A µ-opioid receptor superagonist analgesic with minimal adverse effects | Nature

## Summary
FNZ had high affinity for MOR (inhibition constant ( K i ) = 1.36 ± 0.11 nM (Fig. 1c,d )), relatively slow MOR binding kinetics 13 (on rate ( k on ) = 6.35 × 10 8 M −1 min −1 ; off rate ( k off ) = 0.062 min −1 ; T 1/2 = 11.05 min (Fig. 1e,f )), and activated MOR with high potency and supramaximal efficacy (expressed as percentage of DAMGO activity): G i1 (half-maximal effective concentration (EC 50 ) = 0.79 ± 0.15 nM, maximum effect ( E max ) = 122.0 ± 3.0%), G oA (EC 50 = 0.51 ± 0.09 nM, E max = 118.5 ± 2.6%), β-arrestin 1 (EC 50 = 0.57 ± 0.16 nM, E max = 108.8 ± 3.6%), β-arrestin 2 (EC 50 = 0.54 ± 0.15 nM, E max = 116.3 ± 3.9%) (Fig. 1g–j and Extended Data Fig. 1 ). DFNZ activated MOR with high potency and supramaximal efficacy for G i1 dissociation (EC 50 = 4.07 ± 0.43 nM, E max = 118.7 ± 1.6%), G oA dissociation (EC 50 = 1.66 ± 0.29 nM, E max = 116.5 ± 2.6%), β-arrestin 1 recruitment (EC 50 = 8.49 ± 0.89 nM, E max = 104.2 ± 1.6%) and β-arrestin 2 recruitment (EC 50 = 2.78 ± 0.78 nM, E max = 108.2 ± 3.9%), but showed divergent G-protein versus β-arrestin preference compared with FNZ (Fig. 1g–j and Extended Data Fig. 1 ) and was moderately stable in mouse liver microsomes (Extended Data Fig. 2 ). At β-arrestin 2, FNZ showed significantly greater efficacy than DAMGO for the first 30 min, whereas DFNZ showed significantly higher efficacies than both DAMGO and FNZ during this time, and significantly greater efficacies than DAMGO over the full time course (Fig. 2c and Extended Data Fig. 4 ). Full size image a , Schematic of in vitro BRET kinetic functional assays to show MOR G o activation and β-arrestin 2 (β-arr2) recruitment. b – e , Efficacy as percentage of the DAMGO effect (% DAMGO) ( b , c ) or potency (pEC 50 ) ( d , e ) of FNZ ( n = 9 independent experiments) and DFNZ ( n = 9 independent experiments) over time. b , G o efficacy: two-way analysis of variance (ANOVA), drug–time interaction, F (44, 902) = 3.46, P < 0.001. d , G o potency: two-way ANOVA, drug effect, F (2, 902) = 422.2, P < 0.001. c , β-Arrestin efficacy: two-way ANOVA, drug effect, F (2, 483) = 247.8, P < 0.001. e , β-Arrestin potency: two-way ANOVA, drug effect, F (2, 483) = 2,803, P < 0.001. f , G o −β-arrestin 2 bias of FNZ ( n = 8 independent experiments) and DFNZ ( n = 8 independent experiments) (two-way ANOVA, drug effect, F (1, 322) = 390.2, P < 0.001). g , Schematic of the MOR–APEX approach following activation with DAMGO, FNZ and DFNZ. log 2 FC, log 2 -transformed fold change. h , Location-specific coefficients for each ligand and each time point to model receptor trafficking ( n = 4 replicates per drug and time point). i , Schematic depicting known regulators of MOR signalling, endocytosis and trafficking. j , Heat map comparing log 2 -transformed fold change in proximal labelling for the known regulators of MOR following receptor activation.

## Article Content
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Subjects
Addiction
Neuropathic pain
Pharmacology
Small molecules
Abstract
Developing safe and effective pain medications is an ongoing challenge for human health. Agonists for the µ-opioid receptor (MOR) are essential pain medications, but their high intrinsic efficacy also induces adverse side effects, including respiratory depression, constipation, tolerance, dependence, withdrawal and addiction
1
,
2
,
3
,
4
,
5
,
6
,
7
. Strategies to limit adverse effects traditionally include developing MOR agonists that have low intrinsic efficacy or that preferentially activate G-protein signalling over β-arrestin signalling
8
. Here we identify a novel MOR agonist with supramaximal intrinsic efficacy and a unique pharmacological profile that produced effective analgesia in rodents with minimal adverse effects.
N
-desethyl-fluornitrazene (DFNZ) was derived from a class of synthetic benzimidazole opioids called nitazenes. DFNZ has impaired brain penetrance, a unique spatiotemporal MOR cellular signalling profile, and diminished efficacy at the MOR–galanin 1 receptor (GAL1) heteromer. DFNZ does not induce respiratory depression, tolerance or MOR downregulation after repeated exposure. Compared with other MOR agonists, DFNZ has limited effects on dopamine neurotransmission in nucleus accumbens and weaker reinforcing effects in the drug self-administration procedure. These results provide novel insights about MOR and nitazene pharmacology, have important implications for pain and addiction treatment, and challenge the prevailing dogma that high-efficacy MOR agonists cannot constitute safe and effective therapeutic agents.
Main
The opioid crisis, initially sparked by the overprescription of opioid analgesics and exacerbated by the rise of potent synthetic opioids such as fentanyl, is a public health crisis that has led to many deaths over recent decades
6
. Recently, a class of synthetic opioids—the benzimidazole opioids (nitazenes)—has entered the recreational drug supply
9
. Nitazenes comprise a class of selective MOR agonists with high potency and efficacy
10
. Etonitazene, the most potent compound of this class, and related analogues were synthesized in the 1950s as potential analgesics, but clinical development was abandoned because of their extreme potency and overdose risk. Several nitazene analogues have been associated with human overdose fatalities, prompting their placement into Schedule I by the US Drug Enforcement Administration. Nitazenes show complex structure–activity relationships, with antinociceptive potency ranging from levels comparable to morphine to 1,000-fold higher
10
, yet their pharmacological mechanisms are not well understood. Here we report the identification of a novel nitazene as a potential therapeutic agent for pain and opioid addiction.
FNZ and DFNZ are selective MOR superagonists
Etonitazene has 1,000-fold greater antinociceptive potency than morphine
10
, rendering it unsuitable for clinical use. However, modifications to its alkoxy chain length can alter MOR potency
11
,
12
. We explored whether fluorine substitution at the end of the ethoxy chain would reduce potency and afford radiolabelling with
18
F for positron emission tomography (PET) studies. We synthesized fluornitrazene (FNZ) (Fig.
1a
and Supplementary Fig.
1
) and confirmed its high MOR selectivity (Fig.
1b
and Extended Data Fig.
1
). FNZ had high affinity for MOR (inhibition constant (
K
i
) = 1.36 ± 0.11 nM (Fig.
1c,d
)), relatively slow MOR binding kinetics
13
(on rate (
k
on
) = 6.35 × 10
8
M
−1
min
−1
; off rate (
k
off
) = 0.062 min
−1
;
T
1/2
= 11.05 min (Fig.
1e,f
)), and activated MOR with high potency and supramaximal efficacy (expressed as percentage of DAMGO activity): G
i1
(half-maximal effective concentration (EC
50
) = 0.79 ± 0.15 nM, maximum effect (
E
max
) = 122.0 ± 3.0%), G
oA
(EC
50
= 0.51 ± 0.09 nM,
E
max
= 118.5 ± 2.6%), β-arrestin 1 (EC
50
= 0.57 ± 0.16 nM,
E
max
= 108.8 ± 3.6%), β-arrestin 2 (EC
50
= 0.54 ± 0.15 nM,
E
max
= 116.3 ± 3.9%) (Fig.
1g–j
and Extended Data Fig.
1
).
Fig. 1: FNZ and DFNZ are selective and potent MOR superagonists.
Full size image
a
, Structures of FNZ and DFNZ.
b
, Competitive binding screens of 100 nM FNZ (
n
= 2 independent experiments) and DFNZ (
n
= 2 independent experiments).
c
, Competitive binding of FNZ and DFNZ against [
3
H]DAMGO in rat brain membranes (three independent experiments).
d
, [
3
H]FNZ and [
3
H]DFNZ autoradiography in rat brain.
e
,
f
, Association and dissociation kinetic binding of [
3
H]FNZ and [
3
H]DFNZ to human MOR (two independent experiments).
g
–
j
, BRET assays showing human MOR G-protein activation and G
i1
(
g
), G
oA
(
h
), β-arrestin 1 (
i
) and β-arrestin 2 (
j
) recruitment with DAMGO, FNZ, DFNZ and lofentanil (LFT) (three independent experiments).
k
, Cryo-EM structure of FNZ bound to mouse MOR–G
i
protein complex at 2.3 Å resolution.
l
, Overlays of FNZ-bound MOR structure showing residues in Ballesteros–Weinstein notat

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## Expert Analysis

### Merits
- Download PDF Subjects Addiction Neuropathic pain Pharmacology Small molecules Abstract Developing safe and effective pain medications is an ongoing challenge for human health.
- Here we identify a novel MOR agonist with supramaximal intrinsic efficacy and a unique pharmacological profile that produced effective analgesia in rodents with minimal adverse effects.
- These results provide novel insights about MOR and nitazene pharmacology, have important implications for pain and addiction treatment, and challenge the prevailing dogma that high-efficacy MOR agonists cannot constitute safe and effective therapeutic agents.
- FNZ had high affinity for MOR (inhibition constant ( K i ) = 1.36 ± 0.11 nM (Fig. 1c,d )), relatively slow MOR binding kinetics 13 (on rate ( k on ) = 6.35 × 10 8 M −1 min −1 ; off rate ( k off ) = 0.062 min −1 ; T 1/2 = 11.05 min (Fig. 1e,f )), and activated MOR with high potency and supramaximal efficacy (expressed as percentage of DAMGO activity): G i1 (half-maximal effective concentration (EC 50 ) = 0.79 ± 0.15 nM, maximum effect ( E max ) = 122.0 ± 3.0%), G oA (EC 50 = 0.51 ± 0.09 nM, E max = 118.5 ± 2.6%), β-arrestin 1 (EC 50 = 0.57 ± 0.16 nM, E max = 108.8 ± 3.6%), β-arrestin 2 (EC 50 = 0.54 ± 0.15 nM, E max = 116.3 ± 3.9%) (Fig. 1g–j and Extended Data Fig. 1 ).

### Areas for Consideration
- Download PDF Subjects Addiction Neuropathic pain Pharmacology Small molecules Abstract Developing safe and effective pain medications is an ongoing challenge for human health.
- These results provide novel insights about MOR and nitazene pharmacology, have important implications for pain and addiction treatment, and challenge the prevailing dogma that high-efficacy MOR agonists cannot constitute safe and effective therapeutic agents.
- Etonitazene, the most potent compound of this class, and related analogues were synthesized in the 1950s as potential analgesics, but clinical development was abandoned because of their extreme potency and overdose risk.

### Implications
- DOR has fewer clashes that all result from minor conformational differences.
- TM6 is pushed closer to the binding pocket, bringing V6.55 inward towards the o -fluoroethyl, and K5.39 and Q2.60 occupy different rotamers or positions that may result in additional mild clashes.
- Some of these differences for DOR (and to a lesser extent KOR) might be overcome with conformational rearrangements, albeit with an associated energetic penalty to compound binding.
- Both the fluoroethyl group and the diethyl amine may occupy more than one conformation, as suggested by the presence of adjacent map features near these groups and consistent with the flexible nature of these moieties (Extended Data Fig. 3 ).

### Expert Commentary
This article covers fig, fnz, mor topics. Notable strengths include discussion of fig. Areas of concern are also raised. Readability: Flesch-Kincaid grade 0.0. Word count: 2520.