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Structural characteristics of 5-HT1A receptors and their ligands
Serotonin Receptors and their Ligands
B. Olivier, I. van Wijngaardenand W. Soudijn (Editors) 1997 Elsevier ScienceB.V, All rights reserved.
45
Structural characteristics of 5-HT~A receptors and their ligands W. Kuipers
Solvay Duphar B.V., Dept. of Medicinal Chemistry, P.O.Box 900, 1380 DA Weesp, The Netherlands.
R E C E P T O R STRUCTURE AND MOLECULAR BIOLOGY
Receptor Structure The 5-HT1A receptors belongs to the class of G-protein coupled receptors (GPCRs). The members of this class all share a number of characteristics which are essential for their structure and second-messenger activation. Although no 3D-structures of GPCRs have been published yet, these receptors are believed to consist of 7 membrane-bound a-helices, connected by three extracellular and three cytoplasmic loops (see Figure 1). The amino-terminus is located on the outside of the cell, and the carboxyl-terminus on the inside. The lipophilic part of the transmembrane regions is in contact with the membrane while the more polar and highly conserved residues, located on the inside, form a hydrophilic receptor core. This topology for GPCRs was initially based on sequence and hydrophobicity analyses, and later experimentally corroborated (reviews e.g. Savarese and Fraser [1], Lee and Karlavage [2], Strader et al. [3] and Schwartz [4]). Figure 2 shows the sequences of the putative transmembrane domains of the rat 5-HT m receptor. All GPCRs use an intermediary G-protein, connected to the receptor at the cytoplasmic side to activate the second messenger system. GPCRs have a number of characteristic amino acid patterns in common. These highly conserved residues are believed to play a special role in receptor structure and cell signalling. The residues involved in ligand binding are less conserved, as different receptor subclasses recognize structurally different ligands. The specific function (for structure or ligand-binding) of a number of more or less conserved residues was revealed by mutation studies. Residues involved in receptor structure and function
N-glycosylation sites at the N-terminal end may be important for proper binding of the receptor to the cell membrane [1]. The N-terminus of the 5-HT1A receptor contains three sites with the consensus sequence for N-glycosylation of Asn residues: AsnoXaa-Ser/Thr (i.e. NNTT: both Asn residues, and NVTS, see Figure 1).
45
Figure 1. Schematic representation of the putative seven transmembrane domains of the rat 5-HT~A receptor as defined by Kuipers et al 1994. Solid circles represent amino acids with a special function for receptor conformation or ligand-binding. Amino acids marked Y are putative N-glycosylation sites. Sites marked * are sites that may be phosphorylated by protein kinase C (PKC).
47 In helix I the combination Gly-Asn is highly conserved, like the Leu-Ala-X-XAsp-Leu motif at the cytoplasmic side of helix II. The highly conserved aspartate residue in this last motif was found to influence agonist binding in a number of investigated GPCRs (e.g. Savarese and Fraser [1] and Schwartz [4]), but hardly affects the affinities of the antagonists tested. In analogy, mutation of Asp82 in the 5-HT~A receptor decreases 5-HT (i.e. 5-hydroxytryptamine, serotonin) agonist affinity [5, 6], while the mutant's affinity for the antagonist pindolol equals that of the wild-type [5]. Variations in [Na § and pH were shown to alter agonist affinity via the highly conserved Asp-residue in helix II [7, 8]. This residue probably allosterically modulates the receptor conformation by which the agonist binding site is altered. Oliveira et al. [9] postulated that the highly conserved Asp in helix II may interact with the Arg of the highly conserved 'DRY' motif at the cytoplasmic side of helix III. According to their hypothesis this interaction would prevent coupling of the G-protein. Interestingly, the Asp of 'DRY' was shown to interact with the G-protein in a number of receptors [1]. Decoupling of the G-protein is known to decrease the affinity for the neurotransmitter. Mutagenesis studies have shown that C- and N-terminal sides of the 3~a cytoplasmic loop are important not only for G-protein coupling, but may also determine G-protein selectivity. The exact mechanism of G-protein binding and specificity has not been elucidated yet, as also other parts of the cytoplasmic loops seem to contribute to the binding process [1, 2]. Two highly conserved cysteines appear to be important for proper protein folding, probably by forming a disulfide bond between the second extracellular loop (between helices IV and V) and the N-terminal end of helix III. Mutation studies concerning these residues in ~-adrenergic, bovine rhodopsin and muscarinic acetylcholine receptors provided strong evidence for this hypothesis [10, 11, 12]. In the 5-HT~A receptor, these residues correspond with Cysl09 at the top of helix III at the extracellular side and Cys187 in the second extracellular loop. Highly conserved proline residues are found in helices V, VI and VII of most GPCRs. Several of these prolines are surrounded by a number of highly conserved other residues, like in the PFF motif in helix VI, or the NP motif in helix VII. Prolines are known to produce kinks in a-helices of membrane proteins [13, 14]. Therefore these highly conserved prolines in GPCRs are believed to be required for a proper receptor structure [15]. A highly conserved NP (Asn-Pro) motif is located at the intracellular side of helix VII. The Asn of this motif probably directly interacts with the highly conserved Asp in helix II, which indicates that helices II and VII are located next to each other [16, 17]. The importance of the Asn396 of the NP motif for agonist binding to the 5-HT~A receptor was illustrated by Chanda et al. [6]. In human 5-HT~A receptors only a highly conservative Asn~Gln mutation was allowed without loss of affinity for the agonist 8-OH-DPAT. However, replacements of this Asn396 with Ala, Phe or Val were detrimental for 5-HT~A receptor affinity for 8-OH-DPAT. Other residues known to be involved in ligand binding are located far from the NP motif.
48
5-HT~^ 5-HT~B 5-HTI~ 5-HT2^ 5-HT2c
r r h r r
38
I L S W W
T L L P P
S V A A A
L A V L L
L L V L S
L L L T I
G A S T V
T L V V V
L I I V I
I T T I I
F L L I I
C A A L M
A T T T T
V T V I I
L L L A G
G S S G G
N N N N N
A A A I I
C F F L L
V V A A V I A T V L T T V I M A V I M A
5-HT~A 5-HTIs 5-HTI~ 5-HT2^ 5-HT2c
r r h r r
74
L L L F F
I I I L L
G A G M M
S S S S S
L L L L L
A A A A A
V V T I I
T T T A A
D D D D D
L L L M M
M L L L L
V V V L L
S S S G G
V I I F F
L L L L L
V V V V V
L M M M M
P P P P P
M I I V V
A S S S S
A T I M M
5-HTI^ 5-HTIB 5-HTI~ 5-HT2^ 5-HT2c
r r h r r
i09 C C C C C
D D D A P
L F I I V
F W W W W
I L L I I
A S S Y S
L S S L L
D D D D D
V I I V V
L T T L L
C C C F F
C C C S S
T T T T T
S A A A A
S S S S S
I I I I I
L M L M M
H H H H H
L L L L L
C C C C C
A I V I V I A I A I
5-HTI^ 5-HTIs 5-HTI~ 5-HT2^ 5-HT2c
r r h r r
153 A A A A A
A A A F I
A I T L M
L M M K K
I I I I I
S V A I A
L L I A I
T V V V V
W W W W W
L V A T A
I F I I I
G S S S S
F I I V I
L S C G G
I I I I V
S S S S S
I L I M V
P P P P P
P P P I I
M F L P P
L F F V V
5-HTI^ 5-HTIB 5-HTI~ 5-HT2^ 5-HT2c
r r h r r
195 Y Y Y F F
T T T V V
I V I L L
Y Y Y I I
S S S G G
T T T S S
F V C F F
G G G V V
A A A A A
F F F F F
Y Y Y F F
I L I I I
P P P P P
L T S L L
L L V T T
L L L I I
M L L M M
L I I V V
V A I I I
L L L T T
Y Y Y Y Y
5-HTI^ 5-HTIB 5-HTI~ 5-H%^ 5-HT2c
r r h r r
347
L L L L L
G G G G G
I I I I I
I I I V V
M L L F F
G G G F F
T A A L V
F F F F F
I I I V L
L V I V I
C C C M M
W W W W W
L L L C C
P P P P P
F F F F F
F F F F F
I I V I I
V I V T T
A S S N N
L L L I I
V V V M L
L W L A S
P P P V V
F I I I L
5-HTIA 5-HTIB 5-HTI~ 5-HT2A 5-HT2c
r r h r r
381
L L L L L
G G F L L
A D D N N
I F F V V
I F F F F
N N T V V
W L G Y S N W L G Y L N W L G Y L N W I G Y L S W I G Y V C
S S S S S
L L L A G
L I I V I
N N N N N
P P P P P
V I I L L
I I I V V
Y Y Y Y Y
A T T T T
Y F M S V F LF L F
N N N N N
L M A L L
Y Y Y T T
II
III
IV
VI
VII
Figure 2. Sequences of the putative transmembrane domains of the rat 5-HT~A receptor [28]. Residues marked bold were found in the active site of serotonin in the model of Trumpp-KaUmeyer et al. [51]. Underlined residues play a role in agonists or antagonist binding in the 5-HTIA receptor model by Kuipers et al. [49, 52], and residues printed in italics were found to be part of the binding site of MHA in the model by Hedberg et al. [55]. Therefore, allosteric modulation of the agonist binding site by mutation of Asn396 seems a more likely explanation for the observed effects than a direct involvement of this residue in agonist binding. This hypothesis is in agreement with the putative contact of the Asn of the NP motif with the Asp in helix II, which was also shown to influence agonist binding allosterically. Phosphorylation disrupts the coupling between the receptor and the Gprotein, which is required for high agonist affinity, thus desensitizing the
49 receptor for agonist activation. Phosphorylation by protein kinase C (PKC) of probably two or three sites has been reported as a possible mechanism for desensitization of the 5-HT~A receptor [18]. Its putative PKC-sites, having consensus sequence Serfrhr-X-Arg~ys, are located in the second and third cytoplasmic loop (see also Figure 1).
Residues involved in ligand binding An Asp116 cognate in helix III is conserved in all cationic neurotransmitter receptors, but absent in other GCPR. It is, therefore, believed to interact with the ligand's basic nitrogen via an ionic H-bond interaction. Many mutagenesis experiments have confirmed the importance of this residue for agonist and antagonist binding (reviews e.g. by Savarese and Fraser [1] and Schwartz [4]). In the 5-HT1A receptor, 5-HT affinity was markedly reduced by an Aspll6Asn mutation [5], but surprisingly the affinity of the antagonist pindolol was not affected. For several receptors hydrogen bonding residues in helix V have been shown to be important for agonist affinity. In the 5-HT1A receptor, Ser199Val mutation decreases 5-HT affinity, but has no effect on the affinity for the antagonist pindolol [5]. Thr200Val replacement renders a receptor devoid of measurable 5HT affinity, although still a signal can be obtained by 5-HT stimulation of the mutant receptor. Therefore, both Ser199 and Thr200 on helix V are believed to be involved in binding serotonin to the 5-HT1A receptor [5]. The OH-group of Thr200 is conserved in many other GPCRs. Trumpp-Kallmeyer et al. [15] postulated that aromatic residues that are highly conserved in GPCR may be involved in signal transduction as well as ligand binding. Site-directed mutagenesis studies in the ~2-adrenoceptor and the 5-HT2A receptor showed the importance of the Phe residues in the 'PFF' motif of helix VI for ligand binding [19, 20]. In the rat 5-HT~A receptor these residues correspond with Phe361 and Phe362, respectively. Asn386 in helix VII was shown to be crucial for receptors which bind aryloxypropanol antagonists like pindolol and propranolol (i.e. ~-adrenergic, 5-HT~A and rodent 5-HT~B receptors). In the rat 5-HT~A receptor Asn386Val mutation decreases the receptors affinity for pindolol, while 5-HT binding is not changed [21]. Several other mutation studies have further substantiated the importance of this residue for aryloxypropanol affinity [22-25]. Chanda et al. [6] investigated the importance of a number of serine residues in helix VII for 5-HT1A agonist binding. The mutation Ser393Ala dramatically decreased receptor affinity for 8-OH-DPAT. Apparently, the highly conserved Ser393 is either directly involved in agonist-binding, or is required for a proper receptor conformation. This residue is one turn above the NP motif. No effect on the receptor affinity for 8-OH-DPAT was found by replacement of Ser391 into Ala. Thus this less conserved residue does not appear to be crucial for 5HT~A receptor affinity of 8-OH-DPAT.
Receptor Cloning The human 5-HTIA receptor has been cloned and expressed by Kobilka et al.
50 [26] and has later been characterized by Fargin et al. [27]. This receptor contains 422 amino acid residues, and shows 89% amino acid homology with the rat analogue, which also has 422 residues [6, 28, 29]. The sequence of the 5-HT~A receptor by Fujiwara et al. [29] differs in one residue from that of Albert et al. [28], but this may not cause significant pharmacological differences [30]. The mouse 5-HT~A gene has also been localized [31], but no sequence data have been published. MODELS OF 5-HTL~ LIGANDS AND T H E I R INTERACTION WITH THE RECEPTOR The 5-HT~A receptor is the most intensively studied serotonin receptor, as a result of the early discovery of 8-OH-DPAT, the (R)-enantiomer being a highly potent and selective 5-HT~A agonist. Several models that rationalize affinities and functional properties of 5-HT~A ligands have been published. Experimental studies in GPCRs indicate that agonists and antagonists occupy partially overlapping, but different binding sites on the receptor [1, 32]. Superimposing antagonists on agonists should therefore be avoided, although their structures may look very similar. For agonists, the hypothesis that ligands bind at a similar site at the receptor is supported by experimental data. Mutagenesis data indicate that all amine neurotransmitters bind in a region between the helices III, V and VI, close to the extracellular side (for a review see Schwartz [4]). Structure-affinity relationships (SARs) of most 5-HT1A receptor agonists show highly similar trends between structurally different classes. This allows the superimposition of these ligands in model building procedures. For antagonists, however, this hypothesis seems less substantiated. In several cases it was shown that antagonists may respond differently to receptor mutations or changes in receptor conformation, for instance as a result of the decoupling of the Gprotein (e.g. Neve et al. [8]). These findings indicate that antagonists do not address identical binding regions at the receptor [33]. Therefore it is erroneous to superimpose structurally very different antagonists without taking SARs into consideration.
Agonist models
5-HT1Aagonist pharmacophores Hibert et al. [34] derived a crude 5-HTI^ pharmacophore by fitting potent 5HT1A agonists of different structural classes (aminotetralins, ergotamines, tryptamines, arylpiperazines and RU24969). This pharmacophore gives the relative orientation of an aromatic ring and a basic nitrogen atom (see Figure 3a). These two elements are the only features shared by all 5-HT~A agonists. Both ends of a normal through the centre of the aromatic ring and a dummy atom (O') in the direction of the nitrogen lone pair were used as fitting elements.
51
a
N
-~ § y[ ~
-~ O'
Figure 3. Relative position of the essential benzene ring with respect to the basic nitrogen in 5-HTIA ligand models by a) Hibert et al. [34] and b) Mellin et al.
[35].
a) In the pharmacophore by Hibert et al. [34], for potent agonists the distance between the aromatic centre and the basic nitrogen d= 5.2/~, and the height of the basic nitrogen with respect to the aromatic plane h= 0.9 .~. b) The pharmacophore by Mellin et al. [35] describes the spatial orientation of the aromatic ring with respect to a dummy atom O'. This dummy O' represents an oxygen atom (of an Asp at the receptor) which forms an ionic hydrogen bond with the ligand's protonated basic nitrogen. The distance of O' to the aromatic plane is defined as y. X is the distance between O' and the normal through the centre of the aromatic plane. Vector V results from summation of the two vectors connecting the basic nitrogen atoms of the two enantiomers 15 and 16 with O'. The angle a represents the angle between this vector V, and the N-dummy vector of the compound. The angle ~ is the angle between the aromatic planes of the compound and that of the enantiomers 15 and 16. For potents agonists, the pharmacophore is defined by : x= 5.2-5.7 A. y= 2.1-2.6 .~, a = -,- 28 ~ ~= _+4".
52 These dummy atoms represent putative aromatic and hydrogen bonding groups at the receptor. In potent compounds, the nitrogen atom is nearly coplanar with the aromatic ring, and the electron lone pair is almost perpendicular to it. Mellin et al. [35] further refined this pharmacophore model with affinity data of a number of chiral aminotetralin-derived agonists. She compared the relative position of the aromatic ring and the nitrogen lone pair of each compound with respect to the enantiomers of a semi-rigid six/six fused angular 2-aminotetralin (15 and 16, respectively), as depicted in Figure 3b. Each compound was fitted on these enantiomers by using the same dummy atoms as Hibert et al. [34]: the ends of a normal through the aromatic ring centre, and a dummy atom (O') in the direction of the nitrogen lone pair. In the model by Mellin et al. [35], receptor excluded volume was defined by areas which are unfavourable for aliphatic substituents. Chidester et al. [36] presented a model based on a series of new tricyclic aminotetralin-related compounds. In the defined pharmacophore the hydroxy group of 8-OH-DPAT acts as a hydrogen bond acceptor. For most compounds in their study a relative orientation with respect to the aminotetralin moiety of (R) 8-OH-DPAT was presented. This model was further corroborated by Jain et al. [37], who used similar compounds for the construction of a quantitative model with the Compass method. This method searches for the best conformation and alignment of the molecules investigated. The preferred orientations of the compounds as found by Jain et al. [37] were consistent with the Chidester study [36]. Figure 4a shows the interacting groups which were described in various ligand-fitting models [34, 36, 38]. For high 5-HT~^ receptor affinity, besides an aromatic ring and a basic nitrogen atom, additional interacting groups are required. The hydroxy group in both serotonin and 8-OH-DPAT is essential for high 5-HT~A affinity. This group may be replaced with methoxy without loss of affinity, and probably acts as a hydrogen bond acceptor [36]. The pyrrole ring in serotonin seems to be i m p o ~ t for its high affinity for 5-HT 1 receptors [34]. This ring can have an additional aromatic interaction and the pyrrole NH may form a hydrogen bond. In 8-OH-DPAT, which lacks a pyrrole ring, affinity for the 5-HT~A receptor is regained by one of the N-n-propyl chains. According to ligand models this essential group must be located in a hydr0Phobic pocket that is unique for the 5-HT~^ receptor with respect to other serotonin subtypes. The size of the pocket is limited to non-branched chains smaller than n-butyl.
Modeling of 8-OH-DPAT and analogues The two enantiomers of 8-OH-DPAT both have high affinity for the 5-HT~A receptor, but the (R)-enantiomer 1 is a full agonist, while the (S)-enantiomer 2 is a partial agonist (see page *, Table 3). The two different ways of superimposing the two enantiomers as suggested by Hibert et al. [34] and Mellin et al. [35] are shown in Figure 5.
53
o
-"-',,'*"'-X o ,z" ~ - /
"
Figure 4. Schematic representation of a) pharmacophore elements as defined in various 5-HT~A agonist models [34, 36, 38] and b) the binding site of agonists like serotonin and 8-OH-DPAT in the 5-HT~A receptor model by Kuipers et al. [52].
54
|
Figure 5. Fit of (R) and (S) 8-OH-DPAT according to a) Hibert et al. [34] and b) MeUin et al. [35]. In both fits the nitrogen lone pairs coincide. In a) the hydroxy groups are also fitted, but not the aromatic centers. In contrast, in b) the aromatic centers are fitted, while the hydroxy groups are located on opposite sides of the benzene ring. As a result, in a) the N-substituents of both enantiomers are more distant than in b).
Y OH
eq
O(
essential n-propyl
9
- , e q
7
Figure 6. Schematic representation of (R) 8-OH-DPAT 1 in the putative bioactive conformation. The basic nitrogen is equatorially positioned at the tetralin ring, which is in a half-chair conformation. The preferred torsion angle Hc2C2-N-H N amounts approximately 180 ~ Methyl substituents t r a n s C1, C2, and cis and t r a n s C3 are unfavourable for 5-HTI^ receptor affinity, while a similar substitution at the c/s C1 position is indifferent.
55 In the fit presented by Hibert et al. [34] (see Figure 5a), the hydroxy groups and the dummy atoms in the direction of the lone pairs coincide and Nsubstituents of the (R) and (S) enantiomers are further apart. This fit seems to be in good agreement with 5-HT1A structure-affinity relationships. As an example, effects of substitution at the 8-positions of the enantiomers of dipropylaminotetralins show similar trends [39]. This indicates that the 8positions of both (R) and (S) enantiomers may have a fairly identical surrounding at the receptor. In addition, the effect of N-substitution of 8hydroxy aminotetralin derivatives is not the same for (R) and (S) enantiomers [40]. Possibly the N-alkyl substituents of (R) and (S) 8-hydroxy aminotetralin enantiomers do not occupy similar binding sites at the receptor. In this respect the fit of (R) and (S) 8-OH-DPAT (1 and 2) as presented by Mellin et al. [35] seems to be less satisfactory. In this fit (see Figure 5b) the aromatic parts and the dummy atoms in the direction of the nitrogen lone pairs coincide, while the hydroxy groups are located on opposite sides of the aromatic centre. It is also possible that both fits are partly correct (i.e. two binding modes for the (S)enantiomer), which might account for the partial agonist character of the (S)enantiomer 2. Structure-affinity relationships of the aminotetralin class are rather complex (Page *, Table 3). Substituents may directly affect the interaction with the receptor, but may also cause a conformational change of the compound. For example t r a n s (equatorial) C1 methyl substitution (see also Figure 6) as in compound 5 has a negative effect on 5-HT1A receptor affinity. However, compounds with t r a n s C1 substituents incorporated into a fused ring, like compounds 11 and 15, are considerably more potent than compound 5. Therefore, the C1 methyl group in 5 is unlikely to occupy essential receptor volume. Instead steric repulsion between the C1 substituent and C~-atoms of the propyl chain may be responsible for the low 5-HT~A receptor affinity of compound 5. As a result of this hindrance the compound may not be able to adopt the bioactive conformation. In the bioactive conformation as defined in ligand-fitting models, the nitrogen atom is equatorially positioned at the tetralin ring, which is in a half-chair conformation. The propyl C~-atoms are approximately in the plane of the aromatic ring, and the torsion angle Hcg-C2N-HN amounts 180". Indeed aminotetralins with t r a n s equatorial C1 methyl substituents were shown to prefer conformations with a torsion angle Hc2-C2N-H N of-60" [41, 42]. The aminotetralin t r a n s C1 substituents may also force the 8-hydroxy group into a conformation in which it cannot accept a hydrogen bond. This effect may play a role in the affinity decrease of compounds 5 and 15 when compared to compound 1. C1 substituents positioned c/s (axially) with respect to N, like in compound 3, or the small 4,6,6 and 5,6,6 fused ring analogues 7 and 9, are tolerated or even enhance 5-HT1A receptor affinity and maintain agonist activity. In the bioactive conformation of the aminotetralin moiety as defined in ligand models, these substituents do not interfere with either the N-propyl chains or the 8hydroxy group [42, 43]. The corresponding 6,6,6 fused ring analogue 13 is unexpectedly only weakly active. The calculated (MM2) energy minimum for
55 compound 13 deviates from the calculated (bioactive) conformation of compound 3 by a different direction of the nitrogen lone pair, which may account for its rather low 5-HTIA receptor affinity [42]. 5-HT1^ receptor ~ i t y is lowered by the introduction of axial 2- and 3- (cis) methyl (compound 17) substituents at the aminotetralin ring of (R) 8-OHDPAT, 1. These substituents may occupy essential receptor volume and thus prevent a good fit at the receptor. The 2- and 3- axially positioned methyl groups may also cause hindrance with (e.g. C~ or C~ atoms of) the essential propyl chain in the bioactive conformation. Similar explanations may account for the negative effect on 5-HT~^ receptor affinity of the trans (equatorial) C3 methyl substituents in compound 19. As aforementioned, the torsion angle Hc2C2-N-H N in the putative bioactive conformation amounts approximately 180". However, compounds with equatorial C3 methyl substituents were shown to prefer conformations with a torsion angle Hc2-C2-N-HN of 60 ~ [42]. It is also possible that aminotetralin trans C3 substituents cause direct hindrance with the receptor. This last hypothesis would account for the 10w 5-HT~A receptor affinity of compounds with trans C3 substituents incorporated into a ring system. In compound 27 the conformation of the essential propyl chain is constrained into a fused ring. Apparently, this conformation is not the same as the bioactive conformation of the free propyl chain.
Moon-22 X=CH2
OCH3
Moon-24 X=NH NH
HN
U24969
Figure 7. Structures of 5-HT~^ receptor ligands.
57 As a result, the lipophilic interaction may not be optimal and the ring may even cause steric hindrance with the receptor. Similar arguments may explain the low 5-HT1A receptor affinity of compound 23. In the model by Chidester et al. [36] the fit of several aminotetralin-derived compounds was discussed. The aminotetralin ring of most compounds was oriented similar to that of (R) 8-OH-DPAT. In contrast, ACA ('Angular, Cis, Ring Closure Away from O', see Figure 7) was fitted on the model in the same orientation as the (S) enantiomer of 8-OH-DPAT according to Mellin et al. [35]. In this fit, the 8-OH group of ACA is located close to the indole NH of serotonin. This orientation accounts for the preferred stereochemistry (R) of the compound. The preference for hydroxy over methoxy subsituents indicates that the 8-hydroxy~ group of ACA, like the pyrrole NH of serotonin, may act as a hydrogen bond donor. Compound Moon-24 (Figure 7) was also fitted in this orientation. The racemate of Moon-24 was reported to possess full agonist activity [44]. According to Chidester et al. [36], the activity of Moon-24 probably resides in the (R) enantiomer. In agreement with the role as a hydrogen bond acceptor in the model of Figure 4a, the oxygen atom was shown to increase 5HTIA receptor affinity 20-fold. The amide NH of Moon-24 seems to have no special function, as replacement with CH2 in Moon-22 was shown to be indifferent [44].
Fitting 5-HTIA agonists of other structural classes With the use of the defined common interacting groups, structurally different 5-HT1A agonists can be superimposed. For aminotetralins, tryptamines and ergotamines this can be done rather straightforwardly, as is shown in Figure 8a. The aromatic nuclei, the basic nitrogens and the nitrogen lone pairs of the compounds can be fitted. As a result, the hydroxy groups shown to be essential for high 5-HT1A affinity, occupy the same spacial position. Hibert and co-workers [34] argued that for RU24969 two different orientations with respect to ergotamines are possible. The benzene ring of this compound may be fitted on the benzene or the pyrrole ring of the indole moiety. Substitution patterns at the 2- and the 5-positions of the indole ring of tryptamines are similar to observations for RU24969 analogues [45, 46]. These findings justify a straightforward fit of the indole rings of the two classes, as shown in Figure 8b. The way of fitting other classes may be somewhat more ambiguous. For instance, the structure of MHA (page *, Table 6 (5-HT1A ligands)) can be considered an aminotetralin-analogue. However, MHA cannot be superimposed on 8-OH-DPAT in an atom-to-atom fit with full comprehension of its 5-HT1A profile. Westkaemper and Glennon [38] showed that MHA may well be fitted on 8-OH-DPAT as depicted in Figure 8c. In this orientation, the aromatic nuclei, the basic nitrogens and the nitrogen lone pairs of the two compounds coincide, and the hydroxy groups are located close to each other. The (R) enantiomer of MHA is preferred, as the (S) enantiomer of MHA cannot be superimposed on (R)-8-OH-DPAT 1 satisfying all four criteria. Thus the stereoselectivity of MHA regarding 5-HT~Aaffinity is accounted for.
58 a
b
d
J
F i g u r e 8.
Superimpositions of a) 8-OH-DPAT and 5-HT, b) RU24969 and 5-HT, c) MHA and 8-OH-DPAT, and d) arylpiperazines and 5-HT.
Fitting the class of arylpiperazines also appeared to be somewhat puzzling [47]. Like flesinoxan, many potent agonists that have been reported in this class owe their high 5-HT~^ receptor affinity to a large N4-substituent. Because of their flexibility they were left out of most modelling studies [34, 48]. Without these substituents, however, most compounds show only moderate 5-HT~A receptor affinity. In a recent study by Kuipers et al [49] new highly potent N 4unsubtituted arylpiperazines were reported. It was shown that arylpiperazines that display agonist affinity probably bind to the receptor in a relatively coplanar conformation with a plane angle of approximately 30 ~ between the aryl and piperazine rings. The fit as in Figure 8d was shown to agree best with structure-affinity relationships of bicyclic arylpiperazines.
Receptor-ligand interactions Models of GPCR are used for the study of receptor-ligand interactions, as for no member of the GPCR family experimentally determined high-resolution 3D structures are available. Most of these models are based on the high-resolution structure of bacteriorhodopsin which is not coupled to a G-protein, but shows
59 functional resemblance with the GPCR rhodopsin [4, 33, 50]. In these studies an attempt was made to combine receptor-binding data with knowledge from other experimental data concerning receptor structure and function. The strength and weaknesses of GPCR models were discussed and evaluated by Hibert et al. [33]. Several models that rationalize 5-HT~A receptor-ligand interactions have been published. As an example Trumpp-Kallmeyer et al. [51] presented a docking study of several natural ligands in their corresponding receptor models. The 5-HT~A receptor was one of the subtypes investigated. In the 5-HT~A receptor model Aspll6 in helix III interacts with the agonists basic nitrogen. The indole NH of serotonin points towards Gly164 in helix IV, while its 5-OH group forms a hydrogen bonding interaction with Thr200 of helix V. Aromatic interactions occur between serotonin and Phe204 and Phe362 in helices V and VI, respectively. Kuipers et al. [52] rationalized affinities of 5-HT~A agonists of several structural classes (e.g. tryptamines, aminotetralins, aporphines) with a docking study of these compounds in a 5-HT~A receptor model. Figure 4 shows a comparison of interactions as defined in this model (b) which were based on mutation data, with those from ligand-fitting models (a) [34, 36, 38]. The space in the ligand models that may contain large substituents at the basic nitrogen atom, overlaps with the available space in the core of the 5-HT~A receptor model. Sterically hindered positions in the receptor map derived from ligandfitting, coincide with the positions of the backbones of helices IV and V. The hydroxy group interacts with Thr200 in helix V. This residue has a dual character as it contains a hydroxy and a methyl group which enables the formation of hydrogen bonds and hydrophobic contacts, respectively. Thus methoxy groups in the 5-position of tryptamines and the 8-position of aminotetralins may also have favourable hydrophobic contacts with this residue. The indole NH of tryptamines (e.g. serotonin) as well as the hydroxy groups of compounds homochiral with (S) 8-OH-DPAT may form a hydrogen bonding interaction with Ser199 in helix V. In the model by Kuipers et al. [52], the lipophilic pocket which is essential for the high 5-HT~A receptor affinity of 8-OH-DPAT may consist of the residue combination Valll7-Cysl20 (see Figure 2). In 5-HT2 receptors Cysl20 is replaced by a polar Ser. According to the receptor model, the propyl chain of 8-OH-DPAT does not attribute to affinity for 5-HT 2 receptors because a lipophilic pocket is lacking, in 5-HT1B.D receptors Valll7 is replaced by a more bulky Ile, which may cause steric hindrance. This hypothesis is corroborated by SAR data, which indicate that 8-OH-DPAT has low affinity for 5-HT 2 receptors because it lacks a pyrrole ring, while steric hindrance of its propyl substituents causes low affinity for 5-HT m and 5-HT~D receptors [53, 54]. The area in which aromatic groups are favourable in ligands, is surrounded with aromatic residues in the receptor model. For instance, the second benzene ring of MHA may have an additional aromatic interaction with residues Phe361 and Phe362 in helix VI [52]. The binding mode of MHA was also subject of a docking study by Hedberg et al. [55], who analyzed differences in aporphine binding to 5-HT~A and D2
6O receptors. In this model, the basic nitrogen interacts with Asp116 and the essential benzene ring of MHA has an aromatic interaction with Phe362 in helix VI. The selectivity of MHA for 5-HTI^ receptors is explained by the C10methyl substituent of MHA being located in a lipophilic cavity unique for the 5HT1A receptor. The binding site of agonists appears to be rather similar in all three receptor models, as several residues found in the active sites are the same (see Figure 2). For instance, Phell2, Aspll6 (TM III), Gly164 (TM IV), Ser199, Thr200 (TM V) and Trp358, Phe361, Phe362 (TM VI) are involved in agonist binding in two or all three of the models. However some differences exist. In ligandmodelling studies it was shown that MHA can be fitted on 8-OH-DPAT and serotonin with overlap of the hydroxy groups (see Figure 4). In the model by Hedberg et al. [55] the hydroxy group of MHA interacts with Ser199. However, in the models by Trumpp-Kallmeyer et al. [51] and Kuipers et al. [52] the hydroxy group of the agonists interacts with Thr200. This last interaction accounts for the effect of Thr200-~Ala substitution which completely abolishes serotonin affinity [5]. This effect is not explained by the Hedberg model [55]. In the model by Kuipers et al. [52] Ser199 interacts with the indole NH of serotonin. The occurrence of this interaction is supported by SAR data of tryptamines [56, 57] and mutation data [5]. In this aspect the model differs from that of Trumpp-Kallmeyer et al. [51] in which the indole NH is directed towards Gly164. This last model provides no explanation for the effect of Ser199-~Ala substitution which decreases 5-HT1A receptor affinity for serotonin. The model by Hedberg et al. [55] explains the difference in 5-HT1A/D2 receptor binding profile of MHA. The C10-methyl group of MHA may bind in a lipophilic pocket formed by Ala203 and Ile205 of the 5-HT~A receptor. This lipophilic [5~pocket is absent in D2 receptors because Ala203 is replaced by a Ser residue. However, in the model by Kuipers et al. [52] a similar lipophilic pocket that can accommodate the C10-methyl group may be formed by Aia203, Thr200 and Leu366 [49]. This lipophilic pocket is also absent in Dg. receptors as these residues have been replaced by two polar serines and an isoleucine, respectively. Thus both orientations of MHA as in the models by Hedberg et al. [55] and Kuipers et al. [49, 52] account for its selectivity for 5-HT1A with respect to D2 receptors. Probably further mutation experiments will be required to elucidate the exact binding mode of this compound.
Antagonist models The pharmacophore derived by Hibert et al. [58] by superimposition of antagonists of different structural classes contains the same elements as the agonist pharmacophore, namely an aromatic ring and a basic nitrogen atom. As might be expected, the spacial requirements differ from the agonist pharmacophore: d= 5.6/~ and h= 1.6~. Unfortunately, the model was built with only moderately active compounds. For validation of this model more SARs of the concerning classes should become available.
61
oo
"r
Figure 9. Schematic representation of a) a ligand model for aryloxypropanol amines by Langlois et al. [59] and b) interactions of this class of antagonists in a 5HT~A receptor model by Kuipers et al. [52].
62 Figure 9a shows a schematic representation of a model of aryloxypropanolamines, which are potent ~-adrenoceptor antagonists but also display high affinity for 5-HT~A and (rodent) 5-HT~B receptors [59]. This model seems to be in good agreement with the suggested binding site of these compounds in the 5-HT~^ receptor model by Kuipers et al. [52] (Figure 9b). The preference for lipophilic and aromatic ring substituents R may be explained by the surrounding with aromatic (Tyr96, Tyr390, Trp387 and Phell2) and lipophilic aliphatic (Met92 and Leu43) residues in the receptor model. The sterically unfavourable region in the ligand model coincideswith the backbone of helix I in the receptor model. The (double) interaction of the oxypropanol moiety with the essential Asn386 in helix VII makes it very unlikely that these compounds are capable of occupying the area between the helices III, V and VI in which agonists bind. Thus the antagonistic properties of this class may be explained.
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Savarese TM, Fraser CM. Biochem J 1992 (a); 283; 1-19. Lee NH, Kerlavage NR. Tr Biomed Res 1993; 6; 488-497. Strader CD, Fong TM, Tota MR, Underwood D. Ann Rev Biochem 1994; 63: 101-132. Schwartz T. Curr Opinion Biotech 1994; 5: 434-444. Ho BY, Karschin A, Branchek T, Davidson N, et al. FEBS Lett 1992; 312: 259-262. Chanda PK, Minchin MCW, Davis AR, Greenberg L, et al. Mol Pharmacol 1993; 43: 516-520. Horstman DA, Brandon S, Wilson AL, Guyer CA, et al. J Biol Chem 1990; 35: 21590-21595. Neve KA, Cox BA, Henningsen RA, Spanoyannis A, et al. Mol Pharmacol 1991; 39: 733-739. Oliveira L, Paiva ACM, Sander C, Vriend G. TIPS 1994; 15: 170-172. Dixon RAF, Sigal IS, Candelore MR, Register RB, et al. EMBO J 1987; 6: 3269-3275. Karnik SS, Khorona HG. J Biol Chem 1990; 265: 17520-17524. Savarese TM, Wang CD, Fraser CM. J Biol Chem 1992 (b); 267: 1143911448. Henderson R, Baldwin JM, Ceska TA, Zemlin D, et al. J Mol Biol 1990; 213: 899-929. Heijne von G. J Mol Biol 1991; 218: 499-503. Trumpp-Kallmeyer S, Hoflack J, Bruinvels A, Hibert M. J Med Chem 1992; 35: 3448-3462. Zhou W, Flanagan C, Ballesteros JA, Konvicka K, et al. Mol Pharmacol 1994; 45: 165-170. Sealfon SC, Chi L, Ebersole BJ, Rodic V, et al. J Biol Chem 1995; 270: 16683-16688.
63 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48
Raymond JR. J Biol Chem 1991; 266: 14747-14753. Strader CD, Sigal IS, Dixon RAF. FASEB J 1989 (a); 3: 1825-1832. Choudhary MS, Graigo S, Roth BL. Mol Pharmacol 1993; 43: 755-761. Guan X, Peroutka SJ, Kobilka BK. Mol Pharmacol 1992; 41: 695-698. Suryanarayana S, Daunt DA, Zastrow M von, Kobilka BK. J Biol Chem 1991; 266: 15488-15492. Oksenberg D. Marsters SA, O'Dowd BF, Jin H, et al. Nature 1992; 360: 161-163. Metcalf MA, McGuffin RW, Hamblin MW. Biochem Pharmacol 1992; 44: 1917-1920. Adham N, Tamm JA, Salon JA, Vaysse PJJ, et al. Neuropharmacol 1994; 33: 387-391. Kobilka BK, Frielle T, Collins S, Yang-Feng T, et al. Nature 1987; 329: 7579. Fargin A, Raymond JR, Lohse MJ, Kobilka BK, et al. Nature 1988; 335" 358-360. Albert PR, Zhou Q, Tol HHM van, Bunzow JR, et al. J Biol Chem 1990; 265: 5825-5832. Fujiwara Y, Nelson DL, Kahsihara K, Varga E, et al. Life Sci 1990; 47" 127-132. Peroutka SJ. Synapse 1994; 18: 241-260. Oakey RJ, Caron MG, Lefkowitz RJ, Seldin MF. Genomics 1991; 10: 338344. Strader CD, Candelore MR, Hill WS, Dixon RAF, et al. J Biol Chem 1989 (b); 264: 16470-16477. Hibert, M, Trumpp-Kallmeyer, S, Hoflack, J, Bruinvels A. TIPS 1993; 14: 7-12. Hibert MF, McDermott I, Middlemiss DN, et al. Eur J Med Chem 1989; 24: 31-37. Mellin C, Vallgarda J, Nelson DL, et al. J Med Chem 1991; 34: 497-510. Chidester CG, Lin C-H, Lahti RA, et al. J Med Chem 1993; 36: 1301-1315. Jain AN, Harris NL, Park JY. J Med Chem 1995; 38: 1295-1308. Westkaemper RB, Glennon RA. Pharmacol Biochem Behav 1991; 40: 10191031. Hacksell U, Liu Y, et al. Drug Design Disc 1993; 9: 287-297. BjSrk L, Backlund B, H85k, et al. J Med Chem 1989; 32: 779-783. Mellin C, BjSrk L, Karl~n A, et al. J Med Chem 1988; 31: 1130-1140. WikstrSrn H, Andersson B, et al. J Med Chem 1987; 30: 1567-1573. Arvidsson LE, Johansson AM, et al. J Med Chem 1987; 30: 2105-2109. Moon MW, Morris JK, Heier RF, et al. J Med Chem 1992; 35: 1076-1092. Glennon RA, Naimann NA, Pierson ME, Smith JD, et al. J Med Chem 1989; 32: 1921-1926. Taylor EW, Nikam, SS, Lambert G, Martin AR, et al. Mol Pharmacol 1988; 34: 42-53. Glennon RA. Drug Dev Res 1992; 26: 251-274. Sleight AJ, Peroutka SJ. Naunyn-Schmiedeberg's Arch Pharmacol 1991;
64 343: 109-116. 49 Kuipers W, Van Wijngaarden I, Kruse CG, Ter Horst-Van Amstel M, et al. J Med Chem 1995; 38: 1942-1954. 50 Baldwin JM. EMBO J 1992; 12: 1693-1703. 51 Trumpp-Kallmeyer S, Bruinvels A, Hoflack J, et al. Neurochem Int 1991; 19/4: 397-406. 52 Kuipers W, Van Wijngaarden I, IJzerman AP. Drug Design Disc 1994; 11: 231-249. 53 Glennon RA, Titeler M, Lyon RA, Slusher RM. J Med Chem 1988; 31: 867870. 54 Slaughter JL, Harrington MA, Peroutka SJ. Life Sci 1990; 47: 1331-1337. 55 Hedberg MH, Johansson AM, Nordvall G, Yliniemel~i A, et al. J Med Chem 1995; 38: 647-658. 56 Hoyer D.In: Fozard JR, ed. The peripheral actions of 5-hydroxytryptamine. New York: Oxford University Press, 1989; 72-99. 57 Macor JE, Fox CB, Johnson C, Koe K, et al. J Med Chem 1992; 35: 36253632. 58 Hibert MF, Gittos MW, Middlemiss DN, et al. J Med Chem 1988; 31: 10871093. 59 Langlois M, Br~mont B, Rouselle D, et al. Eur J Pharm - Mol Pharm Sec 1993; 244: 77-87.
B. Olivier, I. van Wijngaardenand W. Soudijn (Editors) 1997 Elsevier ScienceB.V, All rights reserved.
45
Structural characteristics of 5-HT~A receptors and their ligands W. Kuipers
Solvay Duphar B.V., Dept. of Medicinal Chemistry, P.O.Box 900, 1380 DA Weesp, The Netherlands.
R E C E P T O R STRUCTURE AND MOLECULAR BIOLOGY
Receptor Structure The 5-HT1A receptors belongs to the class of G-protein coupled receptors (GPCRs). The members of this class all share a number of characteristics which are essential for their structure and second-messenger activation. Although no 3D-structures of GPCRs have been published yet, these receptors are believed to consist of 7 membrane-bound a-helices, connected by three extracellular and three cytoplasmic loops (see Figure 1). The amino-terminus is located on the outside of the cell, and the carboxyl-terminus on the inside. The lipophilic part of the transmembrane regions is in contact with the membrane while the more polar and highly conserved residues, located on the inside, form a hydrophilic receptor core. This topology for GPCRs was initially based on sequence and hydrophobicity analyses, and later experimentally corroborated (reviews e.g. Savarese and Fraser [1], Lee and Karlavage [2], Strader et al. [3] and Schwartz [4]). Figure 2 shows the sequences of the putative transmembrane domains of the rat 5-HT m receptor. All GPCRs use an intermediary G-protein, connected to the receptor at the cytoplasmic side to activate the second messenger system. GPCRs have a number of characteristic amino acid patterns in common. These highly conserved residues are believed to play a special role in receptor structure and cell signalling. The residues involved in ligand binding are less conserved, as different receptor subclasses recognize structurally different ligands. The specific function (for structure or ligand-binding) of a number of more or less conserved residues was revealed by mutation studies. Residues involved in receptor structure and function
N-glycosylation sites at the N-terminal end may be important for proper binding of the receptor to the cell membrane [1]. The N-terminus of the 5-HT1A receptor contains three sites with the consensus sequence for N-glycosylation of Asn residues: AsnoXaa-Ser/Thr (i.e. NNTT: both Asn residues, and NVTS, see Figure 1).
45
Figure 1. Schematic representation of the putative seven transmembrane domains of the rat 5-HT~A receptor as defined by Kuipers et al 1994. Solid circles represent amino acids with a special function for receptor conformation or ligand-binding. Amino acids marked Y are putative N-glycosylation sites. Sites marked * are sites that may be phosphorylated by protein kinase C (PKC).
47 In helix I the combination Gly-Asn is highly conserved, like the Leu-Ala-X-XAsp-Leu motif at the cytoplasmic side of helix II. The highly conserved aspartate residue in this last motif was found to influence agonist binding in a number of investigated GPCRs (e.g. Savarese and Fraser [1] and Schwartz [4]), but hardly affects the affinities of the antagonists tested. In analogy, mutation of Asp82 in the 5-HT~A receptor decreases 5-HT (i.e. 5-hydroxytryptamine, serotonin) agonist affinity [5, 6], while the mutant's affinity for the antagonist pindolol equals that of the wild-type [5]. Variations in [Na § and pH were shown to alter agonist affinity via the highly conserved Asp-residue in helix II [7, 8]. This residue probably allosterically modulates the receptor conformation by which the agonist binding site is altered. Oliveira et al. [9] postulated that the highly conserved Asp in helix II may interact with the Arg of the highly conserved 'DRY' motif at the cytoplasmic side of helix III. According to their hypothesis this interaction would prevent coupling of the G-protein. Interestingly, the Asp of 'DRY' was shown to interact with the G-protein in a number of receptors [1]. Decoupling of the G-protein is known to decrease the affinity for the neurotransmitter. Mutagenesis studies have shown that C- and N-terminal sides of the 3~a cytoplasmic loop are important not only for G-protein coupling, but may also determine G-protein selectivity. The exact mechanism of G-protein binding and specificity has not been elucidated yet, as also other parts of the cytoplasmic loops seem to contribute to the binding process [1, 2]. Two highly conserved cysteines appear to be important for proper protein folding, probably by forming a disulfide bond between the second extracellular loop (between helices IV and V) and the N-terminal end of helix III. Mutation studies concerning these residues in ~-adrenergic, bovine rhodopsin and muscarinic acetylcholine receptors provided strong evidence for this hypothesis [10, 11, 12]. In the 5-HT~A receptor, these residues correspond with Cysl09 at the top of helix III at the extracellular side and Cys187 in the second extracellular loop. Highly conserved proline residues are found in helices V, VI and VII of most GPCRs. Several of these prolines are surrounded by a number of highly conserved other residues, like in the PFF motif in helix VI, or the NP motif in helix VII. Prolines are known to produce kinks in a-helices of membrane proteins [13, 14]. Therefore these highly conserved prolines in GPCRs are believed to be required for a proper receptor structure [15]. A highly conserved NP (Asn-Pro) motif is located at the intracellular side of helix VII. The Asn of this motif probably directly interacts with the highly conserved Asp in helix II, which indicates that helices II and VII are located next to each other [16, 17]. The importance of the Asn396 of the NP motif for agonist binding to the 5-HT~A receptor was illustrated by Chanda et al. [6]. In human 5-HT~A receptors only a highly conservative Asn~Gln mutation was allowed without loss of affinity for the agonist 8-OH-DPAT. However, replacements of this Asn396 with Ala, Phe or Val were detrimental for 5-HT~A receptor affinity for 8-OH-DPAT. Other residues known to be involved in ligand binding are located far from the NP motif.
48
5-HT~^ 5-HT~B 5-HTI~ 5-HT2^ 5-HT2c
r r h r r
38
I L S W W
T L L P P
S V A A A
L A V L L
L L V L S
L L L T I
G A S T V
T L V V V
L I I V I
I T T I I
F L L I I
C A A L M
A T T T T
V T V I I
L L L A G
G S S G G
N N N N N
A A A I I
C F F L L
V V A A V I A T V L T T V I M A V I M A
5-HT~A 5-HTIs 5-HTI~ 5-HT2^ 5-HT2c
r r h r r
74
L L L F F
I I I L L
G A G M M
S S S S S
L L L L L
A A A A A
V V T I I
T T T A A
D D D D D
L L L M M
M L L L L
V V V L L
S S S G G
V I I F F
L L L L L
V V V V V
L M M M M
P P P P P
M I I V V
A S S S S
A T I M M
5-HTI^ 5-HTIB 5-HTI~ 5-HT2^ 5-HT2c
r r h r r
i09 C C C C C
D D D A P
L F I I V
F W W W W
I L L I I
A S S Y S
L S S L L
D D D D D
V I I V V
L T T L L
C C C F F
C C C S S
T T T T T
S A A A A
S S S S S
I I I I I
L M L M M
H H H H H
L L L L L
C C C C C
A I V I V I A I A I
5-HTI^ 5-HTIs 5-HTI~ 5-HT2^ 5-HT2c
r r h r r
153 A A A A A
A A A F I
A I T L M
L M M K K
I I I I I
S V A I A
L L I A I
T V V V V
W W W W W
L V A T A
I F I I I
G S S S S
F I I V I
L S C G G
I I I I V
S S S S S
I L I M V
P P P P P
P P P I I
M F L P P
L F F V V
5-HTI^ 5-HTIB 5-HTI~ 5-HT2^ 5-HT2c
r r h r r
195 Y Y Y F F
T T T V V
I V I L L
Y Y Y I I
S S S G G
T T T S S
F V C F F
G G G V V
A A A A A
F F F F F
Y Y Y F F
I L I I I
P P P P P
L T S L L
L L V T T
L L L I I
M L L M M
L I I V V
V A I I I
L L L T T
Y Y Y Y Y
5-HTI^ 5-HTIB 5-HTI~ 5-H%^ 5-HT2c
r r h r r
347
L L L L L
G G G G G
I I I I I
I I I V V
M L L F F
G G G F F
T A A L V
F F F F F
I I I V L
L V I V I
C C C M M
W W W W W
L L L C C
P P P P P
F F F F F
F F F F F
I I V I I
V I V T T
A S S N N
L L L I I
V V V M L
L W L A S
P P P V V
F I I I L
5-HTIA 5-HTIB 5-HTI~ 5-HT2A 5-HT2c
r r h r r
381
L L L L L
G G F L L
A D D N N
I F F V V
I F F F F
N N T V V
W L G Y S N W L G Y L N W L G Y L N W I G Y L S W I G Y V C
S S S S S
L L L A G
L I I V I
N N N N N
P P P P P
V I I L L
I I I V V
Y Y Y Y Y
A T T T T
Y F M S V F LF L F
N N N N N
L M A L L
Y Y Y T T
II
III
IV
VI
VII
Figure 2. Sequences of the putative transmembrane domains of the rat 5-HT~A receptor [28]. Residues marked bold were found in the active site of serotonin in the model of Trumpp-KaUmeyer et al. [51]. Underlined residues play a role in agonists or antagonist binding in the 5-HTIA receptor model by Kuipers et al. [49, 52], and residues printed in italics were found to be part of the binding site of MHA in the model by Hedberg et al. [55]. Therefore, allosteric modulation of the agonist binding site by mutation of Asn396 seems a more likely explanation for the observed effects than a direct involvement of this residue in agonist binding. This hypothesis is in agreement with the putative contact of the Asn of the NP motif with the Asp in helix II, which was also shown to influence agonist binding allosterically. Phosphorylation disrupts the coupling between the receptor and the Gprotein, which is required for high agonist affinity, thus desensitizing the
49 receptor for agonist activation. Phosphorylation by protein kinase C (PKC) of probably two or three sites has been reported as a possible mechanism for desensitization of the 5-HT~A receptor [18]. Its putative PKC-sites, having consensus sequence Serfrhr-X-Arg~ys, are located in the second and third cytoplasmic loop (see also Figure 1).
Residues involved in ligand binding An Asp116 cognate in helix III is conserved in all cationic neurotransmitter receptors, but absent in other GCPR. It is, therefore, believed to interact with the ligand's basic nitrogen via an ionic H-bond interaction. Many mutagenesis experiments have confirmed the importance of this residue for agonist and antagonist binding (reviews e.g. by Savarese and Fraser [1] and Schwartz [4]). In the 5-HT1A receptor, 5-HT affinity was markedly reduced by an Aspll6Asn mutation [5], but surprisingly the affinity of the antagonist pindolol was not affected. For several receptors hydrogen bonding residues in helix V have been shown to be important for agonist affinity. In the 5-HT1A receptor, Ser199Val mutation decreases 5-HT affinity, but has no effect on the affinity for the antagonist pindolol [5]. Thr200Val replacement renders a receptor devoid of measurable 5HT affinity, although still a signal can be obtained by 5-HT stimulation of the mutant receptor. Therefore, both Ser199 and Thr200 on helix V are believed to be involved in binding serotonin to the 5-HT1A receptor [5]. The OH-group of Thr200 is conserved in many other GPCRs. Trumpp-Kallmeyer et al. [15] postulated that aromatic residues that are highly conserved in GPCR may be involved in signal transduction as well as ligand binding. Site-directed mutagenesis studies in the ~2-adrenoceptor and the 5-HT2A receptor showed the importance of the Phe residues in the 'PFF' motif of helix VI for ligand binding [19, 20]. In the rat 5-HT~A receptor these residues correspond with Phe361 and Phe362, respectively. Asn386 in helix VII was shown to be crucial for receptors which bind aryloxypropanol antagonists like pindolol and propranolol (i.e. ~-adrenergic, 5-HT~A and rodent 5-HT~B receptors). In the rat 5-HT~A receptor Asn386Val mutation decreases the receptors affinity for pindolol, while 5-HT binding is not changed [21]. Several other mutation studies have further substantiated the importance of this residue for aryloxypropanol affinity [22-25]. Chanda et al. [6] investigated the importance of a number of serine residues in helix VII for 5-HT1A agonist binding. The mutation Ser393Ala dramatically decreased receptor affinity for 8-OH-DPAT. Apparently, the highly conserved Ser393 is either directly involved in agonist-binding, or is required for a proper receptor conformation. This residue is one turn above the NP motif. No effect on the receptor affinity for 8-OH-DPAT was found by replacement of Ser391 into Ala. Thus this less conserved residue does not appear to be crucial for 5HT~A receptor affinity of 8-OH-DPAT.
Receptor Cloning The human 5-HTIA receptor has been cloned and expressed by Kobilka et al.
50 [26] and has later been characterized by Fargin et al. [27]. This receptor contains 422 amino acid residues, and shows 89% amino acid homology with the rat analogue, which also has 422 residues [6, 28, 29]. The sequence of the 5-HT~A receptor by Fujiwara et al. [29] differs in one residue from that of Albert et al. [28], but this may not cause significant pharmacological differences [30]. The mouse 5-HT~A gene has also been localized [31], but no sequence data have been published. MODELS OF 5-HTL~ LIGANDS AND T H E I R INTERACTION WITH THE RECEPTOR The 5-HT~A receptor is the most intensively studied serotonin receptor, as a result of the early discovery of 8-OH-DPAT, the (R)-enantiomer being a highly potent and selective 5-HT~A agonist. Several models that rationalize affinities and functional properties of 5-HT~A ligands have been published. Experimental studies in GPCRs indicate that agonists and antagonists occupy partially overlapping, but different binding sites on the receptor [1, 32]. Superimposing antagonists on agonists should therefore be avoided, although their structures may look very similar. For agonists, the hypothesis that ligands bind at a similar site at the receptor is supported by experimental data. Mutagenesis data indicate that all amine neurotransmitters bind in a region between the helices III, V and VI, close to the extracellular side (for a review see Schwartz [4]). Structure-affinity relationships (SARs) of most 5-HT1A receptor agonists show highly similar trends between structurally different classes. This allows the superimposition of these ligands in model building procedures. For antagonists, however, this hypothesis seems less substantiated. In several cases it was shown that antagonists may respond differently to receptor mutations or changes in receptor conformation, for instance as a result of the decoupling of the Gprotein (e.g. Neve et al. [8]). These findings indicate that antagonists do not address identical binding regions at the receptor [33]. Therefore it is erroneous to superimpose structurally very different antagonists without taking SARs into consideration.
Agonist models
5-HT1Aagonist pharmacophores Hibert et al. [34] derived a crude 5-HTI^ pharmacophore by fitting potent 5HT1A agonists of different structural classes (aminotetralins, ergotamines, tryptamines, arylpiperazines and RU24969). This pharmacophore gives the relative orientation of an aromatic ring and a basic nitrogen atom (see Figure 3a). These two elements are the only features shared by all 5-HT~A agonists. Both ends of a normal through the centre of the aromatic ring and a dummy atom (O') in the direction of the nitrogen lone pair were used as fitting elements.
51
a
N
-~ § y[ ~
-~ O'
Figure 3. Relative position of the essential benzene ring with respect to the basic nitrogen in 5-HTIA ligand models by a) Hibert et al. [34] and b) Mellin et al.
[35].
a) In the pharmacophore by Hibert et al. [34], for potent agonists the distance between the aromatic centre and the basic nitrogen d= 5.2/~, and the height of the basic nitrogen with respect to the aromatic plane h= 0.9 .~. b) The pharmacophore by Mellin et al. [35] describes the spatial orientation of the aromatic ring with respect to a dummy atom O'. This dummy O' represents an oxygen atom (of an Asp at the receptor) which forms an ionic hydrogen bond with the ligand's protonated basic nitrogen. The distance of O' to the aromatic plane is defined as y. X is the distance between O' and the normal through the centre of the aromatic plane. Vector V results from summation of the two vectors connecting the basic nitrogen atoms of the two enantiomers 15 and 16 with O'. The angle a represents the angle between this vector V, and the N-dummy vector of the compound. The angle ~ is the angle between the aromatic planes of the compound and that of the enantiomers 15 and 16. For potents agonists, the pharmacophore is defined by : x= 5.2-5.7 A. y= 2.1-2.6 .~, a = -,- 28 ~ ~= _+4".
52 These dummy atoms represent putative aromatic and hydrogen bonding groups at the receptor. In potent compounds, the nitrogen atom is nearly coplanar with the aromatic ring, and the electron lone pair is almost perpendicular to it. Mellin et al. [35] further refined this pharmacophore model with affinity data of a number of chiral aminotetralin-derived agonists. She compared the relative position of the aromatic ring and the nitrogen lone pair of each compound with respect to the enantiomers of a semi-rigid six/six fused angular 2-aminotetralin (15 and 16, respectively), as depicted in Figure 3b. Each compound was fitted on these enantiomers by using the same dummy atoms as Hibert et al. [34]: the ends of a normal through the aromatic ring centre, and a dummy atom (O') in the direction of the nitrogen lone pair. In the model by Mellin et al. [35], receptor excluded volume was defined by areas which are unfavourable for aliphatic substituents. Chidester et al. [36] presented a model based on a series of new tricyclic aminotetralin-related compounds. In the defined pharmacophore the hydroxy group of 8-OH-DPAT acts as a hydrogen bond acceptor. For most compounds in their study a relative orientation with respect to the aminotetralin moiety of (R) 8-OH-DPAT was presented. This model was further corroborated by Jain et al. [37], who used similar compounds for the construction of a quantitative model with the Compass method. This method searches for the best conformation and alignment of the molecules investigated. The preferred orientations of the compounds as found by Jain et al. [37] were consistent with the Chidester study [36]. Figure 4a shows the interacting groups which were described in various ligand-fitting models [34, 36, 38]. For high 5-HT~^ receptor affinity, besides an aromatic ring and a basic nitrogen atom, additional interacting groups are required. The hydroxy group in both serotonin and 8-OH-DPAT is essential for high 5-HT~A affinity. This group may be replaced with methoxy without loss of affinity, and probably acts as a hydrogen bond acceptor [36]. The pyrrole ring in serotonin seems to be i m p o ~ t for its high affinity for 5-HT 1 receptors [34]. This ring can have an additional aromatic interaction and the pyrrole NH may form a hydrogen bond. In 8-OH-DPAT, which lacks a pyrrole ring, affinity for the 5-HT~A receptor is regained by one of the N-n-propyl chains. According to ligand models this essential group must be located in a hydr0Phobic pocket that is unique for the 5-HT~^ receptor with respect to other serotonin subtypes. The size of the pocket is limited to non-branched chains smaller than n-butyl.
Modeling of 8-OH-DPAT and analogues The two enantiomers of 8-OH-DPAT both have high affinity for the 5-HT~A receptor, but the (R)-enantiomer 1 is a full agonist, while the (S)-enantiomer 2 is a partial agonist (see page *, Table 3). The two different ways of superimposing the two enantiomers as suggested by Hibert et al. [34] and Mellin et al. [35] are shown in Figure 5.
53
o
-"-',,'*"'-X o ,z" ~ - /
"
Figure 4. Schematic representation of a) pharmacophore elements as defined in various 5-HT~A agonist models [34, 36, 38] and b) the binding site of agonists like serotonin and 8-OH-DPAT in the 5-HT~A receptor model by Kuipers et al. [52].
54
|
Figure 5. Fit of (R) and (S) 8-OH-DPAT according to a) Hibert et al. [34] and b) MeUin et al. [35]. In both fits the nitrogen lone pairs coincide. In a) the hydroxy groups are also fitted, but not the aromatic centers. In contrast, in b) the aromatic centers are fitted, while the hydroxy groups are located on opposite sides of the benzene ring. As a result, in a) the N-substituents of both enantiomers are more distant than in b).
Y OH
eq
O(
essential n-propyl
9
- , e q
7
Figure 6. Schematic representation of (R) 8-OH-DPAT 1 in the putative bioactive conformation. The basic nitrogen is equatorially positioned at the tetralin ring, which is in a half-chair conformation. The preferred torsion angle Hc2C2-N-H N amounts approximately 180 ~ Methyl substituents t r a n s C1, C2, and cis and t r a n s C3 are unfavourable for 5-HTI^ receptor affinity, while a similar substitution at the c/s C1 position is indifferent.
55 In the fit presented by Hibert et al. [34] (see Figure 5a), the hydroxy groups and the dummy atoms in the direction of the lone pairs coincide and Nsubstituents of the (R) and (S) enantiomers are further apart. This fit seems to be in good agreement with 5-HT1A structure-affinity relationships. As an example, effects of substitution at the 8-positions of the enantiomers of dipropylaminotetralins show similar trends [39]. This indicates that the 8positions of both (R) and (S) enantiomers may have a fairly identical surrounding at the receptor. In addition, the effect of N-substitution of 8hydroxy aminotetralin derivatives is not the same for (R) and (S) enantiomers [40]. Possibly the N-alkyl substituents of (R) and (S) 8-hydroxy aminotetralin enantiomers do not occupy similar binding sites at the receptor. In this respect the fit of (R) and (S) 8-OH-DPAT (1 and 2) as presented by Mellin et al. [35] seems to be less satisfactory. In this fit (see Figure 5b) the aromatic parts and the dummy atoms in the direction of the nitrogen lone pairs coincide, while the hydroxy groups are located on opposite sides of the aromatic centre. It is also possible that both fits are partly correct (i.e. two binding modes for the (S)enantiomer), which might account for the partial agonist character of the (S)enantiomer 2. Structure-affinity relationships of the aminotetralin class are rather complex (Page *, Table 3). Substituents may directly affect the interaction with the receptor, but may also cause a conformational change of the compound. For example t r a n s (equatorial) C1 methyl substitution (see also Figure 6) as in compound 5 has a negative effect on 5-HT1A receptor affinity. However, compounds with t r a n s C1 substituents incorporated into a fused ring, like compounds 11 and 15, are considerably more potent than compound 5. Therefore, the C1 methyl group in 5 is unlikely to occupy essential receptor volume. Instead steric repulsion between the C1 substituent and C~-atoms of the propyl chain may be responsible for the low 5-HT~A receptor affinity of compound 5. As a result of this hindrance the compound may not be able to adopt the bioactive conformation. In the bioactive conformation as defined in ligand-fitting models, the nitrogen atom is equatorially positioned at the tetralin ring, which is in a half-chair conformation. The propyl C~-atoms are approximately in the plane of the aromatic ring, and the torsion angle Hcg-C2N-HN amounts 180". Indeed aminotetralins with t r a n s equatorial C1 methyl substituents were shown to prefer conformations with a torsion angle Hc2-C2N-H N of-60" [41, 42]. The aminotetralin t r a n s C1 substituents may also force the 8-hydroxy group into a conformation in which it cannot accept a hydrogen bond. This effect may play a role in the affinity decrease of compounds 5 and 15 when compared to compound 1. C1 substituents positioned c/s (axially) with respect to N, like in compound 3, or the small 4,6,6 and 5,6,6 fused ring analogues 7 and 9, are tolerated or even enhance 5-HT1A receptor affinity and maintain agonist activity. In the bioactive conformation of the aminotetralin moiety as defined in ligand models, these substituents do not interfere with either the N-propyl chains or the 8hydroxy group [42, 43]. The corresponding 6,6,6 fused ring analogue 13 is unexpectedly only weakly active. The calculated (MM2) energy minimum for
55 compound 13 deviates from the calculated (bioactive) conformation of compound 3 by a different direction of the nitrogen lone pair, which may account for its rather low 5-HTIA receptor affinity [42]. 5-HT1^ receptor ~ i t y is lowered by the introduction of axial 2- and 3- (cis) methyl (compound 17) substituents at the aminotetralin ring of (R) 8-OHDPAT, 1. These substituents may occupy essential receptor volume and thus prevent a good fit at the receptor. The 2- and 3- axially positioned methyl groups may also cause hindrance with (e.g. C~ or C~ atoms of) the essential propyl chain in the bioactive conformation. Similar explanations may account for the negative effect on 5-HT~^ receptor affinity of the trans (equatorial) C3 methyl substituents in compound 19. As aforementioned, the torsion angle Hc2C2-N-H N in the putative bioactive conformation amounts approximately 180". However, compounds with equatorial C3 methyl substituents were shown to prefer conformations with a torsion angle Hc2-C2-N-HN of 60 ~ [42]. It is also possible that aminotetralin trans C3 substituents cause direct hindrance with the receptor. This last hypothesis would account for the 10w 5-HT~A receptor affinity of compounds with trans C3 substituents incorporated into a ring system. In compound 27 the conformation of the essential propyl chain is constrained into a fused ring. Apparently, this conformation is not the same as the bioactive conformation of the free propyl chain.
Moon-22 X=CH2
OCH3
Moon-24 X=NH NH
HN
U24969
Figure 7. Structures of 5-HT~^ receptor ligands.
57 As a result, the lipophilic interaction may not be optimal and the ring may even cause steric hindrance with the receptor. Similar arguments may explain the low 5-HT1A receptor affinity of compound 23. In the model by Chidester et al. [36] the fit of several aminotetralin-derived compounds was discussed. The aminotetralin ring of most compounds was oriented similar to that of (R) 8-OH-DPAT. In contrast, ACA ('Angular, Cis, Ring Closure Away from O', see Figure 7) was fitted on the model in the same orientation as the (S) enantiomer of 8-OH-DPAT according to Mellin et al. [35]. In this fit, the 8-OH group of ACA is located close to the indole NH of serotonin. This orientation accounts for the preferred stereochemistry (R) of the compound. The preference for hydroxy over methoxy subsituents indicates that the 8-hydroxy~ group of ACA, like the pyrrole NH of serotonin, may act as a hydrogen bond donor. Compound Moon-24 (Figure 7) was also fitted in this orientation. The racemate of Moon-24 was reported to possess full agonist activity [44]. According to Chidester et al. [36], the activity of Moon-24 probably resides in the (R) enantiomer. In agreement with the role as a hydrogen bond acceptor in the model of Figure 4a, the oxygen atom was shown to increase 5HTIA receptor affinity 20-fold. The amide NH of Moon-24 seems to have no special function, as replacement with CH2 in Moon-22 was shown to be indifferent [44].
Fitting 5-HTIA agonists of other structural classes With the use of the defined common interacting groups, structurally different 5-HT1A agonists can be superimposed. For aminotetralins, tryptamines and ergotamines this can be done rather straightforwardly, as is shown in Figure 8a. The aromatic nuclei, the basic nitrogens and the nitrogen lone pairs of the compounds can be fitted. As a result, the hydroxy groups shown to be essential for high 5-HT1A affinity, occupy the same spacial position. Hibert and co-workers [34] argued that for RU24969 two different orientations with respect to ergotamines are possible. The benzene ring of this compound may be fitted on the benzene or the pyrrole ring of the indole moiety. Substitution patterns at the 2- and the 5-positions of the indole ring of tryptamines are similar to observations for RU24969 analogues [45, 46]. These findings justify a straightforward fit of the indole rings of the two classes, as shown in Figure 8b. The way of fitting other classes may be somewhat more ambiguous. For instance, the structure of MHA (page *, Table 6 (5-HT1A ligands)) can be considered an aminotetralin-analogue. However, MHA cannot be superimposed on 8-OH-DPAT in an atom-to-atom fit with full comprehension of its 5-HT1A profile. Westkaemper and Glennon [38] showed that MHA may well be fitted on 8-OH-DPAT as depicted in Figure 8c. In this orientation, the aromatic nuclei, the basic nitrogens and the nitrogen lone pairs of the two compounds coincide, and the hydroxy groups are located close to each other. The (R) enantiomer of MHA is preferred, as the (S) enantiomer of MHA cannot be superimposed on (R)-8-OH-DPAT 1 satisfying all four criteria. Thus the stereoselectivity of MHA regarding 5-HT~Aaffinity is accounted for.
58 a
b
d
J
F i g u r e 8.
Superimpositions of a) 8-OH-DPAT and 5-HT, b) RU24969 and 5-HT, c) MHA and 8-OH-DPAT, and d) arylpiperazines and 5-HT.
Fitting the class of arylpiperazines also appeared to be somewhat puzzling [47]. Like flesinoxan, many potent agonists that have been reported in this class owe their high 5-HT~^ receptor affinity to a large N4-substituent. Because of their flexibility they were left out of most modelling studies [34, 48]. Without these substituents, however, most compounds show only moderate 5-HT~A receptor affinity. In a recent study by Kuipers et al [49] new highly potent N 4unsubtituted arylpiperazines were reported. It was shown that arylpiperazines that display agonist affinity probably bind to the receptor in a relatively coplanar conformation with a plane angle of approximately 30 ~ between the aryl and piperazine rings. The fit as in Figure 8d was shown to agree best with structure-affinity relationships of bicyclic arylpiperazines.
Receptor-ligand interactions Models of GPCR are used for the study of receptor-ligand interactions, as for no member of the GPCR family experimentally determined high-resolution 3D structures are available. Most of these models are based on the high-resolution structure of bacteriorhodopsin which is not coupled to a G-protein, but shows
59 functional resemblance with the GPCR rhodopsin [4, 33, 50]. In these studies an attempt was made to combine receptor-binding data with knowledge from other experimental data concerning receptor structure and function. The strength and weaknesses of GPCR models were discussed and evaluated by Hibert et al. [33]. Several models that rationalize 5-HT~A receptor-ligand interactions have been published. As an example Trumpp-Kallmeyer et al. [51] presented a docking study of several natural ligands in their corresponding receptor models. The 5-HT~A receptor was one of the subtypes investigated. In the 5-HT~A receptor model Aspll6 in helix III interacts with the agonists basic nitrogen. The indole NH of serotonin points towards Gly164 in helix IV, while its 5-OH group forms a hydrogen bonding interaction with Thr200 of helix V. Aromatic interactions occur between serotonin and Phe204 and Phe362 in helices V and VI, respectively. Kuipers et al. [52] rationalized affinities of 5-HT~A agonists of several structural classes (e.g. tryptamines, aminotetralins, aporphines) with a docking study of these compounds in a 5-HT~A receptor model. Figure 4 shows a comparison of interactions as defined in this model (b) which were based on mutation data, with those from ligand-fitting models (a) [34, 36, 38]. The space in the ligand models that may contain large substituents at the basic nitrogen atom, overlaps with the available space in the core of the 5-HT~A receptor model. Sterically hindered positions in the receptor map derived from ligandfitting, coincide with the positions of the backbones of helices IV and V. The hydroxy group interacts with Thr200 in helix V. This residue has a dual character as it contains a hydroxy and a methyl group which enables the formation of hydrogen bonds and hydrophobic contacts, respectively. Thus methoxy groups in the 5-position of tryptamines and the 8-position of aminotetralins may also have favourable hydrophobic contacts with this residue. The indole NH of tryptamines (e.g. serotonin) as well as the hydroxy groups of compounds homochiral with (S) 8-OH-DPAT may form a hydrogen bonding interaction with Ser199 in helix V. In the model by Kuipers et al. [52], the lipophilic pocket which is essential for the high 5-HT~A receptor affinity of 8-OH-DPAT may consist of the residue combination Valll7-Cysl20 (see Figure 2). In 5-HT2 receptors Cysl20 is replaced by a polar Ser. According to the receptor model, the propyl chain of 8-OH-DPAT does not attribute to affinity for 5-HT 2 receptors because a lipophilic pocket is lacking, in 5-HT1B.D receptors Valll7 is replaced by a more bulky Ile, which may cause steric hindrance. This hypothesis is corroborated by SAR data, which indicate that 8-OH-DPAT has low affinity for 5-HT 2 receptors because it lacks a pyrrole ring, while steric hindrance of its propyl substituents causes low affinity for 5-HT m and 5-HT~D receptors [53, 54]. The area in which aromatic groups are favourable in ligands, is surrounded with aromatic residues in the receptor model. For instance, the second benzene ring of MHA may have an additional aromatic interaction with residues Phe361 and Phe362 in helix VI [52]. The binding mode of MHA was also subject of a docking study by Hedberg et al. [55], who analyzed differences in aporphine binding to 5-HT~A and D2
6O receptors. In this model, the basic nitrogen interacts with Asp116 and the essential benzene ring of MHA has an aromatic interaction with Phe362 in helix VI. The selectivity of MHA for 5-HTI^ receptors is explained by the C10methyl substituent of MHA being located in a lipophilic cavity unique for the 5HT1A receptor. The binding site of agonists appears to be rather similar in all three receptor models, as several residues found in the active sites are the same (see Figure 2). For instance, Phell2, Aspll6 (TM III), Gly164 (TM IV), Ser199, Thr200 (TM V) and Trp358, Phe361, Phe362 (TM VI) are involved in agonist binding in two or all three of the models. However some differences exist. In ligandmodelling studies it was shown that MHA can be fitted on 8-OH-DPAT and serotonin with overlap of the hydroxy groups (see Figure 4). In the model by Hedberg et al. [55] the hydroxy group of MHA interacts with Ser199. However, in the models by Trumpp-Kallmeyer et al. [51] and Kuipers et al. [52] the hydroxy group of the agonists interacts with Thr200. This last interaction accounts for the effect of Thr200-~Ala substitution which completely abolishes serotonin affinity [5]. This effect is not explained by the Hedberg model [55]. In the model by Kuipers et al. [52] Ser199 interacts with the indole NH of serotonin. The occurrence of this interaction is supported by SAR data of tryptamines [56, 57] and mutation data [5]. In this aspect the model differs from that of Trumpp-Kallmeyer et al. [51] in which the indole NH is directed towards Gly164. This last model provides no explanation for the effect of Ser199-~Ala substitution which decreases 5-HT1A receptor affinity for serotonin. The model by Hedberg et al. [55] explains the difference in 5-HT1A/D2 receptor binding profile of MHA. The C10-methyl group of MHA may bind in a lipophilic pocket formed by Ala203 and Ile205 of the 5-HT~A receptor. This lipophilic [5~pocket is absent in D2 receptors because Ala203 is replaced by a Ser residue. However, in the model by Kuipers et al. [52] a similar lipophilic pocket that can accommodate the C10-methyl group may be formed by Aia203, Thr200 and Leu366 [49]. This lipophilic pocket is also absent in Dg. receptors as these residues have been replaced by two polar serines and an isoleucine, respectively. Thus both orientations of MHA as in the models by Hedberg et al. [55] and Kuipers et al. [49, 52] account for its selectivity for 5-HT1A with respect to D2 receptors. Probably further mutation experiments will be required to elucidate the exact binding mode of this compound.
Antagonist models The pharmacophore derived by Hibert et al. [58] by superimposition of antagonists of different structural classes contains the same elements as the agonist pharmacophore, namely an aromatic ring and a basic nitrogen atom. As might be expected, the spacial requirements differ from the agonist pharmacophore: d= 5.6/~ and h= 1.6~. Unfortunately, the model was built with only moderately active compounds. For validation of this model more SARs of the concerning classes should become available.
61
oo
"r
Figure 9. Schematic representation of a) a ligand model for aryloxypropanol amines by Langlois et al. [59] and b) interactions of this class of antagonists in a 5HT~A receptor model by Kuipers et al. [52].
62 Figure 9a shows a schematic representation of a model of aryloxypropanolamines, which are potent ~-adrenoceptor antagonists but also display high affinity for 5-HT~A and (rodent) 5-HT~B receptors [59]. This model seems to be in good agreement with the suggested binding site of these compounds in the 5-HT~^ receptor model by Kuipers et al. [52] (Figure 9b). The preference for lipophilic and aromatic ring substituents R may be explained by the surrounding with aromatic (Tyr96, Tyr390, Trp387 and Phell2) and lipophilic aliphatic (Met92 and Leu43) residues in the receptor model. The sterically unfavourable region in the ligand model coincideswith the backbone of helix I in the receptor model. The (double) interaction of the oxypropanol moiety with the essential Asn386 in helix VII makes it very unlikely that these compounds are capable of occupying the area between the helices III, V and VI in which agonists bind. Thus the antagonistic properties of this class may be explained.
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