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Structure-based discovery of opioid analgesics with reduced side effects

Abstract

Morphine is an alkaloid from the opium poppy used to treat pain. The potentially lethal side effects of morphine and related opioids—which include fatal respiratory depression—are thought to be mediated by μ-opioid-receptor (μOR) signalling through the β-arrestin pathway or by actions at other receptors. Conversely, G-protein μOR signalling is thought to confer analgesia. Here we computationally dock over 3 million molecules against the μOR structure and identify new scaffolds unrelated to known opioids. Structure-based optimization yields PZM21—a potent Gi activator with exceptional selectivity for μOR and minimal β-arrestin-2 recruitment. Unlike morphine, PZM21 is more efficacious for the affective component of analgesia versus the reflexive component and is devoid of both respiratory depression and morphine-like reinforcing activity in mice at equi-analgesic doses. PZM21 thus serves as both a probe to disentangle μOR signalling and a therapeutic lead that is devoid of many of the side effects of current opioids.

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Figure 1: Structure based ligand discovery for the μOR.
Figure 2: Discovery of a novel Gi/o-biased μOR agonist.
Figure 3: Structure-guided optimization towards a potent biased μOR agonist.
Figure 4: PZM21 is an analgesic with reduced on-target liabilities.

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Acknowledgements

Supported by the US National Institutes of Health grants GM106990 (B.K.K., B.K.S. and P.G.), DA036246 (B.K.K.), GM59957 (B.K.S.), and the National Institutes of Mental Health Psychoactive Drug Screening Program (B.L.R.) and DA017204 (B.L.R., D.A.), DA035764 (B.L.R.) and the Michael Hooker Distinguished Professorship (B.L.R.) and the German Research Foundation Grants Gm 13/10 and GRK 1910 (P.G). A.M. received support from the Stanford University Medical Scientist Training Program (T32GM007365) and the American Heart Association (12PRE8120001).

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Authors and Affiliations

Authors

Contributions

A.M. and H.L. initiated the project. H.L. performed docking and identified compounds to be tested in the initial and analogue screens. A.M. performed binding studies to identify initial hits and devised structure-guided optimization strategies for subsequent analogues. D.K.A. performed in vivo studies, including analgesia assays, mouse plethysmography, faecal boli accumulation studies, open field locomotor assay, and conditioned place preference. J.D.M., M.F.S. and P.M.G. performed radioligand binding and signalling studies. X.P.H. performed signalling studies and assessed compound activity against the GPCRome. D.De., V.B., S.L. and H.H. synthesized compounds and determined affinities by radioligand binding and performed signalling studies. A.L. and A.M. docked PZM21 and TRV130 and R.C.K. simulated PZM21 binding to μOR. G.C. performed reflexive and affective analgesia studies of μOR knockout mice and was supervised by G.S. D.Du. performed pharmacokinetic studies. The manuscript was written by A.M., H.L. and B.K.S. with editing and suggestions from B.L.R. and input from D.K.A., B.K.K. and P.G. P.G. supervised chemical synthesis of compounds and the separation and identification of diastereomers, B.K.K. supervised testing of initial docking hits, B.L.R. supervised radioligand binding, signalling and in vivo studies and B.K.S. supervised the compound discovery and design. The project was conceived by A.M., H.L., B.K.K., P.G., B.K.S and B.L.R.

Corresponding authors

Correspondence to Brian K. Kobilka, Peter Gmeiner, Bryan L. Roth or Brian K. Shoichet.

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Competing interests

A.M., H.L., P.G., D.D., B.K.K., B.L.R. and B.K.S. have filed a provisional patent on PZM21 and related molecules. A.M., P.G., B.K.K., B.L.R. and B.K.S. are consultants and co-founders of Epiodyne, a company seeking to develop novel analgesics.

Additional information

Reviewer Information Nature thanks G. Henderson, E. Kelly, B. Kieffer and J. Meiler for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Docking poses of active compounds.

Seven of 23 experimentally tested compounds bound to the μOR with micromolar affinity. Their docked poses often occupy sites not exploited by the antagonist β-funaltrexamine. In each case, a canonical ionic interaction with D1473.32 is observed.

Extended Data Figure 2 Stereochemical structure-activity relationship.

a, As with the different stereoisomers of 12, variation of the chiral centres in compound PZM21 results in large changes in efficacy and potency. Data are mean ± s.e.m. of normalized results (n = 3 measurements). b, Structure–activity relationship of compound 12 and 21 stereoisomers with affinities displayed as pKi values and agonist potency and efficacy in a Gi/o Glosensor assay. c, d, PZM21 docked to active μOR shows a more extended conformation as compared to the inactive state. e, In the docked active state, the PZM21 thiophene extends into the specificity-determining region of opioid receptors. Key interacting residues here are highlighted as red lines and corresponding residues at the other human opioid receptors are indicated. f, Docked pose of TRV130 within the μOR site, showing minimal overlap in key pharmacophores with PZM21 besides the ionic interaction between the cationic amine and D1473.32. g, Molecular dynamics simulations of PZM21 in the inactive μOR state (grey and black traces) leads to a stable conformation with the thiophene positioned >10 Å away from N1272.63 (total of 2 μs of simulation time over three independent trajectories). In contrast, PZM21 adopts a more extended pose when simulated with active μOR, with an average distance of 6 Å between the thiophene and N1272.63. Other key interactions between μOR and PZM21 are also highlighted.

Extended Data Figure 3 Structure activity relationship defined by PZM21 analogues.

Eight analogues were synthesized to probe the binding orientation of PZM21 and their efficacy as agonists was tested in a CAMYEL-based Gi/o signalling assay. Analogues were compared to a parent reference compound (PZM22) with similar efficacy and potency to PZM21. In each case, the EC50 value for PZM22 is shown in black (1.8 nM) and the EC50 for the analogue is coloured. The covalent compound PZM29 binds to the μOR:N127C variant irreversibly, as evidenced by wash-resistant inhibition of radioligand binding. Signalling data are mean ± s.e.m. of normalized results (n = 3 measurements).

Extended Data Figure 4 Signalling properties of PZM21 at the opioid receptors.

Displayed are raw luminescence data from a Gi/o Glosensor assay. In agonist mode, agonists decrease luminescence while inverse agonists increase it by diminishing basal signalling. For each opioid receptor, a prototypical well-characterized agonist (black curves) and antagonist (red curves) were used to validate the assay. In antagonist mode, a competition reaction is performed with 50 nM agonist and an escalating amount of tested drug. Here, true antagonists increase the observed signal, consistent with their ability to compete with the agonist but not induce Gi signalling. Data are mean ± s.e.m. of non-normalized results (n = 3 measurements).

Extended Data Figure 5 PZM21 is selective for μOR.

a, Compound PZM21 was screened against 320 non-olfactory GPCRs for agonism in the arrestin recruitment TANGO assay. Each point shows luminescence normalized to basal level at a given GPCR, with vertical lines indicating the standard error of the mean. b, GPCRs for which PZM21 induces an increase in signal twofold over background were further tested in full dose–response mode. Several potential targets (GPR110, MCHR1R, PTGER1) did not show dose-dependent increase in signal and probably represent screening false positives. CXCR7 and SSTR4 did show dose-dependent signals at high concentrations of PZM21, and were further tested in non-arrestin signalling assays. c, PZM21 does not show a dose-dependent change in cAMP inhibition in a Gi/o Glosensor assay measuring SSTR4 activation, indicating that the single elevated point in b is probably a false positive result. d, e, Inhibition assays of hERG (d) and the dopamine transporter (DAT), norepinephrine transporter (NET), and serotonin transporter (SERT) (e) show that PZM21 has weak inhibitory activity ranging from 2–34 μM at these targets. For a, data are mean ± s.e.m. of non-normalized results (n = 4 measurements). For be, data are mean ± s.e.m. of normalized results (n = 3–6 measurements).

Extended Data Figure 6 Signalling profile of PZM21 and other μOR agonists.

a, PZM21 is an efficacious Gi and Go agonist in a GTPγS assay. b, Like other μOR agonists, PZM21 induces a dose-dependent decrease in cAMP levels that is sensitive to pertussis toxin, confirming Gi/o mediated signalling. c, Herkinorin is a Gi/o agonist and robustly recruits arrestin in a BRET assay performed in the absence of GRK2 overexpression. TRV130 or PZM21 show undetectable levels of arrestin recruitment in the same experiement. d, PZM21 and other opioids show no activity in a calcium-release assay, indicating no Gq-mediated second messenger signalling. The positive control TFLLR-NH2 efficiently activates the Gq coupled receptor PAR-1. e, PZM21 and TRV130 induce much decreased receptor internalization versus DAMGO and even morphine. f, Herkinorin and TRV130 are potent agonists of the κOR. PZM21 is a κOR antagonist (see Extended Data Fig. 4). g, In HEK293 cells, GRK2 expression levels have minimal effect on the potency and efficacy of the unbiased agonist DAMGO in a Gi/o activation assay. Increased GRK2 levels change the basal cAMP signal due to increased desentization of μOR, which lowers receptor basal activity and leads to elevated isoproterenol-induced cAMP. In an arrestin-recruitment BRET assay, increased GRK2 expression increases both the potency and maximal efficacy of the unbiased agonist DAMGO. This is likely because GRK2 mediated phosphorylation is required for efficient β-arrestin recruitment. h, Gi activation and arrestin recruitment in cells co-expressing 1.0 μg/15 cm2 of GRK2. Notably, PZM21 induces a higher maximal level of arrestin recruitment as compared to U2OS cells, which express very low levels of GRK2, but this level is significantly lower than morphine. Despite the lower efficacy for arrestin recruitment observed for morphine, TRV130 and PZM21 compared to DAMGO, a formal calculation of bias by the operational models fails to show that this effect is significant. i, Table of pEC50 values and Emax values for various signalling assays. All data are mean ± s.e.m. of results (n = 2–6 measurements).

Extended Data Figure 7 Additional in vivo studies of PZM21.

a, Analgesic responses measured in the hotplate assay were subcategorized into either affective or reflexive behaviours and scored separately. b, Morphine (n = 10 animals) induces changes in both behaviours, while PZM21 (n = 13 animals) only modulates the attending (affective) component. Knockout of the μOR ablates all analgesic responses by morphine and PZM21. c, PZM21 shows minimal cataleptic effect compared to morphine at different time points. The effect of haloperidol was included as a positive control. d, Pharmacokinetic studies of PZM21 (n = 3–4 animals for each time point) show central nervous system penetration of the compound, with a peak level of 197 ng of PZM21 per g of brain tissue. With a concomitant serum concentration of 1,253 ng/ml, this represents a serum:brain concentration ratio of 6.4. These levels are similar to those achieved by morphine, which shows a peak brain concentration of approximately 300 ng/g and a serum:brain concentration ratio of 3.7 30 min after subcutaneous injection75. e, Metabolism of PZM21 over 60 min exposure to mouse liver microsomes. Rotigotine and imipramine serve as positive controls for extensive phase I metabolism. The total amount of PZM21 and metabolite pool is slightly greater than 100% (101.8%) reflecting cumulative error in LC/MS analysis. f, A Gi/o signalling assay shows that none of the metabolites are measurably more potent activators of the μOR versus PZM21 alone. The metabolite pool after the 60-min incubation was used directly in the signalling assay. As a negative control, the pooled material from a reaction carried out in the absence of the key cofactor NADPH was used in the signalling assay. All data are mean ± s.e.m. For e, reactions were run in triplicate and the s.e.m. was calculated from individual measurements of each reaction.

Extended Data Table 1 Molecules with μOR activity identified in the initial screen
Extended Data Table 2 Analogues tested at the μOR
Extended Data Table 3 Binding and signalling properties of compounds 12 and PZM21

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Manglik, A., Lin, H., Aryal, D. et al. Structure-based discovery of opioid analgesics with reduced side effects. Nature 537, 185–190 (2016). https://doi.org/10.1038/nature19112

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