Identification of a Novel SSTR3 Full Agonist for the Treatment of Nonfunctioning Pituitary Adenomas

Abstract

Somatostatin receptor (SSTR) agonists have been extensively used for treating neuroendocrine tumors. Synthetic therapeutic agonists showing selectivity for SSTR2 (Octreotide) or for SSTR2 and SSTR5 (Pasireotide) have been approved for the treatment of patients with acromegaly and Cushing's syndrome, as their pituitary tumors highly express SSTR2 or SSTR2/SSTR5, respectively. Nonfunctioning pituitary adenomas (NFPAs), which express high levels of SSTR3 and show only modest response to currently available SSTR agonists, are often invasive and cannot be completely resected, and therefore easily recur. The aim of the present study was the evaluation of ITF2984, a somatostatin analog and full SSTR3 agonist, as a new potential treatment for NFPAs. ITF2984 shows a 10-fold improved affinity for SSTR3 compared to Octreotide or Pasireotide. Molecular modeling and NMR studies indicated that the higher affinity for SSTR3 correlates with a higher stability of a distorted β-I turn in the cyclic peptide backbone. ITF2984 induces receptor internalization and phosphorylation, and triggers G-protein signaling at pharmacologically relevant concentrations. Furthermore, ITF2984 displays antitumor activity that is dependent on SSTR3 expression levels in the MENX (homozygous mutant) NFPA rat model, which closely recapitulates human disease. Therefore, ITF2984 may represent a novel therapeutic option for patients affected by NFPA.

Keywords: ITF2984; nonfunctioning pituitary adenomas (NFPAs); somatostatin agonists (SSAs); somatostatin receptor 3 (SSTR3).

Figure 1

Structures of somatostatin analogues evaluated in the present study. ITF2984 (A) and Pasireotide (B) are backbone-cyclized hexapeptides, with unnatural amino acids in the most probable active substructure. Approved somatostatin agonists Octreotide (C) and Lanreotide (D) are cyclic octapeptides conformationally restricted by the disulfide bridge. All molecules share the same key dyad D-Trp-Lys, except ITF2984, where D-Trp is replaced by 3,5 dimethoxy D-2-naphtylalanine (3,5 diMeO-D-2Nal).

Figure 2

Somatostatin in SSTR2. (A) Somatostatin-14 (green) in SSTR2, Cryo-EM structure, resolution 2.5 Å (PDB structure 7T10), detail of binding site hosting the key dyad Trp8-Lys9 (receptor residues close to analogue in purple color). (B) Detail of Phe-Trp-Lys-Thr motif of SRIF-14 in best-scored pose (left) and generic sketch summarizing reference values of backbone torsions of I, I’, II and II’ β-turns (right). Measured dihedral angles ϕ and ψ of Trp-Lys dyad are in qualitative agreement with ideal turn type II’.

Figure 3

ITF2984-induced internalization of SSTR3 in HEK293 (A) and U2OS (B) hSSTR-transfected cells. (A) HEK293 cells stably expressing wild-type HA-SSTR2 or HA-SSTR3 were preincubated with anti-HA antibody for 2 h at 4 °C. Afterwards, cells were treated with either 1 µM SST-14 (SRIF-14), Octreotide, Pasireotide or ITF2984 for 30 min at 37 °C. After fixation, the cells were incubated with Alexa488-conjugated secondary antibody and examined by confocal microscopy. Shown images are representative of three independent experiments. Scale bar, 20 µm. (B) Internalization of SSTR3 in U2OS cell line transfected with human receptor (SSTR3-tGFP) following treatments with SST-28, Pasireotide, ITF2984 and Octreotide. Compounds were tested at five different concentrations (10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹ M) in comparison to untreated cells (Control). A concentration of 10⁻⁶ M SST28 was included as positive control. Approximately 500 cells per field were analyzed. Data obtained on SSTR2-tGFP- and SSTR5-tGFP-transfected U2OS cells are shown in Table 1.

Figure 4

SSTR phosphorylation (left panel) and agonist-mediated G protein signaling of SSTRs in HEK293 cells (right panel). (Left panel) ITF2984-selective SSTR3 phosphorylation in HEK293 cells. HEK293 cells stably expressing (A) HA-SSTR3, (B) SSTR2 or (C) HA-SSTR5 were either treated with 10 µM SST-14, Octreotide, Pasireotide or ITF2984 with concentrations ranging from 10⁻⁵ M to 10⁻¹² M for 5 min at 37 °C. Cells were lysed and immunoblotted with the indicated phosphositespecific antibodies. Blots were stripped and reprobed with the phosphorylation-independent anti-HA-tag or UMB antibody to confirm equal loading of the gels. Blots are representative of three independent experiments. The positions of molecular mass markers are indicated on the left (in kDa). (Right panel) Agonist-mediated G protein signaling of SSTR3 in HEK293 cells. The ability of SST14, Octreotide, Pasireotide and ITF2984 to activate GIRK2 channels via SSTR3 (A), SSTR2 (B) or SSTR5 (C) was tested using a fluorescence membrane potential assay. The concentrations used are indicated. Data points represent mean ± S.E.M. The uncropped blots are shown in Figures S9–S11.

Figure 5

Analysis of G protein signaling in mouse AtT-20 cells using a fluorescence-based membrane potential assay. The ability of Octreotide (A) and ITF2984 (B) to activate endogenous GIRK channels in wild-type AtT-20 cells via the endogenously expressed SSTR2 and SSTR5 receptors (black) or in exogenously expressed human SSTR3 receptors (red) was tested. Expression of SSTR3 resulted in a leftward shift of the dose–response curve.

Figure 6

Changes in tumor volume in rats treated s.c. with ITF2984 or placebo (A), proliferation rate evaluation (number of Ki67-positive cells) (B), Sstr gene expression in rat NFPAs (C) at the end of treatment. (A) Changes in tumor volume in rats treated s.c. with ITF2984 or placebo. MENX-affected rats at the age of 5.5 months were injected with ITF2984 1× every 14 days at 12.5 mg/kg body weight. MRI was performed every 14 days and the tumor volume was normalized against the volume at day 0. Male and female rat tumors are shown separately. Data presented are the mean ± SEM. #, not significant; *, p-value < 0.05; **, p-value < 0.001. (B) Proliferation of NFPAs following treatment. Number of Ki67-positive cells per 100,000 µm² in tumors of rats belonging to the 2 groups (ITF2984-treated and control) and both sexes. Shown is the mean ± SEM. *, p-value < 0.05; **, p-value < 0.001. (C) Sstr gene expression in rat NFPAs. Relative expression of the Sstr1,2,3,5 genes in the ITF2984 treatment group compared to the control group, arbitrarily set to 100%. Shown is the average ± SEM. *, p-value < 0.05. ♂, ♀ are male and female gender symbols, respectively.