Computational insight into molecular mechanism of differentiated allosteric modulations at the mu opioid receptor by structurally similar opioid ligands

ABSTRACT: Activating or blocking the mu opioid receptor (MOR) through orthosteric ligands are current methods to treat opioid abuse and addiction. Unfortunately, most MOR orthosteric ligands suffer from significant side effects mainly due to their low selectivity to the kappa and delta opioid receptors. In contrast, some G protein-coupled receptor (GPCR) allosteric ligands have been reported to exhibit high subtype selectivity and can modulate effectively the potency and efficacy of orthosteric ligands. Recently, we identified structurally similar NAQ and its analog NCQ as novel MOR ligands. Interestingly, NAQ acted mainly as a MOR antagonist but NCQ as a MOR partial agonist. In the present work, molecular modeling methods were applied to explore their molecular mechanism on their different efficacy profiles on the MOR. When NAQ and NCQ bound with the inactive state MOR, the ‘address’ moieties of NAQ and NCQ interacted with the allosteric binding site but showed no allosteric modulation to the binding of the ‘message’ moieties of NAQ and NCQ at the orthosteric binding site, similarly to the silent allosteric modulator. In contrary, in the active state MOR, the ‘address’ moieties of NAQ and NCQ showed allosteric modulation function of a positive allosteric modulator at different levels. The “address” moieties of both ligands seemed to be able to positively modulate the efficacy of the ‘message’ moieties of the two ligands at the orthosteric binding site. Owing to the relatively larger ‘address’ moiety of NCQ, its positive allosteric modulation seemed to be much stronger than that of NAQ. These computational results combined with the experimental data helped us understand the allosteric modulation mechanism of NAQ and NCQ, and provided valuable information to develop our next generation of MOR selective ligands with high affinity and subtype selectivity to potentially treat opioid abuse and addiction.

KEYWORDS:NAQ, NCQ, mu opioid receptor, molecular modeling, allosteric binding site, orthosteric binding site

1. INTRODUCTION

America is under an opioid crisis. According to the National Institute on Drug Abuse, every day more than 115 people in the United States died from opioid overdose in 2017.¹Opioid abuse and addiction are the main cause of opioid overdose. Currently, there are three medications approved by the Food and Drug Administration (FDA) to treat opioid abuse and addiction, including opioid agonist methadone, partial agonist buprenorphine, and antagonist naltrexone.² However, due to their side effects, such as abuse liability and high relapse rate concerns associated with methadone and buprenorphine maintenance, patient compliance issues associated with naltrexone treatment, the utilization rates of these pharmacotherapies are low.³Thus, new and effective opioid abuse and addiction treatments still need to be developed.

Mu opioid receptor (MOR) plays a central role in the treatment of opioid abuse and addiction. The abuse and addiction liability of clinically available opioids is mainly caused by their interactions with the MOR.^{4, 5}Hence, activation of the MOR through MOR agonists or partial agonists or keeping the MOR at its inactive state via MOR antagonists may provide potential therapies for opioid abuse and addiction.^6-8 Many MOR agonists, partial agonists, and antagonists also can be called as orthosteric ligands, which target the primary binding site of the MOR, termed as orthosteric binding site.^{9, 10}Among three subtypes of the opioid receptors, the MOR, kappa opioid receptor (KOR), and delta opioid receptor (DOR),their orthosteric binding sites are highly conserved and the residues acting critical roles in the binding with the orthosteric ligands are almost identical.¹¹Therefore, the development of subtype-specific MOR orthosteric ligands is challenging.On the other hand, different from the orthosteric binding site, the allosteric binding site of G-protein coupled receptors (GPCRs) have been reported to locate at the non-conserved regions and can accommodate allosteric ligands, also termed as allosteric modulators.^12-14Hence, allosteric modulators binding with the allosteric binding sites may achieve high subtype selectivity. Moreover, allosteric modulators may have the potentialities to promote or reduce the binding affinity of orthosteric ligands, or modulate the functional response of GPCRs to the binding of orthosteric ligands via inducing long-range conformational changes of the receptors, including the opioid receptors.^15-18

Depending on whether the functional response of the receptor is enhanced, inhibited, or had no effect on the affinity or the efficacy of orthosteric ligands, allosteric modulators can act as a positive allosteric modulator (PAM), a negative allosteric modulator (NAM), or a silent allosteric modulator (SAM), respectively.^{17, 19} On the other hand, these potentialities of allosteric modulators only act when orthosteric ligands bind to the receptor. Recently, several ligands targeting both the orthosteric and allosteric binding sites of GPCRs simultaneously have been identified with enhanced receptor affinity and subtype specificity, e.g. McN-A-343 for the M₁-muscarinic acetylcholine receptor,²⁰R-22 for the Dopamine D₃-like receptor,²¹SPM-242 and SPM-354 for the sphingosine-1-phosphate receptor.^{22, 23} These ligands have been termed as bitopic ligands due to the fact that they carry the characters of both orthosteric and allosteric ligands with unique pharmacological profiles.^24-26Thus, design and development of bitopic ligands selectively targeting the MOR may be a plausible route to treat opioid abuse and addiction.

Figure 1. The chemical structures of NAQ (a) and NCQ (b). The chemical structures with atom notation were derived from the complexes after molecular dynamics (MD) simulations.

As one example, our lab recently reported that 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(2’-indolyl)acetamido]morphinan (INTA) was a MOR agonist and 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(indole-7-carboxamido)morphinan (NAN) was a MOR antagonist.^{27, 28}Molecular modeling studies indicated that both two ligands may be termed as bitopic ligands. Their epoxymorphinan moieties bound to the orthosteric binding site of MOR and their indole rings interacted with different domains of the MOR allosteric binding site. Hence, the indole ring of INTA acted as a PAM to the epoxymorphinan moiety but the indole ring of NAN acted as a NAM.²⁸Recently, based on the ‘message-address’ concept, our lab developed a C(6)-isoquinoline substituted naltrexone derivative NAQ (Figure 1a).²⁹According to the docking study, we found that the ‘message’ moiety of NAQ bound with the same binding sites of the epoxymorphinan moieties of INTA and NAN while its ‘address’ moiety may interact with the allosteric binding site of MOR.³⁰NAQ displayed a high binding affinity for the MOR at K_i = 0.55 nM with about 48-fold selectivity for the MOR over the KOR and about 241-fold selectivity over the DOR.²⁹It acted as a low-efficacy MOR partial agonist compared with DAMGO in the ³⁵S-GTP[γS]-binding assay with MOR expressing CHO cell lines.^31-33In the comparative opioid withdrawal precipitation study, NAQ exhibited less withdrawal effect compared with the well-known opioid antagonists, naloxone and naltrexone.³⁴These properties associated with NAQ may make it serve as a potential lead to develop more potent and selective MOR ligands to treat opioid abuse and addiction.

In this regard, a series of NAQ analogs were synthesized and pharmacologically characterized to understand their structure-activity relationship (SAR) on the MOR. Among them, NCQ (Figure 1b) was the most efficacious MOR ligand. The binding affinity of NCQ to MOR was 0.55 nM with about 40-fold selectivity for the MOR over the KOR and approximately 62-fold selectivity over the DOR.³⁵Notably, Figure 1 showed that the ‘message’ moieties (epoxymorphinan moiety) of NAQ and NCQ were identical. For the ‘address’ moieties (isoquinoline ring) of NAQ and NCQ, there were two substitutions on the 1’- and 4’-position of the isoquinoline ring of NCQ. Based on the MD simulations on another NAQ analog NNQ,³⁵we anticipated that the ‘message’ moiety of NCQ may bind with the same orthosteric binding site of NAQ in MOR. However, compared to NAQ, NCQ acted as a high-efficacy MOR partial agonist in its [³⁵S]GTPγS binding assay (Table 1).³⁵The SAR studies of NAQ analogs on the MOR indicated that the substitutions on the 1’- and/or 4’-position of the isoquinoline ring (the address moiety) seemed to be critical to the efficacies of the MOR ligands.³⁶

Therefore, in the present work, in order to comprehend the molecular mechanism of how the substitutions of the ‘address’ moieties of NAQ and NCQ induced different efficacy profiles on the MOR, NAQ and NCQ were docked into the crystal structures of antagonist-bound and agonist-bound MORs followed by MD simulations. These computational results together with the pharmacological characterization may help us understand the allosteric characteristics of the MOR binding site and the putative mechanism of action for the ‘address’ moieties of both NAQ and NCQ interacting with the MOR. Eventually, such knowledge will provide critical clues to modify the ‘address’ moiety and further design and develop more potent and selective MOR modulators for opioid abuse and addiction treatment.

Table 1. Binding affinities and functional efficacies of NAQ and NCQ^{29, 35}

2. RESULTS AND DISCUSSION

2.1. Docking studies of NAQ and NCQ in the inactive state and active state MORs. In order to gain insights into the binding modes of NAQ and NCQ in theinactive state and active state MORs, NAQ and NCQ were docked into the crystal structures of antagonist-bound (PDB ID: 4DKL)³⁷and agonist-bound (PDB ID: 5C1M)³⁸MORs using GOLD 5.4.^{39, 40} The orientation of the ‘address’ moieties of NAQ and NCQ in the allosteric binding site of the MOR was mainly decided by favorable interactions between the ligands and the protein, that is, the hydrophobic portion of the ligands with the hydrophobic transmembrane helices of the protein and the hydrophilic portion of the ligands with the polar extracellular loop region of the protein,^{29, 31} which can be reflected by different scoring systems. In our operation, the highest CHEM-PLP scores and the reasonable orientations of NAQ and NCQ in the inactive state and active state MORs were considered together to choose the optimal docking poses, and the results were displayed in Figures 2 (inactive state MOR) and 3 (active state MOR).

Similar to other MOR ligands that were designed based on the “message-address” concept,^{30, 41} the epoxymorphinan moieties (‘message’ moiety) of NAQ and NCQ occupied a similar ‘message’ domain of MOR, which also can be termed as the orthosteric binding site: the epoxymorphinan moiety formed hydrophobic interactions with M151, W293, and H297, the dihydrofuran oxygen of the epoxymorphinan moiety formed hydrogen bonding interaction with Y148, and the piperidine quaternary ammonium nitrogen atom formed ionic interactions with D147. Moreover, owing to the amide linker between the ‘message’ moiety and the ‘address’ moiety, the ‘address’ moieties of NAQ and NCQ can reach to the allosteric binding site of the MOR and may occupy different domains of the allosteric binding site in different binding poses. According to the docking results, there were mainly three different domains in the allosteric binding site of the MOR interacting with the ‘address’ moieties of NAQ and NCQ, termed as allosteric binding domain 1 (ABD1); ABD2, and ABD3. As shown in Figures 2 and 3, ABD1 formed between transmembrane helices (TMs) 6 and 7, ABD2 formed between TM 5 and ECL2, and ABD3 formed between TMs 2, 3, and ECL 1. The residues V300^6.55 and W318^7.35 at ABD1, L219^ECL2 and L232^5.38 at ABD2, Q124^2.60, W133^ECL1, and I144^3.29 at ABD3 could directly interact with the isoquinoline rings of NAQ and NCQ in different binding poses. In the inactive state MOR, the ‘address’ moieties of NAQ and NCQ may occupy either ABD1 or ABD2 (Figure 2). In the active state MOR, the ‘address’ moiety of NAQ seemed to interact with ABD1 or ABD3, and the ‘address’ moiety of NCQ may interact with ABD2 or ABD3 (Figure 3), as summarized in Table 2.

Figure 2. The conformations of NAQ (a) and NCQ (b) in the inactive state MOR with the highest CHEM-PLP scores from docking studies. The protein showed as cartoon model in gray; NAQ and NCQ showed as stick model. Carbon atoms: NAQ in NAQ_MOR^inactive_ABD1 complex (cyan); NAQ in NAQ_MOR^inactive_ABD2 complex (orange); NCQ in NCQ_MOR^inactive_ABD1 complex (magentas); NCQ in NCQ_MOR^inactive_ABD2 complex (light-blue); key amino acid residues showed as stick and ball model in green. The surface models in yellow, light-orange, and cyan represented the ABD1, ABD2, and ABD3, respectively.

Figure 3. The conformations of NAQ (a) and NCQ (b) in the active state MOR with the highest CHEM-PLP scores from docking studies. The protein showed as cartoon model in light-green; NAQ and NCQ showed as stick model. Carbon atoms: NAQ in NAQ_MOR^inactive_ABD1 complex (cyan); NAQ in NAQ_MOR^inactive_ABD3 complex (orange); NCQ in NCQ_MOR^inactive_ABD2 complex (magentas); NCQ in NCQ_MOR^inactive_ABD3 complex (light-blue); key amino acid residues showed as stick and ball model in green. The surface models in yellow, light-orange, and cyan represented the ABD1, ABD2, and ABD3, respectively.

Table 2. The binding domains of the ‘address’ moieties of NAQ and NCQ in the allosteric binding site of inactive and active state MORs, and CHEM-PLP scores for the optimal binding poses in the inactive and active state MORs from docking studies.

2.2. Molecular dynamics (MD) simulations of NAQ and NCQ in the inactive state and active state MORs. Previous studies had indicated that molecular dynamics (MD) simulation was a feasible method to evaluate the reliability of the molecular docking results.^{35, 41}Thus, to determine which binding pose may be favored for NAQ and NCQ in the inactive state and active state MORs and understand their binding mechanisms in the MOR, MD simulations were performed on the eight optimal docking poses obtained from docking studies. In order to obtain stable systems, we conducted 10 ns MD simulations on the eight systems within a POPC lipid bilayer membrane system solvated by TIP3 water layers. As described in previous studies, the root-mean-square deviation (rmsd) values of a system smaller than 3.0 Å can be used as a criterion to evaluate the dynamics equilibrium of the system.^{42, 43}Therefore, the rmsd values of all the protein backbone atoms based on the respective starting structures were calculated and the results were displayed in Figure 4. It seemed apparent that the rmsd values of all the eight systems showed minimum variance after 5 ns of MD simulations. The average rmsd values of NAQ_MOR^inactive_ABD1, NAQ_MOR^inactive_ABD2, NCQ_MOR^inactive_ABD1, NCQ_MOR^inactive_ABD2, NAQ_MOR^active_ABD1, NAQ_MOR^active_ABD3, NCQ_MOR^active_ABD2, and NCQ_MOR^active_ABD3 systems were 1.49, 1.46, 1.31, 1.12, 1.34, 1.28, 1.36, and 1.36 Å, respectively. Based on the above analysis, we concluded that all eight systems achieved stability after 5 ns MD simulations. Therefore, it was reasonable and reliable to conduct the energy and distance analyses based on the snapshots extracted from 7-10 ns MD simulations.

Figure 4. The root-mean-square deviation (rmsd) of the protein backbone atoms of the eight systems relative to the respective starting structures.

The average non-bonded interaction energies of the last 4 ns MD simulations between ligands (NAQ and NCQ) and their surrounding environment (including protein and water molecules) within different cutoff distances of the ligands in the eight systems were calculated and the results were summarized in Table 3. As shown in Table 3, the total interaction energies within different cutoff distances followed the trend: 10 Å < 8 Å < 5Å, which meant the cutoff distance of the ligands had a significant effect on the total interaction energies and comparing the total interaction energies between different binding poses had to be within the same cutoff distances. For the inactive state MOR, comparing the total interaction energies of NAQ_MOR^inactive_ABD1 and NCQ_MOR^inactive_ABD1 systems with that of NAQ_MOR^inactive_ABD2 and NCQ_MOR^inactive_ABD2 systems, respectively, we found that the total interaction energies for the binding of NAQ and NCQ with ABD1 and ABD2 of the allosteric binding site at the same cutoff distance followed the trend: ABD2 < ABD1. While in the active state MOR, the total interaction energies of the two different binding poses of NAQ and NCQ at the same cutoff distance showed no significant difference from each other.

Table 3. The interaction energies (kcal/mol) between ligands (NAQ and NCQ) and their surrounding environment (including protein and water molecules) within different cutoff distances of the ligands