Ligand design for somatostatin receptor isoforms 4 and 5

Article abstract of DOI:10.1016/j.ejmech.2018.11.030

The somatostatin receptor (SSTR) isoforms, SSTR-4 and SSTR-5 are targets in numerous disorders and diseases. Although there has been some success in achieving selective isoform inhibition, structure-based drug design and development in this area has faced a challenge, mainly attributed to the lack of availability of SSTR-4 and SSTR-5 crystal structures. Previous structure activity relationship (SAR) studies have included work on non-peptide peptidomimetics or β-turn peptidal peptidomimetics where side chains of lysine, tryptophan, and phenylalanine (i.e. functional epitopes) are presented on a scaffold or molecular framework. However, there could be more structural information that would help design ligands selective for one or more of these isoforms. Here, we include synthesis of new mimetics and include their evaluation as ligands for SSTR-4 and SSTR-5. Inhibitors based on small to larger sized scaffolds (ManNAc, iminosugars, Eannaphane macrocycles, acyclic and cyclised peptide structures) are compared. These scaffolds have been grafted with side chains of lysine, tryptophan, and phenylalanine or similar bioisosteres/pharmacophoric groups. A new macrocycle as well as an iminosugar derivative show 5-fold or greater selectivity for SSTR-4 over SSTR-5. A new glycopeptide presenting GlcNAc showed ～6 fold selectivity for SSTR-5, which contrasted with the non-glycosylated peptide. A number of non-peptide dual inhibitors (K_i values of 0.58 μM to 5 μM) were also identified. Conceivable molecular interactions of these inhibitors were studied with newly constructed homology models of SSTR-4 and SSTR-5 isoforms.

Full text of DOI:10.1016/j.ejmech.2018.11.030

Accepted Manuscript
Ligand design for somatostatin receptor isoforms 4 and 5
Arvind Negi, Jian Zhou, Sinclair Sweeney, Paul V. Murphy
PII:
S0223-5234(18)30989-9
DOI:
https://doi.org/10.1016/j.ejmech.2018.11.030
EJMECH 10887
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 May 2018
Revised Date: 8 November 2018
Accepted Date: 9 November 2018
Please cite this article as: A. Negi, J. Zhou, S. Sweeney, P.V. Murphy, Ligand design for somatostatin
receptor isoforms 4 and 5, European Journal of Medicinal Chemistry (2018), doi: https://doi.org/10.1016/
j.ejmech.2018.11.030.
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ACCEPTED MANUSCRIPT
Ligand Design for Somatostatin Receptor Isoforms 4 and 5
Arvind Negi, Jian Zhou, Sinclair Sweeney, Paul V. Murphy*
School of Chemistry, National University of Ireland Galway, University Road, Galway, H91
TK33, Ireland
*email: paul.v.murphy@nuigalway.ie
Highlights
•
•
•
•
Small to large sized peptidomimetics were synthesized
They all contain Phe, Trp, Lys sidechains or similar bioisosteres
An iminosugar derivative showed 5-fold or greater selectivity for SSTR-4 over SSTR-5
A new glycopeptide presenting GlcNAc and new macrocyclic derivative showed ~5-6
fold selectivity for SSTR-5
•
Homology models of SSTR4 and SSTR-5 were constructed, evaluated and are made
available
Graphical Abstract
Abstract
The somatostatin receptor (SSTR) isoforms, SSTR-4 and SSTR-5 are targets in numerous
disorders and diseases. Although there has been some success in achieving selective isoform
inhibition, structure-based drug design and development in this area has faced a challenge,
mainly attributed to the lack of availability of SSTR-4 and SSTR-5 crystal structures. Previous
structure activity relationship (SAR) studies have included work on non-peptide peptidomimetics
or β-turn peptidal peptidomimetics where side chains of lysine, tryptophan, and phenylalanine

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(i.e. functional epitopes) are presented on a scaffold or molecular framework. However, there
could be more structural information that would help design ligands selective for one or more of
these isoforms. Here, we include synthesis of new mimetics and include their evaluation as
ligands for SSTR-4 and SSTR-5. Inhibitors based on small to larger sized scaffolds (ManNAc,
iminosugars, Eannaphane macrocycles, acyclic and cyclised peptide structures) are compared.
These scaffolds have been grafted with side chains of lysine, tryptophan, and phenylalanine or
similar bioisosteres/pharmacophoric groups. A new macrocycle as well as an iminosugar
derivative show 5-fold or greater selectivity for SSTR-4 over SSTR-5. A new glycopeptide
presenting GlcNAc showed ~6 fold selectivity for SSTR-5, which contrasted with the non-
glycosylated peptide. A number of non-peptide dual inhibitors (Ki values of 0.58 µM to 5 µM)
were also identified. Conceivable molecular interactions of these inhibitors were studied with
newly constructed homology models of SSTR-4 and SSTR-5 isoforms.
Keywords: Somatostatinergic system, Diverse Scaffolds, Synthesis, Ligand Based Design,
Somatostatin Receptor 4, Somatostatin Receptor 5
Introduction
The human somatostatin receptors (SSTRs) belong to the G-protein coupled receptors (GPCRs)
and have 5 isoforms (SSTR-1 to 5), which closely resemble each other in structural homology
and functional efficacy [1-4]. Their high structural resemblance is also linked to their
synchronised roles in numerous cellular homeostases and in several disorders, based on their
tissue specific isoform localisation. Additional interest has been placed in SSTR-4 and SSTR-5
in recent years. Agonism of SSTR-4 is believed to be relevant in Alzheimer disease [5],
influencing memory strategies in the human brain [6]. Targeting SSTR-4 selectively also
represents a promise for non-opioid pain control, the latter successfully shown by clinically
studied agent J2156, which is a potent selective inhibitor of this target [7-9]. SSTR-4 is also
believed to have a role in the migration of hepatic oval cells [10]. On the other hand, SSTR-5 has
found roles in proliferation in pancreatic cancer [11], neuroendocrine tumours [12] and glucose
homeostasis [13]. Therefore, the identification of compounds which have preferential selectivity
or differential binding for these receptors is important and is tied in with the identification of
agonists or antagonists and there are a number of implications [14-16]. These receptors have not
been crystallised to date and this hinders structure-based drug design for them. However, ligand-
based design strategies, have established some key important features that a ligand should
possess, to recognise these receptors. Accordingly, studies on the binding of somatostatins
(SRIFs), especially its tetradecapeptide form (SRIF-14, see figure 1) and its N-terminally
extended peptide form (SRIF-28) with SSTRs, have indicated that tryptophan-8 (trp8) and
lysine-9 (lys9) residues in these structures are recognised by all isomeric forms of SSTR, while
phenylalanine-6 (phe6) is highlighted as being specifically important for SSTR-4 activation [17-
22].

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Figure 1 Structures of SST-14 and selected peptidomimetics with high binding affinities for SSTRs. Important
pharmacophoric groups are shown in blue, purple and red.
To date, various non-peptide, and peptide analogues of somatostatin, with
pharmacophoric groups have been synthesized and evaluated against SSTR-4 and SSTR-5, and
their activity ranges from nM to mM; some of these molecules are shown in Figure 1. It is
unclear why these molecules [23-29], have varying degree of affinities. In this research, a set of
peptidomimetics based on pyranose, iminosugar (multi-hydroxylated piperidine), macrocycle
and peptidyl scaffolds were synthesised. To each scaffold was grafted pharmacophoric groups
which are identical or bioisosteric to those found in amino acid sidechains (Figure 2 and Figure
3). Two of these molecules have show preferential selectivity for SSTR-4 over SSTR5.
Docking to respective homology models of the proteins is included as part of this work.
Results and Discussion
Compounds designed to target SSTRs. Syntheses of some compounds used in this study has been
reported earlier (see Figure 2) and these include the iminosugars [30-33], benzomacrolactones
[34] and pyranoside 3 originally designed by Hirschmann and co-workers [35]. New compounds
based on the pyranose ManNAc, the Eannaphane macrocycle, as well as acyclic/cyclic peptidyl
scaffolds are shown in Figure 3. The basic design concept involved using the functional groups,
inherent in the scaffolds, to graft pharmacophoric groups, and thus defining the points of
attachment for amino acid side chains or their bioisosteres. For the pyranoses or iminosugars
and benzomacrolactones, like 9 and 10, these were inspired by natural product ring systems
found in nature. Whereas 13-15 are not natural products, to the best of our knowledge, their core
scaffolds can be considered to be ‘natural product like’, in that they are chiral macrocycles and
have functional groups found in natural products. The pharmacophoric groups were placed at

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distances from each other on the scaffolds approximating to those in SST-14. Thus there are 3-5
bonds between atoms to where the pharmacophoric groups are attached. In addition, peptidyl
mimetics 16-18 are included, which are similar in structure to octreotide.
Figure 2 Structures of compounds previously synthesised in-house and Hirschman’s pyranoside 3. The colour
codes are used to display the relevant amino acid side chain or their bioisosteres corresponding to those found to be
important in somatostatin ligands.

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Figure 3 Structures of various new potential somatostatin mimetics included herein. The colour codes are used to
display the relevant amino acid side chains or their bioisosteres corresponding to those found important in
somatostatin ligands
Synthesis of somatostatin mimetics based on 2-deoxy-2-acetamido-D-mannopyranose
The earlier work of Hirshmann and co-workers on synthesis of glucopyranose-based
somatostatin ligands encouraged us to synthesize new 2-deoxy-2-acetamido- -mannopyranose
D
(ManNAc) based mimetics (Fig. 3), where the amine of the 2-deoxy-2-aminomannopyranose
could be used for grafting an isostere of the trp sidechain. In addition the C-3 alcohol was used
as the point of attachment for the lys sidechain. The spacing between the trp isostere and lysine
side chain is equal to that found in the benzomacrolactone 10. In addition the STol group can
potentially mimic the phe side chain. The synthesis of the ManNAc derivatives was initiated by
Lewis acid promoted glycosidation reaction of 19 [36] to give the α-thioglycoside 20.

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Subsequent de-O-acetylation and acetalisation gave 21. Next a TIPS group was introduced at the
C-3 OH group to give 22, and its subsequent reduction using the Staudinger reaction gave amine
23, needed for the grafting. In the latter reaction the utilisation of Me3P was found to be more
efficient than the use of Bu3P. The coupling of 23 with a protected indole acetic acid derivative
gave 24. The TIPS group was removed from 24 to give 25. Monoalkylation reactions of 25
were investigated and the use of (E)-1,4-dibromobut-2-ene in the presence of silver oxide and
tetra-N-butyl ammonium iodide (TBAI) gave the desired product 26 in 89% yield; these reagents
and conditions were found superior to the use of NaH in DMF (no product obtained) or the use
of NaH in the presence of TBAI in dichloromethane (50%). Next, we found that attempts to
carry out a Gabriel reaction with PhthK were unsuccessful and led to decomposition. However,
the use of Boc2NK gave the useful intermediate 27 (Scheme 1) [34].
Scheme 1 Synthesis of intermediate 27
The attempted TFA induced removal in one pot of the benzylidene group and Boc groups from
27, resulted in the formation of the Schiff base 28. In contrast, the step by step removal of the
benzylidene group giving 29 followed by Boc removal using TFA afforded the desired
peptidomimetic 11 (Scheme 2).

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Scheme 2 Synthesis of 11
The alkene of 27 was converted to 30 using catalytic hydrogenation and then the stepwise
removal of protecting groups gave the ManNAc derivative 12 via 31 (Scheme 3).
Scheme 3 Synthesis of 12
Synthesis of somatostatin mimetics based on macrocyclic scaffolds with an embedded
monosaccharide
The synthesis of natural product like macrocyclic compounds was carried out via 33, 36 and 37
which were prepared as previously described [37]. Alkylation of the galactose 2-OH group of
33 gave 34/35. Next the Cu(I) catalyzed azide-alkyne cycloaddition of 35 with 36/37, gave 1,4-
triazoles 38/39 [38, 39]. Sequential oxidative cleavage of the alkenes of 38/39, gave
dialdehydes, which then after double reductive amination cyclisation [40] gave 40/41.

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Subsequent removal of the protecting groups, gave 14 and 15 as shown in Scheme 4. Analogous
procedures involving 34, and 36 with tryptamine gave 13 (see supporting information for
details).
Scheme 4 Synthesis of 14 & 15
Somatostatin mimetics based on peptides.
Our investigation also explored peptides, which potentially had the β-turn as found in
somatostatin, along with the incorporation of the key residue sequence Phe-Trp-Lys. Kelly and
co-workers had outlined the stabilization of reverse β-turns with the inclusion of a glycosylated
asparagine (i) two or three places after a phenylalanine residue (i+2 and i+3) through a
carbohydrate-π interaction [41, 42]. For this reason, we synthesized a glycosylated
peptididomimetic 18 (SMS3) with this feature. The glycosylated 18 and other related peptidyl
mimetics were prepared from aspartate derivative 43 [43], prepared according to the previously

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reported procedures in high yield [44-46]. Solid phase peptide synthesis based on the rink amide
resin, was then used to give 16-18 (SMS1-3) (see supplementary information) [47] after
deacetylation of the acetylated glycopeptide and subsequent purification using by high-
performance liquid chromatography.
Scheme 5 Structure of 43
Binding affinities, molecular modelling and structure activity relationship
The binding affinity assays were performed against two somatostatin receptor isoforms (SSTR-4
and SSTR-5) and inhibitory constants (Ki) determined and these are shown in Table 1. Most of
molecules based on the different scaffolds displayed similar affinities, in most cases in the low
micromolar range, for both isoforms with a limited number of exceptions where > fivefold
selectivity differences were observed. The macrocycle 13 showed preferential selectivity for
SSTR-5 over SSTR-4 while the iminosugar 8 had greater selective for SSTR-4 compared to
SSTR-5. The iminosugar 7 and the glycosylated peptide 18 were the only agents which showed
moderate preferential selectivity for SSTR-5 (Table 1). Iminosugar 7 differs from analogues 5
and 6, which have similar affinities for both isoforms, in that 7 contains free hydroxyl groups.
Peptide 18 differs only from 17 due to the presence of the GlcNAc residue linked via the
asparagine and 17 displayed similar activity for both isoforms.

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Table 1 Binding affinities (Ki) of peptidomimetics for SSTRs: SSTR-4 [48]: Binding studies
were carried out at Cerep (www.cerep.fr). According to Cerep’s procedures, they were
performed with cell membranes from transiently transfected COS-1 cells as described [49]. 10
µg of membrane protein was incubated in 10 mM Hepes (pH 7.5), 5 mM. MgCl2, bacitracin (20
jig/ml), 0.5% bovine serum albumin, and 125I-labeled [Tyr11]-somatostatin-14 (30,000 cpm) with
various concentrations of unlabeled somatostatin 14 (1 µg) and compounds of interest for 2 h at
room temperature. Later, scintillation counting method was used for detection; SSTR-5 [50]:
The SSTR-5 gene was cloned into pCMV6c expression vector [51] and transfected into COS1
cells. 20 µg of membrane protein preparation was incubated in 500 µL of Na+-free binding
buffer (10 mM HEPES, 1% bovine serum albumi, 5mM MgCl2, 1mg/ml bacitracin, pH 7.45)
containing approximately 10 pM of [125I-Tyr11]-somatostain-14 alone or with somatostatin-14
and compounds of interest at various concentrations for 2 hrs at room temperature. Later,
scintillation counting was used for detection. (detailed experimental data are provided in
Supplementary Information).
Somatostatin
mimetic
Scaffold
type
Ki, SSTR-4 (µM)
Ki, SSTR-5 (µM)
Pyranose
>100
1.1
>100
2
3 [35]
4
5
Pyranose
not available
5.0 ± 0.66
1.3 ± 0.08
5.1 ± 0.91
23 ± 1.39
>100
Iminosugar
Iminosugar
Iminosugar
Iminosugar
Iminosugar
Macrocycle
Macrocycle
Pyranose
4.4 ± 0.89
1.9 ± 0.37
5.4 ± 0.60
>100
6
7
3.2 ±0.57
0.58 ± 0.23
1.9 ± 0.41
2.1 ± 0.26
6.8 ± 1.02
21 ± 1.47
>100
8
9
1.1 ± 0.25
3.2 ± 0.71
3.9 ± 0.23
12 ± 1.35
4.1 ± 0.88
>100
10
11
12
13
14
15
16
17
18
Pyranose
Macrocycle
Macrocycle
Macrocycle
Peptide
>100
>100
7.2 ± 0.73
20 ± 1.77
>100
>100
10 ± 1.12
3.4 ± 0.52
Peptide
Peptide
In order, to hypothesise how the selectivities of these ligands might be influenced by scaffold
and pharmacophoric groups, homology models of SSTR-4 and SSTR-5 were developed and
utilised in docking. For the homology modelling, template-based modelling was implemented,
which was based on accessed templates obtained from a BLASTp search. A selection was made
based on complementarity with respect to the sequences of SSTR-4 and SSTR-5 [52, 53]. A
protein sequence of the human delta opioid 7-transmembrane receptor (PDB: 4N6H [54],

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resolution = 1.80 Å, 48% sequence identity, 44.01% similarity, 72% coverage) was selected for
SSTR-4 and a sequence of the nociceptin-orphanin FQ peptide receptor (PDB: 5DHH [55],
resolution 3.0 Å, 41.76% sequence identity, 40.08% seq. similarity, 70% coverage) was selected
for SSTR-5.
These were retrieved from the Research Collaboratory for Structural
Bioinformatics - Protein Data Bank (RCSB-PDB).
The built homology models were evaluated for structural consistency in a qualitative manner.
Firstly, the RMSD value was compared between the homology model and the respective
template. These were within acceptable limits (0.887 Å for SSTR-4 and 0.609 Å for SSTR-5,
see Supplementary Information). Next Ramachandran plots were evaluated to investigate the
geometry of amino acid residues in the homology models. For SSTR-4 93.4% of the amino
acids were in the most favoured geometries and for SSTR-5 this was 96% see Supplementary
Information). The ERRAT plots, which are for the determination of errors in model building
indicate a high degree of confidence (83.45% for SSTR4, 91.20% for SSTR-5). The z-score
plots were obtained by the protein structure analysis tool (ProSA), as it evaluated the overall
model quality; a score of -3.87 was obtained for SSTR-4 as compared with -4.51 for the template
PDB file used (4N6H). A score of -3.08 was obtained for SSTR-5 as compared with a score of -
3.23 for the template PDB used (5DHH) [56]. These results provided assurance of a reasonable
structural quality of the constructed homology models.
Table 2 Comparative analysis of key residues within active site domain of individual isoforms
with the template amino acids.
Template SSTR4 SSTR5 Template SSTR4 SSTR5 Template SSTR4 SSTR5
Gln107
Asp110
Ile111
Val67 Leu96
Ser70 Gln99
Tyr131
Gly91 Gly120
Met94 Gln123 Gln280
Phe95 Phe124 Val283 Gln243 Asn268
Cys200 Cys162 Cys186 Leu301 Asn257 Tyr286
Val279 Phe239 Phe264
Met134
Tyr240 Phe265
Ala71 Asn100 Phe135
Trp116
Val126
Ile127
Trp76 Trp105
Val86 Val115
Leu87 Met116
Asp90 Asp119
Ile219
Thr179 Thr205 Arg302
Gly183 Gly209 Thr305 Leu261 Val290
Trp276 Trp236 Trp261 Tyr309 Tyr265 Tyr294
His258 Phe287
Ser223
Asp130
Underlined amino acids are highly conserved through the GPCR family; Italics denote the key residues involved in
antagonist/agonist binding interactions
To identify the active cavity on the surface of both proteins, the Molecular Operating
Environment active site detection tool (MOE 2015.1001) was employed [57, 58]. Nehrung et al
had performed mutation based studies on Asp90 (SSTR-4) and Asp119 (SSTR-5) in
transmembrane domain 3 (TM3) of both SSTR-4/5 isoforms, and this showed that an ionic
interaction with the positively charged lysine amine group in the side chain is important for the

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binding of endogenous somatostatin (SRIF-14) [59, 60]. However, Kontoyianni et al. suggested
two possible ligand binding modes, indicating one which makes a H-bond with Gln243 while the
other mode involves interaction with Asp90 with the latter also indicating that heteroaryl
moieties could engage in π-stacking within the hydrophobic domain constituted by Phe175,
Phe239, Trp171 and Tyr240 in the SSTR4 receptor [61]. Ozenberger and Hadcock reported a
single site tyrosine substitution for Phe265 in the region of transmembrane domain 6 (TM6) of
SSTR-5 and this resulted in altered ligand binding selectivity and loss of the binding preference
of SSTR-5 for SRIF-28 over SRIF-14 [62]. To the best of our knowledge, at the time of writing
of this manuscript, no more structural data information was available for these proteins and their
interactions with their ligands. This led us to compare the active sites in the homology models
with that of the template protein (human delta opioid receptor) used for generating the SSTR-4
homology model in the form where it was co-crystallised with its ligand. The template is also a
GPCR protein, which would help to evaluate whether amino acids in the active site could be
important for the binding of somatostatins and their mimetics (see Table 2). The binding cavity
was found to be relatively large, which is commensurate with its requirement to bind SST-14.
Smaller non-peptidic ligands could thus occupy different parts of this cavity. To explore these
possibilities, docking was investigated.
Firstly, a comparison of glucopyranoside 3 in SSTR-4 with peptidomimetics based on the
iminosugar scaffold was made. The iminosugar derivative 4 retained potency to an extent,
compared to 3, but had low isoform selectivity (Ki, SSTR-4/ SSTR-5 = 1.13, see Table 1). In the
docking, the interaction of 4 with receptor site was found to be highly influenced by π-stacking
interactions of its own aromatic rings (Fig. 4) as well as with residues in the receptor. These
were observed in both isoforms and may explain lack of selectivity observed.
Fig. 4 Compound 3 (green) exhibits a distinct binding mode, via interacting with Asp90 (2.21 Å) and aromatic π-π
interaction with Tyr18. Iminosugar 4 (purple) lacks selectivity, displaying π-π interactions in SSTR-4 (3.52 Å) and
in SSTR-5 (4.07 Å). 2D ligand interaction diagrams are in addition provided in the supplementary information.
A similar loss of preferential isoform selectivity was also observed (Ki, SSTR-4: SSTR-5 = 1.46, see
Table 1) when comparing 4 with 5. The similar π-stacking interactions of benzyl group aromatic
rings for both isoforms are again believed to be the major influence in there being little

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selectivity (see, Fig. 5), although there was a slight variation in the docked binding mode for 5,
when compared to 4. The acetylated analogue 6, containing acetates rather than aromatic
residues, showed low selectivity (Ki, SSTR-4: SSTR-5 = 1.05) to 4 and 5, which may be due to various
interactions in the orthosteric binding sites of both these proteins [63-65].
In contrast, the trihydroxylated 7 displayed selectivity towards SSTR-5 albeit with low potency
(shown in Fig. 5). Molecular modelling of 7 showed that a H-bond donor interaction with one of
the hydroxyl groups of the iminosugar core with the backbone of Asn268 of the receptor and
there was an interaction of the naphthyl group with Phe201; these may explain the selectivity
seen for 7 for SSTR-5. The modelled structure of 8 (more selective for SSTR-4) shows that its
amino group has a shared interaction with the backbone of Asp90-Gly91 (2.82 Å) residues of
SSTR-4 while its indole ring engages in H-bond donor interactions with the amide group of the
Gln243 (2.74 Å) side chain and π-π interactions with Phe239 (3.89 Å). These indicate that the
presence of the indole group is helpful in stabilising/adopting the bound structure of 8 to SSTR-
4; these interactions are not possible for SSTR5 and could account for the selectivity shown for
8.

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Fig. 5 Iminosugars shown in a docked interactive mode. Tri-O-benzylated 5 (Cyan) with SSTR-4 (π-π interactions
with His258 (3.67 Å) & H-bond donor interaction with Leu87 (2.29 Å)) and with SSTR-5 (H-bond donor interaction
with Thr205 (2.95 Å)); Compound 6 (pink) with SSTR-4 (H-bond donor interactions with the backbone of
Gly91(3.05 Å) and Leu87 (2.39 Å), π-π interaction with His258 (4.07 Å)) and with SSTR-5 (H-bonding donor-
acceptor interactions with Ala188 (2.19 Å) & Gln123 (2.32 Å)); Interaction of compound 7 (blue) with SSTR-5
(naphthyl functionality has π-π interaction with Phe201 (3.48 and 3.79 Å) and polar carbohydrate head has H-bond
donor interaction with Asn268 (2.44 Å)); Compound 8 (yellow) with SSTR-4 (H-bond donor interactions with
Asp90-Gly91 and Gln243). 2D ligand interaction diagrams are in addition provided in the supplementary
information.
Fig. 6 Docked mode of ManNAc derivatives: Compound 11 (green) with SSTR-4 (H-bond donor with Asp90 (1.81
Å) & π-π interaction with Phe175 (4.21 Å)) and with SSTR-5 (H-bond donor interaction with Met170 (2.85 Å),

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Gln123 (2.27 & 2.57 Å); Compound 12 (pink) with SSTR4 (H-bond donor interaction with Leu87 (2.81 Å) and
para-OMe-phenyl ring inserted into the hydrophobic cavity, constituted by the Tyr265, Trp236 and Phe239), and
with SSTR-5 (π-π interaction with Trp190 (3.76 Å & 4.05 Å) and H-bond with Gln123 (2.65 Å) respectively). 2D
ligand interaction diagrams are in addition provided in the supplementary information.
The binding modes of ManNAc derivatives, 11 and 12 (Fig. 6) showed interactions with both
receptors consistent with relatively low selectivity (Ki, SSTR-4: SSTR-5 ≈ 1.8, see Table 1). The
SSTR isoform’s active site cavities are large and this may contribute to poor selectivity observed
for these pyranose derivatives [66].
The benzomacrolactone based mimetics 9 and 10 (Fig. 7) which although having improved
affinities compared to iminosugar and pyranose-based scaffolds showed low selectivities for the
SSTR isoforms and this was also consistent with interactions observed in both binding sites.
Changing the structure of the macrocyclic scaffold to that found in 14 and 15 showed a complete
loss of affinity to both isoforms of these receptors, despite these scaffolds presenting naphthyl
and alkyl amine groups found in 5-7. On the other hand if the tryptophan side chain is
incorporated onto this type of macrocycle, even without the apparently required lysine residue as
in 13 then affinity is restored to a degree [67, 68]. Five-fold isoform selectivity was observed for
13 for SSTR-5 (see Fig. 7) and this is proposed to arise due a π-π interaction with its Tyr-294.

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Fig. 7 Interactive mode of macrocycles: (a) 9 (blue) with SSTR-4. The phenol moiety has π-π interaction with
Phe175 (3.60 Å), the butenylamine shows H-bond donation to the COOH group of Asp90 (3.19 Å) and the amide
carbonyl of the indole side chain has a H-bond acceptor interaction with Asn246 (2.93 Å)). For SSTR-5 the phenol
moiety has a π-π interaction with Trp190 (1.96 Å), a H-bond acceptor interaction with the amide group of Asn268
(1.91 Å), a π-π interaction of the benzyl and indole groups with Phe201 (2.58 Å) and Phe265 (2.35 Å & 2.34 Å)
respectively, and there are additional H-bond donor interactions of the butenylamine with the COOH group of
Asp119 side chain (2.13 Å)). (b) 10 (orange) with SSTR4: a benzene residue was parallel to Trp76 in a π-sacking
manner (3.46 Å) and a H-bond donor interaction was observed for the free hydroxyl group with Asp90. This
compound with SSTR-5 showed that the benzene residue has a T-shaped π-π interaction [69] with Trp105 (3.91 Å)
whereas the indole displayed a sandwich type π-π interaction [69] with Tyr286 (3.54 Å and 3.41 Å), whereas the
phenolic (OH) and macrocycle oxygen atom shows H-bond donor and acceptor interactions with Gln99 (2.11 Å)
and phenol of Tyr286 (2.17 Å) respectively); (c) 13 (brown) with SSTR-4 utilised H-bond donor/acceptor
interactions with Asp90 (2.12 Å) and Gln123 (3.09 Å) respectively, while it shows a H-bond donor interaction with
Tyr294 (2.67 Å) and π-π interaction with Tyr286 (4.48 Å) of SSTR-5. 2D ligand interaction diagrams are in addition
provided in the supplementary information.
Next, we compared the larger peptide ligands (16-18). Molecular modelling showed
three features. Firstly 16 occupied a greater space in the receptor than the other ligands shown so
far [70]. Constraining the peptide into a cyclic structure giving 17 reduced the overall size of the
cavity occupied by the peptide and also reduced conformational flexibility leading to improved
binding [71]. The presence of the GlcNAc residue in 18 led to an increase in selectivity for
SSTR-5 [72]. Interestingly, the GlcNAc unit of 18 showed intramolecular H-bond interactions in
both the binding poses to both SSTR-4 and SSTR-5 isoforms, which led 18 to adopt a different
conformation to 17. For SSTR-5, the indole of 18 was proximal to Phe201 (shown in Fig. 13)
and this interaction was not observed for the various non-peptide-based structures described
above. This indicates the potential utilisation of an alternative site which could be explored in
drug design (Fig. 8).

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Fig. 8 Peptides 17 (cyan) and 18 (orange) are docked to SSTR-4 (left) and SSTR-5 (right). In both cases, we found
number of peptide backbone intermolecular H-bond interactions. The amino acid functional epitopes (Lys, Trp, Phe)
along with the glycosylated amino acid (ball and stick representation in the first structure on left, SSTR-4) behaviour
with proteins, was primarily considered. As observed for SSTR-4, 18 and 17 spanned the wide binding cavities and
adopted unrelated binding modes. The 35.ii peptide backbone had H-bond acceptor/donor interactions with Asp90
(2.22 Å & 2.40 Å), Thr179 (2.75 Å), Gln243 (2.29 Å) and its Trp indole group displayed a H-bond donor interaction
with the backbone of Cys162 (2.80 Å). In the case of 18, the peptide backbone also showed H-bond acceptor/donor
interactions with Asp90 (3.02 Å) as well as with Cys162 (2.65 Å), Asn246 (2.54 Å), His258 (2.18 Å). The Lys
sidechain of SMS3 had H-bond acceptor interactions with the backbone of Asp253 (2.47 Å). However, in case of
SSTR-5, there was a similar binding pattern for both 17/18, as their peptide backbone interacted with the same
amino acids of the cavity. The 17 peptide backbone interacts through H-bond acceptor/donor interactions with
Ala188 (2.16 Å), Asn187 (2.99 Å), Tyr286 (2.80 Å), Gln99 (2.34 Å) & Met116 (2.72 Å). On the other hand, the 18
peptide backbone interacts through H-bond acceptor/donor interactions with Gln123 (2.54 Å), Ala188(2.76 Å),
Tyr286 (2.31 Å). The indole ring of 18 had π-stacking interactions with Phe201 (3.82 Å and 4.14 Å) while the
GlcNAc residue had H-bond acceptor interactions with Asn187 (2.80 Å). 2D ligand interaction diagrams are
provided in the supplementary information.
Conclusions and Future Perspectives
Various scaffold or scaffold-based ligand design for somatostatin receptors, including
iminosugars, ManNAc, (glyco)peptides and macrocycles inspired by natural products have been
explored as ligands for somatostatin receptors. Their binding affinities for somatostatin receptors
were determined and hypotheses for their modes of interaction with these receptors generated
using molecular modelling. This has included construction of homology models for SSTR-4 and
SSTR-5, which disclosed some key interactions with certain amino acid residues which, from the
docking experiments, was ligand dependent. The study showed that various pharmacophoric
groups could interact with these key amino acid residues in a wide ranging manner and this was
due to the large active site cavity. Many of these key amino acids are evolutionarily conserved
and only a few have been found to be mutated, and these are comprehensively listed in Table 2.
Because of the size of this cavity, smaller sized ligands, especially those based on the
(imino)sugars and the macrocycles, could adopt various different binding poses [73]. Some

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observations were also made in the case of the docked larger peptidyl somatostatin mimetics.
The peptide backbone of 17 bonded to SSTR-4 more often through H-bond donor interactions
which was very different to its proposed binding to SSTR-5. Study of the glycosylated 18
(shown in Table 3), indicated the possibility of two binding modes to SSTR-4 (indicated in Table
3) [61].
Moreover, it has been postulated previously that the interaction of ligands with His258 of SSTR-
4 could lead to agonist activity. This interaction was seen in the docking of the iminosugars 5
and 6, and glycosylated 18 [61]. However, we have not received bioassay support as of yet for
this for these compounds and this could form the basis for the design of new agonists.
In general, favourable interactions are predicted when the hydroxyl groups on various scaffolds
were not protected allowing these hydrophilic groups interact with polar surface areas of the
receptors. This was seen computationally in case of compounds 7, 8, 11-13 and 18 [74, 75].
This study has contributed to identification of new inhibitors and provides a basis for the design
and synthesis of ligands for SSTR-4 and SSTR-5 as well for generating hypotheses regarding
their modes of binding. The homology models provided can be useful for performing any further
structure-based ligand design for these receptors in lieu of crystal structures. More generally,
ligand-based design has potential to identify new pharmacophoric groups together with new
scaffolds that can identify new chemical entities that target large active sites in proteins.
Supplementary Information
Experimental details concerning synthesis and NMR spectra of final compounds. Somatostatin
receptor affinity Ki plots and measurements, Homology modelling data and related qualitative
plots and figures. 2D Ligand interaction diagrams.
Conflicts of interest
The authors declare no competing financial interest.
Author Contributions
AN generated the homology models and did the docking, interpreted the models and contributed
to drafting and refining the manuscript. JZ synthesised new compounds 11-15 and drafted
experimental data. SS synthesised new peptides 16-18 and drafted experimental data. PVM
planned the work with the other authors and contributed to drafting and finalising the
manuscript.
Acknowledgements
The material described herein was funded, in part, by the Irish Research Council (Government of
Ireland postgraduate scholarships to AN, JZ, SS) and, in part, by Science Foundation Ireland
(06/RFP/CHO032, 12/TIDA/B2371).

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