Agonist properties of putative small-molecule somatostatin sst₂ receptor-selective antagonists

Abstract

The availability of antagonist ligands for somatostatin receptors is very limited, with those that are available often displaying agonist properties or limited receptor subtype selectivity. Hay et al. [Bioorg. Med. Chem. Lett. 11 (2001) 2731] recently described the development of small-molecule somatostatin receptor subtype 2 (sst₂) selective compounds. This study investigates the binding affinity and functional characteristics of two of those antagonists (2 and 3) and the agonist compound, from which they were derived (1). In radioligand binding studies using the agonist radioligands [¹²⁵I][Tyr¹¹]SRIF-14 (Ala-Gly-c[Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-(¹²⁵I-Tyr)-Thr-Ser-Cys]-OH), [¹²⁵I]LTT-SRIF-28 ([Leu⁸,dTrp²²,¹²⁵I-Tyr²⁵]SRIF-28; Ser-Ala-Asn-Ser-Asn-Pro-Ala-Leu-Ala-Pro-Arg-Glu-Arg-Lys-Ala-Gly-c[Cys-Lys-Asn-Phe-Phe-dTrp-Lys-Thr-(¹²⁵I-Tyr)-Thr-Ser-Cys]-OH), [¹²⁵I]CGP 23996 (c[Lys-Asu-Phe-Phe-Trp-Lys-Thr-(¹²⁵I-Tyr)-Thr-Ser]), [¹²⁵I][Tyr³]octreotide (dPhe-c[Cys-(¹²⁵I-Tyr)-dTrp-Lys-Thr-Cys]-Thr-OH) and [¹²⁵I][Tyr¹⁰]cortistatin-14 (Pro-c[Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-(¹²⁵I-Tyr)-Ser-Ser-Cys]-Lys) at human recombinant somatostatin receptors expressed in Chinese hamster lung fibroblast (CCL39) cells and native rat cortex, the compounds bound with high affinity (pK_d 6.8–9.7) and selectivity to human sst₂ receptors. Some affinity was also observed for sst₅ labelled by [¹²⁵I][Tyr³]octreotide and [¹²⁵I]CGP 23996. In functional studies at human sst₂ receptors expressed in Chinese hamster ovary (CHO) cells, both the agonist 1 and the two putative antagonists 2 and 3 concentration dependently inhibited forskolin-stimulated adenylate cyclase and stimulated luciferase reporter gene expression, with similar efficacy to the natural ligand somatotropin release inhibiting factor (SRIF)-14. Compound 1 had similar potency to SRIF-14, which was in the nanomolar range, whereas 2 and 3 were 10–100-fold less potent. The intrinsic activity of 2 and 3 was too high to allow antagonist studies to be carried out. In conclusion, in contrast to previous findings, all three compounds are potent agonists at recombinant human sst₂ receptors.

Introduction

Somatostatin (SRIF, somatotropin release inhibiting factor) is expressed by neuroendocrine, inflammatory, immune and tumour cells, neurones and osteoblasts (for review, see Epelbaum et al., 1994). Its first discovered role was as an inhibitor of growth hormone secretion from the anterior pituitary Krulich et al., 1968, Brazeau et al., 1973, but it has since been shown to have many physiological inhibitory effects including modulation of neurotransmitter release in the brain, inhibition of endocrine and exocrine secretions (such as thyroid-stimulating hormone and prolactin from the anterior pituitary, and insulin and glucagon from pancreas), and inhibition of cell proliferation in normal and tumour cells (for review, see Patel, 1999).

Somatostatin produces its effects via interaction with specific cell-surface G protein-coupled receptors of which five subtypes have so far been cloned (somatostatin receptor subtype 1–5 [sst_1–5]; Hoyer et al., 1995). In rodents, the sst₂ receptor exists in two splice variant forms—sst_2a and sst_2b Vanetti et al., 1992, Schindler et al., 1998. From original operational studies, the receptors were classified into two groups—SRIF₁, with high affinity for the short chain synthetic somatostatin analogues such as SMS 201 995 (octreotide) and MK 678 (seglitide), and SRIF₂, which has high affinity for the analogue CGP 23996 Reubi, 1984, Tran et al., 1985, Raynor and Reisine, 1989, Martin et al., 1991, Raynor et al., 1992. Subsequent molecular cloning and further pharmacological characterisation showed that SRIF₁ consists of sst_{2, 3 and 5} and SRIF₂ of sst_{1 and 4} (Hoyer et al., 1994). Somatostatin receptors modulate multiple cellular effectors via activation of heterotrimeric G-proteins. These include adenylate cyclase, phospholipase C, K⁺ channels, Ca²⁺ channels, Ser/Thr phosphatases and mitogen-activated protein kinase cascades (Patel, 1999).

In any system where a single ligand can modulate numerous receptors and effectors, selective synthetic ligands are essential; both as tools to elucidate physiological and biochemical effects mediated by individual receptor subtypes, and as potential therapeutic compounds. This is especially true for the somatostatin system where it is common for more than one receptor subtype to be expressed in the same cell (Patel, 1997). While somatostatin receptor subtypes have been characterised by molecular cloning and pharmacology, the availability of “selective” ligands for individual subtypes is still relatively limited. Early synthetic peptide analogues of somatostatin, such as octreotide, BIM 23014, RC 160 and MK 678, bind to two or more receptor subtypes with similar high affinity Raynor et al., 1993, Hoyer et al., 1994, Siehler et al., 1999.

Merck scientists recently developed a series of non-peptide agonists with high affinity and selectivity for the individual somatostatin receptor subtypes Rohrer et al., 1998, Rohrer and Berk, 1999, Yang et al., 1998a, Yang et al., 1998b. Of these, the sst₂-selective L-054,522 has been shown to mediate inhibition of growth hormone release from rat anterior pituitary and glucagon release from rat pancreas at nanomolar concentrations (Yang et al., 1998a). The advantage of non-peptidic compounds is that they have potential for greater oral bioavailability than peptides that must be administered by subcutaneous or intravenous injection (Yang et al., 1998b) and, in most cases, have short duration of action and little, if any, brain penetration.

However, receptor-selective antagonists are also needed, to conclusively prove that a particular receptor subtype mediates a particular physiological function of somatostatin. Unfortunately, at the present time, very few somatostatin receptor-subtype selective antagonists have been identified. With regards to sst₂-selective compounds specifically, a number of antagonist ligands have been have been reported Bass et al., 1996, Baumbach et al., 1998, Hocart et al., 1998, Hocart et al., 1999 and have been used to distinguish effects of SRIF which are mediated by sst₂, including inhibition of gastric acid release (Rossowski et al., 1998) and growth hormone release from pituitary cells (Hocart et al., 1999). However, these are all modifications of peptides, making them poor candidates for therapeutic or diagnostic use.

Scientists at Pfizer have recently described a series of small sst₂-selective non-peptide ligands, presented as antagonists (Hay et al., 2001a). These compounds are based on the structure of the Merck sst₂-selective non-peptide agonists such as L-054,522 (Yang et al., 1998a), but have been modified to produce compounds that bind with nanomolar affinity to sst₂ and to behave as antagonists of SRIF-14-mediated inhibition of vasoactive intestinal peptide (VIP)-stimulated cAMP accumulation. Their rationale for this study was that sst₂-selective antagonists might be useful to upregulate growth hormone levels in livestock.

Thus, the present study examines radioligand binding and functional properties of three compounds: 1, 2 and 3 that correspond to compounds 6, 7e and 7a reported by Hay et al. (2001a). Compound 1 is the agonist from which the two putative antagonists 2 and 3 were derived.