Synthesis and Evaluation of a Novel Bivalent Selective Antagonist for

Article Cite This: J. Med. Chem. 2018, 61, 6075−6086

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Synthesis and Evaluation of a Novel Bivalent Selective Antagonist for the Mu-Delta Opioid Receptor Heterodimer that Reduces Morphine Withdrawal in Mice Keith M. Olson,†,‡ Attila Keresztes,† Jenna K. Tashiro,‡ Lisa V. Daconta,‡ Victor J. Hruby,‡ and John M. Streicher*,† Department of Pharmacology, College of Medicine and ‡Department of Chemistry & Biochemistry, College of Science, University of Arizona, Tucson, Arizona 85724, United States

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S Supporting Information *

ABSTRACT: A major limitation in the study of the mu-delta opioid receptor heterodimer (MDOR) is that few selective pharmacological tools exist and no heteromer-selective antagonists. We thus designed a series of variable-length (15−41 atoms) bivalent linked peptides with selective but moderate/low-affinity pharmacophores for the mu and delta opioid receptors. We observed a U-shaped MDOR potency/affinity profile in vitro, with the 24-atom spacer length (D24M) producing the highest MDOR potency/affinity (95%; Table S1). The identity of each compound

We used a highly optimized solid-phase peptide synthesis (SPPS) strategy to create all compounds (see Experimental Section for details). We synthesized our D (red) and M (blue) pharmacophores as diagrammed in Scheme S1. Additionally, we coupled these pharmacophores together with spacers of 15−41 atoms each as diagrammed in Scheme 1 to produce six variable-length compounds (D15M through D41M) as well as 6077

DOI: 10.1021/acs.jmedchem.8b00403 J. Med. Chem. 2018, 61, 6075−6086

Journal of Medicinal Chemistry

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Figure 2. Heterobivalent linked MDOR antagonists produce biphasic inhibition curves in MDOR-CHO cells. All compounds (D15M through D41M) produce similar curves; D24M chosen to display sample representative curves. Concentration curves of D24M or pharmacophore (D, M) and D-spacer (D15) controls used to antagonize the agonist activity of 1 μM CYM51010 in MOR-, DOR-, or MDOR-CHO cells in 35S-GTPγS coupling (see Experimental Section for details). Data normalized to the stimulation caused by CYM51010 alone (100%) or vehicle alone (0%). Data displayed as the mean ± SEM of mean values from N = 3−4 independent experiments performed in duplicate. Biphasic curve fitting performed for bivalent antagonists in MDOR-CHO; monophasic curve fitting for all the rest. (A) D24M, D15, D, and M used to antagonize CYM51010 in MDOR-CHO cells. D24M produced a clear biphasic curve with high (IC50HIGH) and low (IC50LOW) potency sites. The fraction of the total response due to the high site is reported as FHIGH. D15, D, and M all produced monophasic, shallow, low-potency sites in MDOR-CHO cells, suggesting that D24M is targeting the MDOR in the high-potency site. (B) D24M used to antagonize CYM51010 in MOR-, DOR-, and MDOR-CHO cells. D24M produces a biphasic curve with a high-potency site when used in the MDOR cells; D24M produces monophasic lowpotency curves in MOR and DOR cells. There is an 89-fold shift in D24M potency from the DOR IC50 to the MDOR IC50HIGH (arrow). These results also suggest that D24M only produces a biphasic high-potency site in the presence of the MDOR.

Table 2. Functional Antagonistsa

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S-GTPγS Antagonist Activity and Competition Binding Profiles of Heterobivalent Linked MDOR competition binding vs 3H-diprenorphine

GTPγS antagonist activity vs CYM51010 ligand

MDOR IC50HIGH (nM)

MDOR FHIGH (%)

DOR IC50(nM)

MOR IC50(nM)

MDOR/ DOR

MDOR KI‑HIGH (nM)

D15M D18M D21M D24M D30M D41M D15 D M

3.4 ± 0.5 2.1 ± 0.6 1.6 ± 0.3 0.85 ± 0.08 20 ± 7 48 ± 27 170 ± 22 250 ± 45 >1 μM

0.43 ± 0.04 0.39 ± 0.09 0.43 ± 0.06 0.39 ± 0.06 0.41 ± 0.01 0.37 ± 0.01 0.28 ± 0.04b 0.44b (0.43)c

58 ± 14 58 ± 13 68 ± 18 76 ± 5 100 ± 56 94 ± 38 ND 100 ± 33 NC

>1 μM >1 μM >1 μM >1 μM >1 μM >1 μM ND NC >1 μM

17 28 43 89 5 2

2.5 ± 0.8 2.0 ± 0.4 1.3 ± 1.2 0.63 ± 0.14 8.1 ± 4.4 16 ± 8 ND ND ND

MDOR FHIGH (%)

DOR KI, (nM)

MOR KI, (nM)

MDOR/ DOR

± ± ± ± ± ±

270 ± 67 120 ± 33 98 ± 40 84 ± 17 290 ± 68 230 ± 77 250 ± 83 230 ± 45 NC

>1 μM >1 μM >1 μM >1 μM >1 μM >1 μM NC NC >1 μM

110 58 78 133 35 15

0.12 0.12 0.09 0.11 0.15 0.12 ND ND ND

0.02 0.02 0.02 0.01 0.03 0.03

a

NC = not converged. ND = not determined. All assays run in CHO cells expressing MOR, DOR, or MDOR. The lead compound, D24M, showed the highest affinity, potency, and selectivity for MDOR. N= 3−4 independent experiments, reported as mean ± SEM. bIMAX values for single site partial antagonist activity reported. cMaximum inhibition at 10 μM.

was confirmed by MS with no more than ∼10 ppm of error (Table S1). Each compound was designed to minimize differences apart from varying spacer lengths, such as hydrophilicity and rigidity, which could impact MDOR binding and selectivity. All calculated cLogP values fall within 1.5 units of each other, with four compounds falling within 0.21 of each other (Table 1). Similarly, the rigidity as expressed by the ratio of rotatable to nonrotatable bonds (R/N) is within a difference of 1.0 for all compounds (Table 1). Keeping the hydrophilicity and rigidity of the compounds similar to each other minimizes confounding variables, such as lipophilic membrane interactions, that could impact the interpretation of spacer length on MDOR binding affinity/potency and selectivity of the series. In Vitro Evaluation of Compounds. Each compound was evaluated for receptor affinity by competition radioligand binding versus the nonselective opioid antagonist 3Hdiprenorphine and for receptor potency by 35S-GTPγS coupling versus the MDOR-preferring agonist

CYM51010.20,38 All compounds were evaluated in both assays in Chinese hamster ovary (CHO) cells expressing the human MOR, DOR, or MDOR (see Experimental Section for details). In both assays, when the bivalent antagonists competed against 3 H-diprenorphine (binding) or CYM51010 (function) in MDOR-CHO cells, they produced a biphasic competition/ inhibition curve with a high- and low-affinity/potency site (Figure 2A). Multiple lines of evidence indicate that this highaffinity/potency site represents the MDOR. First, only bivalent antagonists produced the high site in MDOR cells. The D and M pharmacophores as well as the D15 monovalent spacer control produced a monophasic, shallow, low-affinity/potency site in MDOR cells, in sharp contrast to the bivalent antagonists (Figure 2A). Second, the bivalent antagonists only produced the high-affinity/potency site in MDOR cells; they did not produce them in MOR or DOR cells. In MOR or DOR cells, the bivalent antagonists only produced a monophasic curve, with moderate affinity/potency at DOR, and low potency/affinity at MOR, in agreement with the relative potencies/affinities of the D and M pharmacophores (Figure 6078



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Figure 3. Additional controls confirm D24M targeting of the MDOR. (A) Schematic of the mixed membrane setup with MOR and DOR expressed separately on membrane fragments in solution vs expressed on the same membrane in MDOR cells. (B) D24M used to antagonize the agonist activity of 1 μM CYM51010 in a mixed membrane preparation of MOR and DOR membrane protein mixed in the same molar ratio as the MDORCHO cells (Figure S2A). Data normalized to the stimulation caused by CYM51010 alone (100%) or vehicle alone (0%). Data displayed as the mean ± SEM of mean values from N = 3 independent experiments performed in duplicate. Comparison curves from MDOR, DOR, and MOR cells included. The mixed-membrane experiment does produce a biphasic curve, but the “high” site is shifted to have far less potency than the high site in MDOR cells. (C) The IC50 values derived from (B) are shown, reported as the mean ± SEM of independent values from N = 3 independent experiments. The “high” site in the mixed membrane experiment has a potency of 89 nM (red); this is similar to the DOR potency of 76 nM (red), but far from the MDOR IC50HIGH potency of 0.85 nM. These results suggest that the mixed-membrane experiment produces two sites that are a mix of monomeric MOR and DOR, while the high-potency site in MDOR cells requires MOR and DOR expression on the same membrane. (D) D24M and the D and M pharmacophores used to antagonize the agonist activity of 1 μM DAMGO in MDOR cells using 35S-GTPγS coupling. Data normalized and displayed as above, N = 3 independent experiments performed in duplicate. IC50 values: D24M = 153 ± 78 nM; D = 36.9 ± 13 nM; M ≥ 3333 nM. D24M produces a low-potency, shallow, monophasic curve vs DAMGO in MDOR cells, suggesting that an MDOR agonist (CYM51010) is required to observe a biphasic high-potency site in MDOR cells. (E) D24M, the D and M pharmacophores, and the positive control norBNI were used to antagonize the agonist activity of 1 μM U50,488 in KOR cells as above; N = 3 independent experiments were performed in duplicate. D24M, D, and M produce no U50,488 antagonist activity, suggesting that they do not interact with the KOR. The norBNI positive control displayed an expected potency value, validating the assay (9.3 ± 1.2 nM).

2B,28,29). Third, we performed saturation radioligand binding in the MDOR cells, which revealed a BMAX of 7.9 pmol/mg of DOR and 1.1 pmol/mg of MOR (Figure S2A); Le Chatelier’s principle tells us the excess DOR will push most of the MOR into the MDOR state, suggesting that MDOR should consist of ∼12% of the total receptor species present. This expected value matches closely with the high site fraction (FHIGH) observed in the competition binding studies (Table 2). Notably, the FHIGH in the 35S-GTPγS experiments was higher, ∼40%; this may be due to the enhanced efficacy produced by CYM51010 at the MDOR versus MOR or DOR (Figure S2B, also ref 20). Lastly, CYM51010, a known MDOR-preferring agonist,20 also produced a biphasic agonist curve in MDOR cells but only a monophasic curve in DOR and MOR cells (Figure S2B). To unambiguously demonstrate that the biphasic response required MOR and DOR expression on the same membrane, we performed a mixed membrane experiment using the MOR and DOR cells. Membrane preparations from MOR-only and DOR-only cells were mixed in the same molar ratio as observed in MDOR saturation binding (Figure S2A) and used

to perform 35S-GTPγS coupling versus CYM51010 as above (Figure 3A). In this assay, D24M did produce a biphasic curve (Figure 3B); however, the curve was shallow, and the “high” site had a potency that closely matched that observed in DOR cells alone, with the “low” site closely matching that observed in MOR cells alone (Figure 3C). This suggests that the results reflect a simple mix of the MOR and DOR cells and further indicates MOR and DOR expression on the same membrane is required to observe the high-potency site, not merely mixed in solution. We also tested D24M and the D and M pharmacophores in the MDOR cells versus the MORmonomer selective agonist DAMGO (see below) instead of the MDOR agonist CYM51010. D24M and both pharmacophores produced shallow, low-potency, monophasic curves versus DAMGO in the MDOR cells, demonstrating how an MDOR agonist (CYM51010) is required to produce biphasic, high-potency curves even in the MDOR cells (Figure 3D). This evidence further confirms that the high-potency biphasic site in binding and GTPγS coupling is the MDOR. Lastly, we tested D24M and both pharmacophores versus the κ-opioid 6079



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Figure 4. Heterobivalent linked antagonists produce a U-shaped spacer length dependency in MDOR cells. (A) The MDOR (IC50HIGH) and DOR (IC50) potencies as well as the MDOR/DOR selectivity ratio all from Table 2 are plotted as a function of spacer length (15−41 atoms). The antagonist series produced a U-shaped spacer length and selectivity dependence in MDOR cells. D24M in the middle of the series has the highest MDOR potency (0.85 nM) and selectivity (89-fold). This relationship is not observed in DOR cells; potency as a function of spacer length is flat. These results are characteristic of bivalent linked compounds targeting heterodimers. (B) Compound MDOR potency as a function of rigidity (R/ N from Table 1) is plotted. Unlike (A), there is no obvious relationship between compound potency and rigidity. (C) Compound MDOR potency as a function of hydrophilicity (cLogP from Table 1) is plotted. Also unlike (A), there is no obvious relationship between compound potency and hydrophilicity; (B, C) further confirm that bivalent antagonist targeting of the MDOR is dependent on spacer length, although the narrow range of values prevents a full analysis and conclusion of the effects of hydrophilicity and rigidity on compound performance.

̈ CD-1 male mice were tested using the 52 °C warm water tail flick assay (10 s cutoff; Figure 5. D24M selectively targets the MDOR in vivo. Naive see Experimental Section). Dose curves of D24M up to 10 nmol or vehicle were icv injected into the mouse brain with a 5 min treatment time. Equi-efficacious (∼A90) doses of agonist or vehicle were then icv injected followed by a 2 h tail flick time course. Data displayed as the mean ± SEM; sample sizes of individual mice per group noted below each graph legend. (A) MDOR-preferring agonist CYM51010; 3.2 nmol. (B) MDOR agonist Deltorphin-II; 3.2 nmol. (C) MOR monomer agonist DAMGO; 0.1 nmol. (D) DOR monomer agonist DSLET; 1 nmol. (E) Vehicle postinjection, no agonist. D24M had no effect alone on tail flick antinociception. (F) The baseline-subtracted peak effect from each dose for each drug normalized to vehicle pretreatment for each drug (100%; no D24M) and used to construct dose−response curves. Reported as the mean ± SEM. Linear regression performed and the A50 for D24M vs CYM51010 (2.04 nmol) and Deltorphin-II (7.77 nmol) calculated. D24M dosedependently antagonized the MDOR agonists CYM51010 and Deltorphin-II without blocking the monomer agonists DAMGO and DSLET. These results suggest that D24M is selective for the MDOR in vivo for doses of at least 10 nmol icv.

receptor (KOR) agonist U50,488 in KOR-expressing cells; the positive control antagonist norBNI blocked U50,488 agonist activity, while D24M and both pharmacophores had no effect, further establishing the opioid receptor selectivity of our series (Figure 3E). In addition, the use of multiple assays and cell lines that demonstrate cell line-specific but not assay-specific results helps to rule out potential pan-assay interference (PAINS) activity by our compounds. Using these established approaches, we quantified every compound as well as the D15, D, and M controls in binding and GTPγS coupling in all three cell lines, using the high site

from the MDOR cells (Table 2). Of the compounds, D24M displayed the highest affinity (0.63 nM), potency (0.85 nM), and MDOR/DOR selectivity ratio (89 for GTPγS; 133 for binding). We further graphed the MDOR and DOR potencies, and the MDOR/DOR selectivity ratio, versus spacer length (Figure 4A). The MDOR potency displayed a classic U-shaped profile, with D24M showing the highest potency and selectivity, and both shorter and longer spacers showing decreased potency and selectivity. This profile is a classic indication of heterodimer binding by a bivalent linked ligand and further verifies that our ligands target the MDOR.31,32 6080



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̈ CD-1 male mice were tested using the 52 °C warm water tail flick assay (10 s cutoff; see Experimental Figure 6. Extended selectivity profile. Naive Section). Doses (10 nmol) of D24M or D or vehicle were icv injected into the mouse brain with a 5 min treatment time. Agonist was then icv or sc injected followed by a 2 h tail flick time course. Data displayed as the mean ± SEM; sample sizes of individual mice per group noted below each graph legend. (A) The D pharmacophore was unable to antagonize antinociception produced by 3.2 nmol of icv CYM51010; p > 0.05. (B) D24M was unable to antagonize antinociception produced by 10 mg/kg of sc SNC80; p > 0.05. (C) D24M had a very minor antagonist effect on the antinociception produced by 3.2 mg/kg of sc morphine; * = p < 0.05 vs same time point D24M group by 2 Way ANOVA with Sidak’s posthoc test.

Figure 7. D24M reduces morphine withdrawal behavior. Acute and chronic models of morphine dependence were established as described in the Experimental Section. Vehicle or 1 nmol D24M was icv injected 5 min prior to naloxone precipitation of withdrawal; a control experiment in acute dependence had no naloxone injection (D24M alone). In both models, jumping behavior was recorded for 20 min after the precipitation of withdrawal, and urine and feces weight was recorded at the end of the observation period. Data reported as the mean ± SEM. (A) Acute dependence model. D24M alone produced no jumps and significantly reduced GI output. One nmol D24M reduced naloxone-precipitated jumping behavior by 53.2% with no effect on naloxone-precipitated GI output. *** = p < 0.001 vs D24M, no Naloxone group; # = p < 0.05 vs vehicle group; both by One Way ANOVA with Fisher’s least significant difference posthoc test. N = 10−19 mice/group. (B) Chronic dependence model. D24M (1 nmol) reduced naloxone-precipitated jumping behavior by 85.2%, with a trend but no significant effect on GI output. ** = p < 0.01 vs vehicle group by unpaired two-tailed t test. N = 10 mice/group.

min treatment time. This was followed by icv administration of equi-efficacious ∼A 90 doses of agonist, and tail flick antinociception in 52 °C water (10 s cutoff) recorded over a 2 h time course. We found that D24M dose-dependently antagonized tail flick antinociception produced by CYM51010, which is known to target the MDOR (Figure 5A). Similarly, D24M dose-dependently antagonized Deltorphin-II, which we hypothesized is an agonist for the MDOR, since MOR knockout (KO) alters Deltorphin-II activity, and because Deltorphin-II activates the DOR-1 subtype, which is likely the MDOR (Figure 5B,16,39,40). In sharp contrast to CYM51010 and Deltorphin-II, D24M did not antagonize the antinociceptive activity of DAMGO up to 10 nmol (Figure 5C). DAMGO is likely selective for the MOR monomer, since DOR KO does not alter DAMGO tail flick antinociception.41 Similarly, D24M was unable to antagonize the effects of DSLET (Figure 5D) up to 10 nmol. DSLET is likely DOR monomer selective, since DSLET is well-established as a Δ-2 DOR subtype-selective agonist, which is likely the DOR monomer.16 Importantly, D24M produced no changes in tail

Importantly, a spacer length dependence is not observed in the DOR cells, which display a flat line of potencies across all spacer lengths (Figure 4A). Spacer length did not significantly alter MOR antagonist activity either, although all potencies were greater than 1 μM in MOR cells. To rule out nonspacer length explanations for our results, we also graphed MDOR potency versus rigidity (Figure 4B) and hydrophilicity (cLogP; Figure 4C). There was no apparent relationship between MDOR potency and either of these factors, although the data range by design was narrow, preventing a full analysis of the effects of hydrophilicity and rigidity on compound performance. In Vivo Evaluation of Compounds. We used our highest potency and selectivity compound D24M to evaluate in vivo MDOR antagonist activity and selectivity. The MDORpreferring agonist CYM51010 produces tail flick antinocicep̈ mice, suggesting that MDOR is active in this tion in naive model at baseline, so we used this model for our testing.20 Dose curves of D24M up to 10 nmol were delivered by the ̈ CD-1 mice with a 5 intracerebroventricular (icv) route to naive 6081



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flick antinociception at any dose when vehicle was injected instead of agonist (Figure 5E). Constructing dose−response curves of these results revealed a D24M A50 of 2.04 nmol versus CYM51010 and 7.77 nmol versus Deltorphin-II, while D24M had no antagonist effect on DAMGO or DSLET (Figure 5F). These results suggest that D24M is selective for the MDOR versus the MOR or DOR monomers at icv doses up to 10 nmol. We further tested this conclusion by icv injection of 10 nmol of the D pharmacophore followed by icv CYM51010 as above. The D pharmacophore was completely unable to block CYM51010 antinociception, further supporting the MDOR selectivity of D24M (Figure 6A). We then tested whether 10 nmol of icv D24M could block tail flick antinociception produced by subcutaneous (sc) administration of the MOR and DOR prototypic small molecule agonists morphine and SNC80, respectively. D24M had no effect on SNC80 antinociception (Figure 6B), and only a very small effect on morphine antinociception (Figure 6C). These results further expand the selectivity profile of D24M and suggest that morphine and SNC80 do not act through the MDOR to produce tail flick antinociception. MDOR Antagonists in Morphine Withdrawal. The MDAN series incorporates a DOR antagonist pharmacophore, may disrupt the MDOR, and produces antinociception with less dependence.18 These results suggest the MDOR may promote opioid dependence and withdrawal, but this has never been directly tested. We thus administered 1 nmol of D24M (established as a selective dose in Figure 5) or vehicle icv, 5 min prior to naloxone precipitation of withdrawal in models of acute (Figure 7A) and chronic (Figure 7B) morphine dependence. We found that D24M significantly reduced jumping behavior in acute dependence by 53.2% (Figure 7A) and in chronic dependence by 85.2% (Figure 7B). D24M had no effect on gastrointestinal (GI) output in acute dependence, while producing a nonsignificant trend to reduce GI effects of withdrawal in chronic dependence (Figure 7B). Importantly, D24M administered alone (no naloxone) in acute dependence produced no jumps and significantly reduced GI output (Figure 7A). This control experiment suggests that D24M is not producing withdrawal on its own or reducing naloxone-precipitated withdrawal by producing withdrawal during the pretreatment period. These results suggest that the MDOR does indeed promote opioid withdrawal and that MDOR antagonists could be used to treat opioid withdrawal.

creation of new derivatives of our lead structure D24M. These could include optimization of MDOR potency and selectivity through modulating pharmacophore affinity and spacer rigidity; the central carboxylate of the spacer also provides a modular and simple platform to incorporate useful moieties. These could include fluorescent motifs for MDOR binding and in vivo labeling studies, or the incorporation of sugar-serine amides to improve systemic stability and blood-brain barrier (BBB) penetration of the ligands.44 These features when combined with demonstrated in vitro (∼90-fold) and in vivo (at least up to 10 nmol icv) MDOR selectivity make these compounds highly useful tools to investigate the role of the MDOR in vivo. Limited in vivo studies have suggested a role for the MDOR in modulating opioid antinociception, tolerance, dependence, anxiety/depression, and reward/drug seeking; these studies also suggest that an MDOR antagonist could be therapeutically useful in enhancing opioid analgesic efficacy and reducing side effects.13,15,16,18−20 However, even within this limited set there are seeming contradictions; for instance, work from Whistler and colleagues suggests that the MDOR acts to repress opioid antinociception, while the compound CYM51010 developed by Devi and colleagues produces antinociception itself (in agreement with our data in Figure 5A15,20). Our ability to resolve these seeming contradictions and controversies and to firmly establish the role of the MDOR in vivo is limited by the availability of selective tools. Our ligands, notably D24M, have the potential to bridge this gap, resolve these controversies, and make a strong impact on the field as a selective tool to probe MDOR function in vivo. In addition, as suggested by the literature in the field, our ligands could form the basis for novel therapeutics to enhance opioid analgesia and reduce side effects. Future studies will use D24M and derived compounds to investigate the role of the MDOR in acute and chronic pain, opioid tolerance, and opioid dependence/withdrawal. We made the first step into studies of this kind with our findings that D24M strongly reduces naloxone-precipitated withdrawal behavior in models of acute and chronic morphine dependence (Figure 7). The results with the MDAN series suggested that the MDOR could promote or at least regulate opioid dependence and withdrawal, but this had never been directly tested.18 Our findings show that our MDOR antagonists can be successfully used to test the role of the MDOR in vivo, further suggesting that the MDOR promotes opioid withdrawal behavior. We did not observe a significant effect on the GI symptoms of withdrawal, which is to be expected given that D24M was given icv, while the main site of opioid withdrawal for GI effects is in the intestines.45 Systemically administered MDOR antagonist may have a greater effect on the GI symptoms of withdrawal. Future studies will further explore the role of the MDOR in dependence and withdrawal using these new tools, and our data further suggest that D24M could be a novel therapeutic to treat opioid withdrawal. There are very few such treatments approved for opioid withdrawal, further highlighting the novelty and potential impact of our compounds. One limitation for our compounds as tools and as potential therapeutics is their peptide structure, which likely limits systemic half-life and BBB penetration, necessitating icv or intrathecal (it) delivery (although the enhanced flexibility of peptides may have helped promote binding to the unique MDOR conformation). However, recent advances have been

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DISCUSSION Our data indicate that we have successfully created a first-inclass MDOR-selective antagonist. Our use of low/moderateaffinity pharmacophores enabled subnanomolar affinity/ potency and a ∼90-fold selectivity ratio at the MDOR in the first generation of compounds. The use of high-affinity pharmacophores by contrast typically permits limited 2−5fold overall selectivity,42 with some high-affinity pharmacophores permitting high selectivity through the use of additional chemical modifications36 or by targeting homodimers.37 Highaffinity pharmacophores are typically highly conformationally constrained, which may provide less conformational freedom to bind the altered heterodimer active-site conformation of the MDOR. The synthetic and biochemical approaches reported here thus may be broadly applicable to other GPCR heteromer systems of interest, such as CB1-DOR, NOP-DOR, etc.43 Our modular SPPS synthetic design also permits the flexible 6082



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peptides an additional 2 × 5 min step was included first to enable diffusion to the core), followed by analogous Fmoc chemistry to extend the spacer and MOR pharmacophore. Reactions were monitored via the Kaiser test or chloranil50 colorimetric analysis; incomplete couplings were coupled with PyBROP. For extending off the ε-amino of the longer D30M/D41M sequences or for deprotection/coupling of Fmoc-Tic and Fmoc-Pro, microwave synthesis was used to heat reactions to 70 °C for 5 min (25 W) on a CEM Discover microwave synthesizer. Crude products were precipitated in ice-cold isopropyl ether, purified by C18 RPLC, and novel compounds were analyzed via highresolution MS-MS using the Bruker 9.4 T Apex-Qh hybrid Fourier transfer ion-cyclotron resonance (FT-ICR), while previously established compounds were characterized via low-resolution MS-MS using the Bruker Ultraflex III MALDI TOF-TOF. Peptides were purified using Shimadzu preparative system using a 20−40% ACN gradient with 0.1% formic acid over 20 min on a Phenomenex brand GeminiNX-C18, 5 υM, 110A, 30 × 50 mm column. After each run the gradient was increased to 95% ACN then down to 20% ACN and reequilibrated before the next run. Each peak collected was more than 95% pure via UV−vis monitored at 214 nm; in addition, only the expected product was identified by MS (Figure S1). After purification, compounds were dissolved in 0.1 N HCl and lyophilized. This process was repeated 2−3 times to facilitate the conversion of peptides to the HCl salt, improving compound solubility. Carboxy peptides DSLET and D (Tyr-Tic−OH) were synthesized by loading the first amino acid onto a 2-Cl-Trt resin. C-terminal amidated peptides Deltorphin-II and M (H-Tyr-Pro-Phe-D1NalNH2) started with the Rink Amide resin (Scheme S1B). Resin and subsequent amino acids were deprotected with 1% 1,8diazabicyclo[5.4.0]undec-7-ene (DBU)/20% piperidine in DMF or NMP followed by subsequent coupling with 3:3:3:6 equiv of PyBOP/ HOAt/Fmoc-AA−OH/DIEA and DIEA at 0.25 M (0.5 M DIEA) in DMF or NMP. Cleavage proceeded via 95% TFA, 2.5% triisopropylsilane (TIS), and 2.5% H2O, precipitated in diethyl ether and characterized as above. cLogP Calculations. cLogP values for the compounds were calculated using Chem Draw Professional 16.0. Materials. 3H-Diprenorphine (NET1121250UC) and 35S-GTPγS (NEG030H250UC) were both obtained from PerkinElmer. CYM51010 was obtained from Cayman Chemical. DAMGO, U50,488, norBNI, SNC80, and naloxone were obtained from Tocris/R&D. Guanosine diphosphate (GDP) for the GTPγS assay was obtained from Alfa Aesar, stored at −20 °C under desiccation, made fresh for each experiment, and discarded after 60 d. Morphine was obtained from the NIDA Drug Supply Program. All compound powders were stored as recommended by the manufacturer or at −80 °C under desiccation for synthesized peptides. Drug stock solutions (10 mM) were made in vehicle and stored at −20 °C for no more than 30 d. Morphine and naloxone were made fresh just prior to every experiment. Standard chemicals and buffers were purchased from Fisher Scientific with a minimum purity of 95%. Vehicle comparisons or normalizations were made for every experiment, with the vehicle concentrations equalized between different drugs or concentrations of the same drug. The vehicles used were: 0.1% dimethyl sulfoxide (DMSO) in assay buffer with 0.1% bovine serum albumin (BSA) for the in vitro experiments; 2% DMSO, 10% Tween80, 88% United States Pharmacopeia (USP) water for all icv D24M, D, and agonist injections; 20% DMSO, 10% Tween80, 70% USP saline for SNC80 injections; and USP saline for morphine and naloxone injections. Cell Lines and Cell Culture. MOR-, DOR-, and KOR-CHO cell lines were created and characterized in our lab and are reported in ref 38. The MDOR-CHO cell line was a kind gift from J. B. Wang at the University of Maryland; reported in ref 51. All receptors were human in origin with various N-terminal tags to facilitate analysis. The MDOR-CHO cell line was further characterized by saturation radioligand binding (Figure S2A). All cells were cultured in 50:50 Dulbecco’s Modified Eagle’s Medium (DMEM)/F12 media with 10% heat-inactivated fetal bovine serum (FBS) and 1X penicillin/ streptomycin supplement (all Gibco/ThermoFisher brand) in a 37

made in improving peptide drugability, which could overcome these difficulties. Work from Polt and others has shown that glycosylating peptides with various sugar moieties can improve systemic stability and BBB penetration, including opioid peptides that penetrate the brain to generate antinociception.44,46 Complementary to this approach, recent advances in peptide formulation with nano/microparticles with or without intranasal delivery has shown that peptides can be made systemically stable, even with delayed release formulations, that achieve considerable central nervous system penetration.47,48 We are currently pursuing these approaches, which may overcome the limitations of our peptide compounds as druglike molecules.

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CONCLUSIONS We have established a novel, first-in-class MDOR-selective antagonist by linking low/moderate-affinity DOR and MOR pharmacophores by a flexible spacer. Our lead compound D24M shows sub-nanomolar affinity/potency for the MDOR, with ∼90-fold selectivity for the MDOR over the DOR and MOR monomers. D24M further shows selectivity for the MDOR in vivo with doses up to 10 nmol icv and can sharply reduce withdrawal behaviors in models of acute and chronic dependence. We have thus shown that our antagonists are selective in vitro and in vivo and can be used to study the role of the MDOR in vivo, potentially resolving significant controversy in the field. Our compounds could also be potentially used to treat opioid withdrawal, among other potential side effects of opioid use and misuse.

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EXPERIMENTAL SECTION

Compound Synthesis by SPPS and Evaluation by HPLC/MS. For peptide synthesis, amino acids, reagents, and resins were purchased from Advanced Chem Tech, Chem-Impex International, AAPPTec, and NovaBiochem. Dimethylformamide (DMF), dichloromethane (DCM), N-methyl-2-pyrrolidone (NMP), and other solvents were purchased from Aldrich and EMD. All other synthetic materials were purchased from VWR. Each compound was evaluated for purity by high-performance liquid chromatography (HPLC) and identity by mass spectrometry (MS), with a minimum purity standard of at least 95%. Additional characterization details below. The bivalent antagonists were synthesized using SPPS with the following conditions. For D41M and D30M, Fmoc-Lys(Mtt) was loaded onto a ChemMatrix poly(ethylene glycol) resin with a Wanglinker using degassed DMF and swelled in 9:1 DCM/DMF (dry and degassed) for 1 h. Fmoc-Lys(Mtt)−OH, N,N′-diisopropylcarbodiimide (DIC), HOBt, and 4-dimethylaminopyridine (DMAP) were dissolved in degassed and dry DMF at a 3:3:3:0.1 equiv for 3 h under a drying tube. Residual hydroxyl groups were capped with 1:1 acetic anhydride/pyridine. Resins were washed 3× DMF, 3× DCM, and 3× MeOH and vacuum-dried overnight. For D15, D15M, D18M, D21M, and D24M, Fmoc-Lys(Mtt)−OH was loaded onto a 1−2% divinylbenzene (DVB) polystyrene resin, 200−400 mesh in a twostep procedure. First 1.5 equiv of SOCl2 in dry DCM was mixed with the Wang resin for 45 min at 4 °C. Then Fmoc-Lys(Mtt)−OH, N,Ndiisopropylethylamine (DIEA), and KI were added and mixed at room temperature for 18−24 h.49 Residual hydroxyl groups were capped with 2:2 acetic anhydride/pyridine and halogen groups with MeOH. Resins were washed 3× DMF, 3× DCM, and 3× MeOH and vacuum-dried overnight. In a general methodology, the spacer and the DOR pharmacophore were extended from the α-amino terminus using standard Fmoc(tBu/ Boc) chemistry with PyBOP+HOAt+DIEA or DIC+HOAt as coupling reagents (Scheme S1A). The terminal DOR Tyr is protected with (Boc/tBu), enabling quasi-orthogonal Mtt deprotection with 2− 3% trifluoroacetic acid (TFA) in DCM 2 × 20 min (for longer 6083



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°C humidified incubator with 5% CO2 atmosphere. Propagation cultures were further maintained with 500 μg/mL G418. Cultures were propagated for no more than 20 passages before discarding. Cell pellets for experiments were prepared by growth in 15 cm plates, harvested with 5 mM ethylenediaminetetraacetic acid (EDTA) in Dulbecco's Phosphate-Buffered Saline (no calcium or magnesium), and the storage of cell pellets at −80 °C prior to use. Radioligand Binding. Saturation and competition radioligand binding experiments were performed as reported in ref 38. Membrane preparations of MOR-, DOR-, or MDOR-CHO cells were combined with a concentration curve of 3H-diprenorphine for saturation binding or with a fixed concentration of 3 H-diprenorphine and a concentration curve of competitor ligand (bivalent antagonists, D, M, D15) for competition binding. These reactions were miniaturized to a 200 μL volume in 96-well plates. The reaction proceeded at 30 °C for 80 min. The reactions were terminated by rapid filtration through 96-well format GF/B filter plates (PerkinElmer) with cold water, washed, dried, and Microscint PS (PerkinElmer) added. The plates were read in a MicroBeta2 96-well format 6 detector scintillation counter (PerkinElmer). KD and BMAX values for saturation binding were determined by one-site saturation binding fit using GraphPad Prism 7.0 after subtraction of nonspecific binding (500 nM of both DMT-Tic-Ala-OH and CTAP). KI values were calculated using the IC50 of each competitor ligand and the previously established KD of 3H-diprenorphine in each cell line (Figure S2A, ref 38). Competition binding in the MDOR cells further used a twosite competition binding model (GraphPad Prism 7.0). 35 S-GTPγS Coupling. 35S-GTPγS coupling also was performed as in ref 38. Membrane preparations of MOR-, DOR-, KOR-, or MDORCHO cells were combined with concentration curves of bivalent antagonists or controls, 1 μM CYM51010 (or DAMGO or U50,488), and 0.1 nM 35S-GTPγS. Antagonists were combined with membrane protein first for 5 min prior to the addition of agonist and 35S-GTPγS. For the agonist mode experiment in Figure S2B, concentration curves of CYM51010 were combined with membrane protein and 0.1 nM 35 S-GTPγS, no pretreatment time. The reaction size was also 200 μL in 96-well plates. The reactions were incubated at 30 °C for 80 min, then collected and measured as for the binding experiments. Threevariable one-site (MOR, DOR, KOR) or two-site (MDOR) antagonist curves were fit using GraphPad Prism 7.0. The IC50HIGH, IC50LOW, and FHIGH values were obtained from MDOR cells and IC50 values from MOR, DOR, and KOR cells. Animals. Male CD-1 mice, 4−5 weeks of age, were obtained from Charles River Laboratories. Mice were housed five per cage and recovered for at least 5 d after shipment prior to experimentation. The animals were maintained on a 12 h light/dark cycle with food (standard chow) and water available ad libitum. The University of Arizona vivarium is AAALAC-accredited, with veterinary staff on hand to monitor the animals for signs of illness or distress. All procedures performed were approved by the University of Arizona IACUC. Tail Flick Assay. The warm water tail flick assay was performed as reported in our published work (52 °C water, latency to withdraw, 10 s cutoff52). The experimenter was blinded to treatment group by the delivery of coded drug vials. Food and water was available ad libitum for the duration of the behavioral experiments. Baseline latencies were recorded, and then a dose range of D24M or vehicle was icv injected, also performed as reported in ref 52, with a 5 min pretreatment time. Agonist or vehicle was then icv or sc injected, with doses reported in the legends for Figures 5 and 6, and tail flick latencies recorded in a 2 h time course. Dose−response curves were generated by normalizing the baseline-subtracted peak effect of each dose to the vehiclepretreated peak for that agonist (100%). The A50 values for D24M versus CYM51010 and Deltorphin-II were calculated after linear regression (GraphPad Prism 7.0) using the equation and method described in ref 52. Morphine Dependence and Withdrawal. Models of acute and chronic dependence were also established and withdrawal precipitated as reported in ref 52. Acute dependence: 100 mg/kg morphine sc, 4 h, then 10 mg/kg naloxone ip. Chronic dependence: 4 d escalating dose protocol with 2×/d sc morphine injections; day 1 at 10 mg/kg, day 2

at 20 mg/kg, day 3 at 35 mg/kg, day 4 a single morning injection of 35 mg/kg, 4 h, then 30 mg/kg naloxone ip. In both models, 1 nmol D24M or vehicle was icv injected as above 5 min prior to the precipitation of withdrawal with naloxone; one control experiment in acute dependence injected D24M with no naloxone injection. After naloxone injection, all animals were observed for 20 min in plexiglass observation cylinders (6″ OD × 0.125″ wall thickness × 16″ long), and the number of jumps was counted. At the end of the period, the urine and feces output was weighed. Data Availability. Detailed protocols are available for all experiments upon request, including optimized synthetic protocols for all compounds. Small amounts of D24M are available to qualified investigators on a collaborative basis after forming a Material Transfer Agreement with the University of Arizona. Raw data for all experiments are available to qualified investigators. Requests can be made to the corresponding author Dr. John Streicher.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b00403. Synthetic schemes for the D and M pharmacophores, sample MS and HPLC traces for all compounds, additional compound analytical data (HRMS data, yield, etc.), and additional characterization of the MDORCHO cell line (PDF) Molecular formula strings (SMILES) for all novel compounds as well as the D and M pharmacophores (CSV)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (520)-6267495. ORCID

John M. Streicher: 0000-0002-4173-7362 Author Contributions

K.M.O. initially conceived and designed the bivalent antagonist series with guidance from V.J.H. and performed most of the chemical syntheses and evaluations. J.K.T. and L.M.D. synthesized several bivalent antagonists and contributed to synthetic development with guidance from K.M.O. and V.J.H.; K.M.O. also performed all in vitro evaluation of the bivalent antagonists with guidance and training from J.M.S.; A.K. performed all of the in vivo evaluation of the ligands with guidance and training from J.M.S.; V.J.H. supervised the chemistry aspects of the project and participated in chemistry experimental design and training. J.M.S. supervised the biology aspects of the project and participated in biology experimental design and training, along with overseeing the overall direction of the project. J.M.S. also wrote the manuscript. All authors had editorial input into the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by NIH P01-DA006284 (V.J.H.) and institutional funds from the Univ. of Arizona (J.M.S.). We would also like to acknowledge the technical assistance of Dr. K. Keck of the Univ. of Arizona Bio5 Institute with peptide purification and LC-MS analysis. 6084



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ABBREVIATIONS USED (MDOR), Mu-Delta Opioid Receptor Heterodimer; (DOR), Delta Opioid Receptor; (MOR), Mu Opioid Receptor; (KOR), Kappa Opioid Receptor; (SPPS), Solid Phase Peptide Synthesis; (HPLC), High Performance Liquid Chromatography; (MS), Mass Spectrometry; (CHO), Chinese Hamster Ovary; (PAINS), Pan-Assay Interference Compounds; (icv), Intracerebroventricular; (it), Intrathecal; (sc), Subcutaneous; (ip), Intraperitoneal; (KO), Knockout; (GI), Gastrointestinal; (IC50), 50% Inhibitory Concentration; (BBB), Blood-Brain Barrier; (PPM), Parts Per Million

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Synthesis and Evaluation of a Novel Bivalent Selective Antagonist for

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