Ring size of somatostatin analogues (ODT-8) modulates receptor selectivity and binding affinity

Article abstract of DOI:10.1021/jm701444y

The synthesis, biological testing, and NMR studies of several analogues of H-c[Cys³-Phe⁶-Phe⁷-DTrp⁸-Lys ⁹-Thr¹⁰-Phe¹¹-Cys¹⁴]-OH (ODT-8, a pan-somatostatin analogue, 1)

Full text of DOI:10.1021/jm701444y

2668
J. Med. Chem. 2008, 51, 2668–2675
Ring Size of Somatostatin Analogues (ODT-8) Modulates Receptor Selectivity and Binding
Affinity
Judit Erchegyi,† Christy Rani R. Grace,‡ Manoj Samant,† Renzo Cescato,§ Veronique Piccand,§ Roland Riek,‡
Jean Claude Reubi,§ and Jean E. Rivier*,†
The Clayton Foundation Laboratories for Peptide Biology and Structural Biology Laboratory, The Salk Institute for Biological Studies,
10010 North Torrey Pines Road, La Jolla, California 92037, and DiVision of Cell Biology and Experimental Cancer Research, Institute of
Pathology, UniVersity of Berne, Berne, Switzerland 3012
ReceiVed NoVember 15, 2007
The synthesis, biological testing, and NMR studies of several analogues of H-c[Cys3-Phe6-Phe7-DTrp8-
Lys9-Thr10-Phe11-Cys14]-OH (ODT-8, a pan-somatostatin analogue, 1) have been performed to assess the
effect of changing the stereochemistry and the number of atoms in the disulfide bridge on binding affinity.
Cysteine at positions 3 and/or 14 (somatostatin numbering) were/was substituted with D-cysteine, norcysteine,
D-norcysteine, homocysteine, and/or D-homocysteine. The 3D structure analysis of selected partially selective,
bioactive analogues (3, 18, 19, and 21) was carried out in dimethylsulfoxide. Interestingly and not
unexpectedly, the 3D structures of these analogues comprised the pharmacophore for which the analogues
had the highest binding affinities (i.e., sst4 in all cases).
Introduction
that binds to all of the five SRIF receptors (ssts) similar to
SRIF.24 DTrp8 and IAmp9 substitutions in the undecapeptide
des-AA1,2,5-SRIF, DAgl(NMe,2naphthoyl),8 or L-threo-ꢀ-Me-
Nal8 in the octapeptide des-AA1,2,4,5,12,13-SRIF resulted in a
selective agonist at sst1,25–29 a selective antagonist at sst3,30,31
and a selective agonist at sst4,24,32–34 respectively, by stabilizing
the active conformation at these receptors.
Somatostatin (SRIF)a is a major endocrine hormone with
multiple physiological actions1–11 modulated by one or more
of the five SRIF receptors.12–16 The biological role, as well as
the cellular distribution of each receptor subtype, is far from
being completely understood. Availability of receptor subtype-
selective ligands is still critical to our understanding of SRIF’s
physiological role. Numerous analogues are presently being used
in the clinic to manage a number of pathophysiological
conditions4,17–19 and as ligands for radiotherapy.20,21
The present study describes the synthesis, biological testing,
and NMR studies of several analogues of the des-AA1,2,4,5,12,13
-
[DTrp8]-SRIF (ODT-8, 1) scaffold. D-amino acids and/or
unnatural amino acids, which reduce or increase the flexibility
of the cyclic backbone, aiming to obtain ligands with high
binding affinity at the different receptors and sst-selectivity, were
used. We have synthesized a series of ODT-8 analogues in which
Cys at positions 3 and/or 14 (SRIF numbering) was substituted
with DCys, norcysteine (Ncy), D-norcysteine (DNcy),35 ho-
mocysteine (Hcy), and D-homocysteine (DHcy). NMR studies
of selected analogues identified structural features that may be
responsible for binding to more than one receptor.
Constraints which reduce the flexibility of peptide-backbones
or side-chain orientation have been identified, under certain
circumstances, to generate analogues with enhanced biological
potency and duration of action or selectivity, stability against
proteolytic breakdowns, and improved biodistribution and
bioavailability. Such conformational flexibility could allow
binding by an induced-fit mechanism, and the analogue could
exhibit a conformation accommodating two or more pharma-
cophores simultaneously.
We have shown that the introduction of DTrp8 in the
octapeptide des-AA1,2,4,5,12,13-SRIF22,23 resulted in an analogue
Results
Peptide Synthesis and Determination of the Stereo-
chemistry of Ncy in the Peptides. All of the analogues shown
in Table 1 were synthesized either manually or automatically
on a chloromethylated (CM) resin by using the t-butoxycarbonyl
(Boc)-strategy. Boc-Ncy(Mob)-OH, Boc-D/LNcy(Mob)-OH,35
Boc-Hcy(Mob)-OH, and Boc-DHcy(Mob)-OH were synthesized
in our laboratory;36 see details in the Experimental Section.
Peptides containing Ncy at the N-terminus were unstable during
hydrogen fluoride cleavage/workup conditions and were acety-
lated by an excess of acetic anhydride in dichloromethane on
the resin before hydrofluoric acid cleavage. All of the Ncy-
containing Ac-ODT-8 analogues (9–16) were synthesized by
incorporating racemic Boc-Ncy(Mob)-OH35 or on racemic Boc-
Ncy(Mob)-CM resin. The diastereomeric peptides were sepa-
rated by reversed-phase high-performance liquid chromatogra-
phy (RP-HPLC), and the absolute stereochemistry of Ncy in
the Ac-ODT-8 analogues was confirmed by the comparison of
the HPLC retention times and coelution of each diastereomer
with those of the analogues synthesized separately by using
* Corresponding author. Phone: (858) 453-4100. Fax: (858) 552-1546.
E-mail: jrivier@salk.edu.
†
The Clayton Foundation Laboratories for Peptide Biology, The Salk
Institute for Biological Studies.
‡
Structural Biology Laboratory, The Salk Institute for Biological Studies.
Institute of Pathology, University of Berne.
§
a Abbreviations: The abbreviations for the common amino acids are in
accordance with the recommendations of the IUPAC-IUB Joint Commission
on Biochemical Nomenclature (Eur. J. Biochem. 1984, 138, 9-37). The
symbols represent the L-isomer except when indicated otherwise. Additional
abbreviations: Boc, t-butoxycarbonyl; Bzl, benzyl; Z(2Cl), 2-chlorobenzy-
loxycarbonyl; CM, chloromethylated; CZE, capillary zone electrophoresis;
CYANA, combined assignment and dynamics algorithm for NMR applica-
tions; DHcy, D-homocysteine; DMF, dimethylformamide; DMSO, dimeth-
ylsulfoxide; DNcy, D-norcysteine; Hcy, homocysteine; HOBt, 1-hydroxy-
benzotriazole; IAmp, 4-(N-isopropyl)-aminomethylphenylalanine; IBMX,
3-isobutyl-I-methylxanthine; Mob, 4-methoxybenzyl; Ncy; norcysteine;
NOE, nuclear Overhauser enhancement; 3D, three-dimensional; OBzl,
benzyl ester; PROSA, Processing algorithms; rmsd, root-mean-square
deviation; RP-HPLC, reversed-phase high-performance liquid chromatog-
raphy; SAR, structure-activity relationship; SRIF, somatostatin; ssts, SRIF
receptors; TEA, triethylamine; TEAP, triethylammonium phosphate.
10.1021/jm701444y CCC: $40.75
2008 American Chemical Society
Published on Web 04/12/2008

Ring Size of Somatostatin Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9 2669
Table 1. Physico-chemical Properties and sst1–5 Binding Affinities (IC50, nM) of the Analogues and Control Peptide ODT-8 (1)
purity
MSc IC50 (nM)d
no. of
atoms in
the cycle
ID
compd
HPLCa CZEb Mcalc M + Hobs
sst1
sst2
sst3
sst4
sst5
SRIF-28
1e ODT-8 [H-c(Cys3-Phe6-Phe7-
99
95
99 3145.45 3146.46 3.2 ( 0.3 (16) 2.6 ( 0.3 (18) 3.8 ( 0.6 (16) 3.4 ( 0.3 (16) 3.2 ( 0.5 (16)
38
26
98 1078.45 1078.90 27 ( 3.4 (4)
41 ( 8.7 (6) 13 ( 3.2 (4) 1.8 ( 0.7 (4) 46 ( 27 (3)
DTrp8-Lys9-Thr10-Phe11
-
Cys14)-OH](SRIF numbering)
[DCys3]-ODT-8
2
99
99
99
99
99
99
99
97
99
99
98
97
99
99
97
97
96
99
94
97
98
99 1078.44 1079.48 14 (11; 17)
99 1078.44 1079.23 >1K (2)
99 1078.44 1079.36 >1K (2)
99 1120.45 1121.29 22 ( 8.8 (3)
97 1120.45 1121.23 16 (19; 13)
99 1120.45 1121.26 >1000 (4)
99 1120.45 1121.43 >1000 (4)
98 1106.44 1107.20 79 (107; 50)
97 1106.44 1107.23 416 ( 126 (4) 42 ( 11 (5)
98 1106.44 1107.18 358 (540; 175) 41 (36; 45)
99 1106.44 1107.18 >1000 (4)
96 1106.44 1107.48 540 ( 171 (3) 18.5 (18; 19) 30 (31; 29)
14.5 (15; 14) 4.5 (4.4; 4.6) 1.0 (1.2; 0.8) 9.5 (11; 7.9)
26
26
26
26
26
26
26
25
25
25
25
25
25
25
25
24
24
24
24
27
27
3e [DCys14]-ODT-8
18 (17; 19)
11 (9; 12)
49 ( 9 (4)
8.0 (6.4; 8.6) 19 (7.8; 30)
4.9 (5.3; 4.4) 12 (5.5; 18)
18.5 (20; 17) 8.2 (9; 7.3)
6.3 (6; 6.6) 2.6 (2.8; 2.4) 8.7 (8.6; 8.8)
7.1 ( 0.9 (3) 1.3 ( 0.2 (4) 26 ( 12 (3)
27 (29; 24)
4
5
6
7
8
9
[DCys3, DCys14]-ODT-8
Ac-ODT-8
[DCys3]Ac-ODT-8
[DCys14]Ac-ODT-8
[DCys3, DCys14]Ac-ODT-8
[Ncy3]Ac-ODT-8
0.7 ( 0.1 (3) 9.1 (8.9; 9.3)
4.5 (4.7; 4.3) 11 (12; 10)
3.25 (3.3; 3.2) 7.4 (6.2; 8.5)
1.9 ( 0.6 (3) 9.0 (5.9; 12)
9 (11; 7)
33 (39; 26)
15 (11; 18)
6.0 (3.9; 8)
14 ( 2.6 (4) 3.1 ( 0.9 (5) 46 ( 16 (4)
20 (13; 26)
10 [DNcy3]Ac-ODT-8
11 [Ncy14]Ac-ODT-8
2.0 ( 0.5 (3) 50 (39; 61)
6.0 (8.1; 3.9) 82 (38; 126)
1.5 ( 0.5 (3) 51 (84; 18)
12 [DNcy14]Ac-ODT-8
5.4 (3.6; 7.1) 53 (22; 83)
13 [DCys3, Ncy14]Ac-ODT-8
14 [DCys3, DNcy14]Ac-ODT-8
15 [Ncy3, DCys14]Ac-ODT-8
16 [DNcy3, DCys14]Ac-ODT-8
17 [Ncy3, DNcy14]Ac-ODT-8
18e [DNcy3, Ncy14]Ac-ODT-8
19e [Ncy3, Ncy14]Ac-ODT-8
20 [DNcy3, DNcy14]Ac-ODT-8
21e [Hcy3]-Ac-ODT-8
99 1106.44 1107.48 >1000 (4)
99 1106.44 1107.44 >1000 (4)
97 1106.44 1107.45 >1000 (5)
98 1092.42 1093.39 >1000 (4)
99 1092.42 1093.58 >1000 (2)
4.8 (4.3; 5.3) 36 (45; 27)
11 (9; 13) 69 (21; 117) 4.4 (5.1; 3.6) 50 (15; 85)
3 ( 0.8 (3)
17 (25; 9.8)
13 ( 2.7 (5) 28 ( 6.1 (4) 5.0 ( 0.6 (4) 50 ( 7.9 (3)
3.9 (6.2; 1.3) 50 (84; 16)
2.6 ( 0.7 (3) 60 (93; 26)
2.7 ( 0.3 (3) 107(163; 51)
1.6 ( 0.2 (3) 57 (73; 40)
2.2 ( 0.6 (3) 21 (29; 12)
0.9 (0.61; 1.1) 28 (17; 38)
1.4 (1.3; 1.5) 90 (57; 122)
109 ( 8.5 (3) 45 (32; 58)
99 1092.42 1093.37 302 (381; 222) 34 (32; 36)
19 (16; 22)
99 1092.42 1093.53 >1000 (4)
98 1134.47 1135.88 96 (103; 89)
13 ( 2.5 (3) 31 (23; 38)
6.5 (6.1; 6.9) 38 (59; 17)
22 [DHcy3]-Ac-ODT-8
99 1134.47 1135.64 218 (306; 130) 40 (48; 32)
69 (74; 63)
a Percent purity determined by HPLC by using buffer system: A ) TEAP (pH 2.5) and B ) 60% CH3CN/40% A with a gradient slope of 1% B/min, at
a flow rate of 0.2 mL/min on a Vydac C18 column (0.21 × 15 cm, 5-µm particle size, 300 Å pore size). Detection at 214 nM. b Capillary zone electrophoresis
(CZE) was done by using a Beckman P/ACE System 2050 controlled by an IBM Personal System/2 Model 50Z and by using a ChromJet integrator. Field
strength of 15 kV at 30 °C, mobile phase: 100 mM sodium phosphate (85:15, H2O:CH3CN) pH 2.50, on a Supelco P175 capillary (363 µm o.d. × 75 µm
i.d. × 50 cm length). Detection at 214 nM. c Calculated m/z of the monoisotope compared with the observed [M + H]+ monoisotopic mass. d The IC50
values (nM) were derived from competitive radioligand displacement assays reflecting the affinities of the analogues for the cloned SRIF receptors by using
the nonselective 125I-[Leu8,DTrp22,Tyr25]SRIF-28 as the radioligand. Mean value ( SEM when N g 3 (shown in parentheses). In the other cases, mean is
shown with single values in parentheses. e 3D NMR structures of these analogues are presented in this paper.
resolved Boc-Ncy(Mob)-OH. This approach of determining
stereochemistry was successful in analogues containing Ncy
residue at position 3 (9, 10, 15, and 16) but not in analogues
containing Ncy at the C-terminus (11, 12, 13, and 14). Our
attempts to link the Boc-Ncy(Mob)-OH in optically active form
to the CM resin failed because racemization occurred during
the attachment step (2.0 equiv of KF per mmol of amino acid
in dimethylformamide (DMF) at 80 °C for 15 h). It is
noteworthy to mention here that optical integrity of the resolved
Boc-Ncy(Mob)-OH35 in peptides 9 and 15 was significantly
reduced when coupling was mediated under basic conditions
(TBTU/diisopropylethylamine/1-hydroxybenzotriazole (HOBt)
in DMF) instead of neutral conditions (N,N′-diisopropylcarbo-
diimide/HOBt in DMF). Hence, to determine the stereochemistry
of Ac-ODT-8 analogues containing Ncy (11–14) at the C-
terminus, we used an alternative method28 described by our
group for the synthesis of NR-methylated SRIF analogues. On
the basis of the premise that failure to attach Boc-Ncy(Mob)-
OH in optically active form to the CM resin was due to
susceptibility of Ncy residue to racemization under standard
coupling conditions, we first synthesized lysine-elongated pep-
tides, that is, [Ncy14]Ac-ODT-8-Lys and [DCys3, Ncy14]Ac-
ODT-8-Lys, starting with Boc-Lys(2-Cl-Z)-CM resin. The
enzymatic hydrolysis of the purified peptides [Ncy14]Ac-ODT-
8-Lys and [DCys3, Ncy14]Ac-ODT-8-Lys with a carboxypep-
tidase B enzyme preparation37 resulted in the desired nonlysine-
containing 11 and 13, respectively. RP-HPLC studies revealed
that 9, 11, 13, and 15 synthesized with the resolved Boc-
Ncy(Mob)-OH coeluted with the first eluting diastereomer of
the pairs 9 + 10, 11 + 12, 13 + 14, and 15 + 16, respectively.
Analogues 17–20 were synthesized by using racemic Boc-
Ncy(Mob)-OH on racemic Boc-Ncy(Mob)-CM resin. The four
diastereomers (17–20) with retention times of 11.1, 12.3, 13.1,
and 14.1 min were separated and purified by RP-HPLC. The
stereochemistry of the diastereomers 17, 18, and 19 was
confirmed by comparison of the HPLC retention times and
coelution of each diastereomer with those of the analogues
obtained from the enzymatic hydrolysis of lysine-extended
peptides [Ncy3, DNcy14]Ac-ODT-8-Lys, [DNcy3, Ncy14]Ac-
ODT-8-Lys, and [Ncy3, Ncy14]Ac-ODT-8-Lys, respectively.
This confirmed that analogue 20 eluting (tR) at 14.1 min on
analytical HPLC contained DNcy at positions 3 and 14.
Purification and Characterization of the Analogues (see
Table 1). Purification was carried out by using multiple HPLC
steps.38 Characterization and purity of the analogues were
established by HPLC,38 capillary zone electrophoresis,39 and
mass spectrometry. The C-terminus-extended purified target
peptides (RP-HPLC and CZE purity >96%) could be hydro-
lyzed with carboxypeptidase B, resulting in the desired ana-
logues. The measured masses obtained by matrix-assisted laser
desorption ionization mass spectrometry were within 100 ppm
of those calculated for the protonated molecule ions and are
given in Table 1.
Receptor Binding. The compounds were tested for their
ability to bind to the five human sst receptor subtypes (ssts) in
competitive experiments by using 125I-[Leu8,DTrp22,Tyr25]SRIF-
28 as radioligand. Cells stably expressing the five cloned human
ssts were grown as described previously.3 Cell membrane pellets
were prepared, and receptor autoradiography was performed as
previously depicted in detail.3 The binding affinities are
expressed as IC50 values that are calculated after quantification
of the data by using a computer-assisted image processing
system as described previously3,40 and are summarized in
Table 1.

2670 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9
Erchegyi et al.
Table 2. Characterization of the NMR Structures of the Analogues Studied by NMRa
residual restraint violations on
distances dihedral angles
CFF91 energies (kcal/mol)
NOE
distance
CYANA
target
angle
backbone
overall
rmsd (Å)
total
energy
van der
Waals
ID restraints restraintsb functionc rmsd (Å)
electro-static no g 0.1 Å max (Å) no g 1.5° max (°)
1
3
18
19
21
116
163
110
88
22
18
16
24
29
0.001 0.65 ( 0.12 1.39 ( 0.21 192.7 ( 18 100.5 ( 3
30.2 ( 2
71.6 ( 3
45.7 ( 1
48.3 ( 8
49.5 ( 2
0.1 ( 0.1 0.04 ( 0.03
0.9 ( 0.1 0.12 ( 0.04
0 ( 0
0 ( 0
0 ( 0
0 ( 0
0.08
0.08
0.11
0.18 ( 0.09 0.62 ( 0.12 214.3 ( 4 142.7 ( 2
0.13 ( 0.04 0.72 ( 0.15 178.2 ( 2 132.5 ( 2
0.72 ( 0.20 1.12 ( 0.15 185.5 ( 6 137.2 ( 6
0.8 ( 0.0 0.09 ( 0.00 0.9 ( 0
0.9 ( 0.04
1.0 ( 0.2 0.15 ( 0.06 0.5 ( 0.5 0.4 ( 0.4
0.3 ( 0.1 0.50 ( 0.07
111
0.015 0.17 ( 0.07 0.79 ( 0.21 193.2 ( 4 143.8 ( 2
0 ( 0
0 ( 0.04
a The bundle of 20 conformers with the lowest residual target function was used to represent the NMR structures of each analogue. b Meaningful NOE
distance restraints may include intraresidual and sequential NOEs.42 c The target function is zero only if all the experimental distance and torsion angle
constraints are fulfilled and all nonbonded atom pairs satisfy a check for the absence of steric overlap. The target function is proportional to the sum of the
square of the difference between calculated distance and isolated constraint or van der Waals restraints, and similarly isolated angular restraints are included
in the target function. For the exact definition see ref 42.
Analogue 1 binds to the five receptors of SRIF with low
nanomolar affinity and has maximum affinity for sst4. Analogue
2 differs from 1 by a DCys at position 3 and binds to all of the
SRIF receptors with higher affinity than analogue 1. Analogue
3 differs from 1 by a DCys at position 14 and does not bind to
sst1. Acetylation of ODT-8 (1) results in 5 that binds to all of
the ssts similar to ODT-8 (1). The acetylation of 2 and 3 resulting
in 6 and 7 has some influence on the binding affinity of these
peptides at sst2 but not at the other ssts compared with the parent
peptides. Analogue 8 differs from 6 by having a DCys at
position 14, and this peptide does not bind to sst1 but binds to
all of the other receptors with a binding affinity similar to that
of 6. Ncy and DNcy substitutions at position 3 for Cys in Ac-
ODT-8 (5) result in 9 and 10 that bind to all ssts but sst1 similar
to 5. Analogues 11 and 13 differ from 5 and 6 by having Ncy
at position 14. Both peptides bind to sst1 with significantly less
affinity than the parent analogues. DCys substitution at position
14 has resulted in 15-17 showing enhancement in binding
affinity for sst2 compared to 5. Similarly, DNcy substitution at
position 14 resulted in 12, 14, 17, and 20, which bind to sst2
with higher affinity than 5. Analogue 18 differs from 1 by having
DNcy at position 3 and Ncy at position 14 and has an acetylated
1, 3, 18, 19, and 21 (Table 1) obtained by solution-state NMR
studies in the solvent DMSO.
Assignment of Proton Resonances, Collection of Struc-
tural Restraints, and Structure Determination. Almost all
chemical shift assignments of proton resonances (Table S2) for
all of the analogues (Table 1, assignment and structural
characterization of analogue 1 (ODT-8) have been taken from
Grace et al.34) have been carried out by using 2D NMR
experiments by applying the standard procedure described in
the Experimental Section. The N-terminal amino protons for 1
and 3 have not been observed because of fast exchange with
the solvent. The N-acetyl terminal amide protons, however, are
observed for 18, 19, and 21.
A large number of experimental nuclear Overhauser enhance-
ments (NOEs) have been observed for all of the five analogues
in the NOESY spectrum measured with a mixing time of 100
ms, leading to over 100 meaningful distance restraints per
analogue and concomitantly ∼10 restraints per residue (Table
2). These structural restraints were used as input for the structure
calculation with the program combined assignment and dynam-
ics algorithm for NMR applications (CYANA)42 followed by
restrained energy minimization by using the program DIS-
COVER.43 The resulting bundle of 20 conformers per analogue
represents the 3D structure of each analogue in DMSO. For
each analogue, the small residual constraint violations in the
distances for the 20 refined conformers (Table 2) and the
coincidence of the experimental NOEs and short interatomic
distances (data not shown) indicate that the input data represent
a self-consistent set, and that the restraints are well satisfied in
the calculated conformers (Table 2). The deviations from ideal
geometry are minimal, and similar energy values have been
obtained for all of the 20 conformers for each analogue. The
quality of the structures determined is furthermore reflected by
the small backbone root-mean-square deviation (rmsd) values
relative to the mean coordinates of ∼0.5 Å (see Table 2 and
Figure 2).
N-terminus. It binds to sst4 in low nanomolar affinity (IC50
)
2.7 nM) and with moderate affinity to receptors sst2, sst3, and
sst5 (IC50 ) 110, 45, and 107 nM, respectively) but does not
bind to sst1. Analogue 19 is similar to 18 except for Ncy residues
at position 3. Analogue 19 binds to sst4 with high affinity (IC50
) 1.6 nM) and shows moderate affinity for receptors sst2, sst3,
and sst5, (IC50 ) 34, 19, and 57 nM, respectively) and weak
binding affinity to sst1 (IC50 ) 302 nM). Analogue 21 differs
from 1 by having Hcy at position 3 and has an acetylated
N-terminus. It shows high binding affinity for sst2 and sst4 (IC50
≈ 6 and 0.8 nM, respectively) but only moderate affinity for
the other three receptors. The substitution of Hcy by DHcy in
analogue 21 results in analogue 22 that shows lower binding
affinity for all of the ssts than 21.
To get insights into the action of such nonselective analogues,
structural studies were carried out in dimethylsulfoxide (DMSO).
The conformation preferred in DMSO may be difficult to
interpret because of the nonselective binding of the analogues.
3D Structure of H-c[Cys3-Phe6-Phe7-DTrp8-Lys9-Thr10-
Phe11-Cys14]-OH (ODT-8) (1). As we published earlier for
analogue 134 (Table 1), the backbone torsion angles indicate
that a ꢀ-turn of type-VIII′ conformation is present around Phe7-
DTrp8. A turn-like structure is also supported by the medium
range dNN(i, i + 2) distance restraints (Figure 1). Although the
unshifted resonance for the amide proton of Lys9 (293-318
K) is indicative of an expected hydrogen bond Lys9NH-O′Phe6,
the carbonyl CdO of Phe6 is not in a favorable orientation to
be involved in a hydrogen bond. The side chain of Phe6 is in
the gauche+ rotamer, Phe7 is in the trans rotamer, DTrp8 is in
the gauche- rotamer, and Lys9 is in the gauche+ rotamer (Table
S1). Conclusively, the side chains of DTrp8 and Phe7 are
Analogues studied here bind nonselectively to more than one
receptor, and hence, it is interesting to compare their 3D
structures in DMSO with the available receptor-selective
pharmacophores.29,34,41 Analogues 1, 3, 18, 19, and 21 (Table
1) have been selected for these studies. They have similar
structures except for the stereochemistry of the bridgeheads and
the number of atoms involved in the cycle.
NMR Studies. We present details about the chemical shift
assignment of proton resonances and structural information for

Ring Size of Somatostatin Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9 2671
Figure 1. Survey of characteristic NOEs describing the secondary
structure of the five analogues studied by NMR (i.e., analogues 1, 3,
18, 19, and 21 as indicated). Thin, medium, and thick bars represent
weak (4.5-6 Å), medium (3-4.5 Å), and strong (<3 Å) NOEs
observed in the NOESY spectrum. The medium-range connectivities
dNN(i, i + 2), dRN(i, i + 2), and dꢀN(i, i + 2) are shown by lines starting
and ending at the positions of the residues related by the NOE. Residues
Ncy, DNcy, Hcy, DHcy, DCys, and DTrp refer to norcysteine,
D-norcysteine, homocysteine, D-homocysteine, D-cysteine, and D-
j
tryptophan denoted by the symbols, C, c, C, jc, c, and w, respectively.
adjacent to each other in the plane of the analogue backbone,
whereas the side chains of Phe6 and Phe11 are pointing away
from the plane of the peptide backbone.34
3D Structure of H-c[Cys3-Phe6-Phe7-DTrp8-Lys9-Thr10-
Phe11-DCys14]-OH (3). For analogue 3 (Table 1), the backbone
torsion angles do not fit into any of the classified reported ꢀ-turns
in literature. The side chains of Phe6, Phe7, DTrp8, Lys9, and
Phe11 are in the gauche+ rotamer (Table S1). This configuration
orients Phe7, Phe11, and Lys9 pointing away from the backbone
on one side, whereas the side chains of Phe6 and DTrp8 point
on the opposite side (Figure 2). Ring current of Phe11 resulted
in slightly upfield-shifted resonances for the γ-protons of Lys9
(Table S2).
3D Structure of Ac-c[DNcy3-Phe6-Phe7-DTrp8-Lys9-
Thr10-Phe11-Ncy14]-OH (18). For analogue 18 (Table 1), the
backbone torsion angles indicate a ꢀ-turn of type-II conforma-
tion around Phe7-DTrp8 (Table S1). The side chains of Phe6,
DTrp8, and Lys9 are in the gauche+ rotamer, Phe7 is in the trans
rotamer, and Phe11 is in the gauche- rotamer (Table S1). Hence,
the side chains of Phe7 and DTrp8 adjacent to each other are in
the plane of the peptide backbone, whereas the side chains of
Lys9 and Phe6 are in close proximity. The side chain of Phe11
is far away from the DTrp8-Lys9 pair behind the peptide
backbone (Figure 2). Ring current of Phe6 has resulted in slightly
upfield-shifted resonance for the γ-protons of Lys9. Hydrogen
bonds are observed in all of the calculated 20 conformers
between the amide protons of Lys9, Thr10, and the carbonyl of
Phe6 and also between the amide proton of Phe6 and the
carbonyl of Thr10. Experimentally measured small temperature
coefficients of -0.02, -0.004, and -0.01 ppm/K for the amide
protons of Phe6, Lys9, and Thr10, respectively, confirm that these
amide protons are involved in the hydrogen bond.
Figure 2. 3D structures of the five analogues studied by NMR (i.e.,
analogues 1, 3, 18, 19, and 21 as indicated). For each analogue, 20
energy-minimized conformers with the lowest target function are used
to represent the 3D NMR structure. The bundle is obtained by
overlapping the CR atoms of all the residues. The backbone and the
side chains are displayed including the disulfide bridge. The following
color code is used: gray, (1) H-c[Cys-Phe-Phe-DTrp-Lys-Thr-Phe-Cys]-
OH, ODT-8 taken from Grace et al.;34 navy-blue, (3) H-c[Cys-Phe-
Phe-DTrp-Lys-Thr-Phe-DCys]-OH; violet, (18) Ac-c[DNcy-Phe-Phe-
DTrp-Lys-Thr-Phe-Ncy]-OH; royal blue, (19) Ac-c[Ncy-Phe-Phe-DTrp-
Lys-Thr-Phe-Ncy]-OH; and dark green, (21) Ac-c[Hcy-Phe-Phe-DTrp-
Lys-Thr-Phe-Cys]-OH. The amino acid side chains which are proposed
to be involved in binding to the various SRIF receptors are highlighted:
light green, DTrp at position 8; blue, Lys at position 9; and yellow,
Phe at positions 6, 7, and 11. The disulfide bridges are shown in orange
for clarity.
3D Structure of Ac-c[Hcy3-Phe6-Phe7-DTrp8-Lys9-
Thr10-Phe11-Cys14]-OH (21). The backbone torsion angles from
the 3D structures show that an inverse γ-turn is present around
DTrp8 for analogue 21 (Table 1). The side chains of Phe6, Phe7,
Lys9, and Phe11 are in gauche+ rotamer, and that of DTrp8 is
in gauche- rotamer (Table S1). This orients the side chains of
Phe6, Phe7, and Lys9 on one side of the peptide backbone,
whereas the side chains of DTrp8 and Phe11 are on the other
side (Figure 2). Close proximity between the amide group of
Lys9 and the carbonyl of Phe7, as well as the amide group of
Thr10 and the carbonyl of the acetyl group at the N-terminus,
shows possible hydrogen bonds between these groups. Experi-
mentally measured small temperature coefficients of -0.01 and
-0.002 ppm/K for the amide protons of Phe7 and Thr10,
respectively, confirm that these amide protons are involved in
a hydrogen bond.
3D Structure of Ac-c[Ncy3-Phe6-Phe7-DTrp8-Lys9-Thr10-
Phe11-Ncy14]-OH (19). For analogue 19 (Table 1), the backbone
torsion angles do not fit into any of the classified ꢀ-turns reported
in the literature. The side chains of Phe6, Phe7, DTrp8, Lys9,
and Phe11 are in the gauche+ rotamer (Table S1). This orients
the side chains of Phe6, Lys9, and Phe11 on one side of the
peptide backbone, with Phe11 in close proximity to Lys9. Side
chains of Phe7 and DTrp8 are in the plane of the peptide
backbone (Figure 2).
Discussion
All of the five analogues studied here by NMR in DMSO
bind nonselectively to more than one receptor, and hence, it is
interesting to compare their structures with receptor-selective

2672 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9
Erchegyi et al.
Figure 3. Schematic drawings of agonist pharmacophores for receptor-selective analogues binding to the SRIF receptors. (A) sst1, (B) sst2, (C)
sst2/5, and (D) sst4. The amino acid side chains, which are part of the pharmacophores, and the distances between the corresponding Cγ atoms of
the side chains are shown and are also listed in Table 3.
pharmacophores. However, because of the nonselective binding
of these analogues, the elucidation of such a multiple structure-
activity relationship (SAR) is not trivial, and a step-by-step
analysis is required. Therefore, we first review the structural
parametersproposedforthereceptor-selectivepharmacophores29,34,41,44
and then discuss the 3D structures of the nonselective analogues
of interest.
Pharmacophores of SRIF Receptors. Receptor-selective
agonistic analogues binding selectively to SRIF receptors 1, 2,
and 4 have been obtained by using both chemical modifications
of different amino acid side-chain groups in short SRIF
analogues26,45 and structural design to optimize the pharma-
cophore.29,34,41 The pharmacophore for sst1 binding has been
shown to be the DTrp8-IAmp9 (4-(N-isopropyl)-aminometh-
ylphenylalanine) pair of residues in addition to two aromatic
side chains at positions 7 and 11.29 On the basis of the
pharmacophore and other experimental findings,25,46 the ratio-
nale is that the longer side chain of IAmp can only be
accommodated by SRIF receptor 1, whereas, in the other SRIF
receptors, the binding cavity is limited to accommodating a
smaller side chain as that of Lys. In contrast to the role of the
IAmp in receptor selectivity, the two aromatic side chains at
positions 7 and 11 are important to enhance the binding to sst1
(Figure 3A).
Design of an sst4-selective agonist was more straightfor-
ward.32 The 3D structures of several sst4-selective analogues
identified the pharmacophore for sst4 to have one aromatic ring
of Phe at position 6 or 11 close to Lys9 in addition to the crucial
DTrp8 and Lys9 pair34 (Figure 3D). The development of a
strictly sst2-selective analogue appears to be more demanding
because analogues binding with nanomolar affinity to SRIF
receptor 2 often also bind with high affinity to SRIF receptor 5
and sometimes to receptor 3.47,48 Most of these partially selective
analogues are based on the structure of a SRIF analogue named
octreotide and have a type-II′ ꢀ-turn in their structure.44 The
octreotide pharmacophore requires two aromatic side chains at
positions 7 and 2 in addition to the DTrp8-Lys9 pair. It has also
been shown that octreotide-type analogues undergo conforma-
tional change in their backbone from ꢀ-sheet to R-helix, resulting
in two different positions for the Phe/DPhe/Tyr at position 2.47
This conformational exchange complicates the basis for receptor
selectivity and suggests that two conformations are necessary
for an analogue to fit into the two different receptor-selective
pharmacophores for receptors 2 and 5 (Figure 3C). Recently,
we published data showing that removal of the aromatic side
chain at position 7 from an octreotide analogue led to sst2
selectivity.41 Hence, the pharmacophore of a sst2-selective
agonist indeed appears to be a subset of the octreotide
pharmacophore with one aromatic side chain of Phe2 in addition
to the side chains of the DTrp8-Lys9 pair. Furthermore, in
contrast to octreotide, where Phe2 undergoes conformational
exchange, the sst2-selective pharmacophore has Phe2 at a single
position (Figure 3B).
Although sst3-selective agonists have not been reported yet,
there is a report on sst3-selective antagonists, which have
aminoglycine at position 830 instead of Trp/DTrp, and the
structure of this analogue has been reported in water.31 Similarly,
SRIF agonists or antagonists selectively binding to sst5 have
not been described. In summary, selective binding to a receptor
requires a unique pharmacophore for the analogues having Trp8
and Lys9 (or an amino acid with a different side chain) and a
specific number of aromatic residues at the correct distances
with respect to each other (Figure 3, Table 3).
Comparison of the 3D Structures of Nonselective SRIF
Analogues with Receptor-Selective Pharmacophores. Because
shortening or lengthening the side chains of Cys at positions 3
and 14 of Ac-ODT-8 resulted in loss of binding to some
receptors, we decided to compare the 3D structures of these
nonselective analogues in DMSO to those of the corresponding
receptor-selective pharmacophores determined earlier.29,34,41 All
structural data reported above support the fact that the backbone
conformation is not responsible for the binding of peptides; it
rather acts as a scaffold in orienting the side chains of the
analogues to interact efficiently with the receptor. Such conclu-
sions had also been derived on the basis of the NMR structure
of astressin (a corticotropin releasing factor antagonist) in
DMSO51 anditsboundconformationtotheECD1ofCRF-R2.49,50
Hence, the spatial orientations of the amino acid side chains
for the different analogues are compared here with those of the
receptor-selective pharmacophores. To explain the nonselective
binding of an analogue to multiple receptors, either conforma-
tional flexibility is necessary to allow binding by an induced-
fit mechanism or the analogue prefers a conformation accom-
modating two or more pharmacophores simultaneously. In
particular, the conformation preferred by nonselective analogues
in DMSO will not evidently fit all of the pharmacophores
simultaneously as proposed.
Comparison of the 3D Structures of the Nonselective
Analogues with the sst1-Selective Pharmacophore. A com-
parison of the 3D structures of the nonselective analogues with
the sst1-selective pharmacophore shows the following: the
introduction of Ncy in the ODT-8 structure (i.e., in 3, 18, and
19) has resulted in loss of binding to receptor 1. Their 3D
structures determined in DMSO show that, among the three
aromatic residues, two of them are oriented on the front side of
the peptide backbone. Because the sst1 pharmacophore requires
two aromatic side chains at the backside of the peptide backbone
in addition to Trp8 and Lys9 side chains as shown in Figure

Ring Size of Somatostatin Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9 2673
Table 3. Distances between Cγ Atoms (in Å) of Selected Residues for the Analogues Studied by NMR and the Selective sst-Pharmacophores
analogue
F6-F7
F6-DW8
10.0-10.2 7.9-9.6
8.4-8.6
10.0-10.1 5.4-5.5
F6-K9
F6-F11
7.6-9.7
F7-DW8
F7-K9
F7-F11
DW8-K9 DW8-F11
K9-F11
1
3
18
19
21
7.2-8.0
7.6-7.7
8.6-8.6
5.1-7.5
4.8-5.1
4.7-4.9 9.3-10.8 11.8-14.5 7.4-8.9 11.7-13.7
5.9-10.6
5.0-5.2
11.0-11.7 13.2-13.6 8.0-8.1 5.6-6.5
8.4-8.9 5.5-5.5 9.4-9.4
7.1-8.2
8.7-9.9
5.7-5.9
8.9-9.4
7.5-7.5 12.0-13.0 11.5-11.6
7.8-8.6
8.7-8.9
5.3-7.1
7.3-8.0
6.6-10.2 7.3-8.2 9.1-10.3 12.6-13.4 5.9-6.2
12.6-13.8 6.4-6.4 5.5-5.7
6.0-7.5 9.0-11.0
8.9-10.7
6.6-9.5
9.5-12.0
5.5-9.5
4.4-6.0
10.1-10.5
8.0-10.0
4.5-6.5
11.9-13.7 7.0-7.1
6.0-7.5
sst1 pharmacophore
sst4 pharmacophore
7.0-8.0
4.5-6.5
5.5-9.5
F2-DW8
4.5-6.5
F2-K9
F2-F7
sst2 pharmacophore
octreotide pharmacophore 5.0-11.0 11.0-15.0 12.0-15.0
12.0-13.5 12.5-15.0
4.0-5.0
5.0-5.0
7.0–9.0
9.0–11.0
3A29 and Table 3, the sst1 pharmacophore is not observed in 3,
18, and 19. This may explain why 3, 18, and 19 do not bind to
receptor 1. In contrast, the 3D structure of ODT-8 (1), which
binds to all of the five receptors, has the sst1 pharmacophore as
well.
Comparison of the 3D Structures of the Nonselective
Analogues with the Sst2,4-Selective Pharmacophores. The sst2-
selective pharmacophore requires one aromatic side chain far
from the Trp8-Lys9 pair in addition to these two side chains, as
shown in Figure 3B41 and Table 3. Although 1, 3, 18, 19, and
21 bind to receptor 2 with nanomolar affinity, the 3D NMR
structures of the analogues in DMSO show that these analogues
do not have the sst2-selective pharmacophore (Figure 4). In 1,
18, and 21, a small conformational change of the side chain of
either Phe6 or Phe11 can establish the partially selective
pharmacophore (Figure 4). But in 3 and 19, the side chain of
Phe6 has to undergo a large conformational change to fit the
sst2-selective pharmacophore. Therefore, the 3D structures of
the analogues determined in DMSO are not sufficient to explain
the sst2,5 nonselective pharmacophore.
Figure 4. Superposition of receptor-specific pharmacophore with the
3D NMR structure of the analogues 1, 18, and 21. The sst2 pharma-
cophore41 is shown in green. The octreotide pharmacophore proposed
by Melacini et al.44 is shown in yellow. In both pharmacophores, only
the side chains of the amino acids that are involved in binding to the
receptor are shown. For analogues 1, 18, and 21, the conformer with
the lowest energy is used to represent the 3D structures. The analogues
are color-coded as in Figure 2. The side chains of the amino acids,
which are proposed to be involved in receptor binding, are labeled.
Phe6 in analogue 1 and Phe11 in analogues 18 and 21 should undergo
a change in their conformation to fit either the sst2 or octreotide
pharmacophores.
Also DCys or DNcy substitution at position 14 has resulted
in analogues (3, 4, 7, 8, 15, and 16 or 12, 14, 17, and 20)
showing enhanced binding affinity for sst2 compared to 5. A
D-amino acid, part of the cycle at the C-terminus, changes the
backbone conformation, probably bringing the side chains of
Phe11 close to the sst2 pharmacophore (Figure 4). In addition,
all of these analogues do not bind to sst1, probably because of
the positioning of the side chain of Phe11, critical for sst1 binding
(Figure 3A).
Figure 5. Superposition of sst4 receptor-specific pharmacophore with the 3D NMR structures of the analogues 1, 3, 18, 19, and 21. The sst4
pharmacophore34 is represented by amino acid side chains colored in red. The conformer with the lowest energy represents the 3D structures of the
analogues, and they are color-coded as in Figure 2. In addition, for each analogue, the amino acid side chains proposed to be involved in receptor
binding are labeled.

2674 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9
Erchegyi et al.
1
DMSO-d6. The H NMR spectra were recorded on a Bruker 700
MHz spectrometer operating at a proton frequency of 700 MHz as
reported previously.29,34,41,51
However, these analogues have the highest affinity for
receptor 4, and the sst4 pharmacophore is present in their
conformations. The sst4-selective pharmacophore requires one
aromatic side chain close to Lys9 in addition to the side chains
of Trp8 and Lys9, as shown in Figure 3C. The distances between
the Cγ atoms of the side chains to fit the sst4 pharmacophore
are given in Table 3. The 3D structures of 1, 3, 18, 19, and 21
have the sst4 pharmacophore as shown in Figure 5, with either
Phe11 (1, 3, and 19) or Phe6 (18 and 21) involved in sst4 binding
in addition to the necessary DTrp8 and Lys9 side chains.
Structure Determination. The chemical shift assignment of the
major conformer (the population of the minor conformer was
<10%) was obtained by the standard procedure by using double
quantum filtered correlation spectroscopy and total correlation
spectroscopy spectra for intraresidual assignment, and the NOESY
spectrum was used for the sequential assignment.52 The collection
of structural restraints was based on the NOEs assigned manually
3
and vicinal JNHR couplings. Dihedral angle constraints were
obtained from the 3JNHR couplings, which were measured from the
1D 1H NMR spectra and from the intraresidual and sequential
NOEs, along with the macro GRIDSEARCH in the program
CYANA.42 The calibration of NOE intensities versus 1H-1H
distance restraints and appropriate pseudoatom corrections to the
nonstereo specifically assigned methylene, methyl, and ring protons
were performed by using the program CYANA. On average,
approximately 100 NOE constraints and 20 angle constraints were
utilized to calculate the conformers (Table 2). A total of 100
conformers were initially generated by CYANA, and a bundle
containing 20 CYANA conformers with the lowest target function
values were utilized for further restrained energy minimization by
Conclusions
Synthesis, binding, and 3D NMR structure of nonselective
analogues of H-c(Cys-Phe-Phe-DTrp-Lys-Thr-Phe-Cys)-OH
(ODT-8, 1) containing different side chain lengths for the Cys
at positions 3 and 14 have been presented here. These analogues
bind nonselectively to all of the five SRIF receptors, and the
elucidation of a SAR is challenging. In our attempt to establish
such a relationship between the 3D structures of the analogues
determined in DMSO and their receptor specificity, it has been
observed that the 3D structures mostly have the pharmacophore
for which the analogues have the highest binding affinities. All
of these analogues bind to sst4 with the highest binding affinity,
and their 3D structures have the sst4 pharmacophore. However,
the pharmacophores of the other receptors are often not covered
by the 3D structure determined in DMSO, and their pharma-
cophores can only be established by reorientation of some of
the amino acid side chains.
using the CFF91 force field53 with the energy criteria fit 0.1 kCal/
54
mol/Å
in the program DISCOVER with steepest decent and
conjugate gradient algorithms.55 The resulting energy-minimized
bundle of 20 conformers was used as a basis for discussing the
solution conformation of the different SRIF analogues. The
structures were analyzed by using the program MOLMOL.56
Acknowledgment. This work was supported in part by NIH
grant R01 DK059953. We are indebted to R. Kaiser and C.
Miller for technical assistance in the characterization of the
peptides, Dr. W. Fisher and W. Low for mass spectrometric
analysis of the analogues, and D. Doan for manuscript prepara-
tion. J.R. is the Dr. Frederik Paulsen Chair in Neurosciences
Professor.
The establishment of receptor-selective and nonselective
analogues of SRIF by introducing small chemical modifications
to the peptides highlights the complexity of a binding event.
The structure determination of these analogues in DMSO
elucidates thereby, at least in part, the nature of this complexity.
Experimental Section
Supporting Information Available: Starting materials, peptide
synthesis, cleavage and deprotection with HF, and cyclization are
reported in Supporting Information along with Table S1 (torsion
angles ꢁ, Ψ, and ꢂ1 (in °) for the bundle of 20 energy-minimized
conformers) and Table S2 (proton chemical shifts of the analogues
studied by NMR). This material is available free of charge via the
Internet at http://pubs.acs.org.
Determination of the Stereochemistry of Ncy14 in the
Analogues. The fully protected Ac-Ncy(Mob)3-Phe6-Phe7-DTrp8-
Lys(2-Cl-Z)9-Thr(Bzl)10-Phe11-Ncy(Mob)14-Lys(2-Cl-Z)15-CM resin
(SRIF numbering) was synthesized, cleaved from the resin by
hydrofluoric acid, cyclized and purified on preparative RP-HPLC.
The purified peptide Ac-[Ncy3-Phe6-Phe7-DTrp8-Lys9-Thr10-Phe11-
Ncy14]-Lys15-OH (20 mg, 16.30 µM) was hydrolyzed in 0.1 M
NaCl-0.05 M Tris buffer at pH 7.6 (20 mL) with undiluted
carboxypeptidase B enzyme solution (50 U) at room temperature.
The hydrolysis was complete in 30 min, resulting in analogue 19
(Table 1). The product was desalted by preparative RP-HPLC, and
pure peptide 19 (12 mg, 10.9 µM) was obtained (yield of hydrolysis:
66.87%). Analogues 11, 13, 17, and 18 were obtained from the
lysine-extended peptides [Ncy14]Ac-ODT-8-Lys, [DCys3, Ncy14]Ac-
ODT-8-Lys, [Ncy3, DNcy14]Ac-ODT-8-Lys, and [DNcy3, Ncy14]Ac-
ODT-8-Lys, respectively, in comparable yields by using this
protocol.
Cell Culture. CHO-K1 cells stably expressing human sst1 and
sst5 were kindly provided by Dr. T. Reisine and Dr. G. Singh
(University of Pennsylvania, Philadelphia, PA), and CCL39 cells
stably expressing human sst1, sst2, sst3, and sst4 were provided by
Dr. D. Hoyer (Novartis Pharma, Basel, Switzerland). Cells were
grown as described previously.3 All culture reagents were supplied
by Gibco BRL (Life Technologies, Grand Island, NY).
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