Application of the Message-Address Concept to the Docking of Naltrexone and Selective Naltrexone-Derived Opioid Antagonists Into Opioid Receptor Models*

Neurochemical Research, VoL 21, No. 11, 1996, pp. 1287-1294

Application of the Message-Address Concept to the Docking of Naltrexone and Selective Naltrexone-Derived Opioid Antagonists into Opioid Receptor Models*

Thomas G. Metzger, 1 M. Germana Paterlini, 1 Philip S. Portoghese, ~ and David M. Ferguson ~,2

Accepted August 1, 1996

A binding site model for the opioid family of G-protein coupled receptors (GPCRs) is proposed based on the message-address concept of ligand recognition. Using ligand docking studies of the universal opioid antagonist, naltrexone, the structural basis for 'message' recognition is explored across all three receptor types, la, g, and K. The binding mode proposed and basis for selectivity are also rationalized using the naltrexone-derived ligands, naltrindole (NTI) and norbinaltorphimine (nor BNI). These ligands are docked to the receptor according to the common naltrexone core or message. The resulting orientation places key 'address' elements in close proximity to amino acid residues critical to selectivity among receptor types. Selectivity is explained by sequence differences in the It, g, and K receptors at these recognition points. Support for the model is derived from site directed mutagenesis studies and ligand binding data for the opioid receptors and other related GPCRs.

KEY WORDS: Opioid receptor; ligand docking; naltrexone.

INTRODUCTION while naltrindole is selective for 8-type receptors. This selectivity has been rationalized in terms of a message- Opioid receptors are the primary site of interaction address model in which the naltrexone pharmacophore of morphine and related opioid alkaloids as welt as the serves as the 'message' that is specifically recognized by various endogenous opioid peptides (l). Due to the dif- all three types of opioid receptor. The selective ligands, ferences in the pharmacological profiles of the three NTI and norBNI, possess additional 'address' elements known receptor types, Ix, 8, and K, a great deal of effort to their structure that enhance selectivity by increasing has been devoted to the development of selective ago- affinity for their preferred receptor type or decreasing nists and antagonists (2). Two prototypical selective an- affinity for other types (3). tagonists, norbinaltorphimine (norBNI) and naltrindole The relatively recent cloning and determination of (NTI), are derivative of the universal opioid antagonist the amino acid sequences of the opioid receptors has, naltrexone (3). NorBNI is selective for K-type receptors for the first time, provided insight to the structure-based design of opioid ligands (4-11). Sequence analyses have 1 Department of Medicinal Chemistry and Minnesota Supercomputer revealed these receptors belong to a superfamily of pro- Institute 308 Harvard St., SE, University of Minnesota, Minneapolis, teins known as G-protein coupled receptors (GPCRs). MN 55455. Although a high resolution crystallographic structure has 2 To whom to address reprint requests. Fax: 612 626 4429; e-mail: [email protected] not yet been reported for a GPCR, various models have * Special issue dedicated to Dr. Eric J. Simon been proposed and applied to rationalize ligand binding. 1287 0364-3190/96/1100-128750950/0 1996 PlenumPublishing Corporation 1288 Metzger, Paterlini, Portoghese, and Ferguson

MaloneyHuss and Lybrand developed a model of the [3- RESULTS AND DISCUSSION adrenergic receptor that explained the stereoselectivity of epinephrine and other related ligands (12). Hibert et 'Message' Binding Site. Although a universal rec- al. model built a series of GPCR structures using bac- ognition site among opioid receptors has been postulated teriorhodopsin (BR) as a template (13,14). Binding site for some time, the structural basis for ligand binding is models were proposed for the serotonin, adrenergic, not yet understood. Clues to the location of a message muscarinic, and dopamine receptors based on ligand af- binding site can be found in the non-selective binding finities and sequence analyses. More recently, Teeter et of naltrexone and other related ligands. Naloxone, a uni- al. have proposed an alternative binding site model for versal opioid antagonist of almost of identical structure the dopamine D2 receptor (15). Using a direct sequence to naltrexone, has been shown to bind both wild type alignment to BR and docking studies of rigid and semi- receptors and chimeric receptors (~t/K (25), ~/K (26), ~t/~ rigid ligands, different binding modes are suggested for (27)) with nanomolar affinity. This durability of high agonists and antagonists within the receptor cavity. affinity binding suggests that naloxone and, by relation, Here, we present a docking study of opioid alka- naltrexone recognition occurs within regions common to loids naltrexone, NTI, and norBNI using 3-dimensional all three receptor types. Such regions exist within the models of the opioid receptor family. In the sections that transmembrane domain of the receptors. Sequence align- follow, a binding site model is developed to explain the ments show that the highest identity among in this do- structure-activity relationships of these and other related main while the extracellular loops and N-terminal region ligands. A particular emphasis is placed on determining are extremely variable (as indicated in Fig. 1). the structural basis for ligand binding and selectivity Mutations of residues within the transmembrane among receptor types. The key interactions proposed are helices have also been shown to effect naloxone binding, supported by results derived from site directed mutage- further narrowing the range of possible binding sites. In nesis and ligand binding studies of opioid receptors and particular, mutation of His(VI:17) to alanine in the ~t other related GPCRs. receptor resulted in a substantial reduction of binding of [3H]naloxone (28). (See Figure 1 for a description of the numbering scheme.) The position of this histidine cor- responds to a phenylalanine in adrenergic receptors be- EXPERIMENTAL PROCEDURE lieved to be important for catecholamine recognition (as discussed below). In the ~ receptor, single point muta- Figure 1 displays a serpentine model of the ~-opioid receptor. The 3-dimensional structure of the transmembrane domain was taken tions of Tyr(III:09) and Trp(VI:13) to alanine were from our previous work on the K-opioid receptor (18). Sequences for found to cause significant reductions in naloxone bind- the ~ and K receptors were obtained from SWISSPROT (19) and ing (29). aligned manually. Due to the high sequence homology in the TM When considering possible docking modes within region between these receptor types, no gross structural changes were transmembrane domain of the opioid receptors, it may made to the K model in the generation of the ~ receptor. The torsion angles of all side chains, however, were rotated to appropriate values be useful to draw analogies to ligand docking studies of in accordance with the backbone dependent rotamer library of Dun- the [3 adrenergic receptor (30). In the past, this receptor brack and Karplus (20). Close contacts between residue sidechains has been linked with opioid receptors based on sequence were removed by minimization with the SANDER module of the AM- analysis. Although the endogenous ligands for opioid re- BER (21) suite of programs. The calculations were performed using ceptors are peptides, they may also be considered recep- the Cornell et al. force field (22) with a constant dielectric of 2.0. To reduce the size of the computation, all non-bonded interactions were tors that recognize phenethylamines when one considers truncated past 8.0/~. Further details of these modeling procedures and the prototypical opioid structures. The tyramine moiety K-receptor model-building are given in ref. 18. found in many opioids bears considerable structural sim- Receptor ligands, naltrexone, naltrindole, and norBNI were model ilarity to catecholamines. It is possible that the general built with the PREP module of the AMBER. The structures of these orientation of the tyramine moiety in opioid receptors opioid alkaloids are shown in Fig. 2. Atom types and parameters were chosen by analogy to the those found in the Cornell et al. force field may be similar to that of the phenethylamine moiety of (22). Partial charges for the ligands were generated by electrostatic adrenergic ligands in its receptor. Based on several site potential fitting using the STO-3g basis set in Gaussian 92 (23). Each directed mutagenesis studies, it has been proposed that ligand was energy minimized and subsequently docked to the receptor the epinephrine binding pocket is located within the model via interactive graphics using MidasPlus (24). Close steric con- transmembrane helices. In particular, the cationic amine tacts between ligand and receptor were removed by visual inspection followed by side chain rotation via interactive graphics. The entire of epinephrine was shown to interact with an aspartate ligand-receptor complexes were energy minimized using the SANDER (11I:08) conserved among monoamine neurotransmitter module of AMBER as specified above. receptors (31) while specific interactions between the Docking of Opioid Antagonists to Receptor Models 1289

N-Terminus

EL-I EL-II

Vll

IL-I

IL-H IL-IH

C-Terminus

Fig. 1. Serpentine model of the 8 opioid receptor. The black lines represent the boundaries of the membrane. Filled circles indicate the residues that are conserved among ~, K and ~t types. Each transmembraneregion is indicated with a Roman numeral. The arabic numbers in the membrane denote the position of each residue from the N-terminal end of each region using the generic GPCR numbering scheme defined in Refs. 16 and 17. For example,Pro VII:17 indicates the 17th position from the N-terminalend of TM VII. Glycosylationsites on the N-terminusand palmitoylation site on the C-terminus are indicated. IL = Intracellular loop. EL = Extracellular loop.

catechol hydroxyls were found to exist with serine res- cluding Trp(VI: 13) and Phe(V: 13). All four of these residues (V:09 and V:12) (32). A phenylalanine (VI:16) idues have been shown to be important in naloxone was also found to be important for interaction with the binding via site directed mutagenesis experiments aromatic ring of adrenergic ligands (33). Thus, it was (28,29) and additionally, are conserved among opioid proposed that these residues, located approximately one types. The proposed docking mode also reveals several third of the way down the membrane, comprise the es- hydrophobic residues, not yet studied by mutagenesis, sential features of catecholamine recognition. A com- that may also be involved in ligand binding. In partic- parable binding site has also been proposed to exist in ular, Ile(VI:16), Ile(VI:20) and Ile(VII:06) may form a other amine neurotransmitter receptor systems (e.g. do- hydrophobic pocket capable of interacting with the li- pamine, serotonin, histamine) (13,14). gand. Ile(VI:20) is changed to a valine in the ~ receptor A similar binding mode is postulated here for nal- while the other two isoleucines are conserved among all trexone. The docking orientation shown in Fig. 3 shares three types. Another conserved residue, Met(III:12), is several structural features noted in catecholamine bind- positioned just below the naltrexone tyramine and may ing to adrenergic receptors. In particular, the tyramine be part of a hydrophobic floor to the binding site. or 'message' moiety docks into a pocket formed by the A second recognition point is also evident in the transmembrane helices. The cationic amine is directed receptor-ligand complex. One potential binding site el- toward helices I, II, III and VII while the aromatic por- ement that opioid receptors possess in common with tion binds to a pocket formed by aromatic residues in adrenergic receptors is the presence of an aspartate in helices IV, V, and VI. The aromatic ring of the tyramine helix III (II1:08). This residue is the presumed counterion moiety is positioned between Tyr(III:09) and His(Vl: for the cationic amine of the small molecule neurotrans- 17). These residues lie just above a network of aromatic mitters. Mutation of this aspartate in the ~t opioid to sidechains that are highly conserved among GPCRs in- alanine, asparagine, or glutamate yielded decreases in 1290 Metzger, Paterlini, Portoghese, and Ferguson

variety of opioid ligands to the K receptor upon mutation of this Asp(III:08) to asparagine have been reported (35). Thus, while the role of this residue is not precisely clear, it appears to play some role in ligand binding in all three opioid receptor types. "Address' Binding Sites. NTI and norBNI represent H 0 prototypical g and K naltrexone-derived antagonists. Ac- cording to the message-address concept, these ligands Naltrexone should satisfy the general constraints of non-selective binding (through the naltrexone core) as well as the specific requirements for selective binding to the g and K receptors. The docking modes shown in Figs. 4 and 5 meet this criteria. A direct overlay of the naltrexone cores of these compounds places the second naltrexone of norBNI and the indole moiety of NT! in close proximity to transmembrane helices VI and VII and extra- H 00~ cellular loop III (EL III). This latter region is highly variable among receptor types and has been implicated I in the selective binding of these and other related li- H gands. Binding profiles of norBNI to both ~t/K and g/K chimera consistently indicate K EL-III to be necessary Naltrindole (NTI) for high affinity binding and K selectivity of this ligand (25,26,36). A single point mutation in this loop (Glu(VI: 23):Lys) has also been found to dramatically effect binding. This mutant receptor shows a 140 fold decrease in norBNI binding while the less selective antagonists, di- .: prenorphine and naloxone, bind both native and mutant receptors with similar affinities (25). This glutamate at the top of helix VI is specific to the K receptor. Given the restrictions required by the specific orientation of HO u" -- -m i U -OH naltrexone in the proposed 'message' binding site, it is I significant that the second cationic amine of norBNI is H ideally positioned to form an ion pair with Glu(VI:23) in the binding site model given in Fig. 4. There is also evidence that the presence of a second Nor-binaltorphimine (norBNI) cationic group in norBNI is the dominant feature in de- Fig. 2. Structures of the ligands used in the docking study. termining K selectivity. Other naltrexone-derived alkaloids with a second basic nitrogen that lack the bivalent character of norBNI have been synthesized (37). In one case, attachment of a basic group to the indole moiety binding affinity for [3H]naloxone (28). More recently, of naltrindole caused a remarkable change in selectivity however, a detailed study of this residue in the g recep- by yielding a K selective compound (38). This result is tor has led to the conclusion that the aspartate is not the consistent with a common docking mode for NTI in the anionic counterpart to the cationic amine of opioid li- g receptor and norBNI in the K receptor. The basic group gands in the g receptor (34). This conclusion was based attached to the indole ' address' of naltrindole would be on the result that mutation of aspartate to alanine in the suitably located to interact with Glu(VI:23) in the K re- receptor caused no change in binding affinity to nal- ceptor based on the docking mode given for naltrindole. oxone as well as non-selective and g-selective agonists It is not known if this naltrindole-derived K-selective and antagonists. By contrast, mutation to asparagine compound displayed a similar binding profile to mutant caused dramatic decreases in binding affinity to the same proteins as that of norBNI. set of ligands tested in the Asp ~ Ala mutant (34). As In the case of NTI, the trend of binding to chimeric in the kt and g cases, reduced binding affinities for a receptors is similar to that found for norBNI. In a study Docking of Opioid Antagonists to Receptor Models 1291

Fig. 3. Naltrexone docked into the proposed 'message' binding pocket in the transmembrane region. Naltrexone is colored yellow with the exception of the amine nitrogen (light blue) and oxygen (red) atoms. Residues in magenta are conserved among the opioid receptor types. Residues in green are Trp(VI: 13) and Phe(V: 13). These two residues are highly conserved among GPCRs.

Fig. 5. Naltrindole docked into the ~-opioid receptor model. Naltrin- dole is shown in yellow with nitrogen atoms in blue and oxygen atoms in red. Receptor residues shown in green and magenta are the same Fig. 4. NorBNI docked into the K-opioid receptor model. NorBNI is as in Figure 3 (with the addition of Ile (VI:06) in magenta). The la- shown in yellow with nitrogen atoms in blue and oxygen atoms in beled residues in the figure compose of hydrophobic pocket which red. Receptor residues shown in green and magenta are the same as interacts with the 'address' of naltrindole. Trp (VI:23) and Leu (VII: in Fig. 3. Glu (V1:23), unique to the K receptc_, is shown in red. 02) (light blue) are unique to the ~ receptor. 1292 Metzger, Paterlini, Portoghese, and Ferguson of g/K chimera, it was found that high affinity of NTI tion point is also evident in our docking model shown was retained as long as EL-III of the g receptor was in Fig. 3. The cationic amine of the tyramine is posi- present (26). More recently, it has been shown that nal- tioned in close proximity to an aspartate in helix III. trindole binding to the g receptor is significantly reduced Although site directed mutagenesis data has suggested when 17 amino acids from EL-III of the g receptor are this interaction may not be significant in binding the ty- substituted for those of the g receptor (39). When docked ramine pharmacophore of opioid ligands (34), it is im- into the g receptor model as shown in Fig. 5, the indole portant to point out the environment surrounding this portion of NTI is directed toward a hydrophobic pocket aspartate is polar. It is therefore difficult to gauge the formed by residues Ile(VII:06), Leu(VII:02), Val(VI:20) strength of this potential interaction in the overall picture and Trp(VI:23). Ile(VII:06) is conserved among all three of pharmacophore binding. Ion-pair interactions are not receptor types while Val(VI:20) is changed to an isoleu- necessarily strong in this case, especially if the desol- cine in the K receptor. Leu(VII:02) and Trp(VI:23) are vation of the cationic center is considered in forming the unique to the g receptor. It is noteworthy that position salt link within the receptor cavity. This interaction may VI:23 is occupied by a lysine in the g receptor and a in fact be weak which could explain the differences in glutamate in the K receptor (noted in norBNI binding ligand binding profiles for the Asp(III:08):Ala and above). It is possible that the hydrophobic character of Asp(III:08):Asn mutants (see Results and Discussion). this portion of the receptor is important in conferring the The binding site model has also provided insight high affinity and selectivity of NTI. In this case, the into the structural basis for selectivity among opioid re- presence of charged residues at position VI:23 would ceptors. The common docking of norBNI and NTI corrupt the hydrophobic nature of this proposed 'ad- within the message pocket places the variable address dress' binding pocket. In any event, while it is evident elements of these ligands within a second binding do- that the g selectivity of NTI correlates with the presence main formed by helices VI, VII, and EL-III. Based on of EL-III of the g receptor, it is not clear whether ele- the results of site directed mutagenesis studies and the ments of EL-III of the 8 receptor enhance binding of orientation of the second cationic amine of norBNI, an naltrindole or portions of EL-III of g and K receptors address recognition site is proposed in the K-receptor. cause diminished naltrindole binding. The position of glutamate (VI:23) in our model is ideally suited to stabilize this cationic center in norBNI and other similarly derived K-antagonists. Unfortunately, this CONCLUSION address site does not appear to be universal in determining K-selectivity as demonstrated by the dependence dy- This report has presented an application of the mes- norphin binding shows on the presence of EL-II sage-address concept to the docking of naltrexone, NTI, (26,36,40). It is reasonable to conclude that variable ad- and norBNI to 3-dimensional models of the opioid re- dress sites may function in the selective binding of dif- ceptors. The unique structural relationship displayed by ferent ligands to the K-receptor. In the case of the these ligands has allowed several key questions to be g-receptor, there is much less detailed information. Al- addressed with regard to ligand binding and selectivity. though EL-III has been implicated in the selective bind- Using naltrexone as a model message compound, we ing of NTI, a specific recognition site has not yet been have shown that non-selective binding is most likely established. Our model indicates the indole moiety of conferred within the receptor cavity. The docking mode this ligand may bind selectively based partly on a hy- proposed places the phenolic group of the tyramine moi- drophobic interaction with Trp(VI:23). This residue is ety within an aromatic pocket formed by transmembrane not only specific to the g-receptor but also occupies the helices III, IV, V and VI. It is reasonable to conclude same position as the Glu noted in norBNI binding above. these aromatic residues are involved in stabilizing nal- Further experimental work, however, is required to de- trexone binding and may, in part, be responsible for rec- termine the precise role this residue plays in recognizing ognition. This hypothesis is supported by results derived the address of NTI. from site directed mutagenesis studies of opioid and The overall convergence of the results presented other GPCRs. Substitutions in aromatic groups within here is encouraging. Although our study has been lim- the receptor cavity have been recently shown to reduce ited to a relatively narrow range of alkaloid structures, ligand binding in certain cases (29). Considering that -rr- the binding models proposed and interactions noted -rr interactions are known to be the driving force behind should provide new insight to the design of opioid an- a wide variety of molecular processes, including ligand tagonists with variable selectivities. Extensions of the binding, this result is not surprising. A second recogni- message-address concept to rationalize agonist binding Docking of Opioid Antagonists to Receptor Models 1293 and selectivity are also underway using these three di- by Docking of Agonists and Tricyclic Antagonists. J. Med. Chem. 37:2874-2888. mensional models and related alkaloid compounds. 16. Schwartz, T. W., Gether, U., Schambye, H. T., and Hjorth, S. A. 1995 Molecular Mechanism of Action in Non-peptide Ligands for Peptide Receptors. 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