5-HT1E and 5-HT1F receptors

Serotonin Receptors and their Ligands B. Olivier, I. van Wijngaarden and W. Soudijn (Editors) 9 1997 Elsevier Science B.V. All rights reserved.

141

5-HTm and 5-HT1FReceptors G. McAllister and J.L. Castro Merck, Sharp & Dohme Research Laboratories, Neuroscience Research Centre, Terlings Park, Eastwick Road, Harlow, Essex CM20 2QR, United Kingdom.

INTRODUCTION The recent advances in gene cloning techniques has led to an explosion of information about the design of the nervous system, and has altered the approach of scientists to the process of drug discovery. The ability to isolate individual genes encoding particular receptors has revealed a level of complexity in the brain not always appreciated by the traditional techniques of pharmacology. It seems that most neurotransmitters have not just one target receptor but many, and as this book demonstrates, serotonin, in particular, has a plethora of receptors to interact with. The challenge for scientists today are to understand why so many receptors have evolved, understand their function in vivo, and use this information to develop novel drugs to interact with these receptors in a more directed, selective way than has been possible until now. As discussed in previous chapters five 5HTl-like receptors, termed 5-HTIA, 5-HT1B, 5-HT1D- 5-HTIF have been described. The 5-HTlc subtype is generally agreed to belong in the 5-HT2 family and the 5HTIB receptor in rodents is the species homologue of the 5-HTID~ receptor in man [for review see 32]. All of these 5-HTl-like receptors have relatively high affinity for serotonin and when activated can inhibit forskolin stimulated adenylyl cyclase activity in transfected mammalian cells. The question of why nature has evolved several receptors that have similar affinity for the endogenous ligand, serotonin, and apparently couple to the same effector system is particularly intriguing and awaits the development of subtype selective ligands to find an answer. This chapter will deal with two of these 5-HTl-like receptors: 5-HTIE and 5HT1F. Radioligand binding studies initially identified a putative receptor, termed 5-HT1E, in human cortex and putamen which was able to bind [3H]5-HT even in the presence of concentrations of5-carboxamidotryptamine (5-CT) and mesulergine that would block 5-HTIA.D sites [1]. Recently, two receptor subtypes, 5-HT m [2,3,4] and 5-HTIF[5,6,7] that have pharmacological profiles similar to this 5-HT m binding site have been cloned by several groups. RECEPTOR STRUCTURE

The discovery of the existence of a G protein coupled receptor superfamily [8] has greatly facilitated the isolation and characterisation of serotonin receptor clones. With the exception of 5-HT3 receptors (see chapter 7), all of the cloned

142 serotonin receptors are members of this family and analysis of their primary structures reveals the characteristic seven putative transmembrane domains and large regions of sequence homology indicating their common evolutionary origins. The 5-HT1A receptor was the first of the 5-HTl-like receptors to be cloned and this was achieved because of its homology with the [~2-adrenergic receptor (see chapter 2). Since then several groups, have used variations of homology cloning to obtain the other 5-HTl-like receptor clones. All of these receptors are encoded by intronless genes, a feature which distinguishes them from the 5-HT 2 receptors, and a feature which has allowed the direct amplification of novel receptors from genomic DNA using polymerase chain reaction (PCR) techniques. This has accelerated the characterisation of this subfamily of receptors because clones can be isolated without identifying a tissue source that contains the receptor. The first group to isolate a 5-HT1E receptor clone did so by homology screening of a human genomic DNA library using oligonucleotide probes derived from the cloned 5-HT~A and 5-HTlc receptors [3]. The authors termed this human gene $31 and demonstrated that when expressed in mammalian cells it mediated the inhibition of adenylyl cyclase activity. However, no radioligand binding data accompanied this finding so $31 was not identified as a 5-HTIE receptor initially. However, soon afterwards it emerged that several groups had independently isolated this gene and confirmed that its pharmacology was very similar to the 5HT~E binding site found in human brain [2,4,9]. More recently another receptor was cloned, again by several groups, which also had a 5-HT1E-like pharmacology, but was clearly encoded by a separate gene. The mouse version of this gene was termed 5-HT1E~ [5], whereas the human gene was termed 5-HTIF [6] or MR77, a 5-HT~E-like receptor [7]. However, based on sequence comparisons some differences in the pharmacology of this receptor it has been proposed that it is sufficiently different to warrant its own subtype therefore it will be referred to as the 5-HTIF receptor in the rest of this chapter. As can be seen from the dendrogram in figure 1 the 5-HT~E and 5-HTIF receptors are more closely related to each other than to other 5-HT receptors. The 5-HTIE receptor, within the highly conserved transmembrane (TM) domains, exhibits approximately 52%, 40%, 64% and 70% amino acid identity to the 5-HT1A, 5-HT m, 5-HT1D subtypes, and 5-HTIF receptors, respectively. The 5-HT~F receptor exhibits approximately 53%, 40%, 63% and 70% amino acid identity to the 5-HT1A, 5-HTlc, 5-HT1D subtypes, and 5-HT1E receptors, respectively. Both the 5-HT~p. and the 5-HTLF receptors are of similar length (365 and 366 amino acids respectively) and share other common features with the serotonin receptor family, such as conserved aspartate residues in transmembrane (TM) regions 2 and 3 and a single conserved serine residue in TM5, potential glycosylation sites in the NH2-terminal domain, and consensus phosphorylation sites particularly in the third intracellular loop regions. Comparing the sequences of the serotonin family as a whole it can be seen that those most related at the amino acid sequence level (eg. 5-HT~D~and 5-HT1D~ or 5-HT 2 and 5-HTlc) also share similar properties (eg. same affinity for 5-HT, same effector systems, similar pharmacological profile). These closely related members (or subtypes) display amino identity values of 75% or more in their TM regions, whereas less related members (eg. 5-HT1A and 5-HT2) display

143 values closer to 45% amino acid identity. The 70% identity between the 5-HTI~. and 5-HTIv receptors makes it difficult to decide on structural criteria alone whether they belong to the same subtype or represent two new subtypes. Indeed, following this analysis, the 5-HT m and 5-HT~ receptors could both be considered distantly related members of the 5-HT~D subtype.

5-HT2

[2A]

5-HT1c [2C] 5-HT1D 5-HT1B 5-HT1F 5-HT1E 5-HT1A Figure 1. Dendrogram. The human 5-HT1A [10], 5-HTI~. [2], 5-HTIF [6], 5HTID~[ll], rat 5-HTIB [12], 5-HT~c [13] and 5-HT~ [14] receptors were compared based on their sequence similarity. The relative lengths of the bars are inversely proportional to their sequence homology.

This raises the interesting question of why so many 5-HTi-like receptors have evolved and been maintained in the genomes of several different species. There are two schools of thought on this subject. The first suggests thatbecause they exist, they must be doing something important or there would be many more pseudogenes of the family. This implies distinct functions for each subtype despite the fact that they are often expressed in the same regions and even cell types (see later). Possible differences in function may arise from different midpoints of activation by serotonin under physiological conditions, different efficiencies of coupling to adenylyl cyclase inhibition, coupling to other effector pathways (eg. directly to ion channels) or discrete spatial or temporal expression. A second way of looking at this question turns the argument on its head. Perhaps there are so many different receptors because it is relatively easy to duplicate intronless genes

144 and any one of the receptors can replace the others functionally. It has been postulated that many of the genes encoding the G protein linked receptor family evolved from a single precursor gene (possibly an opsin gene) that lost its introns approximately 1 billion years ago [15]. Since then, gene duplication events have resulted in many related genes. These intronless genes are so small that they are more likely to be functional, when duplicated, than large intron-containing genes and have, therefore, diverged into a large related family of functional receptors. These events would, therefore, allow an increase in the diversity of receptors available and correspondingly in the level of complexity possible, particularly in the brain, conferring an evolutionary advantage. The extended subfamily of 5-HT~like receptors m a y represent duplicated genes that have not yet evolved to have differentfunctions, or that they represent duplicated genes with discrete functions. The development of subtype specific ligands that can dissect the function(s) of these receptors in vivo will enable us to decide which of these two arguments is correct.

R E C E P T O R LOCALIZATION

As mentioned earlier,5-HT~z receptors were firstdescribed as a binding site in h u m a n cortical tissue and putamen homogenates that displayed high affinity for [3H]5-HT even in the presence of concentrations of 5-CT and mesulergine that would block binding to the other 5-HTl-like receptors [1]. Taking advantage of this, Beer and colleagues carried out autoradiography studies in rat and guinea pig brain (see figure 2) indicating that 5-CT insensitive 5-HT~-like receptors (5HTlz / 5-HTI~), as defined with [3H]5-HT in the presence of 100nM 5-CT and 300nM mesulergine, are most densely concentrated in the caudate putamen and olfactory tubercle [16, 33]. Interestingly,in the guinea pig but not the rat, these receptors are also abundant in the claustrum, a littlestudied brain area thought to be involved in visual attention [17]. This study cannot distinguish between 5HT~z and 5-HT~F receptors as both have low affinity for 5-CT and mesulergine. [aH]Sumatriptan however, having a high affinityfor the 5-HT~F receptor and low affinity for 5-HTIz receptors (Table 2) can be used to distinguish between both receptors [34-37]. A comparison with the 5-HTIE site in h u m a n cortex which also shows low affinityfor sumatriptan suggests that this siteis predominantly 5-HTIE rather than 5-HT~F [2].Another quantitative autoradiography study [18],looking at 5-CT sensitive and insensitive sitesin h u m a n brain regions indicated that while 5-HTID and 5-HT~z sites were relativelyequal and abundant in frontal cortex and globus pallidus (140 - 220 fmol/mg) there was ten times more 5-HTIE sites (224 fmol/mg) than 5-HT~D sitesin the putamen (28 frnol/mg).As pointed out above this binding site is likely to represent 5-HT~E binding rather than 5-HTIF binding but does not rule out the possibility of yet more as yet uncharacterised receptors contributing to the total binding sites.In situ hybridization studies of the 5-HT~z receptor in h u m a n brain revealed expression in cortical areas, caudate, putarnen and amygdala areas [19]. All these areas have been shown to contain 5-CTinsensitive 5-HT~-like binding sites.

145 More information is available for the 5-HTI~ receptor as its mRNA expression has been examined using both PCR and in situ hybridization techniques. Amlaiky and colleagues reported that the mouse 5-HT1F receptor mRNA (5-HT1E~) was not detectable on Northern blots of poly(A)*RNA suggesting a relatively low level of expression in mouse brain [5]. However, using more sensitive PCR techniques, a signal was observed in spinal cord and brain, predominantly in forebrain. Further analysis of the mouse brain, using in situ hybridization techniques, showed that a signal was only found in the pyramidal neurons of the CA1-3 layers of the hippocampus. In contrast, Adham and colleagues carried out in situ hybridization studies in the guinea pig [6], and found 5-HTI~ mRNA in lamina V of frontal cortex, again in large pyramidal cells as well as moderate labelling in the hippocampus. Moderate labelling was also detected over layer VI nonpyramidal neurons. In layer V and VI, the strongest signal was found in dorsal sensorimotor neocortex and in cingulate and retrosplenal cortices. Pyramidal cells in the piriform cortex and large neurons in the raphe nuclei were also heavily labelled and in contrast to the mouse study some labelling was seen in the granule cells of the dentate gyrus. The differences in distribution observed by these two groups may represent species differences or differences in sensitivity in their respective in situ hybridization studies. The regional distribution of guinea pig 5-HT~F receptor mRNA is very similar to that of 5-HTI~ receptors labelled with [3H]sumatriptan [34, 35]. The detection of 5-HTa~ transcripts in the dorsal raphe nucleus indicates a possible role as an autoreceptor regulating neurotransmitter release. However, 5-HTm~ and 5-HTm~ transcripts are also expressed in this nucleus. Whether one or all of these receptors can be autoreceptors will be answered only when selective ligands for these receptors become available. In the same study, the authors also examined the distribution of 5-HT~F mRNA in various human tissues by PCR techniques. Intriguingly, as well as in brain, they also found transcripts in the uterus and the mesentery. The possible role of this receptor in uterine or vascular function is very interesting, particularly as the 5-HT~ receptor has such high affinity for the antimigraine drug, sumatriptan. The mechanisms involved in a migraine attack are still unknown but the intense unilateral and throbbing headache characteristic of migraine is likely to be vascular origin as the brain itself is largely insensitive to pain. Two models have been proposed suggesting that either migraine is caused by vasodilation of intra and/or extracranial arteries leading to activation of sensory nerves and pain, or that the initiating factor is a neuronal disorder leading to neurogenic inflammation of the same blood vessels (reviewed in 20). In both cases the 5-HT~D receptor subtypes have been implicated as the target of efficacious antimigraine compounds such as sumatriptan based on a correlation of their affinities at the 5HTID receptors and their clinically effective doses. However, the discovery of the 5-HT~F receptor and its expression in at least some vascular tissues raise the possibility that 5-HT1~ receptors may also play a role in this disorder and could therefore be a potential target for novel, more selective antimigraine drugs [38].

146

Figure 2. Autoradiography of the distribution of 5-CT-insensitive sites in a coronal section of guinea pig brain. Sites were labelled with [3H]5-HT in the presence of 100nM 5-CT and 300nM mesulergine to block out other 5-HT,-like receptors. Highest density of labelling was observed in the claustrum (C1), olfactory tubercle (Tu) and caudate putamen (Cpu). Data provided by MS Beer. R E C E P T O R BINDING ASSAYS Receptor binding assays of the 5-HT,E and 5-HT,F receptors using tissue preparations are made difficult because no selective compound is available for use as a radioligand. Analysis of the cloned receptors expressed in mammalian cells is much simpler because the cell lines chosen for expression have no endogenous 5-HT,-like receptors present. Therefore it is possible to use a non-discriminating radioligand to characterise the pharmacological profile of the receptor. These studies are very useful, of course, because they may allow the experimenter to identify binding conditions specific for a particular subtype that would be useful for tissue studies. In the case of the 5-HT,E / 5-HT,F (or 5-CT insensitive) binding site, the binding assay predated the characterisation of the cloned receptors by

147 some three years. As mentioned previously, Leonhardt and colleagues first suggested in 1989 the existence of 5-HT1E receptors when they found evidence of heterogeneity in the pharmacology of the 5-HT m binding site in human brain [1]. They carried out radioligand binding studies using [~H]5-HT at a concentration (2nM) that would allow binding to all of the 5-HTl-like receptors known at the time (i.e. 5-HT1A - 5-HT1D). To examine the 5-HT,D binding only, they included in their assay lmM pindolol (to block 5-HT,A and 5-HT m receptors) and 100nM mesulergine (to block 5-HTIc and 5-HT 2 receptors). However, two binding sites were observed in the presence of these blockers. One of these sites demonstrated high affinity for 5-CT and ergotamine, consistent with the known pharmacology of the 5-HT1D site and the second site demonstrated low affinity for these two compounds. The high affinity (or 5-CT sensitive) site represented some 55% of the total specific [3H]5-HT binding in these human cortical tissue homogenates and the low affinity (5-CT insensitive) site comprised the other 45% of binding sites. Further analysis of the low affinity site, termed 5-HT m was carried out by replacing pindolol with 100nM 5-CT in the binding assay. This concentration of 5CT would prevent binding to 5-HT m sites as well as to 5-HT1A and 5-HT m sites. These studies demonstrated that the 5-HTm binding site displayed a Ka of 5.3nM for [3H]5-HT, was GTP- but not ATP-sensitive and had a unique pharmacological profile, the most distinguishing feature being a relatively low affinity for 5-CT and ergotamine. Table 1 A comparison of the I~ values (nM) of serotoninergic ligands at the cloned human 5-HTm and 5-HTI~ receptors and the 5-HT,~ binding site in human cortex.

5-HT1E

5-HT 5-CT Sumatriptan Methysergide Methiothepin Ergotamine Metergoline

6 3300 2090 220 120 540 776

5-HT1F*

Human Cortex

10 717 23 34 650 171 341

6 2000 1300 170 1500 800 426

Values taken from McAllister et al. [2] and Adham et al. *[6]. A comparison of published I~. values for the cloned 5-HTIE [2,4,9] and 5-HT1F [5,6,7] receptors and the values originally found for the human cortex 5-HT1E binding site [1] shows that either or both cloned receptors could in principle

148 correspond to the native receptor. However, McAllister and colleagues extended the pharmacological analysis of the human cortex site in direct comparison with the cloned human 5-HT~E receptor [2]. In particular, this study demonstrated that in cortex the 5-HTIE site had a relatively low affinity for the antimigraine drugs sumatriptan ( ~ 1300nM) and ergotamine (I~. 800nM), a profile much closer to the cloned 5-HT~ receptor than to the cloned 5-HTI~ receptor as shown in Table 1. As previously noted, sumatriptan has approximately 100-fold higher affinity for the 5-HTI~ receptor than the 5-HT1~ receptor suggesting that inclusion of 200nM sumatriptan in future autoradiography studies would eliminate the potential problem of also labelling the 5-HT1F receptor. Further evidence that the "5-HTI~" site labelled in tissue is predominantly 5-HT~F. rather than 5-HT~F comes from similar displacement studies carried out by Beer and colleagues on a variety of species [21]. They demonstrated that in contrast to 5-HT which was mono-phasic, 5-CT and sumatriptan displayed very similar biphasic distribution curves when they were used to displace ['~H]5-HT binding (in the presence of cyanopindolol and mesulergine to block 5-HTIA, 5-HT~B and 5-HT~c receptors) in the cortex and caudate of dog, guinea pig, human, hamster, rabbit, pig and calf. The high affinity component of these biphasic curves corresponds to 5-CT and sumatriptan binding to 5-HT1D receptors and the low affinity component is likely to correspond to 5HT m receptors as 5-CT and sumatriptan show similar displacements. The proportion of sites with low affinity for 5-CT and sumatriptan would be different if significant numbers of 5-HT~p receptors were present. A contribution of the more recently discovered 5-HT receptors (5-HTn.~, 5-HT~b, 5-HT6 and 5-HT 7) to 5-HTm binding site can be ruled out based on their pharmacological profiles (see later Chapters). However, other, as yet undiscovered, subtypes obviously cannot be discounted. It is not immediately obvious how a similar strategy could be used to specifically label the 5-HTI~ sites in tissue preparations, so direct visualization of native 5-HT~.. receptor binding sites will require the development of more specific ligands. LIGANDS Although no selective ligands for 5-HTI,~ or 5-HT1F receptors have been reported so far, several interesting trends in structure-affinity relationships can be extracted from the published binding of several tryptamine derivatives and related analogues. For the purpose of the present discussion, comparisons will be made, where appropriate, with the 5-HT~D,~and 5-HT~t~ receptors because they present the highest homology with the 5-HT1~:.11.~receptors within the TM domains and, as mentioned earlier, the 5-HT1F receptor has been suggested as a potential target for the antimigraine drug sumatriptan. Selectivity with respect to other 5-HT receptors will not be discussed. Inspection of the data in Table 2 reveals that 5-HT remainsthe highest affinity ligand for both 5-HT1~~ and 5-HTI~.~ receptors reported to date, and that simple modifications of this structure can result in dramatic changes in affinity. Particularly notable is the 100-fold reduction in affinity on methylation of the 5-

149 Table 2 A p p a r e n t dissociation c o n s t a n t s (Ki values; nM) of various drugs for cloned h u m a n 5-HT1E, 5-HT1F, 5-HTIDa and 5-HT1D~ receptors. . Compound a

5-HTIE b

5-HT1F c

5-HT1Da d

5-HT1D~ d

5.0 (11")

10

3.9

4.3

Tryptamine

316

2409

86

521

5-MeOT

630

1166

4.8

34

5-BnOT

794

9.6

19

5-MeO-DMT

100

37

4.4

21

a-Me-5-HT

121"

184

211

133

2-Me-5-HT

817"

413

915

860

5-CT

3980

717

0.70

1.6

> 10,000 *

1613

13

42

1995

23

3.4

7.7

5-HT

DP- 5- CT Sumatriptan RU-24,969

63

..........

TFMPP

1995

1002

64

114

1-NP

207*

54

7.4

12

NAN-190

.....

203

194

652

> 10,000

73

5198

Ketanserin

> 10,000

Mianserin

100

Metitepin

126

Cyproheptadine

790

8-OH-DPAT

.......... 652

11

25

..........

3160

1772

120

260

89*

31

0.86

2.9

200 (228*)

34

3.6

25

Ergotamine

125

171

..........

Dihydroergotamine

316

..........

> 10,000

..........

Methylergonovine Methysergide

Bromocriptine Yohimbine

398

92

22

27

a For the s t r u c t u r e s of the compounds discussed in this article see Figure 3. b Values t a k e n from B e e r et al. [17]. c Values t a k e n from A d h a m et al. [6]. d Values t a k e n from W e i n s h a n k et al. [22]. * Ki values t a k e n from Zgombick et al.

[4].

150

/ MeO~~

NH2

NMe2

NH2

N

H 5-MeOT

N

H 5-BnOT

5-MeO-DMT

NH2

NH2

NR2

.o.~~~Me

O HO

H2N

Me H

H 5-CT: R= H DP-5-CT: R= npr

H ~XN

NMe2 MeHN._A ~

N a.Me-5-NT

2-Me-5-HT

jJ

.L

MeO ~~N

H

H RU-24,969

H Sumatriptan

Pindolol

H HO

H

TFMPP

8-OH-DPAT

1-NP

H _Nr--~N__~

F

~

N~N

k__/\ \OMe

O

O O

Ketanserin

NAN-190

Figure 3" Structures of compounds discussed in this chapter

151

N

SMe

~NMe Metitepin

Mianserin

Me Cyproheptadine ,,,~

HO.

0

H ~ N ~

,170 HC~ O."~N ~ N " Me H

N'MeH

H

Ergotamine

..,,,~ ipr

~

,.Me

H

R Methylergonovine: R= H Methysergide: R=Me

He O~I~'N~ ip~ H

Ph

O

N"•Me

.,,~ Ph

~N

..Me H

Br H

H

Dihydroergotamine

Bromocriptine

H

MeOOC OH Yohimbine

Figure 3 (continued)" Structures of compounds discussed in this chapter

152 hydroxy group of 5-HT (to give 5-methoxytryptamine, 5-MeOT) for both 5-HT,z and 5-HT,v , a transformation which has little consequence for 5-HT,D receptors. The fact that tryptamine (T) binds with the same affinity as 5-MeOT suggests that the 5-hydroxy functionality in 5-HT is acting as a hydrogen bond donor (and not an acceptor) group at 5-HT,~.,I.~ receptors (with Ser186 of 5-HT,E or Ser185 of 5HT1F in TM V?) [23-25] but as a hydrogen bond acceptor group at 5-HTID receptors [26] (compare 5-HT, T and 5-MeOT). In marked contrast to 5-HT,D receptors, 5carboxamidotryptamine (5-CT) also has low affinity for 5*HT,E,,F receptors, a result which would appear to indicate that the excellent hydrogen bond acceptor capability of its carboxamido group is being utilized when binding to 5-HT1D receptors but is not relevant for binding at 5-HT1E,,F. Interestingly, sumatriptan, which has comparably low affinity to 5-CT for the 5-HT,~ receptor, binds with high affinity to 5-HT~. Thus, at least in the latter case, effective complementarily (hydrogen bond interactions?) can be achieved with functionalities which are further away from C~ of the tryptamine. It is also noteworthy that large arylalkyl groups are tolerated at C~ of the tryptamine (compare 5-BnOT and 5-MeOT) although, by virtue of the similar affinities, the benzyl group of 5-BnOT does not contribute to binding. By direct analogy to 5-HT,D receptors, 2-methylation of the indole nucleus, as in 2-Me-5-HT, greatly reduces the affinity for both 5-HT,E and 5-HT,F receptors (70 to 80-fold) whereas ~-methylation of the ethylamino side chain (compare a-Me5-HT and 5-HT) is somewhat less detrimental (10 to 20-fold). Assuming that the ergot derivatives bind at the same site in the receptor as 5-HT, comparison of methylergonovine and methysergide, would appear to suggest that N 1methylation of tryptamines might be slightly unfavourable (2-fold) for 5-HT,E receptors but of little consequence for 5-HT,F. Similar trends were reported for 5-HT1D receptors [27] and should be easy to confirm with commercially available 1methyltryptamine. There is the indication, however, that not all modifications result in reduced affinities. In particular, N,N-dimethylation of 5-MeOT to give 5-MeOT-DMT improves the affinity to 5-HT,E by 6-fold to 5-HT,~ by 30-fold. The slightly detrimental effect of larger, N,N-di-alkyl groups for 5-HT~E,~ (compare 5-CT and DP-5-CT) could either reflect a limited space being available for binding at this part of the receptor (steric) or be a direct consequence of the increased conformational freedom of these groups (entropic). Moreover, replacement of the ethylamino side chain by a 1,2,5,6-tetrahydropyridine moiety affords a 10-fold improvement in 5-HT~E binding affinity (compare RU-24,969 and 5-MeOT). The binding of RU-24,969 to 5-HT,~ receptors has not been reported and is awaited with great interest. The fact that ergotamine and dihydroergotamine bind to 5-HT,E and 5-HT,F receptors, although with less affinity than to 5-HT1D, shows that there are regions of bulk tolerance at both receptors. The poor affinity of bromocryptine for 5-HT~z receptors may reflect the detrimental effect of 2-substitution on the indole nucleus as noted above. The presence of an indole moiety does not appear to be a requirement in order to produce moderate to high affinity 5-HTjE.,F receptor ligands. Thus, although a

153 simple arylpiperazine such as TFMPP has micromolar affinities for both receptors, the combination of a naphthyl nucleus and a piperazine ring as in 1napthylpiperazine (1-NP) results in a good mimic of the tryptamine core (compare 1-NP and T). In the case of the 5-HTI~. receptor, this replacement even leads to some 40-fold increase in affinity. The more elaborate 2-methoxyphenylpiperazine analogue NAN-190 also has respectable (200nM) affinity for the 5-HT1F receptor. Other unselective, non-indolic 5-HT receptor ligands which also bind with moderate affinity to 5-HT~E include the structurally related tricyclic/tetracyclic compounds metitepin, mianserin and cyproheptadine. Finally, ~-adrenergic agents such as pindolol bind with very low affinity to 5HTI~,~F receptors. This is perhaps not surprising in view of the fact that, in contrast to 5-HT~A and 5-HT~ receptors which bind [~-adrenergic antagonists with high affinity [28], the 5-HTiE and the 5-HT~.~ receptors lack a key residue in the seventh transmembrane domain (Asn385 in 5-HT1A and Asn351 in 5-HTm) which has been suggested to participate in hydrogen bond interactions with the aryl oxygen of [3-blockers. In the 5-HTtE receptor this Asn residue is replaced by Thr330 and by Ala333 in the 5-HT~ receptor. Indeed, it has recently been shown [29] that replacement of these two residues by Ash affords 5-HT~ and 5-HT~ mutants which bind pindolol and other ~-blockers with significantly improved affinities (>100-fold), although the binding of the endogenous neurotransmitter 5-HT is not affected. In conclusion, although no selective ligands are yet available for either 5-HT m or 5-HT~ receptors, the steadily increasing understanding of their molecular architectures through the combined utilization of pharmacophore mapping, receptor modelling and site directed mutagenesis studies will no doubt lead to the discovery of useful pharmacological tools in the near future. FUNCTIONAL ASSAYS Based on the high degree of sequence homology among the 5-HTl-like receptors and the characteristic long third intracellular loop and short carboxyl-terminal domain of both the 5-HT~ and 5-HT~ receptors it would be predicted that both subtypes are negatively coupled to adenylyl cyclase activity. This prediction was supported by the original characterisation of the cloned 5-HT1E and 5-HTI~ receptors expressed heterologously in various mammalian cell lines. Activation of 5-HT~E receptors in Ltk', Y-1 or HEK cells resulted in a relatively weak (20-35%) inhibition of forskolin-stimulated cAMP levels [2,4,9]. This weak inhibition may be due to a lack, or low levels, of the appropriate G-protein or other component of the signal transduction system being present in these cell lines. Indeed, increased levels of inhibition were observed by reducing the levels of free Mg ~ [9] or by expressing the 5-HTj~ receptor in BS-C-1 cells [30]. Intriguingly, in BS-C-1 cells expressing high levels of the receptor (5 pmol/mg of protein) activation of the receptor led to both the inhibition and potentiation of forskolin-stimulated cAMP accumulation. Pretreatment of cells with pertussis toxin or cholera toxin eliminated agonist induced inhibition and potentiation of cAMP levels respectively.

154 The potentiation of forskolin-stimulated cAMP accumulation appears to be a direct effect as no changes in PI metabolism or Ca2+mobilization were observed. Agonists displayed higher affinity for the inhibitory response suggesting an interesting potential mechanism of regulation of these receptors in which higher 5-HT concentrations would counter the initial inhibition of cAMP levels by stimulating cAMP production. The physiological significance of this finding is unclear. It seems to be a receptor density-dependent feature as cell lines expressing somewhat less receptors (2 pmol/mg of protein) only couple to the inhibition of cAMP levels. However, as the authors point out, there is likely to be both a high concentration of endogenous ligand and a high density of receptors present at the synapse. This is not the first description of 5-HTl-like receptors apparently coupling to more than one second messenger system. For example, cloned 5-HTxD receptors were recently reported to couple to both inhibitory adenylyl cyclase activity and the elevation of intracellular Ca2§ levels via pertussis toxin-sensitive G-proteins [31]. Further enlightenment awaits the development of sub-type specific ligands. The overlapping pharmacology of the 5-HT1D and 5-HTxE / 5-HT~F receptors makes it impossible, at the moment, to unambiguously identify the in vivo function of 5HT~z and 5-HT1F receptors. This problem is compounded by the fact that most ligands developed for these receptors have been agonists and are subject to the problems of receptor reserve in interpreting data. Ideally, subtype specific antagonists will be developed to give a clearer understanding of the functional roles of these receptors. Alternatively, the effects of antagonists can be mirrored by the development of transgenic mice devoid of particular receptors or by the application of antisense oligonucleotides to investigate the function(s) of these receptors in vivo. THERAPEUTIC APPLICATIONS Agonists The successful clinical use of s u m a t r i p ~ as an acute treatment for migraine headache has intensified interest in its mode of action. It was originally thought to be a relatively selective 5.HT~D receptor agonist (see previous chapter). However, that hypothesis turned out to be an oversimplification as sumatriptan also demonstrated affinity for the 5-HTu~, 5-HT m and 5-HT~v receptors in addition to the two 5-HT~D subtypes. It has poor affinity for 5-HTaE receptors so its action is unlikely to be mediated by that receptor subtype. The recent development of the 5-HT1D receptor antagonist, GR127935, may help clarify the role of these various receptor subtypes although the selectivity of this antagonist over the 5-HT~z and 5-HT~F receptors has not yet been reported. However, it may yet be that 5-HT~E receptor agonists are also useful in the treatment of migraine. The full elucidation of which 5-HT receptor subtypes are present on the target tissues of an antimigraine drug and their role(s) in mediating the proposed desirable effects of such a drug (cerebral vasoconstriction, inhibition of plasma extravasation) remains to be discovered. The role of 5-HT1v receptors in particular will be interesting as it has been shown to have a high affinity for sumatriptan (I~. 23nM) and a vascular

155 distribution [6]. It may also be that some of the less desirable effects of sumatriptan (coronary vasoconstriction etc.) could be reduced by avoiding activation of certain subtypes, therefore the distribution of 5-HT receptors in nontarget tissues such as coronary artery will also be of great interest.

Antagonists There are no clear therapeutic indications for 5-HT1E or 5-HTI~ antagonists so far. However, as this chapter has emphasized, the lack of selective antagonists makes it difficult to assign particular functions to a given receptor subtype. In general then, it appears that anything a 5-HT1D antagonist might be proposed for may also be a potential target for a selective 5-HTm or 5-HTIF compound. It is thought that treatment with selective serotonin re-uptake inhibitors (SSRIs), such as paroxetine or fluoxetine, leads to the facilitation of 5-HT neurotransmission. This is the proposed mechanism of action for the antidepressant properties of this class of drug. An alternative method of facilitating 5-HT neurotransmission is to block the inhibitory terminal 5-HT autoreceptor, normally activated by the release of 5-HT. It is proposed that blockade of this autoreceptor would stop the inhibition of 5-HT release, thus increasing synaptic 5-HT concentration and facilitating neurotransmission. The question is, which of these receptor subtypes can act as an autoreceptor? There is some evidence suggesting that a 5-HT~D receptor subtype is the autoreceptor (see previous chapter) and the expression of mRNA encoding both the 5-HT1D~ and 5-HTIDI3 receptors in the guinea pig dorsal raphe nucleus adds support to this idea. However, 5-HTI,.~ mRNA has also been found in this nucleus and the presence of 5-HT~E has not been excluded [14]. Therefore, it is possible that both 5-HT~,~ and 5-HT1~ receptors may act as autoreceptors and are still potential therapeutic targets for a novel antidepressant. However, no mutations in the human 5-HT~, receptor gene of patients suffering from schizophrenia and bipolar affective disorder were detectable, indicating that 5HTlr receptors are not commonly involved in the etiology of these diseases [39]. Interestingly, it appears that fluoxetine is now being successfully used to treat some patients suffering from anxiety. The mechanism of action of this effect is unclear. It could be that an autoreceptor antagonist could mimic this effect or it may be that fluoxetine treatment is causing down regulation of a postsynaptic receptor. Whichever is the case, the possible role(s) of 5-HTm and 5-HT~F receptors in anxiety should also be investigated.

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