However, since these antagonists were based on octreotide, they bind to sst2/3/5 receptors and hence were non-selective. binding to a family of five different receptors, sst1-sst5 (ssts) with nanomolar (nM) affinity. The important role of SRIF and its short duration of action due to rapid proteolytic degradation conformational change in Agl8.25; and Grace et al., unpublished results Non-peptide antagonists binding selectively to each the five somatostatin receptor have also been reported.26,27 It is the aim of the present work to elucidate the pharmacophore for sst2-selective antagonists. Such antagonists are based on octreotide with an L-aromatic amino acid at position 2 and DCys at position 3.28,29 Numerous examples30 suggest that such substitutions (an L-aromatic residue at position Rabbit Polyclonal to CDC25A 2 and DCys at position 3) are crucial for inhibiting the signaling at the receptors. However, since these antagonists were based on octreotide, they bind to sst2/3/5 receptors and hence were nonselective. Based on our knowledge of sst2-selective agonists that this aromatic side chain at position 7 is critical only for sst3/5 receptors, we designed sst2-selective antagonist analogs.31 These antagonists have a longer aromatic side chain at position 7, in addition to DCys at position 3. Here we present the 3D NMR structures of seven potent sst2-selective antagonists and propose the first sst2-selective antagonist pharmacophore model. Analogs 1-7 were tested for antagonism using an calcium release assay and an immunohistochemical sst2 internalization assay as described in detail in Cescato et al.31 Results In this section, we present general details about the chemical shift assignment and the description of the 3D NMR structure for each SRIF antagonists 1 – 7 given in Table 1. Table 1 Sst1-sst5 binding affinities (IC50, nM) of analogs studied by NMR configuration and the side chain of Nal15 is in the configuration (Table S1). Three-dimensional structure of Ac-4NO2Phe2-c[Dcys3-Tyr7-DTrp8-Lys9-Thr10-Cys14]-Dtyr15-NH2 (2) Analog 2 differs from analog 1 by 4NO2Phe2, Tyr7 and Dtyr15 as well as THZ531 the N-terminal acetylation (Table 1) and binds selectively to sst2 with high affinity. The 3D NMR structure shows that the backbone has a -turn of type II around DTrp8 THZ531 and Lys9 (Physique 2, Table S1). The turn is supported by the poor dNN(configuration, the side chains of Tyr7, Lys9 are in the configuration (Table S1). Open in a separate window Physique 1 Survey of characteristic NOEs used in CYANA for structure calculation for analogs 1-7. Thin, medium and thick bars represent poor (4.5 to 6 ?), medium (3 to 4 4.5 ?) and strong ( 3 ?) NOEs observed in the NOESY spectrum. The medium-range NOEs dNN(isomerization of the side chain of Agl(NMe,Benzoyl)7. The 3D structure of the major conformer is determined here and is assumed to be the active conformation. Since the number of assigned NOEs is much less compared to the NOEs observed for the other THZ531 analogs with a single conformation, THZ531 the 3D structure of analog 3 is usually poorly defined compared to the structures of the other analogs (Physique 2, Table 3). Though two conformations were observed, the number of intra-molecular NOEs observed for the minor conformer were few and hence were not sufficient to perform the structure calculation. Hence the major conformation was presumed to be bioactive, based on its conformational similarity with that of the other analogs. The 3D structure shows that the backbone has a -turn of type-II around DTrp8 and Lys9 (Table S1), which is usually supported by the poor sequential dN(configuration, the side chain of DTrp8 is in the configuration and the side chains of Lys9, Nal15 are in the configuration and the side chain of Nal15 is in the configuration (Table S1). Three-dimensional structure of DOTA-Cpa2-c[Dcys3-Tyr7-DAph(Cbm)8-Lys9-Thr10-Cys14]-Nal15-NH2 (5) Analog 5 is usually identical to analog 4 except for the N-terminal DOTA group, which enhances its selectivity for receptor 2 (Table 1). The 3D NMR structure shows that the backbone has a type-II -turn around DAph(Cbm)8 and Lys9 (Physique 2, Table S1), which is usually supported by the poor sequential dN(configuration, the side chain of Lys9 is in the configuration (Table S1). Three-dimensional structure of Cbm-Phe2-c[Dcys3-Aph(Cbm)7-DTrp8-Lys9-Thr10-Cys14]-Nal15-NH2 (6) Analog 6 differs from analog 1 by the N-terminal carbamoylation and the Phe2 substitution. Analog 6 has low binding affinity to sst2 and has moderate binding affinities for sst3, sst4 and sst5 and does not bind to sst1 (Table 1). The 3D structure shows that the backbone has a type-I -turn around DTrp8 and Lys9 (Physique 2, Table S1), which is usually supported by the poor sequential dNN(configuration (Table S1). Three-dimensional structure of Cbm-Phe2-c[Dcys3-Aph(Cbm)7-DTrp8CLys9-Thr10-Cys14]-Thr15-NH2 (7) Analog 7 is similar to analog 6 except for the C-terminal amino acid Thr and it binds with.