(R)-MTD and (S)-MTD preferentially bind and activate MOR
Each enantiomer was tested for its ability to competitively inhibit binding or activity at a panel of 98 receptors and enzymes that are known targets for drugs of abuse and CNS medications. At 10 µM, (R)-MTD inhibited binding at several receptors (Fig. 1a), while at 100 nM, (R)-MTD inhibited binding only at MOR (98%) and SERT (68%). At 10 µM, (S)-MTD inhibited binding at several receptors, while at 100 nM, (S)-MTD inhibited binding only at MOR (79%). We derived each enantiomer’s affinity at MOR using inhibition of [3H]DAMGO binding in rat brain tissue. The Ki values obtained were 15.6 ± 0.1 nM for (R,S)-MTD, 7.5 ± 0.1 nM for (R)-MTD and 60.5 ± 0.1 nM for (S)-MTD (Fig. 1b).
Agonist-stimulated [35S]GTPγS autoradiography in rat brain sections was used to examine the ability of (R,S)-MTD and its enantiomers to activate MOR (Fig. 1c). At 100 nM, only (R)-MTD increased [35S]GTPγS recruitment in the caudate putamen (CPu) (171%) and nucleus accumbens (NAc) (151%) (Fig. 1d, e). By contrast, at 1 μM all drugs increased [35S]GTPγS recruitment in CPu (R,S: 199%; R: 270%; S: 144%) and NAc (R,S: 145%; R: 164%; S: 120%) (Fig. 1d, e). At the 1 µM concentration, (R)-MTD showed significantly greater [35S]GTPγS recruitment compared to (R,S)-MTD (P = 0.01) and (S)-MTD (P < 0.001) in CPu. Additionally, (R,S)-MTD showed greater [35S]GTPγS recruitment compared to (S)-MTD (P = 0.028). Finally, the regional distribution of (S)-MTD-induced [35S]GTPγS recruitment was blocked by naloxone (10 μM) indicating opioid receptor involvement (Fig. 1c).
(S)-MTD exhibits similar analgesic efficacy as (R)-MTD and (R,S)-MTD
The hot plate test was used to evaluate analgesic effects in rats. (R,S)-MTD, (R)-MTD, and (S)-MTD demonstrated full agonistic activity, with ED50 values (%MPE, maximum possible effect) of 1.2, 0.5 and 17.9 mg/kg, respectively (Figs. 2a, Supplementary Fig. 1). Conversely, when evaluating catalepsy score in rats, (S)-MTD behaved as a partial agonist, unable to achieve maximal cataleptic effects, even at 100 mg/kg. The high-dose effect was ~60% of the maximal cataleptic effects of both (R)-MTD and (R,S)-MTD, which were observed at 3 and 10 mg/kg, respectively (Figs. 2b, Supplementary Fig. 1). The cataleptic ED50 values (%MPE) for (R,S)-MTD, (R)-MTD and (S)-MTD were 2.1, 0.9 and 59.4 mg/kg, respectively, which were two- to three-fold higher than their analgesic ED50 values. For the three drugs, the maximal cataleptic effect corresponded to the minimal dose required to produce significant hypothermia (Supplementary Fig. 1). (S)-MTD-induced catalepsy did not observably saturate, and toxic higher doses were not employed. Overall, these experiments demonstrate a MOR agonistic profile of (S)-MTD, with a lower potency and a possibly lower intrinsic efficacy compared to (R)-MTD. Finally, (R)-MTD and (S)-MTD did not differ in their propensity to interact with efflux transporters or in relation to CYP-dependent metabolism (Supplementary Fig. 2), indicating that the two enantiomers would demonstrate similar metabolic profiles in vivo.
(S)-MTD exhibits lower abuse liability than (R)-MTD and (R,S)-MTD
There is evidence that (R,S)-MTD is self-administered in humans17 and rats18. However, the intravenous self-administration (IVSA) of (R)-MTD and (S)-MTD has not been reported. Moreover, depending on the dose administered, (R,S)-MTD can have either rewarding or aversive effects in rats19. IVSA, the standard preclinical approach for predicting abuse liability of drugs in humans20, was used to evaluate the reinforcing effects of (R,S)-MTD and its enantiomers in rats. First, we performed dose finding experiments to determine the dose of each drug that maintained IVSA. Rats exposed to various doses of (R)-MTD readily self-administered 50 µg/kg/infusion and consumed a maximum of ~2 mg/kg at the highest dose (Supplementary Fig. 3). Rats exposed to (S)-MTD never acquired IVSA, even at high unit doses, and did not show any evidence of dose response. Nevertheless, when (R)-MTD-trained rats were switched to (S)-MTD, they showed reliable IVSA at 500 µg/kg/infusion (S)-MTD. The switched rats consumed a cumulative dose of ~30 mg/kg at the highest (S)-MTD dose (Supplementary Fig. 3).
Next, we performed IVSA studies on another cohort of rats trained on either (R)-MTD (50 µg/kg/infusion), (R,S)-MTD (100 µg/kg/infusion), or (S)-MTD (500 µg/kg/infusion) (Fig. 2c-e). For the first 10 days of training, rats were on a fixed-ratio 1 (FR1) schedule. During this time, rats in all three groups learned to discriminate the active from inactive lever. On the 11th session, the schedule was increased to FR5 (5 presses for 1 infusion). Whereas rats trained on (R)- and (R,S)-MTD adjusted lever press rates to maintain stable infusion rates, rats trained on (S)-MTD did not. We then performed a dose response assessment of IVSA (Fig. 2f). Rats trained on (R)- or (R,S)-MTD displayed the typical inverted-U shaped dose-response curve, but rats trained on (S)-MTD showed no evidence of dose response. Rats given (R)-MTD showed peak infusion rates at 25 μg/kg, while rats given (R,S)-MTD peaked at 50 μg/kg. Notably, rats trained on (R,S)-MTD had more infusions at the peak unit dose than those on (R)-MTD, and the (R,S)-MTD curve was significantly shifted to the right indicating that larger drug amounts were required to reach the same level of reinforcement.
(R)-MTD and (S)-MTD preferentially bind to MOR in vivo
Based on drug exposure levels from the studies noted above, and prior reports on antidepressant-like doses of (S)-MTD used in rats6,7, we assessed the binding of (R,S)-MTD and its enantiomers at MOR and NMDAR in vivo. Rats were injected with saline (1 ml/kg, sc), (R,S)-MTD (4 mg/kg, sc), (R)-MTD (2 mg/kg, sc), or (S)-MTD (30 mg/kg, sc) 30 min before decapitation, blood collection, and brain extraction. Brains were split into two hemispheres. One hemisphere was used to assess drug amounts whereas the other was sectioned (20 µm) and subjected to autoradiography using [3H]DAMGO or [3H]MK-801 to examine occupancy at MORs or NMDARs, respectively (Fig. 2g). After 2 mg/kg of (R)-MTD, total/estimated free21,22 drug concentration was 640/19.2 nM ± 136/4.1 nM in plasma and 1.1/0.03 µM ± 0.15/0.005 µM in brain. After 30 mg/kg of (S)-MTD, total/free drug was 5/0.15 µM ± 0.6/0.017 µM in plasma and 11.5/0.35 µM ± 1.5/0.046 µM in brain. Finally, after 4 mg/kg of (R,S)-MTD, total/free (R)-MTD was 551/16.5 nM ± 119/3.6 nM in plasma and 1.3/0.04 µM ± 0.3/0.008 µM in brain, while total/free (S)-MTD was 580/17.4 nM ± 111/3.3 nM in plasma and 1.4/0.04 µM ± 0.3/0.009 µM in brain. The free (i.e., unbound) drug concentration provides the most accurate measure of biophase drug concentration able to engage pharmacological targets in plasma or brain23. Since the free concentration of (R,S)-MTD and its enantiomers is reported to be ~3% of total concentration21,22, it is unlikely that (R,S)-MTD or its enantiomers reach sufficient concentration to engage with NMDAR in vivo. By contrast, the free concentrations shown here align well with each drug’s Ki at MOR. As predicted by the free concentrations of each drug, we found that 4 mg/kg (R,S)-MTD, 2 mg/kg (R)-MTD, and 30 mg/kg (S)-MTD produced near total (99%, 91%, and 79% respectively) occupancy of MORs 30 min after injection (Fig. 2h, i). Importantly, none of the drugs produced any NMDAR occupancy (Fig. 2j, k).
(R)-MTD and (S)-MTD do not produce MOR desensitization
Decreases in MOR density and desensitization contribute to the development of opioid tolerance24. In contrast to other MOR agonists, (R,S)-MTD does not produce tolerance, due to its ability to induce MOR internalization and recycling of re-sensitized MOR25. Thus, we examined to what extent repeated exposure to (R)-MTD (2 mg/kg, sc), (R,S)-MTD (4 mg/kg, sc), or (S)-MTD (30 mg/kg, sc) lead to changes in MOR density and G protein activation using [3H]DAMGO and DAMGO-stimulated [35S]GTPγS autoradiography. We found that neither (R,S)-MTD nor its enantiomers produced any effect on MOR density or G protein activity (Supplementary Fig. 4).
Divergent pharmacodynamic effects of (R)-MTD and (S)-MTD at MOR in the VTA
In view of the apparent lower reinforcing efficacy of (S)-MTD in rats, we next examined effects of the (R,S)-MTD and its enantiomers on locomotor activity in mice. In contrast to rats, which become cataleptic following opioid exposure, mice display dose-dependent increases in locomotion26-30. This opioid-induced hyperlocomotion is dependent on dopaminergic activation31-33, namely the activation of MORs expressed on GABA afferents onto VTAdopamine neurons34. Additionally, locomotor activation can distinguish between full and partial MOR agonists, with partial agonists producing graded increases dependent on efficacy35. We found that (R,S)-MTD and (R)-MTD increased locomotion, but (S)-MTD did not (Fig. 3a-d). Specifically, after 60 minutes of habituation, (R,S)-MTD produced a significant locomotor activation at 10 mg/kg (sc) but not at 3 mg/kg (sc), and 30 mg/kg (sc) was less effective than 10 mg/kg. An inverted U shape effect was also observed with (R)-MTD, which was more potent and effective at 3 mg/kg (sc). (S)-MTD did not produce any significant locomotor-activating effects, even at 100 mg/kg (sc; Fig. 3d). Moreover, when administered 15 min before (R)-MTD, (S)-MTD (10, 30 mg/kg, sc) dose-dependently counteracted the locomotor-stimulating effect of (R)-MTD (10 mg/kg, sc; Fig. 3e-f).
Repeated administration of opioids in rodents leads to psychomotor sensitization, which is classically known to depend on activation of MOR localized in the VTA36. We habituated mice to open-field chambers for two days before giving repeated injections of (R)-MTD (2, 5, or 10 mg/kg, ip), (R,S)-MTD (4, 10, or 20 mg/kg, ip), or (S)-MTD (20, 30, or 40 mg/kg, ip) for three days (Fig. 3g-j). (R)-MTD at 5 or 10 mg/kg and (R,S)-MTD at 10 or 20 mg/kg led to significant acute locomotion each day. Only (R)-MTD produced psychomotor sensitization at 10 mg/kg (D1 vs D2: P = 0.01). Mice treated with (R,S)-MTD and (S)-MTD failed to show sensitization at any dose. We also investigated whether (S)-MTD pretreatment (10 or 30 mg/kg, ip) would prevent (R)-MTD-induced (10 mg/kg, ip) sensitization (Fig. 3k-l). As before, we found that (S)-MTD dose-dependently decreased acute locomotion produced by (R)-MTD, however, it did not prevent psychomotor sensitization ((S)-MTD 0 mg/kg: D1 vs D3: P = 0.0003; (S)-MTD 10 mg/kg D1 vs D3: P < 0.0001; (S)-MTD 30 mg/kg: D1 vs D3: P < 0.0001).
In view of the apparent lower reinforcing efficacy of (S)-MTD in rats we next examined whether the enantiomers of (R,S)-MTD could stimulate MOR receptors in the VTA which are involved in opioid reinforcement. In particular, the weak interaction of (R,S)-MTD with MOR-Gal1R heteromers in rat VTA is thought to underlie its reduced dopaminergic activation and lower abuse liability16. Thus, we studied the effects of (R)-MTD and (S)-MTD perfusion into the rat VTA, using in vivo microdialysis. We recently showed that the intracranial perfusion of (R,S)-MTD in the VTA was less potent and efficacious than other opioids (e.g., morphine, fentanyl and DAMGO) at eliciting somatodendritic dopamine release16. This reduced effect was attributed to (R,S)-MTD’s weak activation of MOR-Gal1R. Here, we show that local perfusion of (R)-MTD into the VTA produced a concentration-dependent increase in extracellular dopamine, with a significant increase at 3 μM and a larger increase at 10 μM (Fig. 3m, n), showing similar potency to that previously obtained with morphine16. In contrast, (S)-MTD did not induce any significant effect on extracellular dopamine levels when perfused up to 100 μM (Fig. 3n). Notably, the 100 µM concentration of (S)-MTD completely counteracted the effect of (R)-MTD on dopamine release (Fig. 3o).
In rat brain slices containing the VTA, 1 μM (R)-MTD significantly increased [35S]GTPγS recruitment (121.6%, P = 0.0003), which was prevented by preincubation with (S)-MTD. As shown in Fig. 3p, 1 μM (S)-MTD + 1 μM (R)-MTD significantly increased [35S]GTPγS recruitment (115%, P = 0.012), while 10 μM (S)-MTD + 1 μM (R)-MTD (107%) did not. The ability of (S)-MTD (10 μM) to reduce [35S]GTPγS recruitment by (R)-MTD (1 μM) was significant (P = 0.039). The [35S]GTPγS results also demonstrate a qualitatively different profile for (R)-MTD and (S)-MTD when comparing effects across different brain areas, with (S)-MTD showing a detectable efficacy in the striatum (Fig. 1d, e) and significantly counteracting the effect of R-MTD in the VTA, but not in the CPu or NAc (Supplementary Fig. 5).
Divergent pharmacodynamic effects of (R)-MTD and (S)-MTD at the MOR-Gal1R heteromer
We next investigated the possibility of divergent pharmacodynamic effects MTD enantiomers at MOR-Gal1R, which could explain their divergent effects on the VTA MOR. First, we evaluated possible differences in binding affinity of (R,S)-MTD, (R)-MTD, and (S)-MTD. We performed radioligand binding experiments in membrane preparations from HEK-293 cells stably transfected with human MOR alone and with human MOR-Gal1R16,37. The results of competitive inhibition experiments using the MOR antagonist [3H]naloxone (1.7 nM) versus increasing concentrations of the ligands (Supplementary Fig. 6) were analyzed with the ‘dimer receptor model’ (see Methods). In both cell lines and for the three compounds, a significantly better fit was obtained for biphasic versus monophasic curves (p < 0.05 in all cases), indicating the preferred dimeric structure of MOR, forming heteromers or not forming heteromers with Gal1R, as previously shown37. Supplementary Table 1 shows that (R,S)-MTD, (R)-MTD and (S)-MTD bind MOR with two different affinities and negative cooperativity, both in MOR and MOR-Gal1R cells. None of the obtained binding parameters show significant differences between MOR and MOR-Gal1R cells for any of the ligands, indicating that the MOR-Gal1R-dependent changes in the pharmacodynamic properties of (S)-MTD are not related to changes in its affinity for the MOR, but likely to its intrinsic efficacy. As expected, (S)-MTD had 14 times lower affinity than (R)-MTD in both cell types.
BRET experiments were performed to evaluate differences in the intrinsic efficacy of (R,S)-MTD, (R)-MTD and (S)-MTD at the MOR (Fig. 4a). MOR-Rluc and Gi-YFP constructs were transiently co-transfected to HEK-293T cells, and concentration-response curves of (R,S)-MTD, (R)-MTD, and (S)-MTD were analyzed for Emax and EC50 values (Fig. 4b-d). As expected, Emax for (S)-MTD was significantly lower than for R-MTD (about 30% lower, Fig. 4c), and EC50 for (S)-MTD was significantly higher than for R-MTD (about 10 times; Fig. 4d). Thus, relative to (R)-MTD, (S)-MTD is a partial and less potent MOR agonist.
CODA-RET experiments were then performed to determine whether MOR-Gal1R heteromerization might determine the specific pharmacodynamic profile of (S)-MTD (see Methods) (Fig. 4g). HEK-293T cells were co-transfected with MOR fused to nRLuc (MOR-nRLuc), Gal1R was fused to cRLuc (Gal1R-cRLuc) and Gi-YFP (Fig. 4h-j). In the presence of Gal1R, no detectable increase of response (BRET ratio) could be obtained with (S)-MTD, while the dose-response curve of (R,S)-MTD was shifted to the right, with an EC50 value significantly higher than for R-MTD (~10-fold; Fig. 4j). These results, therefore, indicate that (S)-MTD, but not (R)-MTD, changes its pharmacological profile and loses its efficacy for the MOR when forming heteromers with Gal1R. This implies that the changes in the pharmacological profile of (R,S)-MTD within the MOR-Gal1R heteromer, as previously described16,37, depend on the modified pharmacodynamic properties of (S)-MTD. Consistent with this, increasing concentrations of (S)-MTD progressively counteracted the effect of a minimal concentration with maximal effect of (R)-MTD (10 μM) (Fig. 4k). At the highest concentration of (S)-MTD (1 mM), the effect of (R)-MTD was completely blocked, and CODA-RET measurements were not significantly different from basal values (Fig. 4l). As a control, the same design was applied with BRET experiments with the MOR alone. In this case, the highest concentration of (S)-MTD (1 mM) did not counteract the effect of (R)-MTD (10 μM) (Fig. 4e, f), and only decreased its effect to the expected maximal level of efficacy of (S)-MTD. These results, therefore, complement those obtained with in vivo and ex vivo experiments in the VTA (microdialysis and [35S]GTPγS) and with locomotor activation and psychomotor sensitization in mice, and provide strong evidence for their mediation by MOR-Gal1R heteromers.
Molecular mechanism of the MOR-Gal1R-dependent pharmacodynamic profile of (S)-MTD
The recently reported structure of MOR in complex with fentanyl38 can be used as a template to understand the pharmacological differences among the enantiomers of (R,S)-MTD at the molecular level. We first performed five replicas of unbiased 1 μs molecular dynamics (MD) simulations of (S)-MTD and (R)-MTD docked into the MOR monomer (see Methods). Root-mean-square deviations (rmsd) of the simulations show that the proposed docking models of (S)-MTD and (R)-MTD remained highly stable (Supplementary Fig. 7). In these models, the protonated amine of (S)-MTD and (R)-MTD forms the conserved ionic interaction with D1493.32, and both phenyl groups adopt a “V” shaped conformation in the orthosteric binding site but, importantly, with significant differences (Fig. 4m, n). In (R)-MTD, both phenyl rings point up to form T-shaped aromatic interactions with H2996.52 and W3207.35, whereas the phenyl rings of (S)-MTD point down to interact with W2956.48 in a “sandwich” mode in which the aromatic Trp ring is between both phenyl rings (Supplementary Fig. 7 shows a detailed analysis of the binding modes). We suggest that the phenyl ring of (S)-MTD positioned between W2956.48 and TM 5, absent in (R)-MTD, restricts the necessary movement of W2956.48 for activation38,39, which explains the decreased ability of (S)-MTD to activate MOR.
To understand the inability of (S)-MTD to activate MOR in the presence of Gal1R, at the molecular level, we first needed to computationally model the MOR-Gal1R heteromer (Supplementary Fig. 8). Previously reported bimolecular fluorescence complementation (BiFC) and total internal reflection fluorescence (TIRF) microscopy experiments, in the presence of synthetic peptides corresponding to different TM domains of MOR and Gal1R, revealed that the interface for the MOR-MOR homodimer changed from the TM 5/6 to the TM 4/5 interface in the absence and presence, respectively, of Gal1R37. Thus, we hypothesized that the MOR-MOR homodimer interacting via the TM 4/5 interface disables (S)-MTD to activate MOR. To test this hypothesis, we performed five replicas of unbiased 1 μs MD simulations of the MOR-MOR homodimer, constructed via both the TM 5/6 (not interacting with Gal1R) and TM 4/5 (interacting with Gal1R) interfaces, in complex with Gi (see Methods and Supplementary Fig. 8). These simulations showed that, in contrast to the TM 5/6 interface, TM 5 of the active Gi-bound protomer moved the extracellular part of TM 5 inward in the TM 4/5 interface. Importantly, this movement of TM 5 relocated the position of the key V2385.42 (Supplementary Fig. 8). Fig. 4o, p summarizes these findings. In the TM 5/6 interface (Fig. 4o), W2956.48 is only partially restricted (depicted as flexible ellipses) by the phenyl ring of (S)-MTD because the dynamic behavior of the ligand is not fully constrained by the partner protomer (depicted as flexible arrows). In contrast, in the TM 4/5 interface (Fig. 4p), the inward movement of V2385.42 fully constrained (depicted as a single arrow) the phenyl ring of (S)-MTD, maintaining W2956.48 in the inactive conformation (depicted as a single ellipse).