Somatostatin Receptor 1 Selective Analogues: 4. Three-Dimensional Consensus Structure by NMR Christy Rani R. Grace, † Lukas Durrer, † Steven C. Koerber, ‡ Judit Erchegyi, ‡ Jean Claude Reubi, § Jean E. Rivier,* ,‡ and Roland Riek † Structural Biology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037, The Clayton Foundation Laboratories for Peptide Biology, 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 Received June 18, 2004 The three-dimensional NMR structures of six analogues of somatostatin (SRIF) are described. These analogues with the amino acid 4-(N-isopropyl)-aminomethylphenylalanine (IAmp) at position 9 exhibit potent and highly selective binding to human SRIF subtype 1 receptors (sst 1 ). The conformations reveal that the backbones of these analogues have a hairpin-like structure similar to the sst 2 -subtype-selective analogues. This structure serves as a scaffold for retaining a unique arrangement of the side chains of D-Trp 8 , IAmp 9 , Phe 7 , and Phe 11 or m-I-Tyr 11 (m-I- Tyr ) mono-iodo-tyrosine). The conformational preferences and results from biological analyses of these analogues 1,2 allow a detailed study of the structure-activity relationship of SRIF. The proposed consensus pharmacophore of the sst 1 -selective analogues requires a unique set of distances between an indole/2-naphthyl ring, an IAmp side chain, and two aromatic rings. This motif is necessary and sufficient to explain the binding affinities of all of the analogues studied and is distinct from the existing models suggested for sst 4 as well as sst 2 /sst 5 selectivity. Introduction Somatostatin (somatotropin release inhibiting factor, SRIF, H-Ala 1 -Gly 2 -c[Cys 3 -Lys 4 -Asn 5 -Phe 6 -Phe 7 -Trp 8 - Lys 9 -Thr 10 -Phe 11 -Thr 12 -Ser 13 -Cys 14 ]-OH), a cyclic tetra- decapeptide, inhibits the release of several hormones including growth hormone (GH), glucagon, insulin, secretin, and gastrin. 3,4 It also plays a vital role in neurotransmission and neuromodulation 5,6 and has antiproliferative effects, regulating cell proliferation and differentiation. SRIF elicits its effects via high-affinity interactions with a family of five different receptors, sst 1-5 . Sequence homology is 39-57% among the five subtypes, and each subtype is highly conserved across different species. The functional significance of the endogenous receptors in mediating the diverse effects of SRIF requires the availability of subtype-selective agonists and antagonists, and hence SRIF continues to be a target for the development of subtype-specific analogues. 7-11 Since the discovery of SRIF, numerous peptide and nonpeptide analogues have been described. With the characterization of the five sst, analogues can be tested for their biological activity in terms of affinity and selectivity. 7-9,12-14 Early structure-activity relationship (SAR) studies using in vitro functional assays suggested a central involvement of the side chains of residues Trp 8 - Lys 9 for biological recognition. 15-17 Furthermore, exten- sive structural studies including NMR and X-ray dif- fraction 18 have been carried out to elucidate the pharmacophore and the consensus structural motif of analogues binding predominantly to sst 2 /sst 5 and sst 4 receptors. According to the proposed model for sst 2 /sst 5 binding, 19-22 the side chains and the relative spatial arrangement of Phe 7 , D-Trp 8 , and Lys 9 constitute the most essential elements (Figure 4D). The side chain of D-Trp 8 is in close proximity to the side chain of Lys 9 (∼4 Å), whereas the side chain of Phe 7 is about 7-9 Å away from the side chain of D-Trp 8 and 9-11 Å from the side chain of Lys 9 . Recently, our group studied the structures of sst 4 -selective SRIF analogues from three different families by NMR in dimethyl sulfoxide (DMSO) and proposed a pharmacophore model for these analogues. 23 Although these analogues have different backbone conformations, the relative spatial arrangement of the side chains of D-Trp 8 , Lys 9 , and Phe 6 or Phe 11 were unique, and on this basis it was proposed that these residues and their observed proximity were important for binding. This binding motif mainly differs from the binding motif for sst 2 /sst 5 -selective receptors in the proximity of the side chains of Lys and Phe, which is 4.5-6.5 Å in the sst 4 and 9-11 Å in the sst 2 /sst 5 binding motifs (Figure 4C and D). These studies furthermore strongly support the idea of Nicolaou et al., who sug- gested that the backbone is not required for receptor binding but serves as a scaffold for supporting the important side chains. 24 In this paper, the studies of Grace et al. 23 on the characterization of the sst 4 pharmacophore and of Falb et al. 20 on the sst 2 pharmacophore have been further extended to propose a pharmacophore model for sst 1 - selective analogues. In the preceding two papers, the synthesis and biological characterization of two families of structurally constrained sst 1 -selective SRIF analogues * Author to whom correspondence should be addressed. Phone: (858) 453-4100. Fax: (858) 552-1546. E-mail: jrivier@salk.edu. † Structural Biology Laboratory, The Salk Institute for Biological Sciences. ‡ The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Sciences. § Division of Cell Biology and Experimental Cancer Research, Institute of Pathology, University of Berne. 523 J. Med. Chem. 2005, 48, 523-533 10.1021/jm049518u CCC: $30.25 © 2005 American Chemical Society Published on Web 12/23/2004