. Davis, Stephen J. Briddon, Stephen J. Hill, Christopher J. H. Porter, Nigel W. Bunnett, Michelle L. Halls, Nicholas A. Veldhuis,
Tópico(s)Pain Mechanisms and Treatments
ResumoG-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 β-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 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 β-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 G-protein-coupled receptors (GPCRs) are tractable therapeutic targets because they have druggable sites on the cell surface and control most pathophysiological processes (1Hauser A.S. Attwood M.M. Rask-Andersen M. Schioth H.B. Gloriam D.E. Trends in GPCR drug discovery: New agents, targets and indications.Nat. Rev. Drug Discov. 2017; 16: 829-842Crossref PubMed Scopus (946) Google Scholar). However, many GPCRs can also signal from intracellular compartments, including endosomes, the Golgi, mitochondria, and the nucleus (2Luttrell L.M. Ferguson S.S. Daaka Y. Miller W.E. Maudsley S. Della Rocca G.J. Lin F. Kawakatsu H. Owada K. Luttrell D.K. Caron M.G. Lefkowitz R.J. 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During this time, GPCR kinases rapidly phosphorylate the NK1R leading to association with β-arrestins and receptor endocytosis to early endosomes ( 20 min). It is these sustained signals from the intracellular NK1R that mediate persistent excitation of spinal neurons and sustained central pain transmission (7Murphy J.E. Padilla B.E. Hasdemir B. Cottrell G.S. Bunnett N.W. Endosomes: A legitimate platform for the signaling train.PNAS. 2009; 106: 17615-17622Crossref PubMed Scopus (263) Google Scholar, 14Jensen D.D. Halls M.L. Murphy J.E. Canals M. Cattaruzza F. Poole D.P. Lieu T. Koon H.W. Pothoulakis C. Bunnett N.W. Endothelin-converting enzyme 1 and beta-arrestins exert spatiotemporal control of substance P-induced inflammatory signals.J. Biol. Chem. 2014; 289: 20283-20294Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 15Cottrell G.S. Padilla B.E. Amadesi S. Poole D.P. Murphy J.E. Hardt M. Roosterman D. Steinhoff M. Bunnett N.W. Endosomal endothelin-converting enzyme-1: A regulator of beta-arrestin-dependent ERK signaling.J. Biol. Chem. 2009; 284: 22411-22425Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Ligands can have spatially specific or "location biased" pharmacological actions in cells (16Irannejad R. Pessino V. Mika D. Huang B. Wedegaertner P.B. Conti M. von Zastrow M. Functional selectivity of GPCR-directed drug action through location bias.Nat. Chem. Biol. 2017; 13: 799-806Crossref PubMed Scopus (91) Google Scholar). We have previously assessed the potential for drug delivery strategies to locally deliver NK1R antagonists to endosomes. This includes pH-responsive nanoparticles that deliver and release the NK1R antagonist aprepitant directly into the endosomes (17Ramirez-Garcia P.D. Retamal J.S. Shenoy P. Imlach W. Sykes M. Truong N. Constandil L. Pelissier T. Nowell C.J. Khor S.Y. Layani L.M. Lumb C. Poole D.P. Lieu T. Stewart G.D. et al.A pH-responsive nanoparticle targets the neurokinin 1 receptor in endosomes to prevent chronic pain.Nat. Nanotechnol. 2019; 14: 1150-1159Crossref PubMed Scopus (44) Google Scholar) and lipid-anchored NK1R antagonists that accumulate in endosomal membranes (5Jensen D.D. Lieu T. Halls M.L. Veldhuis N.A. Imlach W.L. Mai Q.N. Poole D.P. Quach T. Aurelio L. Conner J. Herenbrink C.K. Barlow N. Simpson J.S. Scanlon M.J. Graham B. et al.Neurokinin 1 receptor signaling in endosomes mediates sustained nociception and is a viable therapeutic target for prolonged pain relief.Sci. Transl. Med. 2017; 9: eaal3447Crossref PubMed Scopus (87) Google Scholar). Both of these approaches improved drug efficacy in preclinical models of pain (2–5-fold more effective antinociception, 2–4-fold longer duration of action compared with free drug) (5Jensen D.D. Lieu T. Halls M.L. Veldhuis N.A. Imlach W.L. Mai Q.N. Poole D.P. Quach T. Aurelio L. Conner J. Herenbrink C.K. Barlow N. Simpson J.S. Scanlon M.J. Graham B. et al.Neurokinin 1 receptor signaling in endosomes mediates sustained nociception and is a viable therapeutic target for prolonged pain relief.Sci. Transl. Med. 2017; 9: eaal3447Crossref PubMed Scopus (87) Google Scholar, 17Ramirez-Garcia P.D. Retamal J.S. Shenoy P. Imlach W. Sykes M. Truong N. Constandil L. Pelissier T. Nowell C.J. Khor S.Y. Layani L.M. Lumb C. Poole D.P. Lieu T. Stewart G.D. et al.A pH-responsive nanoparticle targets the neurokinin 1 receptor in endosomes to prevent chronic pain.Nat. Nanotechnol. 2019; 14: 1150-1159Crossref PubMed Scopus (44) Google Scholar). The localized delivery of an NK1R antagonist to endosomes using nanoparticles is a selective approach that bypasses any effects on receptors at the cell surface. In contrast, lipid-anchored NK1R antagonists first partition into the plasma membrane, before they are trafficked to endosomes (5Jensen D.D. Lieu T. Halls M.L. Veldhuis N.A. Imlach W.L. Mai Q.N. Poole D.P. Quach T. Aurelio L. Conner J. Herenbrink C.K. Barlow N. Simpson J.S. Scanlon M.J. Graham B. et al.Neurokinin 1 receptor signaling in endosomes mediates sustained nociception and is a viable therapeutic target for prolonged pain relief.Sci. Transl. Med. 2017; 9: eaal3447Crossref PubMed Scopus (87) Google Scholar). It is therefore possible that lipid-anchored antagonists also affect the signaling and trafficking of plasma membrane-localized NK1R, in addition to their later antagonism of endosomal receptors. This dual antagonism—initial blockade of plasma membrane receptors during partitioning into the plasma membrane and then prolonged blockade of the pathophysiologically relevant signal from endosomes—could enhance therapeutic efficacy. In the current investigation, we used live cell imaging and biophysical approaches to assess NK1R signaling and trafficking in subcellular compartments, in conjunction with behavioral assays of nociception to investigate the mechanisms by which a cholestanol-anchored antagonist, Spantide I (Span-Chol), inhibits endosomal signaling. We used a cholestanol-anchored fluorescent probe (Cy5-Chol) to model the lipid-dependent translocation of the antagonist. We observed that the lipid anchor allows an initial enrichment of probe concentration at the plasma membrane, which correlates with an increased antagonist potency at proximal signaling pathways (i.e., Ca2+ mobilization). The lipid-anchored antagonist also inhibits cell surface NK1R-β-arrestin recruitment and NK1R endocytosis. Over time, Cy5-Chol travels from the plasma membrane to early and late endosomes. This movement deeper into the endosomal network correlates with inhibition of endosomal-selective NK1R signaling pathways (5Jensen D.D. Lieu T. Halls M.L. Veldhuis N.A. Imlach W.L. Mai Q.N. Poole D.P. Quach T. Aurelio L. Conner J. Herenbrink C.K. Barlow N. Simpson J.S. Scanlon M.J. Graham B. et al.Neurokinin 1 receptor signaling in endosomes mediates sustained nociception and is a viable therapeutic target for prolonged pain relief.Sci. Transl. Med. 2017; 9: eaal3447Crossref PubMed Scopus (87) Google Scholar) by Span-Chol, including sustained cytosolic cAMP and cytosolic PKC activity. Consistent with these findings, the lipid-anchored antagonist has long-lasting antinociceptive actions in preclinical models of pain (>9 h). We find that lipid anchors increase the local membrane concentration of GPCR antagonists, cause inhibition of receptor trafficking from the plasma membrane, and prolong the inhibition of signaling from endosomes. This three-pronged mechanism allows lipid-anchored antagonists to very effectively target endosomally derived GPCR signaling pathways of pathophysiological importance. Inspired by prior studies using lipid–drug conjugates (18Rajendran L. Schneider A. Schlechtingen G. Weidlich S. Ries J. Braxmeier T. Schwille P. Schulz J.B. Schroeder C. Simons M. Jennings G. Knolker H.J. Simons K. Efficient inhibition of the Alzheimer's disease beta-secretase by membrane targeting.Science. 2008; 320: 520-523Crossref PubMed Scopus (216) Google Scholar, 19Linning P. Haussmann U. Beyer I. Weidlich S. Schieb H. Wiltfang J. Klafki H.W. Knolker H.J. Optimisation of BACE1 inhibition of tripartite structures by modification of membrane anchors, spacers and pharmacophores - development of potential agents for the treatment of Alzheimer's disease.Org. Biomol. Chem. 2012; 10: 8216-8235Crossref PubMed Scopus (10) Google Scholar), we previously synthesized a series of lipid-anchored probes comprising the sterol cholestanol as a lipid conjugate for anchoring a cargo to membranes via a flexible polyethylene glycol linker (PEG4-PEG3-PEG4) (5Jensen D.D. Lieu T. Halls M.L. Veldhuis N.A. Imlach W.L. Mai Q.N. Poole D.P. Quach T. Aurelio L. Conner J. Herenbrink C.K. Barlow N. Simpson J.S. Scanlon M.J. Graham B. et al.Neurokinin 1 receptor signaling in endosomes mediates sustained nociception and is a viable therapeutic target for prolonged pain relief.Sci. Transl. Med. 2017; 9: eaal3447Crossref PubMed Scopus (87) Google Scholar). For the cargo we used Cyanine 5 (Cy5), to generate a fluorescent reporter of lipid-anchor location (Cy5-Chol), or the NK1R antagonist, Spantide I, to generate a lipid-anchored antagonist (Span-Chol) (Fig. S2). We also generated control probes including a nonlipidated fluorescent probe (ethyl-ester group, PEG linker, Cy5; Cy5-OEt) and a lipid anchor control probe (cholestanol group, PEG linker, biotin; Chol). Fluorescence correlation spectroscopy (FCS) enables measurement of the concentrations of fluorescent molecules within a small defined volume (<0.2 fl) (20Briddon S.J. Hill S.J. Pharmacology under the microscope: The use of fluorescence correlation spectroscopy to determine the properties of ligand-receptor complexes.Trends Pharmacol. Sci. 2007; 28: 637-645Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 21Stoddart L.A. Vernall A.J. Denman J.L. Briddon S.J. Kellam B. Hill S.J. Fragment screening at adenosine-A(3) receptors in living cells using a fluorescence-based binding assay.Chem. Biol. 2012; 19: 1105-1115Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). We used this approach to determine the concentration of Cy5 probes (Cy5-Chol or the control, Cy5-OEt) in the extracellular fluid immediately above the plasma membrane and at increasing distances above the cell (30–200 μm). We chose the Cy5 probes as the simplest example of how a lipid anchor could affect the plasma membrane concentration of a cargo, independent of any receptor-dependent effects on ligand distribution (22Gherbi K. Briddon S.J. Charlton S.J. Micro-pharmacokinetics: Quantifying local drug concentration at live cell membranes.Sci. Rep. 2018; 8: 3479Crossref PubMed Scopus (23) Google Scholar). Consistent with our previous studies (5Jensen D.D. Lieu T. Halls M.L. Veldhuis N.A. Imlach W.L. Mai Q.N. Poole D.P. Quach T. Aurelio L. Conner J. Herenbrink C.K. Barlow N. Simpson J.S. Scanlon M.J. Graham B. et al.Neurokinin 1 receptor signaling in endosomes mediates sustained nociception and is a viable therapeutic target for prolonged pain relief.Sci. Transl. Med. 2017; 9: eaal3447Crossref PubMed Scopus (87) Google Scholar), brightfield and fluorescence confocal imaging confirmed that Cy5-Chol rapidly incorporated into the plasma membrane of HEK293 cells (Fig. 1A), but Cy5-OEt remained in extracellular fluid (Fig. 1B). We then used FCS to quantify the concentration of Cy5 fluorescence at the plasma membrane of cells incubated with a nominal concentration of probe (10 nM). The concentration of Cy5-Chol in the extracellular fluid at 5 μm above the plasma membrane was 23.8 ± 7.1 nM, which decreased more than fourfold to 5.6 ± 1.4 nM at 30 μm (mean ± SEM from n = 4) (Fig. 1, C and E). In contrast, the measured concentration of Cy5-OEt was 6.5 ± 1.1 nM at 5 μm above the plasma membrane, which increased more than threefold to 21.8 ± 3.8 nM at 200 μm (mean ± SEM from n = 4) (Fig. 1, D and E). A comparison of probe concentrations at increasing distances (5 μm intervals) above the plasma membrane suggested that there was an enrichment of Cy5-Chol proximal to the plasma membrane, while the Cy5-OEt reporter molecule could freely diffuse through the extracellular fluid (Fig. 1E). Therefore, the addition of a lipid anchor results in an enhanced association of a probe with cell membranes. This creates a high local concentration of probe at the cell surface. To determine whether the addition of a lipid anchor influences the affinity and potency of an NK1R antagonist, we compared unconjugated ("free") Spantide I (Span) and Span-Chol. A high-content imaging competition binding assay was used to evaluate the capacity of these antagonists to disrupt the binding of SP labeled with fluorescent tetramethylrhodamine (SP-TAMRA) to the NK1R stably transfected in HEK293 cells. Cells were analyzed using an established granularity algorithm to provide a measure of total cell binding (includes both cell surface and intracellular) (21Stoddart L.A. Vernall A.J. Denman J.L. Briddon S.J. Kellam B. Hill S.J. Fragment screening at adenosine-A(3) receptors in living cells using a fluorescence-based binding assay.Chem. Biol. 2012; 19: 1105-1115Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 23Stoddart L.A. Vernall A.J. Briddon S.J. Kellam B. Hill S.J. Direct visualisation of internalization of the adenosine A3 receptor and localization with arrestin3 using a fluorescent agonist.Neuropharmacology. 2015; 98: 68-77Crossref PubMed Scopus (24) Google Scholar). We assessed antagonist affinity at two time points following antagonist addition: 30 min, when FCS data show Cy5-Chol enrichment at the plasma membrane (Fig. 1); and 4 h, when Span-Chol accumulates, and is pharmacologically active, within endosomal compartments (5Jensen D.D. Lieu T. Halls M.L. Veldhuis N.A. Imlach W.L. Mai Q.N. Poole D.P. Quach T. Aurelio L. Conner J. Herenbrink C.K. Barlow N. Simpson J.S. Scanlon M.J. Graham B. et al.Neurokinin 1 receptor signaling in endosomes mediates sustained nociception and is a viable therapeutic target for prolonged pain relief.Sci. Transl. Med. 2017; 9: eaal3447Crossref PubMed Scopus (87) Google Scholar). To assess competition binding after 30 min, HEK-NK1R cells were coincubated with an EC50 concentration of SP-TAMRA (0.5 nM) and increasing concentrations of Span or Span-Chol and equilibrated for 30 min. The affinity of Span-Chol and Span for NK1R was similar with pIC50 values of 6.28 ± 0.09 and 5.99 ± 0.13, respectively (Fig. 2A). Therefore, peptide modification by attachment of a PEG12 linker and cholestanol anchor does not diminish the affinity of Spantide for the NK1R. To assess ligand binding after 4 h, HEK-NK1R cells were preincubated with antagonist for 3.5 h, then with SP-TAMRA for a further 30 min (4 h total). The affinity of Span for the NK1R was significantly reduced compared with that of Span-Chol (pIC50 5.55 ± 0.17 vs 6.50 ± 0.12, p = 0.0018, unpaired t-test) (Fig. 2B). However, there was no significant change in the relative affinities of Span or Span-Chol for the NK1R over time (p = 0.1121 and p = 0.6378, respectively; one-way ANOVA with Tukey's multiple comparisons test). This suggests that the addition of a lipid anchor improves the kinetic properties of Span by sustaining its ability to compete with SP-TAMRA at the NK1R over a 4 h period. Our previous studies indicated no difference in stability of these ligands in spinal cord membranes (5Jensen D.D. Lieu T. Halls M.L. Veldhuis N.A. Imlach W.L. Mai Q.N. Poole D.P. Quach T. Aurelio L. Conner J. Herenbrink C.K. Barlow N. Simpson J.S. Scanlon M.J. Graham B. et al.Neurokinin 1 receptor signaling in endosomes mediates sustained nociception and is a viable therapeutic target for prolonged pain relief.Sci. Transl. Med. 2017; 9: eaal3447Crossref PubMed Scopus (87) Google Scholar). We therefore propose that this apparent improvement in affinity of Span-Chol for the NK1R is due to the accumulation of Span-Chol in endosomes, allowing Span-Chol to access both plasma membrane and endosomal pools of NK1R. To determine if lipid conjugation influenced the potency of Span, we compared the ability of Span and Span-Chol to inhibit SP-stimulated Ca2+ signaling in HEK-NK1R cells at different time points after addition. In initial experiments, HEK-NK1R cells were preincubated with increasing concentrations of Span or Span-Chol for 30 min, prior to challenge with an EC80 concentration of SP (1 nM). Ca2+ transients were measured for 90 s poststimulation. Preincubation of cells with Span or Span-Chol caused a concentration-dependent inhibition of Ca2+ flux (Fig. 2C). A comparison of the pIC50 values of Span and Span-Chol (4.87 ± 0.33 and 6.25 ± 0.19, respectively) revealed a significant increase in the potency of the lipidated antagonist (p = 0.0112). This is consistent with FCS experiments (Fig. 1) and may be due to the lipid anchoring of the antagonist to the plasma membrane, thereby effectively increasing the local concentration of the antagonist near the receptor even at acute time periods (24Vauquelin G. Charlton S.J. Long-lasting target binding and rebinding as mechanisms to prolong in vivo drug action.Br. J. Pharmacol. 2010; 161: 488-508Crossref PubMed Scopus (203) Google Scholar, 25Sykes D.A. Parry C. Reilly J. Wright P. Fairhurst R.A. Charlton S.J. Observed drug-receptor association rates are governed by membrane affinity: The importance of establishing "micro-pharmacokinetic/pharmacodynamic relationships" at the beta2-adrenoceptor.Mol. Pharmacol. 2014; 85: 608-617Crossref PubMed Scopus (73) Google Scholar). While lipid-anchored fluorescent probes initially partition into the plasma membrane, they are then quickly trafficked into endosomal compartments (5Jensen D.D. Lieu T. Halls M.L. Veldhuis N.A. Imlach W.L. Mai Q.N. Poole D.P. Quach T. Aurelio L. Conner J. Herenbrink C.K. Barlow N. Simpson J.S. Scanlon M.J. Graham B. et al.Neurokinin 1 receptor signaling in endosomes mediates sustained nociception and is a viable therapeutic target for prolonged pain relief.Sci. Transl. Med. 2017; 9: eaal3447Crossref PubMed Scopus (87) Google Scholar). As such, the continuous removal of lipidated antagonists from the plasma membrane by constitutive endocytosis could affect the relative potency of Span-Chol compared with soluble Span over time. To assess this possibility, we compared continuous exposure to the antagonists for 4 h to a "pulsed" administration whereby the cells were preincubated with antagonist for 30 min, washed to remove any excess ligand, and then left at 37°C for 3.5 h (4 h total). In both protocols, cells were challenged with 1 nM SP 4 h after the initial antagonist addition. There was no change in the pIC50 of the antagonists when the cells were continuously incubated with Span or Span-Chol for 4 h (5.11 ± 0.76 and 6.36 ± 0.17, respectively) compared with the 30 min preincubation (Fig. 2D). After pulsed administration, only Span-Chol retained its ability to antagonize SP-stimulated Ca2+ signaling at 4 h (pIC50 6.15 ± 0.11) (Fig. 2D). This is likely due to the wash step (after the initial 30 min incubation with antagonist) decreasing the available concentration of free Span in the extracellular fluid. In contrast, the potency of Span-Chol was not lost following the wash, confirming that lipidation causes an increased association of the antagonist with the cell membrane. Notably, the potency of Span-Chol was sustained over 4 h despite the increasing internalization of lipid-anchored probes over time (5Jensen D.D. Lieu T. Halls M.L. Veldhuis N.A. Imlach W.L. Mai Q.N. Poole D.P. Q
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