Ring size in octreotide amide modulates differently agonist versus antagonist binding affinity and selectivity

Article abstract of DOI:10.1021/jm701445q

H-DPhe²-c[Cys³-Phe⁷-DTrp ⁸-Lys⁹-Thr¹⁰-Cys¹⁴]-Thr ¹⁵-NH₂ (1) (a somatostatin agonist, SRIF numbering) and H-Cpa²-c[DCys³-Tyr⁷-DTrp⁸-Lys ⁹-Thr¹⁰-Cys¹⁴]-Nal¹⁵-NH₂ (4) (a somatostatin antagonist) are based on the structure of octreotide that binds to three somatostatin receptor subtypes (sst_2/3/5) with significant binding affinity. Analogues of 1 and 4 were synthesized with norcysteine (Ncy), homocysteine (Hcy), or D-homocysteine (DHcy) at positions 3 and/or 14. Introducing Ncy at positions 3 and 14 constrained the backbone flexibility, resulting in loss of binding affinity at all sst_s. The introduction of Hcy at positions 3 and 14 improved selectivity for sst ₂ as a result of significant loss of binding affinity at the other sst_s. Substitution by DHcy at position 3 in the antagonist scaffold (5), on the other hand, resulted in a significant loss of binding affinity at sst₂ and sst₃ as compared to the different affinities of the parent compound (4). The 3D NMR structures of the analogues in dimethylsulfoxide are consistent with the observed binding affinities.

Full text of DOI:10.1021/jm701445q

2676
J. Med. Chem. 2008, 51, 2676–2681
Ring Size in Octreotide Amide Modulates Differently Agonist versus Antagonist Binding
Affinity and Selectivity
Christy Rani R. Grace,‡ Judit Erchegyi,† 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
H-DPhe2-c[Cys3-Phe7-DTrp8-Lys9-Thr10-Cys14]-Thr15-NH2 (1) (a somatostatin agonist, SRIF numbering)
and H-Cpa2-c[DCys3-Tyr7-DTrp8-Lys9-Thr10-Cys14]-Nal15-NH2 (4) (a somatostatin antagonist) are based
on the structure of octreotide that binds to three somatostatin receptor subtypes (sst2/3/5) with significant
binding affinity. Analogues of 1 and 4 were synthesized with norcysteine (Ncy), homocysteine (Hcy), or
D-homocysteine (DHcy) at positions 3 and/or 14. Introducing Ncy at positions 3 and 14 constrained the
backbone flexibility, resulting in loss of binding affinity at all ssts. The introduction of Hcy at positions 3
and 14 improved selectivity for sst2 as a result of significant loss of binding affinity at the other ssts.
Substitution by DHcy at position 3 in the antagonist scaffold (5), on the other hand, resulted in a significant
loss of binding affinity at sst2 and sst3 as compared to the different affinities of the parent compound (4).
The 3D NMR structures of the analogues in dimethylsulfoxide are consistent with the observed binding
affinities.
Introduction
sst5. Recently, we published that the replacement of Phe at
position 7 by an Ala in the octreotide scaffold resulted in an
agonist (H-DPhe2-c[Cys3-Ala7-DTrp8-Lys9-Thr10-Cys14]-Thr15-
NH2), which showed unique sst2 selectivity.4 Structure-activity
relationship (SAR) studies of such analogues suggested that the
aromatic side chain at position 7 was not necessary for sst2
binding but was crucial for sst3 and sst5 binding. The 3D
structure of this peptide also had a ꢀ-turn of type-II′ for the
backbone conformation, which oriented the side chain of DTrp8,
the amino alkyl group of Lys9, and the aromatic ring of DPhe2
outside the cycle in their respective positions, which were crucial
for effective receptor-ligand binding. On the basis of these
results, we have proposed an sst2-selective pharmacophore for
the SRIF agonists.4
The development of a strictly somatostatin (SRIF)a receptor
2 (sst2)-selective analogue remains a challenge, because ana-
logues reported with high binding affinity to sst2 often also bind
with high binding affinity to sst5 and sometimes to sst3.1,2 Most
of these partially selective analogues are based on the structure
of octreotide (H-DPhe2-c[Cys3-Phe7-DTrp8-Lys9-Thr10-Cys14]-
Thr15-ol, SRIF numbering) and have a type-II′ ꢀ-turn in their
structure.3 The nonselective sst2/5 pharmacophore requires two
aromatic side chains at positions 2 and 7 in addition to the
DTrp8-Lys9 pair. It has also been shown that octreotide-like
analogues undergo conformational changes in their backbone,
from ꢀ-sheet to R-helix, resulting in two different locations for
the Phe/DPhe/Tyr at position 2.1 This conformational transition
complicates the interpretation of a particular structure as the
bioactive conformation for a particular receptor (i.e., complicates
the definition of the pharmacophore). In fact, it suggests that
two conformations are necessary for the analogue to fit into
the two different subtype-selective pharmacophores for sst2 and
Bass et al. reported that changing the chirality of H-DPhe2-
LCys3- to H-LPhe2-DCys3- in the octreotide scaffold resulted
in an SRIF antagonist.5 Similar to the octreotide-based agonists,
these analogues were binding to sst2/5 and sometimes to sst3 as
well. On the basis of our SAR studies with sst2-selective
agonists, we have also designed sst2-selective antagonists having
a longer side chain at position 7 (in preparation). The 3D NMR
structures of these antagonists identified the pharmacophore for
sst2-selective antagonists, very similar to the pharmacophore for
sst2-selective agonists.4 Here, we present a novel approach,
based on the agonistic and antagonistic octreotide scaffold where
the number of the methylene units involved in the disulfide
bridge is reduced or increased by the substitutions of Cys at
positions 3 and 14 with norcysteine (Ncy) or homocysteine
(Hcy). The influence of these modifications on receptor selectiv-
ity and binding affinity seems to be different for agonists and
antagonists. These data are reported along with the 3D NMR
structures of the analogues, which correlate well with the
proposed sst2-selective agonist pharmacophore.
* 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; CZE, capillary zone electrophoresis; CYANA, combined
assignment and dynamics algorithm for NMR applications; DHcy, D-
homocysteine; DIC, N,N′-diisopropylcarbodiimide; DIPEA, diisopropyl-
ethylamine; DQF-COSY, double quantum filtered correlation spectroscopy;
Hcy, homocysteine; HOBt, 1-hydroxybenzotriazole; Mob, 4-methoxybenzyl;
Ncy, norcysteine; NOE, nuclear Overhauser enhancement; NOESY, NOE
spectroscopy; 3D, three-dimensional; OBzl, benzyl ester; rmsd, root-mean-
square deviation; SAR, structure-activity relationship; SRIF, somatostatin;
ssts, SRIF receptors; TEA, triethylamine; TEAP, triethylammonium phos-
phate; TOCSY, total correlation spectroscopy.
Results
Peptide Synthesis. All of the peptides shown in Table 1 were
synthesized automatically on an MBHA resin by using the
10.1021/jm701445q CCC: $40.75
2008 American Chemical Society
Published on Web 04/12/2008

Ring Size in Octreotide Amide
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9 2677
Table 1. Physico-chemical Properties, sst1–5 Binding Affinities (IC50s, nM), and Functional Studies of the Analogues and Control Peptide Octreotide
Amide (1)
sst2
purity
MSc
IC50 (nM)d
sst3
no. of
atoms in
functional
assay sst2
ID
compd
HPLCa CZEb Mcalc M + Hobs
sst1
sst2
sst4
sst5
the cycle internalizatione
SRIF-28
99
95
99
99
3146.5
1031.4
3147.3
1032.1
2.2 ( 0.2
>1000
2.4 ( 0.2 2.7 ( 0.4 2.7 ( 0.3 2.5 ( 0.2
38
20
agonist
agonist
1f H-DPhe2-c[Cys3-Phe7-DTrp8-
1.9 ( 0.3 39 ( 14
>1000
5.1 ( 1.1
Lys9-Thr10-Cys14]-Thr15
NH2 octreotide amide
(SRIF numbering)
-
2
H-DPhe2-c[Ncy3-Phe7-DTrp8-
Lys9-Thr10-Ncy14]-
Thr15-NH2
99
99
99
98
99
99
99
98
1003.4
1059.4
1177.4
1205.4
1004.3
1060.6
1178.4
1205.4
>1000
>1000
>1000
337 ( 60 >1000
214 ( 61 >1000
18
22
20
22
nd
3f H-DPhe2-c[Hcy3-Phe7-DTrp8-
Lys9-Thr10-Hcy14]-
4.9 ( 1.7 452 ( 245 115 ( 34 109 ( 39
5.7 ( 1.5 112 ( 32 296 ( 19 218 ( 63
agonist
antagonist
antagonist
Thr15-NH2
4
H-Cpa2-c[DCys3-Tyr7- DTrp8-
Lys9-Thr10-Cys14]-
Nal15-NH2
28
5f H-Cpa2-c[DHcy3-Tyr7- DTrp8-
Lys9-Thr10-Hcy14]-
763 ( 208 267 ( 60 359 ( 169 174 ( 41 199 ( 35
Nal15-NH2
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 OD × 75 µm
ID × 50 cm length). Detection at 214 nm. c Calculated m/z of the monoisotope compared with the observed [M + H]+ monoisotopic mass. d Values
represent the IC50 in nM (mean ( SEM, n g 3). e Tested in vitro in HEK-sst2 cells (n g 2); nd, not determined. f 3D NMR structures of these analogues
are presented in this paper.
t-Butoxycarbonyl (Boc) strategy. Boc-Ncy(Mob)-OH, Boc-D/
L-Ncy(Mob)-OH,6 Boc-Hcy(Mob)-OH, and Boc-DHcy-(Mob)-
OH were synthesized in our laboratory.7 The peptides were
cleaved and fully deprotected with hydrogen fluoride. Cycliza-
tion of the cysteines/norcysteines/homocysteines was mediated
by iodine in an acidic milieu.8
Purification and Characterization of the Analogues (see
Table 1). Purification was carried out by using multiple
preparative reversed-phase high-performance liquid chroma-
tography (RP-HPLC) steps.9 Purity and identity of the analogues
were established by analytical RP-HPLC,9 capillary zone
electrophoresis,10 and mass spectrometry. The purity of the
peptides was >95%. The observed monoisotopic mass (M +
H)+ values of each peptide matched the calculated mass (M +
H)+ values and are given in Table 1.
Receptor Binding. All of the peptides were tested for their
ability to bind to the five human SRIF receptor subtypes in
competitive experiments by using 125I-[Leu8,DTrp22,Tyr25]SRIF-
28 as radioligand. Cell membrane pellets were prepared, and
receptor autoradiography was performed as described in detail
previously.11 The binding affinities are expressed as IC50 values
that are calculated as described previously.11,12
We have introduced Ncy and Hcy at positions 3 and 14 to
the octreotide scaffold to gain insight into the structure of the
peptide and the influence of the number of atoms in the cysteine
side chain involved in the disulfide bond on receptor binding
and activation. Analogue 2 differs from 1 (a SRIF agonist) in
that the two cysteines are substituted by Ncy at positions 3
and 14, resulting in a disulfide bridge with 18 atoms in the
cycle instead of 20 atoms. This peptide does not bind to any
of the ssts. Analogue 3 differs from 1 in that the two cysteines
are substituted by Hcy at positions 3 and 14, resulting in a
disulfide bridge with 22 atoms in the cycle instead of 20 atoms.
Whereas 1 binds to the sst2/5 receptors with high binding affinity
(IC50 ) 1.9 and 5.1 nM, respectively) and to sst3 with moderate
binding affinity (IC50 ) 39 nM), 3 binds more selectively to
sst2 with a high binding affinity comparable to that of 1 (IC50
) 4.9 nM) but with much less binding affinity to sst3 and sst5
(IC50 ) 452 nM and 109 nM, respectively). On the other hand,
3 also binds to sst4 to some extent (IC50 ) 115 nM) (Table 1).
Analogue 5 differs from 4 (a reference SRIF antagonist) by the
presence of DHcy at position 3 and Hcy at position 14. This
analogue binds 50-fold less to sst2 than 4 (Table 1). 3D structures
of 1, 3, and 5 were determined by NMR and compared with
the sst2-selective pharmacophore.
Functional Studies: Receptor Internalization. As seen in
Table 1, 1 was found to be a potent sst2 agonist and had no
antagonistic properties; 3 was an sst2 agonist, less potent than
1, and had no antagonistic properties either; conversely, 4 and
5 had no agonistic properties up to 10 000 nM. However, they
were sst2 antagonists, because they could completely inhibit the
[Tyr3]-octreotide-induced sst2 internalization.
NMR Studies. In this section, we report the chemical shift
assignment of various proton resonances and structural informa-
tion for the selected analogues 1,4 3, and 5 (Table 1) by using
NMR techniques in the solvent dimethylsulfoxide (DMSO).
Assignment of Proton Resonances, Collection of Struc-
tural Restraints, and Structure Determination. The nearly
complete chemical-shift assignments of proton resonances
(Table S2) for 1, 3, and 5 have been carried out by using 2D
NMR experiments by applying the standard procedure as
described in the Experimental Section. Assignment and struc-
tural characterization of 1 (octreotide amide) have been taken
from our previously published paper.4 A large number of
experimental nuclear Overhauser enhancements (NOEs) is
observed for all of the three analogues in the NOE spectrum
(NOESY) 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 are used as input for the structure calculation
with the program combined assignment and dynamics algorithm
for NMR applications (CYANA)13 followed by restrained
energy minimization by using the program DISCOVER.14 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

2678 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9
Grace et al.
Table 2. Characterization of the NMR Structures of the Analogues Studied by NMRa
residual restraint violations on
CFF91 energies (kcal/mol)
total van der
energy Waals electro-static no g 0.1 Å max (Å) no g 1.5° max (°)
distances
dihedral angles
NOE
distance
CYANA
target
angle
backbone
overall
rmsd (Å)
ID# restraints restraintsb functionc rmsd (Å)
1
3
5
115
90
107
24
14
15
0.002 0.56 ( 0.14 1.06 ( 0.28 210 ( 14 121 ( 10
89 ( 8
59 ( 3
43 ( 2
0.4 ( 0.1 0.11 ( 0.02
0.4 ( 0.1 0.15 ( 0.00
0.7 ( 0.1 0.15 ( 0.03
0 ( 0
0 ( 0
0 ( 0
0 ( 0
0 ( 0
0.0 ( 0.05
0.04
0.09
0.70 ( 0.24 1.67 ( 0.34 172 ( 6 113 ( 4
0.51 ( 0.27 1.55 ( 0.53 215 ( 4 172 ( 5
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.1 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 13.
3D Structure of H-Cpa2-c[DHcy3-Tyr7-DTrp8-Lys9-
Thr10-Hcy14]-Nal15-NH2 (5). Analogue 5 differs from 3 by Cpa
at position 2, DHcy at position 3, Tyr at position 7, and Nal at
position 15 and has completely lost its binding affinity to
receptor 2 (Table 1). The backbone of this analogue has an
inverse γ-turn around residue DTrp8 (Table S1), different from
3 and also from other octreotide-based agonists and antagonists.
The torsion angles show that the side chains of Cpa2, Tyr7,
Figure 1. Survey of characteristic NOEs describing the secondary
structure of the three analogues studied by NMR (i.e., 1, 3, and 5 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 Hcy, DHcy,
DPhe, DTrp, and Nal refer to homocysteine, D-homocysteine, D-
phenylalanine, D-tryptophan, and naphthylalanine and are denoted by
the symbols, c, C, f, w, and X, respectively.
DTrp8, Lys9, and Nal15 are in the gauche+ rotamer. This
configuration orients the side chains of Tyr7, DTrp8, Lys9, and
Nal15 in the plane of the peptide backbone, and the side chain
of Cpa2 is oriented away from the plane of the backbone (Figure
2). No hydrogen bond stabilizing the structure is observed in
all of the 20 conformers calculated.
Discussion
Influence of the Ring Size on Receptor Binding Affin-
ity. Comparing the three analogues studied from a structural,
chemical, and biological point of view, it is observed that the
number of atoms in the disulfide bridge had a major impact on
both the conformation and the receptor selectivity. The introduc-
tion of Ncy in the octreotide-based sst2 agonist (1) resulted in
2. The shorter side chain of Ncy constrained the peptide
backbone so tightly that the analogue lost binding affinity to
all of the receptors. Introduction of Hcy, which has a longer
side chain than Cys, resulted in 3 with partial selectivity for
sst2. In contrast, DHcy with a different chirality at position 3
resulted in 5 with a complete loss of binding to sst2. From a
structural perspective, Hcy3/14 substitutions (3, the number of
atoms in the cycle changed from 20 to 22 compared to 1) modify
the conformation of the peptide backbone because of the
flexibility in the disulfide bridge, thereby slightly changing the
relative orientation of the amino acid side chains (Figure 2).
But the introduction of DHcy3 alters the backbone conformation
from a type-II ꢀ-turn to a γ-turn, thereby significantly changing
the relative orientation of the amino acid side chains (Figure
2). All of these observations 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. Hence, the
spatial orientation of the amino acid side chains for the analogues
is compared with the sst2-selective pharmacophore in the following
section.
conformers (Table 2). The deviations from ideal geometry are
minimal, and similar energy values are 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).
3D Structure of H-DPhe2-c[Cys3-Phe7-DTrp8-Lys9-Thr10-
Cys14]-Thr15-NH2 (1). Analogue 1 is very similar to octreotide
(Thr-ol is substituted by Thr-NH2) and binds to the sst2/3/5
receptors with moderately high binding affinity (Table 1). As
reported earlier,4 the torsion angles indicate a type-II′ ꢀ-turn
conformation for the backbone, as evidenced by the presence
of the medium range dRN(i, i + 2) NOE observed between DTrp8
and Thr10 (Figure 1), as well as the hydrogen bond observed
between Thr10NH-O′Phe7 in all of the 20 structures. The
unshifted amide proton resonance of Thr10 at 7.58 ppm (from
298 to 313K) confirms that this amide proton is involved in a
hydrogen bond. The side chain of Phe7 and DTrp8 are in the trans
rotamer and that of Lys9 is in the gauche+ rotamer (Table S1).
3DStructureofH-DPhe2-c[Hcy3-Phe7-DTrp8-Lys9-Thr10-
Hcy14]-Thr15-NH2 (3). Analogue 3 differs from 1 by Hcy at
positions 3 and 14 and shows selective binding for sst2 (Table
1). The backbone torsion angles indicate a slightly distorted
ꢀ-turn of type-II′ conformation around DTrp8-Lys9 (Figure 2
and Table S1). The side chains of DPhe2 and DTrp8 are in the
gauche- rotamer and those of Phe7 and Lys9 are in the gauche+
rotamer (Table S1). Hence, the side chains of DPhe2, Phe7, and
DTrp8 are in the plane of the backbone of the analogue, whereas
the side chain of Lys9 is pointing away from the plane of the
peptide backbone. In all of the 20 conformers calculated, there
is a hydrogen bond between the amide proton of Lys9 and the
carbonyl of Phe7. Experimentally observed small temperature
coefficient of -0.002 ppm/K for the amide proton of Lys9
suggests that it could be involved in a hydrogen bond.
Comparison of the 3D Structures of 3 and 5 with the
sst2-Selective Pharmacophore. The sst2-selective pharmacoph-
ore requires one aromatic side chain far from the side chains of
DTrp8-Lys9, as shown in Figure 34 and Table 3. Figure 3 shows
octreotide in two different conformations, and the one shown
in magenta represents the structure required for the sst2-selective
pharmacophore. Analogue 3 binds to sst2 with the affinity of

Ring Size in Octreotide Amide
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9 2679
Figure 2. 3D structures of 1, 3, and 5 studied by NMR. 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: magenta, (1) H-DPhe-c[Cys-Phe-DTrp-Lys-Thr-Cys]-Thr-NH2;
cyan, (3) H-DPhe-c[Hcy-Phe-DTrp-Lys-Thr-Hcy]-Thr-NH2; and red, (5) H-Cpa-c[DHcy-Tyr-DTrp-Lys-Thr-Hcy]-Nal-NH2. 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, DPhe or Cpa at position 2, Phe or Tyr at position 7, and Nal at position 15.
Figure 3. Superposition of receptor-specific pharmacophores of octreotide with the 3D NMR structures of 3 and 5. The sst2 pharmacophore4 of
octreotide is shown in magenta. The octreotide pharmacophore proposed by Melacini et al.3 is represented in yellow. For both pharmacophores,
only the amino acid side chains that are involved in binding to the receptor are shown. For each of the analogues 3 and 5, the conformer with the
lowest energy is used to represent the 3D structure of the analogue. The analogues are colored as in Figure 2, and for each analogue, the amino acid
side chains proposed to be involved in receptor binding are labeled for clarity.
Table 3. Distances between Cγ Atoms (in Å) of Selected Residues in the Different Pharmacophores Compared with those Found in the Analogues
Studied by NMR
analogue
F2-F7
F2-DW8
F2-K9
F7-DW8
F7-K9
DW8-K9
sst2 pharmacophore4
12.0-13.5
11.0-15.0
13.5-14.6
10.9-14.3
11.5-14.6
12.5-15.0
12.0-15.0
13.5-14.8
11.9-14.9
9.6-13.7
4.0-5.0
5.0-5.0
5.3-5.7
6.8-8.1
6.3-7.6
octreotide pharmacophore1
5.0-11.0
8.1-9.9
6.3-8.9
8.6–10.3
7.0-9.0
6.7-8.0
5.5-7.0
4.3-7.2
9.0-11.0
10.0-11.2
9.5-10.1
10.2-10.7
1
3
5
4.9 nM, and the 3D NMR structure of this analogue in DMSO
shows that it prefers a conformation which has the sst2-selective
pharmacophore (Figure 3). Analogue 3 has the type-II′ ꢀ-turn
similar to the sst2-selective analogues, and the side chains of
DPhe2, DTrp8, and Lys9 are in the plane of the backbone of the
analogue, just like in 1 (Figure 3). The flexibility in the side
chain of DPhe2 along with the backbone flexibility due to the
longer Hcy at positions 3 and 14 explains the low binding
affinity of this analogue to sst4 and sst5 receptors. Analogue 5,
with DHcy at position 3, has an inverse γ-turn around residue
DTrp8. Superimposing the structure of 5 on that of octreotide
shows that the side chain of Cpa2 lies in between sst2- and sst5-
selective pharmacophores (Figure 3). Because of the available
conformational flexibility in the backbone of the analogue with
DHcy at position 3 and Hcy at position 14, one would expect
the aromatic side chain of Cpa2 to fit both sst2 and
sst5pharmacophores. The binding data of 5 compared to those
of 4 show a 50-fold loss of binding affinity to sst2, equal affinity
to sst5, a 3-fold loss to sst3, and some gain of binding affinity
to sst1 and sst4 (Table 1). Hence, the binding data could only
be explained on the basis of the 3D structure, which shows that
the bulkier Nal group at the C-terminus extends further away
in the plane of the peptide backbone, which probably prevents
the analogue from binding to both sst2 and sst5. Analogues 4
and 5 are antagonists at sst2 on the basis of their ability to inhibit
the [Tyr3]octreotide-induced sst2 internalization. The 3D NMR
structures of antagonist analogues very similar to that of
analogue 4 show that the position of the aromatic side chain at

2680 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9
Grace et al.
NOESY spectrum was used for the sequential assignment.25 The
collection of structural restraints was based on the NOEs assigned
manually and vicinal JNHR couplings. Dihedral angle constraints
position 2 is very crucial for sst2 binding. In addition, the
position of the Nal group at the C-terminus is within the
framework of the peptide backbone for most of the analogues.15
3
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.13 The calibration of NOE intensities versus 1H-1H
distance restraints and appropriate pseudoatom corrections to the
nonstereospecifically assigned methylene, methyl, and ring protons
were performed by using the program CYANA. On average,
approximately 100 NOE constraints and 15-20 angle constraints
were utilized to calculate the conformers (Table 2). A total of 100
conformers was 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
using the program DISCOVER with steepest decent and conjugate
gradient algorithms.26 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.27
Conclusions
The synthesis, binding, and 3D NMR structural characteriza-
tion of octreotide-based analogues with different numbers of
atoms in the cysteine side chain involved in the disulfide bond
is reported. Reducing the number of atoms by using Ncy resulted
in tremendous loss in binding affinity because of the restriction
in the backbone flexibility. Increasing the number of atoms in
the cycle had different effects for the agonist and the antagonist.
Although Hcy at position 3 enhanced selectivity for sst2, DHcy
replacement at position 3 resulted in dramatic loss in affinity
compared to the parent compound. The 3D NMR structures
identified the presence and absence of the sst2-selective phar-
macophore in the analogues, which explains the binding data.
The current data highlight the indirect role of changes in size
of the disulfide bridge in inducing the backbone conformation,
which in turn, orients the side chains of the residues involved
in receptor interaction.
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 synthesis and characteriza-
tion of the peptides. We thank Dr. W. Fisher and W. Low for
mass spectrometric analyses of the analogues and D. Doan for
manuscript preparation. J.R. is the Dr. Frederik Paulsen Chair
in Neurosciences Professor.
Experimental Section
Functional Studies: Receptor Internalization. Immunofluo-
rescence microscopy-based internalization assays with HEK-sst2
cells were performed as previously described by Cescato et al.16
Briefly, cells were treated either with [Tyr3]-octreotide, 1, 3, 4, or
5 at concentrations ranging from 100 to 10-000 nM or, to evaluate
potential antagonism, with 100 nM [Tyr3]-octreotide in the presence
of a 100-fold excess of 1, 3, 4, or 5 for 30 min at 37 °C and 5%
CO2 in growth medium and then processed for immunofluorescence
microscopy by using the polyclonal sst2-specific R2-88 antibody
(provided by Dr. A. Schonbrunn, University of Texas Medical
School, Houston, TX) at a dilution of 1:1000 as first antibody and
Alexa Fluor 488 goat antirabbit IgG (H + L) at a dilution of 1:600
as secondary antibody. The cells were imaged as described pre-
viously.16
Supporting Information Available: Starting materials, peptide
synthesis, cleavage and deprotection with HF and cyclization,
purification and chemical characterization of the analogues, cell
culture, and receptor binding data are reported in Supporting
Material. Similarly, Table S1 (torsion angles ꢁ, Ψ, and ꢂ1 (in °) of
the bundle of 20 energy-minimized conformers) and Table S2
(proton chemical shifts of the analogues studied by NMR) are
reported in Supporting Material. This material is available free of
charge via the Internet at http://pubs.acs.org.
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