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

Abstract

The synthesis, biological testing, and NMR studies of several analogues of H-c[Cys (3)-Phe (6)-Phe (7)-DTrp (8)-Lys (9)-Thr (10)-Phe (11)-Cys (14)]-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., sst 4 in all cases).

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 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 Ncy, dNcy, Hcy, dHcy, dCys, dTrp refer to norcysteine, d-norcysteine, homocysteine, d-homocysteine, d-cysteine and d-tryptophan denoted by the symbols, C, c, $\stackrel{‒}{\mathrm{C}}$, $\stackrel{‒}{\mathrm{c}}$, c and w, respectively.

Figure 2

The NMR structures of the five analogues studied by NMR (i.e., analogues 1, 3, 18, 19 and 21 as indicated). 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: grey (1) H-c[Cys-Phe-Phe-dTrp-Lys-Thr-Phe-Cys]-OH, ODT-8 taken from Grace et al.; 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; 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: dTrp at position 8 in light green, Lys at position 9 in blue, and Phe at positions 6, 7 and 11 yellow. The disulphide bridges are shown in orange for clarity.

Figure 3

Schematic drawings of agonist pharmacophores for receptor-selective analogues binding to the somatostatin receptors: (A) Sst₁, (B) Sst₂, (C) Sst_2/5 and (D) Sst₄. The amino acid side chains, which are part of the pharmacophores and the distances between the corresponding C_γ atoms of the side chains are shown and are also listed in Table 3.

Figure 4

Superposition of receptor-specific pharmacophore with the 3D NMR structure of the analogues 1, 18 and 21. The sst₂ pharmacophore is shown in green. The octreotide pharmacophore proposed by Melacini et al. is shown in yellow. In both the pharmacophores only the side chains of the amino acids are shown that are involved in binding to the receptor. 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, Phe¹¹ in analogues 18 and 21 should undergo a change in their conformation to fit either the sst₂ or octreotide pharmacophores.

Figure 5

Superposition of sst₄ receptor-specific pharmacophore with the 3D NMR structures of the analogues 1, 3, 18, 19 and 21. The sst₄ pharmacophore 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.