Measuring ligand efficacy at the mu-opioid receptor using a conformational biosensor
- University of Michigan Medical School, United States
- University of Michigan, United States
- University of California San Francisco, United States
- University of California San Diego School of Medicine, United States
Figures
Figure 1
Cartoon schematic of interferometry assay to detect µ-OR:Nb39 interactions.
Briefly, (a) Biotin-conjugated rHDL particles containing µ-OR are loaded on streptavidin-coated tips and incubated with saturating ligand (or vehicle) for 10 min. (b) Probe is exposed to Nb39 for …
Figure 2
Orthosteric ligand-mediated Nb39 association and dissociation in µ-OR-rHDL.
As described in the Materials and methods, the association and dissociation of Nb39 (1 µM) was measured using an OctetRed® instrument. Shown is a representative experiment comparing four orthosteric …
Figure 3
Correlation of association times of Nb39 with various measures of agonist efficacy.
The t1/2 of association of Nb39 in the presence of saturating agonist was measured and is plotted against (a) maximal stimulation of GTPγ35S binding by agonist, (b) the calculated Ehlert efficacy (Eh…
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Figure 3—source data 1
List of values used to construct correlation graphs.
Ligand number refers to the list in legend to Figure 3; the t1/2 for association of NB39 is from Table 1; Maximum stimulation of [35S]GTPγS binding by each ligand is taken from Livingston and Traynor (2014); Ehlert’s efficacy value (e, Ehlert, 1985) determined for each ligand using [35S]GTPγS assay data and ligand affinities, taken from Livingston and Traynor (2014); Shift in ligand affinity in the presence of 100 mM NaCl and 10 μM GTPγS, from Livingston and Traynor (2014); Efficacy (τ) values for stimulation of [35S]GTPγS binding (Livingston and Traynor, 2014) calculated according to Black and Leff (1983); Efficacy (τ) values for arrestin recruitment taken from McPherson et al. (2010).
- https://doi.org/10.7554/eLife.32499.006
Figure 4
Association and dissociation of a range of Nb39 concentrations induced by various agonists.
The association of six different concentrations of Nb39 was measured in the presence of saturating ligand concentrations. Utilizing global regression analysis, the Kd of Nb39 (nM) for ligand bound …
Figure 5
Allosteric modulation of µ-OR-rHDL by small molecule PAMs.
Structures of (a) BMS-986187 and (b) BMS-986122. The ability of BMS-986122 (c) or BMS-986187 (d) to enhance the binding affinity of L-methadone was measured using displacement of the orthosteric …
Figure 6
Effects of allosteric ligands on binding kinetics of Nb39.
The association and dissociation of Nb39 (1 µM) was measured as described in the Materials and methods. (a) Shown is a representative experiment of data in Table 1, comparing two allosteric agonists …
Figure 7
Association and dissociation of heterotrimeric G protein and the effect of GDP on dissociation.
(a) Utilizing a global fit analysis the affinity (Kd) of G protein for μ-receptor was determined as 13 ± 1 nM for the BU72-bound receptor and 2.9 ± 0.8 nM for the morphine-bound receptor, though …
Tables
Table 1
Association and dissociation kinetics of Nb39 to µ-OR-rHDL in the presence of various agonists.
kobs and koff were determined for each independent experiment (number of individual experiments indicated in ‘n’ column) and averaged. One-phase association and single-phase exponential decay models …
| Ligand | kobs ± SEM (min−1) | t1/2ass (sec) | koff (min−1) | t1/2dis (sec) | N |
|---|---|---|---|---|---|
| BU72 | 0.20 ± 0.01 | 3.5 | 0.031 ± 0.001 | 22 | 14 |
| DAMGO | 0.179 ± 0.008 | 3.9 | 0.030 ± 0.001 | 23 | 6 |
| Leu-Enk | 0.098 ± 0.02 | 7.1 | 0.031 ± 0.001 | 23 | 9 |
| L-Methadone | 0.19 ± 0.02 | 3.6 | 0.052 ± 0.003 | 13 | 6 |
| Morphine | 0.08 ± 0.01 | 8.5 | 0.033 ± 0.003 | 21 | 7 |
| Nalbuphine | 0.023 ± 0.008 | 30 | 0.036 ± 0.004 | 19 | 11 |
| PZM21 | 0.064 ± 0.004 | 11 | 0.043 ± 0.002 | 16 | 6 |
| Endomorphin 2 | 0.101 ± 0.002 | 6.9 | 0.036 ± 0.002 | 19 | 6 |
| Loperamide | 0.208 ± 0.009 | 3.2 | 0.044 ± 0.003 | 16 | 6 |
| Oxycodone | 0.044 ± 0.001 | 16 | 0.028 ± 0.001 | 25 | 9 |
| Etorphine | 0.180 ± 0.007 | 3.9 | 0.026 ± 0.001 | 27 | 6 |
| Fentanyl | 0.075 ± 0.005 | 9.2 | 0.035 ± 0.002 | 20 | 6 |
| Met-Enk | 0.131 ± 0.004 | 5.3 | 0.027 ± 0.001 | 26 | 6 |
| Hydrocodone | 0.042 ± 0.004 | 17 | 0.029 ± 0.001 | 24 | 6 |
| Buprenorphine | 0.058 ± 0.009 | 12 | 0.025 ± 0.001 | 27 | 6 |
| Naloxone | n/a | ---- | n/a | ---- | 3 |
| Diprenorphine | n/a | ---- | n/a | ---- | 3 |
| BMS-986122 | 0.012 ± 0.001 | 56 | 0.027 ± 0.003 | 25 | 10 |
| BMS-986187 | 0.025 ± 0.006 | 28 | 0.037 ± 0.005 | 19 | 11 |
Table 2
Alteration in Nb39 kinetics in the presence of µ-PAMs
https://doi.org/10.7554/eLife.32499.010| Morphine (1 µM Nb39) | ||||||
|---|---|---|---|---|---|---|
| kobs (min−1) | t1/2Assoc (sec) | koff (min−1) | t1/2Diss(sec) | N | ||
| Vehicle | 0.08 ± 0.01 | 8.5 | 0.033 ± 0.001 | 21 | 3 | |
| BMS-986122 | 0.11 ± 0.001 | 6.4 | 0.032 ± 0.0002 | 22 | 3 | |
| BMS-986187 | 0.13 ± 0.01 *** | 5.3 | 0.024 ± 0.0004 | 29 | 3 | |
| L-Methadone (100 nM Nb39) | ||||||
| kobs (min−1) | t1/2Assoc (sec) | koff (min−1) | t1/2Diss(sec) | n | ||
| Vehicle | 0.087 ± 0.009 | 8.0 | 0.050 ± 0.004 † | 14 | 7 | |
| BMS-986122 | 0.077 ± 0.007 | 9.0 | 0.047 ± 0.004 ++ | 15 | 7 | |
| BMS-986187 | 0.055 ± 0.004 * | 13 | 0.033 ± 0.002 ** | 21 | 7 | |
| DAMGO (100 nM Nb39) | ||||||
| kobs (min−1) | t1/2Assoc (sec) | koff (min−1) | t1/2Diss(sec) | n | ||
| Vehicle | 0.051 ± 0.003 | 14 | 0.035 ± 0.003 ‡ | 20 | 7 | |
| BMS-986122 | 0.050 ± 0.01 | 13 | 0.029 ± 0.003 | 24 | 7 | |
| BMS-986187 | 0.050 ± 0.004 | 14 | 0.022 ± 0.001* | 31 | 7 | |
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Values are means from independent experiments (number of individual experiments indicated in ‘n’ column). Analyses were performed by two-way ANOVA with a Tukey post-hoc test.
*Indicates significance compared to vehicle condition for each orthosteric ligand (*p<0.05, **p<0.01, ***p<0.001). ++Indicates p<0.01 as compared to L-methadone/BMS-986187 combination.
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†Indicates p<0.01 as compared to morphine/vehicle combination.
‡Indicates p<0.01 as compared to DAMGO/vehicle combination.
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Peptide, recombinant protein | mu-opioid receptor | Manglik et al. (2012) | OPRM1 | |
| Peptide, recombinant protein | Gbetagamma | Iñiguez-Lluhi et al., 1992 | ||
| Peptide, recombinant protein | Myristoylated Gαi1 | Greentree and Linder, 2004 | ||
| Peptide, recombinant protein | Apolipoprotein-AI | Kuszak et al., 2009 | ||
| Peptide, recombinant protein | [D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin acetate salt (DAMGO) | Sigma | E7384 | CAS#100929-53-1 |
| Peptide, recombinant protein | Leu-Enkephalin | Sigma | L9133 | |
| Peptide, recombinant protein | Met-Enkephalin | Sigma | M6638 | CAS#82362-17-2 |
| Peptide, recombinant protein | Endomorphin 2 | Sigma | SCP0133 | |
| Peptide, recombinant protein | Nanobody 39 (Nb39) | Huang et al., 2015 | ||
| Chemical compound, drug | 3H-diprenorphine | Perkin Elmer | NET1121250UC | |
| Chemical compound, drug | Morphine sulfate | National Institute on Drug Abuse, NIH. Drug Supply Catalog | 9300–001 | CAS # 6211-15-0 |
| Chemical compound, drug | (R)-Methadone | National Institute on Drug Abuse, NIH. Drug Supply Catalog | 9250–005 | CAS# 125-58-6 |
| Chemical compound, drug | Buprenorphine | National Institute on Drug Abuse, NIH. Drug Supply Catalog | 9064–110 | CAS# 53152-21-9 |
| Chemical compound, drug | BU72 | Huang et al. (2015) | ||
| Chemical compound, drug | Diprenorphine | Other | CAS# 14357-78-9: Opioid Research Center, U Michigan | |
| Chemical compound, drug | Etorphine | Other | CAS# 14521-96-1: Opioid Research Center, U Michigan | |
| Chemical compound, drug | Fentanyl | National Institute on Drug Abuse, NIH. Drug Supply Catalog | 9801–001 | CAS# 1443-54-5 |
| Chemical compound, drug | Hydrocodone | Other | CAS# 125-29-1:Opioid Research Center, U Michigan | |
| Chemical compound, drug | Loperamide | Other | CAS # 34552-83-5: Opioid Research Center, U Michigan | |
| Chemical compound, drug | Nalbuphine | Other | CAS# 23277-43-2: Opioid Research Center, U Michigan | |
| Chemical compound, drug | Naloxone | Sigma | PHR1802 | CAS# 51481-60-8 |
| Chemical compound, drug | Oxycodone | Other | CAS# 76-42-6: Opioid Research Center, U Michigan | |
| Chemical compound, drug | PZM21 | Manglik et al. (2016) | ||
| Chemical compound, drug | BMS-986187 | Bristol Myers Squib; | Burford et al. (2015) | CAS# 684238-37-7 |
| Chemical compound, drug | BMS-986122 | Bristol Myers Squib; | Burford et al. (2013) | CAS# 313669-88-4 |
| Software, algorithm | GraphPad Prism 6.0 | GraphPad, La Jolla, CA | https://www.graphpad.com/scientific-software/prism/ | |
| Software, algorithm | Octet Data Analysis 7.0 software | Pall Forte Bio | https://shop.fortebio.com/site-license-octet-data-analysis-software-version-7.x.html |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.32499.012
