1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics
Download icon

Measuring ligand efficacy at the mu-opioid receptor using a conformational biosensor

  1. Kathryn E Livingston
  2. Jacob P Mahoney
  3. Aashish Manglik
  4. Roger K Sunahara
  5. John R Traynor  Is a corresponding author
  1. University of Michigan Medical School, United States
  2. University of Michigan, United States
  3. University of California San Francisco, United States
  4. University of California San Diego School of Medicine, United States
Research Article
Cite this article as: eLife 2018;7:e32499 doi: 10.7554/eLife.32499
7 figures, 3 tables and 1 additional file

Figures

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 five min in the presence of ligand or vehicle until steady state is reached. (c) In the presence of ligand, probe is moved to well containing no Nb39 to monitor dissociation for 5 min.

https://doi.org/10.7554/eLife.32499.002
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 agonists at 30 µM (a) Using GraphPad Prism 6.02, one-phase association lines were fit and the calculated kobs (b) and koff (c) for each ligand are plotted (±s.e.m.). Rate constants are means of multiple independent experiments as listed in see Table 1. *Dissociation of Nb39 from the methadone-bound receptor is statistically different from all other ligands (one-way ANOVA with Tukey’s post-hoc test).

https://doi.org/10.7554/eLife.32499.003
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 (Ehlert, 1985) values for each agonist to activate G protein, (c) the shift in affinity of the agonist as measured by radioligand competition binding in the absence or presence of Na+/Guanine nucleotide, and (d) the τ value using data from GTPγ35S assays (Livingston and Traynor, 2014) or (e) β-arrestin recruitment assays; McPherson et al., 2010), analyzed with the Black-Leff operational model (Black and Leff, 1983). Data used to compile correlation graphs is listed in the source data table. The ligands are: (1) BU72, (2) DAMGO, (3) Leu-Enk, (4) L-methadone, (5) Morphine, (6) Nalbuphine, (7) Endomorphin 2, (8) Loperamide, (9) Oxycodone, (10) Etorphine, (11) Fentanyl, (12) Met-Enk, (13) Hydrocodone, (14) Buprenorphine, (15) Morphine + BMS-986122, (16) Morphine + BMS-986187. Correlation analysis was performed using GraphPad Prism 6.02.

https://doi.org/10.7554/eLife.32499.005
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
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 receptor was calculated as follows: BU72 (144 ± 4), DAMGO (194 ± 9), Morphine (944 ± 13), Methadone (580 ± 3). The Kon values (min−1, M−1 × 10−5) were BU72 (2.35 ± 0.45), DAMGO (17.8 ± 0.5), Morphine (0.43 ± 0.05), and Methadone (1.21 ± 0.07) and the Koff values (min−1) BU72 (0.034 ± 0.0003), DAMGO (0.034 ± 0.003), morphine (0.04 ± 0.003), methadone (0.07 ± 0.0002).

https://doi.org/10.7554/eLife.32499.007
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 antagonist 3H-diprenorphine. The dotted line in (c) shows the effect previously obtained in membranes from C6μ cells (Livingston and Traynor, 2014). The effect of BMS-986187 on L-methadone affinity is plotted in (e). Enhancement of the affinity of DAMGO or morphine in the presence of 10 µM BMS-986122 is shown in (f) and (g), respectively. All plotted points are means ±s.e.m. of three (morphine) or five independent experiments (all other), each in duplicate. Nonlinear regression analysis with GraphPad Prism 6.02 was utilized to determine the affinity of the ligands. Hill slopes were not significantly different from unity.

https://doi.org/10.7554/eLife.32499.008
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 at 30 µM. (b) Representative experiment comparing the kinetics of Nb39 binding in the presence of morphine in the absence (black), or presence of 30 µM BMS-986122 (green), or 30 µM BMS-986187 (blue).

https://doi.org/10.7554/eLife.32499.009
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 these are pseudo-affinity constants as the G protein binding was nearly irreversible on the time-scale studied. Only in the presence of nucleotide (b) did the BU72-bound heterotrimer rapidly dissociate.

https://doi.org/10.7554/eLife.32499.011

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 were used. Half-time values (t1/2) numbers were calculated from the respective k values (t1/2 = 0.693/k). A one-way ANOVA was performed followed by a Tukey post-hoc test. Methadone was found to be statistically different compared to all other orthosteric ligands other than loperamide and PZM21. Both loperamide and PZM21 were also found to be statistically different from several other ligands, though not as many as methadone.

https://doi.org/10.7554/eLife.32499.004
Ligandkobs ± SEM (min−1)t1/2ass (sec)koff (min−1)t1/2dis (sec)N
BU720.20 ± 0.013.50.031 ± 0.0012214
DAMGO0.179 ± 0.0083.90.030 ± 0.001236
Leu-Enk0.098 ± 0.027.10.031 ± 0.001239
L-Methadone0.19 ± 0.023.60.052 ± 0.003136
Morphine0.08 ± 0.018.50.033 ± 0.003217
Nalbuphine0.023 ± 0.008300.036 ± 0.0041911
PZM210.064 ± 0.004110.043 ± 0.002166
Endomorphin 20.101 ± 0.0026.90.036 ± 0.002196
Loperamide0.208 ± 0.0093.20.044 ± 0.003166
Oxycodone0.044 ± 0.001160.028 ± 0.001259
Etorphine0.180 ± 0.0073.90.026 ± 0.001276
Fentanyl0.075 ± 0.0059.20.035 ± 0.002206
Met-Enk0.131 ± 0.0045.30.027 ± 0.001266
Hydrocodone0.042 ± 0.004170.029 ± 0.001246
Buprenorphine0.058 ± 0.009120.025 ± 0.001276
Naloxonen/a----n/a----3
Diprenorphinen/a----n/a----3
BMS-9861220.012 ± 0.001560.027 ± 0.0032510
BMS-9861870.025 ± 0.006280.037 ± 0.0051911
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
Vehicle0.08 ± 0.018.50.033 ± 0.001213
BMS-9861220.11 ± 0.0016.40.032 ± 0.0002223
BMS-9861870.13 ± 0.01 ***5.30.024 ± 0.0004293
L-Methadone (100 nM Nb39)
kobs (min−1)t1/2Assoc (sec)koff (min−1)t1/2Diss(sec)n
Vehicle0.087 ± 0.0098.00.050 ± 0.004 147
BMS-9861220.077 ± 0.0079.00.047 ± 0.004 +157
BMS-9861870.055 ± 0.004 *130.033 ± 0.002 **217
DAMGO (100 nM Nb39)
kobs (min−1)t1/2Assoc (sec)koff (min−1)t1/2Diss(sec)n
Vehicle0.051 ± 0.003140.035 ± 0.003 207
BMS-9861220.050 ± 0.01130.029 ± 0.003247
BMS-9861870.050 ± 0.004140.022 ± 0.001*317
  1. 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.

  2. †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
DesignationSource or referenceIdentifiersAdditional information
Peptide,
recombinant protein
mu-opioid receptorManglik et al. (2012)
OPRM1
Peptide,
recombinant protein
GbetagammaIñiguez-Lluhi et al., 1992
Peptide,
recombinant protein
Myristoylated Gαi1Greentree and Linder, 2004
Peptide,
recombinant protein
Apolipoprotein-AIKuszak et al., 2009
Peptide,
recombinant protein
[D-Ala2, N-Me-Phe4,
Gly5-ol]-Enkephalin
acetate salt
(DAMGO)
SigmaE7384CAS#100929-53-1
Peptide,
recombinant protein
Leu-EnkephalinSigmaL9133
Peptide,
recombinant protein
Met-EnkephalinSigmaM6638CAS#82362-17-2
Peptide,
recombinant protein
Endomorphin 2SigmaSCP0133
Peptide,
recombinant protein
Nanobody 39 (Nb39)Huang et al., 2015
Chemical
compound, drug
3H-diprenorphinePerkin ElmerNET1121250UC
Chemical
compound, drug
Morphine sulfateNational Institute on Drug
Abuse, NIH. Drug Supply
Catalog
9300–001CAS # 6211-15-0
Chemical
compound, drug
(R)-MethadoneNational Institute on Drug
Abuse, NIH. Drug Supply
Catalog
9250–005CAS# 125-58-6
Chemical
compound, drug
BuprenorphineNational Institute on Drug
Abuse, NIH. Drug Supply
Catalog
9064–110CAS# 53152-21-9
Chemical
compound, drug
BU72Huang et al. (2015)
Chemical
compound, drug
DiprenorphineOtherCAS# 14357-78-9:
Opioid Research Center,
U Michigan
Chemical
compound, drug
EtorphineOtherCAS# 14521-96-1: Opioid
Research Center,
U Michigan
Chemical
compound, drug
FentanylNational Institute on Drug
Abuse, NIH. Drug Supply Catalog
9801–001CAS# 1443-54-5
Chemical
compound, drug
HydrocodoneOtherCAS# 125-29-1:Opioid
Research Center,
U Michigan
Chemical
compound, drug
LoperamideOtherCAS # 34552-83-5: Opioid
Research Center,
U Michigan
Chemical
compound, drug
NalbuphineOtherCAS# 23277-43-2: Opioid
Research Center,
U Michigan
Chemical
compound, drug
NaloxoneSigmaPHR1802CAS# 51481-60-8
Chemical
compound, drug
OxycodoneOtherCAS# 76-42-6: Opioid
Research Center,
U Michigan
Chemical
compound, drug
PZM21Manglik et al. (2016)
Chemical
compound, drug
BMS-986187Bristol Myers Squib;Burford et al. (2015)

CAS# 684238-37-7
Chemical
compound, drug
BMS-986122Bristol Myers Squib;Burford et al. (2013)CAS# 313669-88-4
Software,
algorithm
GraphPad Prism 6.0GraphPad, La Jolla, CAhttps://www.graphpad.com/scientific-software/prism/
Software,
algorithm
Octet Data Analysis
7.0 software
Pall Forte Biohttps://shop.fortebio.com/site-license-octet-data-analysis-software-version-7.x.html

Additional files

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)