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Journal of Molecular Structure: THEOCHEM 816 (2007) 73–76

Comparison of the binding modes of TT-232 insomatostatin receptors type 1 and 4

Agnes Simon a,*, Gyorgy Keri b, Julianna Kardos a

a Department of Neurochemistry, Chemical Research Center, Hungarian Academy of Sciences, Pusztaszeri ut 59-67, H-1025 Budapest, Hungaryb Peptide Biochemistry Research Group of Hungarian Academy of Sciences in the Department of Medical Chemistry,

Molecular Biology and Pathobiochemistry, Semmelweis University, POB 260, H-1444 Budapest, Hungary

Received 7 February 2007; received in revised form 26 March 2007; accepted 2 April 2007Available online 7 April 2007

Abstract

The somatostatin analogue cyclopeptide TT-232 was docked to homology models of somatostatin receptors type 1 and 4. Calcula-tions have been performed by applying H-bonding (subtype 1: Asp137-Lys5, Gln291- DPhe1, Gln291-Cys2; subtype 4: Asp122-Lys5)and distance (subtype 4: His294-Thr7) constraints with the GOLD docking procedure. Docking showed overlapping TT-232 backboneresidues. Differences were found, however, in the position of aromatic amino acids DTrp4, DPhe1 and Tyr3 of TT-232, allowing fordifferent binding modes and functions to be performed by somatostatin receptors type 1 and 4. In accordance, TT-232 did not affectbasal GABA release associated with somatostatin receptor type 1 function.� 2007 Elsevier B.V. All rights reserved.

Keywords: TT-232; Somatostatin receptor type 1; Somatostatin receptor type 4; Docking; GPCR

1. Introduction

D-Phe-c[Cys-Tyr-D-Trp-Lys-Cys]-Thr-NH2 (TT-232), acyclopeptide analogue of somatostatin is a drug candidatein phase II clinical trials [1]. Somatostatin receptor sub-types 1–5 are members of the G protein coupled receptorsuperfamily and mediate inhibitory effects of somatostatinon secretion and proliferation, varying according to thereceptor subtype and tissue localization [2,3].

Somatostatin receptor subtypes are classified accordingto their binding profiles. Somatostatin analogues such asoctreotide, lanreotide, vapreotide and MK678 bind withhigh affinity to subtype 2 and with intermediate affinityto subtypes 3 and 5 (subgroup I). By contrast, their bindingaffinities for subtypes 1 and 4 (subgroup II) are low [3,4].Ligand binding induces the activation of G-proteins fol-lowed by the activation of various pathways [3,4].

0166-1280/$ - see front matter � 2007 Elsevier B.V. All rights reserved.

doi:10.1016/j.theochem.2007.04.003

* Corresponding author. Tel.: +36 1 438 1100/263; fax: +36 1 325 7554.E-mail address: simonagi@chemres.hu (A. Simon).

All somatostatin receptor types are able to inhibitadenylyl cyclase, thereby decreasing cAMP levels, via apertussis toxin-sensitive protein (Gia1–3). Peptide secre-tion is inhibited by somatostatin through a decrease inintracellular calcium ion levels. This effect is either dueto the opening of potassium channels or to the closureof voltage-dependent calcium ion channels. Somatostatinreceptor subtypes 2, 3, 4 and 5 act as activators of theinward-rectifying potassium ion channel via Gia2 or

Gia3, while somatostatin type 1 rather mediates its inhi-bition. Subtypes 1, 2 and 5 negatively couple to volt-age-dependent calcium ion channels. Finally, allreceptor types are coupled to various phospholipase Cisoforms [3,4]. Antisecretory activity of somatostatin alsoshows subtype-specificity, as type 1 triggers the inhibitionof growth hormone prolactin and calcitonin, type 2 isresponsible for inhibiting secretion of growth hormoneadrenocorticotropin, insulin, glucagon, interferon-gammaand type 5 inhibits the secretion of growth hormone,adrenocorticotropin, insulin, glucagon-like peptide-1 andamylase [3,4].

74 A. Simon et al. / Journal of Molecular Structure: THEOCHEM 816 (2007) 73–76

It is proposed, that the antitumour and the antiinflam-matory actions of TT-232 are mediated through somato-statin receptor subtypes 1 [1,5] and 4 [3,6], respectively.In the rat central nervous system somatostatin receptorsubtype 1 is localized primarily presynaptically [7] modu-lating neurotransmitter secretion [8] and references cited],while somatostatin receptor subtype 4 is dominantly post-synaptic [7]. The effect of TT-232 on tumour growth hasbeen compared among various tumour types [9], andamong different application routes [10]. The mechanismof antitumour activity of TT-232 was recently shown toinvolve nuclear translocation of pyruvate kinase M2 lead-ing to apoptotic cell death [11]. The involvement of sst4receptors in the antiinflammatory activity of TT-232 hasbeen further confirmed using the recently developed non-peptide inhibitor, acting selectively on sst4 receptors [12].

Somatostatin binding to cloned somatostatin receptorsexpressed in HEK 293 cells had been characterized in mea-surements of [125I]Tyr11-somatostatin displacement givingIC50 values of 0.36 ± 0.08 nM, 0.22 ± 0.04 nM,0.35 ± 0.09 nM and 1.5 ± 0.2 nM for subtypes 1, 2, 3 and4, respectively [13]. Somatostatin receptor subtypes 1–5expressed in CHO cells showed different somatostatin affin-ity profile, characterized by binding inhibition constantvalues of 1.3 ± 0.4 lM, 3.6 ± 0.9 lM, 3.1 ± 0.8 lM,0.20 ± 0.01 lM, 1.5 ± 0.3 lM, respectively [14]. Somato-statin and TT-232 showed similar and high-affinity binding(IC50 � 0.1 nM) to synaptic somatostatin receptors isolatedfrom purified rat hippocampal synaptosomal membranefractions [15], suggesting that native and HEK 293-expressed somatostatin receptor subtypes 1 and 4 may havesimilar binding profiles.

A better elucidation of the different binding modes pos-sibly engaged by the somatostatin receptors type 1 and 4has been attempted by comparing binding interactionsbetween TT-232 and somatostatin receptors type 1 and 4at the molecular level. To characterize somatostatin recep-tor type 4 binding and its relation to somatostatin receptortype 1, TT-232 was docked to homology models of somato-statin receptors type 1 and 4. The fact, that all somatostatinreceptor subtypes share an Asp residue in the third trans-membrane domain found essential in binding somatostatinanalogues, reviewed by Patel, [16] has been used in calcula-tions performed using the GOLD docking procedure.Associated with a presynaptic somatostatin receptor type1 [7] function, basal [3H]GABA release from rat hippocam-pal synaptosomes [8] and the effect of TT-232 thereon havealso been explored.

2. Materials and methods

High resolution crystal structures of somatostatin recep-tors have not been resolved as yet therefore they are mod-elled based on the high resolution crystal structure ofbovine rhodopsin [17]. Three-dimensional models of 277human G-protein coupled receptors have been builtrecently, using an automated procedure, called GPCRmod

[18]. These models were constructed on the basis of thebovine rhodopsin template, containing transmembranehelices without loop regions. Homology models of somato-statin receptors type 1 and 4 of this database were kindlyprovided by Prof. D. Rognan.

Previously determined three-dimensional structures ofTT-232 disclosed by NMR measurements performed inH2O–D2O mixture [15] have been used. NMR data showeda common overall structure and a rigid backbone for the12 lowest energy conformations of the cyclopeptide. Toobtain a starting conformation for docking in a non-aqueousenvironment, one of these structures was energy minimizedin SYBYL, using a dielectric constant of 4 and a 15 A

0cut-

off for non-bonding interactions, until the RMS gradientreached 0.1 kcal/mol/A

0. This structure was docked into

the homology model of type 1 and type 4 somatostatin recep-tors, using the Genetic Optimisation for Ligand Docking(GOLD) program (GOLD, CCDC Software Ltd., Cam-bridge, UK) with default settings. To rank docking resultsthe GoldScore scoring function was used. A similar protocolhas been successfully applied recently for docking metabo-tropic glutamate receptor ligands into the receptor [19] aswell as catalytic site inhibitors into phosphodiesterase 6[20]. The binding site of TT-232 was centred on Asp137OD1 atom of somatostatin receptor type 1 (and the corre-sponding Asp122 of somatostatin receptor type 4) as it isknown to be essential for binding of the Lys residue ofsomatostatin analogues in all somatostatin receptor sub-types, (reviewed by Patel [16]). The Asp-Lys H-bond con-straint was explicitly included in the docking runs: all Hatoms of the Lys side-chain were offered to form H-bondwith both side chain oxygen atoms of the Asp residue inthe receptor. Additional constraints were applied to selectthe most likely position: H-bonding constraints were appliedin somatostatin receptor type 1 (Gln291 side chain O-DPhe1amid NH, Gln291 side chain NH-Cys2 main chain O); whilea distance constraint was applied for somatostatin receptortype 4 (the distance between His294 ND1 and Thr7 sidechain O was set to remain between 3 A and 4 A, using thedefault spring constant of 5). The highest scoring ligand incomplex with its receptor (scores 47 and 44 in somatostatinreceptors type 1 and 4, respectively) were subjected to a shortenergy minimization in each case, to remove bad contacts.Energy minimization was performed in SYBYL, using theMMFF94 force-field with MMFF94 charges, a dielectricconstant of 4 to mimic a non-aqueous environment [15]and 12 A as the cut-off for non-bonding interactions, untilthe RMS gradient reached 0.05 kcal/mol/ A.

Effects of somatostatin, (R)-baclofen and TT-232 onbasal [3H]GABA release has been measured in purified syn-aptosomal fraction as described earlier [8].

3. Results and discussion

The highest scoring positions of TT-232 in somato-statin receptor types 1 and 4 are compared on Fig. 1.Backbone atoms of TT-232 overlap in the two cases,

Fig. 1. Comparison of TT-232 in the homology models of somatostatinreceptor subtypes 1 and 4. Green: TT-232 docked into the homologymodel of somatostatin receptor subtype 1. Blue: TT-232 docked into thehomology model of somatostatin receptor subtype 4. Red: Asp137,Asp122 of somatostatin receptor subtypes 1 and 4, respectively. (Forinterpretation of color mentioned in this figure the reader is referred to theweb version of the article.)

Table 1Effect of drugs on the basal [3H]GABA release from rat hippocampalsynaptosomes (N = 4)

Drug Basal [3H]GABA releasea

None (control) 0.301 ± 0.00210 lM (R)-Baclofen 0.279 ± 0.002b

10 lM SST 0.283 ± 0.003b

10 lM SST + 100 lM (R)-Baclofen 0.281 ± 0.003b

10 lM TT-232 0.299 ± 0.00210 lM TT-232 + 100 lM (R)-Baclofen 0.282 ± 0.003b

a [3H]GABASUPERNATANT/([3H]GABASUPERNATANT + [3H] GABAPELLET)[8].

b P < 0.05 compared to the control.

A. Simon et al. / Journal of Molecular Structure: THEOCHEM 816 (2007) 73–76 75

however, the positions of the side chains show differ-ences, most obviously in the case of aromatic residues,especially DTrp4 (marked on Fig. 1). Tyr 3 interactswith residues of helices 3, 4, 5 and 6 of somatostatinreceptor type 1 and those of helices 4 and 5 of somato-statin receptor type 4. DTrp4 interacts with helices 3, 6and 7 when bound to somatostatin receptor type 1 andinteracts with residues on helices 3, 5 and 6 when boundto somatostatin receptor type 4. DPhe1 interacts withhelices 5 and 6 in both cases.

Besides the somatostatin receptor-specificAsp137(Asp122)-ligand(Lys5) H-bonding interaction,additional constraints for receptor–ligand binding interac-tions have been introduced to enrich the runs in high scor-ing hits (see also Section 2). These were: H-bondingconstraints for Gln291-DPhe1 and Gln291-Cys2 in subtype1 and a distance constraint for His294-Thr7 in subtype 4.Interestingly, Gln291 of helix 6 was previously shown tobe important in differentiating type 2 and type 1 somato-statin receptor ligands [21,22].

In our previous attempt to delineate binding crevice res-idues for TT-232, it has been docked to a homology modelof rat somatostatin receptor type 1 [15] prepared using theSwiss-Model server [23]. The most important differencesbetween the current method and the previously appliedone reside in the use of H-bonding and distance con-straints, built in the GOLD docking program. Also, thelength of the helical regions differs slightly in the two cases,

however, the two models are similar in their backbone con-formations (RMSBACKBONE = 1.41). Somatostatin recep-tor models obtained from the Rognan laboratory werechosen in the current study, because the orientation ofthe side-chain residues were carefully optimised using theGPCRmod procedure [18]. This is clearly reflected in ahigher RMS value, when all atoms of the overlappingregions are overlayed (RMSALL ATOMS = 2.51).

In spite of differences between the present and the previ-ously applied homology models of somatostatin receptortype 1 including the length of the helices and the positionsof the protein side-chains, Asp137, Gln291, Phe287 andTyr313 have been recognised as binding crevice residuesin both types of homology models. Binding interactionsdelineated above suggest that both receptors can serve astargets for TT-232 actions.

Although loop residues showing low sequence homol-ogy with the template may also participate in ligand bind-ing, only the highly homologous helical regions were usedfor model building [10,13]. In this study, models of somato-statin receptor types 1 and 4 are based on the experimen-tally determined bovine rhodopsin structure [17], whichwas crystallized in its ‘‘inactive’’ (cis-retinal-bound) form.As a consequence, the models likely represent the antago-nist binding state of somatostatin receptors, whose helicesmay undergo a rearrangement when agonist ligands arebound.

Effect of TT-232 on basal [3H]GABA release wasexplored and compared with the effects of somatostatinand (R)-baclofen. While basal [3H]GABA release was sig-nificantly decreased by adding somatostatin (10 lM) or(R)-baclofen (10 lM), TT-232 (10 lM) did not alter basal[3H]GABA release (Table 1). Also, effects of somatostatinand TT-232 on basal [3H]GABA release were distinguish-able by co-application of saturating concentration(100 lM) of (R)-baclofen (Table 1). These findings suggestthat TT-232 does not affect release-regulating presynapticsomatostatin receptor type 1 [7,8] in the hippocampus.The result is in accordance with the distinguishable accom-modation of TT-232 in the binding crevices of somatostatinreceptors type 1 and 4, allowing for no functional responseof presynaptic somatostatin receptors type 1 to be elicitedby TT-232.

76 A. Simon et al. / Journal of Molecular Structure: THEOCHEM 816 (2007) 73–76

4. Conclusions

TT-232 is able to bind to somatostatin receptor types 1and 4 and occupies a similar binding crevice in these recep-tor types. Despite the overlapping peptide backbones,differences in the accommodation of aromatic amino acidsDTrp4, DPhe1 and Tyr3 can also be observed, when thebest scores are compared. These results highlight theimportance of the positions of the aromatic amino acidsof somatostatin receptor ligands in distinguishing bindingmodes of receptor subtypes and allowing different func-tions to be activated. In accordance with these dockingresults, TT-232 did not affect experimentally determinedbasal GABA release mediated by somatostatin receptortype 1, although it binds to it.

Acknowledgements

We thank Prof. Didier Rognan (Universite Louis Pas-teur, Strasbourg) for providing the somatostatin receptormodels and Dr. Karoly Antal (Department of Neurochem-istry) for assistance. Financial support of Grants 1/A/005/2004 NKFP MediChem2, OTKA T049478 and Trans-porter Explorer AKF-050068 are acknowledged.

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