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

Download PDF

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 addiction1,2,3,4,5,6,7. 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 signalling8. 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 decades6. Recently, a class of synthetic opioids—the benzimidazole opioids (nitazenes)—has entered the recreational drug supply9. Nitazenes comprise a class of selective MOR agonists with high potency and efficacy10. 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 higher10, 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 morphine10, rendering it unsuitable for clinical use. However, modifications to its alkoxy chain length can alter MOR potency11,12. We explored whether fluorine substitution at the end of the ethoxy chain would reduce potency and afford radiolabelling with 18F 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 (Ki) = 1.36 ± 0.11 nM (Fig. 1c,d)), relatively slow MOR binding kinetics13 (on rate (kon) = 6.35 × 108 M−1 min−1; off rate (koff) = 0.062 min−1; T1/2 = 11.05 min (Fig. 1e,f)), and activated MOR with high potency and supramaximal efficacy (expressed as percentage of DAMGO activity): Gi1 (half-maximal effective concentration (EC50) = 0.79 ± 0.15 nM, maximum effect (Emax) = 122.0 ± 3.0%), GoA (EC50 = 0.51 ± 0.09 nM, Emax = 118.5 ± 2.6%), β-arrestin 1 (EC50 = 0.57 ± 0.16 nM, Emax = 108.8 ± 3.6%), β-arrestin 2 (EC50 = 0.54 ± 0.15 nM, Emax = 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 [3H]DAMGO in rat brain membranes (three independent experiments). d, [3H]FNZ and [3H]DFNZ autoradiography in rat brain. e,f, Association and dissociation kinetic binding of [3H]FNZ and [3H]DFNZ to human MOR (two independent experiments). g–j, BRET assays showing human MOR G-protein activation and Gi1 (g), GoA (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–Gi protein complex at 2.3 Å resolution. l, Overlays of FNZ-bound MOR structure showing residues in Ballesteros–Weinstein notation with active-state structures of κ- and δ-opioid receptors (KOR and DOR). For K