Synthesis and Evaluation of a Novel Bivalent Selective Antagonist for

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Synthesis and Evaluation of a Novel Bivalent Selective Antagonist for the MuDelta 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 J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00403 • Publication Date (Web): 25 Jun 2018 Downloaded from http://pubs.acs.org on July 1, 2018

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Journal of Medicinal Chemistry

Synthesis and Evaluation of a Novel Bivalent Selective Antagonist for the Mu-Delta Opioid Receptor Heterodimer that Reduces Morphine Withdrawal in Mice

Keith M. Olson1,2, Attila Keresztes1, Jenna K. Tashiro2, Lisa V. Daconta2, Victor J. Hruby2, and John M. Streicher1*

1

Department of Pharmacology, College of Medicine, and 2Department of Chemistry & Biochemistry, College of Science; University of Arizona, Tucson, AZ 85724 USA

Running Title: Selective Mu-Delta Opioid Heterodimer Antagonist

Keywords: Mu Opioid Receptor; Delta Opioid Receptor; Heterodimer/Heteromer; Bivalent Antagonist; Dependence; Withdrawal

Conflicts of Interest: None to disclose.

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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 (MOR) and delta (DOR) 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 was confirmed by MS with no more than ~10 parts per million 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 4 compounds falling within 0.21 of each other (Table 1). Similarly, the rigidity as expressed by the ratio of rotatable to non-rotatable 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 vs. the nonselective opioid antagonist 3H-diprenorphine and for receptor potency by

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S-GTPγS coupling vs. the MDOR-

preferring agonist CYM5101020, 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 were competed against 3H-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 high affinity/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 5 ACS Paragon Plus Environment


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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 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

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S-GTPγS experiments was higher, around ~40%; this may be due to the enhanced

efficacy produced by CYM51010 at the MDOR vs. MOR or DOR (Figure S2B, also20). Lastly, CYM51010, a known MDOR-preferring agonist20, 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

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S-GTPγS coupling vs. 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 vs. the MOR-monomer selective agonist DAMGO (see below) instead of the MDOR agonist CYM51010. D24M and both pharmacophores produced shallow, low potency, monophasic curves vs. 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 vs. the kappa opioid receptor (KOR) agonist U50,488 in KOR-expressing cells; the 6 ACS Paragon Plus Environment

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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 which 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 3 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, vs. 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 MDOR31,

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. 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 > 1 µM in MOR cells. To rule out non-spacer length explanations for our results, we also graphed MDOR potency vs. 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 MDOR-preferring agonist CYM51010 produces tail flick anti-nociception in naïve mice, suggesting that MDOR is active in this model at baseline, so we used this model for our testing20. Dose curves of D24M up to 10 nmol were delivered by the intracerebroventricular (icv) route to naïve CD-1 mice with a 5-minute treatment time. This was followed by icv administration of equi-efficacious ~A90 doses of agonist, and tail flick anti-nociception in 52°C water (10 sec cutoff) recorded over a 2-hour time course. We found that 7 ACS Paragon Plus Environment


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D24M dose-dependently antagonized tail flick anti-nociception 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 anti-nociceptive 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 anti-nociception41. 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 delta-2 DOR subtype-selective agonist, which is likely the DOR monomer16. Importantly, D24M produced no changes in tail flick antinociception at any dose when Vehicle was injected instead of agonist (Figure 5E). Constructing doseresponse curves of these results revealed a D24M A50 of 2.04 nmol vs. CYM51010 and 7.77 nmol vs. Deltorphin-II, while D24M had no antagonist effect on DAMGO or DSLET (Figure 5F).

These results suggest that D24M is selective for the MDOR vs. 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 anti-nociception produced by subcutaneous (sc) administration of the MOR and DOR prototypic small molecule agonists morphine and SNC80, respectively. D24M had no effect on SNC80 anti-nociception (Figure 6B), and only a very small effect on morphine anti-nociception (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 anti-nociception.

MDOR Antagonists in Morphine Withdrawal The MDAN series incorporates a DOR antagonist pharmacophore, may disrupt the MDOR, and produces anti-nociception with less dependence18. 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 8 ACS Paragon Plus Environment

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(established as a selective dose in Figure 5) or Vehicle icv, 5 minutes 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 non-significant 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.

Discussion Our data indicates that we have successfully created a first-in-class MDOR-selective antagonist. Our use of low-moderate affinity pharmacophores enabled sub-nanomolar 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 2-5 fold overall selectivity,42 with some high affinity pharmacophores permitting high selectivity through the use of additional chemical modifications36 or by targeting homodimers37. High affinity 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 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 ligands44.



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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 anti-nociception, 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 effects13, 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 anti-nociception, while the compound CYM51010 developed by Devi and colleagues produces anti-nociception itself (in agreement with our data in Figure 5A,15, 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 have 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 tested18. 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 intestines45. 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 suggests that D24M could be a novel therapeutic to


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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 made in improving peptide drugability which could overcome these difficulties. Work from Robin Polt and others has shown that glycosylating peptides with various sugar moieties can improve systemic stability and BBB penetration, including opioid peptides which penetrate the brain to generate anti-nociception44, 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 CNS penetration47,

48

. We are

currently pursuing these approaches, which may overcome the limitations of our peptide compounds as druglike molecules.

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.

Experimental Section Compound Synthesis by SPPS and Evaluation by HPLC/MS 11 ACS Paragon Plus Environment


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For peptide synthesis, amino acids, reagents, and resins were purchased from Advanced Chem Tech (Louisville, KY), Chem-Impex International (Wood Dale, IL), AAPPTec (Louisville, KY), and NovaBiochem (Darmstadt, Germany). DMF, DCM, NMP and other solvents were purchased from Aldrich (St. Louis, MO) and EMD (Darmstadt, Germany). All other synthetic materials were purchased from VWR (West Chester, PA). Each compound was evaluated for purity by high performance liquid chromatography (HPLC) and identity by mass spectrometry (MS), with a minimum purity standard of ≥ 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 polyethylene glycol resin with a Wang-linker using degassed DMF and swelled in 9:1 DCM:DMF (dry and degassed) for 1 hour. Fmoc-Lys(Mtt)-OH, DIC, HOBt, and DMAP were dissolved in degassed and dry DMF at a 3:3:3:0.1 equivalents for 3 hours under a drying tube. Residual hydroxyl groups were capped with 1:1 acetic anhydride:pyridine. Resins were washed 3x DMF, 3x DCM, and 3x MeOH and vacuum dried overnight. For D15, D15M, D18M, D21M and D24M, Fmoc-Lys(Mtt)OH was loaded onto a 1-2% DVB polystyrene resin, 200-400 mesh in a two-step procedure. First 1.5 eq of SOCl2 in dry DCM was mixed with the Wang resin for 45 minutes at 4ºC. Then Fmoc-Lys(Mtt)-OH, DIEA and KI were added and mixed at room temperature for 18-24 hours49. Residual hydroxyl groups were capped with 2:2 acetic anhydride: pyridine and halogen groups with MeOH. Resins were washed 3x DMF, 3x DCM and 3x 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%TFA in DCM 2 x 20 minutes (for longer peptides an additional 2 x 5 minutes 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


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sequences or for deprotection/coupling of Fmoc-Tic and Fmoc-Pro, microwave synthesis was used to heat reactions to 70°C for 5 minutes (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 High-Resolution 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 LowResolution 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 minutes on a Phenomenex brand Gemini-NX-C18, 5 υM, 110A, 30x50 mm column. After each run the gradient was increased to 95% ACN then down to 20% ACN and re-equilibrated before the next run. Each peak collected was >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-D1Nal-NH2) started with the Rink Amide resin (Scheme S1B). Resin and subsequent amino acids were deprotected with 1% DBU:20% piperidine in DMF or NMP followed by subsequent coupling with 3:3:3:6 eq. 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% 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 3

H-diprenorphine (NET1121250UC) and

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S-GTPγS (NEG030H250UC) were both obtained from

PerkinElmer. CYM51010 was obtained from Cayman Chemical. DAMGO, U50,488, norBNI, SNC80 and 13 ACS Paragon Plus Environment


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naloxone was obtained from Tocris/R&D. 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 days. 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. 10 mM drug stock solutions were made in vehicle and stored at -20°C for no more than 30 days. 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% DMSO in assay buffer with 0.1% BSA for the in vitro experiments; 2% DMSO, 10% Tween80, 88% 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 in38. The MDOR-CHO cell line was a kind gift from Jia Bei Wang at the University of Maryland; reported in51. 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 DMEM/F12 media with 10% heat-inactivated FBS and 1X penicillin/streptomycin supplement (all Gibco/ThermoFisher brand) in a 37°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, harvest with 5 mM EDTA in dPBS (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 in38. Membrane preparations of MOR-, DOR-, or MDOR-CHO cells were combined with a concentration curve of 3

H-diprenorphine for saturation binding, or with a fixed concentration of 3H-diprenorphine and a concentration 14 ACS Paragon Plus Environment

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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 minutes. 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 1 site saturation binding fit using GraphPad Prism 7.0 after subtraction of non-specific 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,38). Competition binding in the MDOR cells further used a 2 site competition binding model (GraphPad Prism 7.0).

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S-GTPγS Coupling 35

S-GTPγS coupling also was performed as in38. Membrane preparations of MOR-, DOR-, KOR-, or

MDOR-CHO cells were combined with concentration curves of bivalent antagonists or controls, 1 µM CYM51010 (or DAMGO or U50,488), and 0.1 nM

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S-GTPγS. Antagonists were combined with membrane

protein first for 5 minutes prior to the addition of agonist and

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S-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 pre-treatment time. The reaction size was also 200 µL in 96 well plates. The reactions were incubated at 30°C for 80 minutes, then collected and measured as for the binding experiments. 3-variable 1 site (MOR, DOR, KOR) or 2 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 5 per cage, and recovered for at least 5 days after shipment prior to experimentation. The animals were maintained on a 12 hour 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. 15 ACS Paragon Plus Environment


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Tail Flick Assay The warm water tail flick assay was performed as reported in our published work (52°C water, latency to withdraw, 10 second cutoff,

52

). 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, then a dose range of D24M or Vehicle was icv injected, also performed as reported in 52

, with a 5-minute pre-treatment time. Agonist or Vehicle was then icv or sc injected, with doses reported in the

Legends for Figures 5-6, and tail flick latencies recorded in a 2-hour time course. Dose-response curves were generated by normalizing the baseline-subtracted peak effect of each dose to the Vehicle-pre-treated peak for that agonist (100%). The A50 values for D24M vs. CYM51010 and Deltorphin-II were calculated after linear regression (GraphPad Prism 7.0) using the equation and method described in 52.

Morphine Dependence and Withdrawal Models of acute and chronic dependence were also established and withdrawal precipitated as reported in

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. Acute dependence: 100 mg/kg morphine sc, 4 hours, then 10 mg/kg naloxone ip. Chronic

dependence: 4 day escalating dose protocol with 2x/day 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 hours, then 30 mg/kg naloxone ip. In both models, 1 nmol D24M or Vehicle was icv injected as above 5 minutes prior to the precipitation of withdrawal with naloxone; 1 control experiment in acute dependence injected D24M with no naloxone injection. After naloxone injection, all animals were observed for 20 minutes in plexiglass observation cylinders (6” OD x 0.125” wall thickness x 16” long) and the number of jumps 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


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basis after forming a Material Transfer Agreement with the University of Arizona. Raw data for all experiments is available to qualified investigators. Requests can be made to the corresponding author Dr. John Streicher.

Corresponding Author: John M. Streicher, Ph.D. Department of Pharmacology, College of Medicine University of Arizona Box 245050, LSN563 1501 N. Campbell Ave. Tucson, AZ 85724 [email protected] (520)-626-7495

Author Contributions: KMO initially conceived and designed the bivalent antagonist series with guidance from VJH, and performed most of the chemical syntheses and evaluations. JKT and LMD synthesized several bivalent antagonists and contributed to synthetic development with guidance from KMO and VJH. KMO also performed all in vitro evaluation of the bivalent antagonists with guidance and training from JMS. AK performed all of the in vivo evaluation of the ligands with guidance and training from JMS. VJH supervised the chemistry aspects of the project, and participated in chemistry experimental design and training. JMS supervised the biology aspects of the project, and participated in biology experimental design and training, along with overseeing the overall direction of the project. JMS also wrote the manuscript. All authors had editorial input into the manuscript.

Acknowledgments



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This work was supported by NIH P01-DA006284 (VJH) and institutional funds from the University of Arizona (JMS). We would also like to acknowledge the technical assistance of Dr. Kristen Keck of the University of Arizona Bio5 Institute with peptide purification and LC-MS analysis.

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

Supporting Information: The Supporting Information associated with this manuscript consists of 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 MDOR-CHO cell line. Molecular Formula Strings (SMILES) for all novel compounds as well as the D and M pharmacophores are also available.

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Scheme 1: SPPS Synthesis Scheme for the Representative Heterobivalent Linked MDOR Antagonist D18M.



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Table 1: Physiochemical Properties of Heterobivalent Linked MDOR Antagonists. Hydrophilicity (cLogP) was calculated for the spacer alone and for the combined pharmacophores and spacer for each compound. Rigidity was expressed as the ratio of rotatable:non-rotatable bonds (R:N). MW = molecular weight.

Table 2: Functional

35

S-GTPγS Antagonist Activity and Competition Binding Profiles of Heterobivalent

Linked MDOR Antagonists. NC = not converged. ND = not determined. 1IMAX values for single site partial antagonist activity reported. 2Maximum inhibition at 10 µM. N = 3-4 independent experiments, reported as mean ± SEM. All assays run in CHO cells expressing MOR, DOR, or MDOR. The lead compound, D24M, showed the highest affinity, potency, and selectivity for MDOR (in red).


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Figure Legends

Figure 1: Chemical Structures of Pharmacophores and Heterobivalent Linked MDOR Antagonists. The structures of the D (Tyr-Tic-OH; red) and M (H-Tyr-Pro-Phe-D1Nal-NH2; blue) pharmacophores are shown, as well as the linked antagonists with spacer lengths from 15-41 atoms (D15M through D41M). A D-spacer control was also created with a 15 atom spacer with no M pharmacophore (D15).

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 low potency 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.

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 27 ACS Paragon Plus Environment


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a mixed membrane preparation of MOR and DOR membrane protein mixed in the same molar ratio as the MDOR-CHO 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 2 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

35

S-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 = >3,333 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 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).

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 is 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, 28 ACS Paragon Plus Environment

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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 and 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.

Figure 5: D24M Selectively Targets the MDOR In Vivo. Naïve CD-1 male mice were tested using the 52°C warm water tail flick assay (10 second cutoff; see Experimental Section). Dose curves of D24M up to 10 nmol or Vehicle were icv injected into the mouse brain with a 5 minute treatment time. Equi-efficacious (~A90) doses of Agonist or Vehicle were then icv injected followed by a 2 hour 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 post-injection, no agonist. D24M had no effect alone on tail flick anti-nociception. F) The baseline-subtracted peak effect from each dose for each drug normalized to Vehicle pre-treatment 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 dose-dependently 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.

Figure 6: Extended Selectivity Profile. Naïve CD-1 male mice were tested using the 52°C warm water tail flick assay (10 second cutoff; see Experimental Section). 10 nmol doses of D24M or D or Vehicle were icv injected into the mouse brain with a 5 minute treatment time. Agonist was then icv or sc injected followed by a 2 hour 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 anti-nociception produced by 3.2 nmol of icv CYM51010; p > 0.05. B) D24M was unable to antagonize anti-nociception produced by 10 mg/kg of sc SNC80; p > 0.05. C) D24M had a very minor antagonist effect on the anti29 ACS Paragon Plus Environment


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nociception produced by 3.2 mg/kg of sc morphine; * = p

Synthesis and Evaluation of a Novel Bivalent Selective Antagonist for

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