Ring Size in Octreotide Amide Modulates Differently Agonist versus Antagonist Binding Affinity and Selectivity

Abstract

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 (sst_2/3/5) with significant binding affinity. Analogues of 1 and 4 were synthesized with norcysteine (Ncy), homocysteine (Hcy) or dHcy at positions 3 and/or 14. Introducing Ncy at positions 3 and 14 constrains 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 sst₂ 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 DMSO are consistent with the observed binding affinities.

Introduction

The development of a strictly somatostatin receptor 2 (sst₂)-selective analogue remains a challenge, since analogues 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' β-turn in their structure.³ The non-selective sst_2/5 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 β-sheet to α-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 sst₅, respectively. 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 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 β-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. Based on this, we have proposed an sst₂-selective pharmacophore for the somatostatin agonists.⁴

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 sst_2/5 and sometimes to sst₃ as well. Based on 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 Ncy (norcysteine) or Hcy (homocysteine). The influence of these modifications on receptor-selectivity 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 correlates well with the proposed sst₂-selective agonist pharmacophore.

Results

Peptide Synthesis

All of the peptides shown in Table 1 were synthesized automatically on an MBHA resin using the 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. Cyclization of the cysteines/norcysteines/homocysteines was mediated by iodine in an acidic milieu.⁸

Table 1. Physico-Chemical Properties, Sst_1-5 Binding Affinities (IC₅₀s, nM) and Functioonal studies of the Analogues and Control Peptide Octreotide Amide (1).

ID#	Compound	Purity		MSc		IC50 nMd					Number of atoms in the cycle	Sst2 functional assay sst2 internalizatione
		HPLCa	CZEb	Mcalc	M+Hobs	sst1	sst2	sst3	sst4	sst5
	SRIF-28	99	99	3146.5	3147.3	2.2 ± 0.2	2.4 ± 0.2	2.7 ± 0.4	2.7 ± 0.3	2.5 ± 0.2	38	Agonist
1*	H-DPhe2-c[Cys3-Phe7-DTrp8-Lys9-Thr10-Cys14]-Thr15-NH2 octreotide amide (SRIF numbering)	95	99	1031.4	1032.1	>1000	1.9 ± 0.3	39 ± 14	>1000	5.1 ± 1.1	20	Agonist
2	H-DPhe2-c[Ncy3-Phe7-DTrp8-Lys9-Thr10-Ncy14]-Thr15-NH2	99	99	1003.4	1004.3	>1000	337 ± 60	>1000	214 ± 61	>1000	18	nd
3*	H-DPhe2-c[Hcy3-Phe7-DTrp8-Lys9-Thr10-Hcy14]-Thr15-NH2	99	99	1059.4	1060.6	>1000	4.9 ± 1.7	452 ± 245	115 ± 34	109 ± 39	22	Agonist
4	H-Cpa2-c[DCys3-Tyr7- DTrp8-Lys9-Thr10-Cys14]-Nal15-NH228	99	99	1177.4	1178.4	>1000	5.7 ± 1.5	112 ± 32	296 ± 19	218 ± 63	20	Antagonist
5*	H-Cpa2-c[DHcy3-Tyr7- DTrp8-Lys9-Thr10-Hcy14]-Nal15-NH2	98	98	1205.4	1205.4	763 ± 208	267 ± 60	359 ± 169	174 ± 41	199 ± 35	22	Antagonist

^*3D NMR structures of these analogs are presented in this paper.

^aPercent purity determined by HPLC using buffer system: A = TEAP (pH 2.5) and B = 60% CH₃CN/40% A with a gradient slope of 1% B/min, at 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.

^bCapillary zone electrophoresis (CZE) was done using a Beckman P/ACE System 2050 controlled by an IBM Personal System/2 Model 50Z and 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.

^cThe calculated m/z of the monoisotope compared with the observed [M + H]⁺ monoisotopic mass.

^dValues represent the IC₅₀ in nM (mean ± SEM, n ≥ 3)

^eTested in vitro in HEK-sst₂ cells (n ≥ 2); nd, not determined.

Purification and Characterization of the Analogues (see legend of Table 1)

Purification was carried out using multiple preparative RP-HPLC steps.⁹ Purity and identity of the analogues were established by analytical RP-HPLC,⁹ capillary zone electrophoresis¹⁰ 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 somatostatin receptor subtypes in competitive experiments 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 somatostatin agonist) in that the two cysteines are substituted by norcysteines (Ncy) at position 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 sst_s. Analogue 3 differs from 1 in that the two cysteines are substituted by homocysteines (Hcy) at position 3 and 14 resulting in a disulfide bridge with 22 atoms in the cycle instead of 20 atoms. While 1 binds to the sst_2/5 receptors with high binding affinity (IC₅₀ = 1.9 nM and 5.1 nM, respectively) and to sst₃ with moderate binding affinity (IC₅₀ = 39 nM), 3 binds more selectively to sst₂ with comparable high binding affinity as 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 somatostatin 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). Three-dimensional 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, as 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 information for the selected analogues 1,⁴ 3 and 5 (Table 1) using NMR techniques in the solvent DMSO.

Assignment of Proton Resonances, Collection of Structural Restraints and Structure Determination

The nearly complete chemical shift assignments of proton resonances (Table S2) for 1, 3 and 5 have been carried out using two-dimensional (2D) NMR experiments applying the standard procedure as described in the Materials and Methods section. Assignment and structural characterization of 1 (octreotide amide) have been taken from our previously published paper.⁴ A large number of experimental NOEs is observed for all of the three 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 are used as input for the structure calculation with the program CYANA¹³ followed by restrained energy minimization 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 inter-atomic 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 are obtained for all of the 20 conformers for each analogue. The quality of the structures determined is furthermore reflected by the small backbone RMSD values relative to the mean coordinates of ∼0.5 Å (see Table 2 and Figure 2).

Table 2. Characterization of the NMR Structures of the Analogues Studied by NMR^*.

ID#	NOE distance restraints	Angle restraints ***	cyana Target function**	Backbone RMSD (Å)	Overall RMSD (Å)	CFF91 energies (Kcal/mol)			Residual restraint violations on
						Total energy	Van der Waals	Electrostatic	Distances		Dihedral Angles
									No. ≥0.1 Å	Max (Å)	No. ≥1.5 deg	Max (deg)
1	115	24	0.002	0.56 ± 0.14	1.06 ± 0.28	210 ± 14	121 ± 10	89 ± 8	0.4 ± 0.1	0.11 ±0.02	0 ± 0	0 ± 0
3	90	14	0.04	0.70 ± 0.24	1.67 ± 0.34	172 ± 6	113 ± 4	59 ± 3	0.4 ± 0.1	0.15 ± 0.00	0 ± 0	0 ± 0
5	107	15	0.09	0.51 ± 0.27	1.55 ± 0.53	215 ± 4	172 ± 5	43 ± 2	0.7 ± 0.1	0.15 ± 0.03	0 ± 0	0.0±0.05

^*The bundle of 20 conformers with the lowest residual target function was used to represent the NMR structures of each analogue.

^**The target function is zero only if all the experimental distance and torsion angle constraints are fulfilled and all non-bonded 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 reference.¹

^***Meaningful NOE distance restraints may include intra-residual and sequential NOEs.¹

Figure 2.

3D NMR structures of 1, 3 and 5 studied by NMR. For each analogue, twenty energy-minimized conformers with the lowest target function are used to represent the 3D NMR structure. The bundle is obtained by overlapping the C^α 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; 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 somatostatin receptors are highlighted: dTrp at position 8 in light green, Lys at position 9 in blue and dPhe or Cpa at position 2, Phe or Tyr at position 7 and Nal at position 15 in yellow.

Three-Dimensional 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 sst_2/3/5 receptors with moderately high binding affinity (Table 1). As reported earlier,⁴ the torsion angles indicate a type-II' β-turn conformation for the backbone, as evidenced by the presence of the medium range d_αN(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 un-shifted amide proton resonance of Thr¹⁰ at 7.58 ppm (from 298K 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).

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 to 6 Å), medium (3 to 4.5 Å) and strong (<3 Å) NOEs observed in the NOESY spectrum. The medium-range connectivities d_NN(i,i+2), d_αN(i,i+2), and d_βN(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, c̄, C̅, f, w and X, respectively.

Three-dimensional Structure of H-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 β-turn of type-II' conformation around dTrp⁸-Lys⁹ (Figure 2 and Table S1). The side chains of dPhe², dTrp⁸ are in the gauche^- rotamer and that of Phe⁷, 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 twenty 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.

Three-dimensional 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, 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 the twenty conformers calculated.

Discussion

The Influence of the Ring Size on Receptor Binding Affinity

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 as well as the receptor selectivity. The introduction of Ncy in the octreotide-based sst₂ agonist (1) resulted in 2. The shorter side chain of Ncy constrains 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, Hcy^3/14 substitutions (3, the number of atoms in the cycle changed from 20 to 22 compared to 1) modifies 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 β-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 in the binding of peptides, rather it 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.

Comparison of the 3D Structures of 3 and 5 with the Sst₂-Selective Pharmacophore

The sst₂-selective pharmacophore 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 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' β-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). With 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 sst₅ pharmacophores. 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 based on 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 based on their ability to inhibit the [Tyr³]octreotide-induced sst₂ internalization. The 3D NMR structures of antagonist analogues very similar to analogue 4 show that the position of the aromatic side chain at 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

Figure 3.

Superposition of receptor-specific pharmacophores of octreotidewith 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 are shown that are involved in binding to the receptor. For each of the analogue 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 that 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	12.5-15.0	-	-	4.0-5.0
octreotide pharmacophore1	5.0-11.0	11.0-15.0	12.0-15.0	7.0-9.0	9.0-11.0	5.0-5.0
1	8.1-9.9	13.5-14.6	13.5-14.8	6.7-8.0	10.0-11.2	5.3-5.7
3	6.3- 8.9	10.9-14.3	11.9-14.9	5.5-7.0	9.5-10.1	6.8-8.1
5	8.6-10.3	11.5-14.6	9.6-13.7	4.3-7.2	10.2-10.7	6.3-7.6

Conclusions

The synthesis, binding and 3D NMR structural characterization 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 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. While 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 pharmacophore 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.

Materials and Methods

Functional Studies: Receptor Internalization

Immunofluorescence 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 nM 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 using the polyclonal sst₂-specific R2-88 antibody (provided by Dr. A. Schonbrunn, University of Texas Medical School, Houston, TX) at a dilution of 1:1,000 as first antibody and Alexa Fluor 488 goat anti-rabbit IgG (H+L) at a dilution of 1:600 as secondary antibody. The cells were imaged as described previously.¹⁶

NMR Studies

NMR samples were prepared by dissolving 2 mg of the analogue in 0.5 mL of DMSO-d₆. The ¹H NMR spectra were recorded on a Bruker 700 MHz spectrometer operating at proton frequency of 700 MHz. Chemical shifts were measured using DMSO (δ = 2.49 ppm) as an internal standard. The 1D spectra and all the 2D spectra were acquired at 298 K. Resonance assignments of the various proton resonances have been carried out using total correlation spectroscopy (TOCSY);^17,18 double-quantum filtered spectroscopy (DQF-COSY)¹⁹ and nuclear Overhauser enhancement spectroscopy (NOESY).^20-22 The TOCSY experiments employed the MLEV-17 spin-locking sequence suggested by Davis and Bax,¹⁷ applied for a mixing time of 50 ms. The NOESY experiments were carried out with a mixing time of 100 ms. The TOCSY and NOESY spectra were acquired using 800 complex data points in the ω₁ dimension and 1024 complex data points in the ω₂ dimension with t_1max = 33 ms and a t_2max = 43 ms and were subsequently zero-filled to 1024 × 2048 before Fourier transformation. The DQF-COSY spectra were acquired with 1024 × 4096 data points and were zero-filled to 2048 × 4096 before Fourier transformation. The TOCSY, DQF-COSY and NOESY spectra were acquired with 8, 8 and 16 scans, respectively, with a relaxation delay of 1 s. The signal from the residual water of the solvent was suppressed using pre-saturation during the relaxation delay and during mixing time. The TOCSY and NOESY data were multiplied by 75° shifted sine-function in both dimensions. All the spectra were processed using the software PROSA.²³ The spectra were analyzed using the software X-EASY.²⁴

Structure Determination

The chemical shift assignment of the major conformer (the population of the minor conformer was < 10%) was obtained by the standard procedure using DQF-COSY and TOCSY spectra for intra-residual assignment and the NOESY spectrum was used for the sequential assignment.²⁵ The collection of structural restraints was based on the NOEs assigned manually and vicinal ³J_NHα couplings. Dihedral angle constraints were obtained from the ³J_NHα couplings, which were measured from the 1D ¹H NMR spectra and from the intra-residual 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 pseudo-atom corrections to the non-stereo specifically assigned methylene, methyl and ring protons were performed using the program CYANA. On an average, approximately 100 NOE constraints and 15 to 20 angle constraints were utilized while calculating 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, 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 using the program MOLMOL.²⁷

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-chlorobenzyloxycarbonyl

CZE

capillary zone electrophoresis

CYANA

Combined assignment and dynamics algorithm for NMR applications

DIC

N,N′-diisopropylcarbodiimide

DIPEA

diisopropylethylamine

DMF

dimethylformamide

DMSO

dimethylsulfoxide

DQF-COSY

double quantum filtered correlation spectroscopy

Hcy

homocysteine

HOBt

1-hydroxybenzotriazole

Mob

4-methoxybenzyl

Ncy

norcysteine

NMR

nuclear magnetic resonance

NOESY

nuclear Overhauser enhancement spectroscopy

3D

three-dimensional

OBzl

benzyl ester

PROSA

Processing algorithms

RMSD

root mean square deviation

SAR

structure activity relationships

SRIF

somatostatin

sst_s

SRIF receptors

TEA

triethylamine

TEAP

triethylammonium phosphate

TFA

trifluoroacetic acid

TOCSY

total correlation spectroscopy