Gisbert Weckbecker

Opportunities in somatostatin research: biological, chemical and therapeutic aspects.

Somatostatins — also known as somatotropin-release inhibiting factors (SRIFs) — are a family of cyclopeptides that have broad inhibitory effects on the secretion of hormones such as growth hormone, insulin and glucagon. These effects have formed the basis for the clinical use of SRIF analogues in the treatment of acromegaly and endocrine tumours. The discovery of the five SRIF receptor subtypes in the 1990s further enhanced our understanding of the biological roles of SRIFs, and paved the way for new therapeutic opportunities. Here we review recent advances in SRIF biology, the chemistry of SRIF agonists and antagonists, and the therapeutic potential of such compounds in a wide range of established and novel indications.

Somatostatin analogs for diagnosis and treatment of cancer

Somatostatin (SRIF) is a cyclic tetradecapeptide hormone initially isolated from ovine hypothalami. It inhibits endocrine and exocrine secretion, as well as tumor cell growth, by binding to specific cell surface receptors. Its potent inhibitory activity, however, is limited by its rapid enzymatic degradation and the consequent short plasma half-life. Octreotide is a short SRIF analog with increased duration of action compared to SRIF. Octreotide is approved for the treatment of acromegaly, amine precursor uptake and decarboxylation-omas, complications of pancreatic surgery and severe forms of diarrhea. Preclinical studies have focussed on the anticancer effects of octreotide and the related SRIF analogs BIM 23014 and RC-160. In vitro at nanomolar concentrations, these analogs inhibit the growth of tumor cells that express high affinity SRIF receptors. Accordingly, SRIF analogs, such as octreotide, potently inhibit the growth of SRIF receptor-positive tumors in various rodent models, and, in particular, xenotransplanted human tumors in nude mice. The range of cancers susceptible to octreotide and related SRIF analogs includes mammary, pancreatic, colorectal and lung malignancies. Moreover, an indirect antiproliferative effect of SRIF analogs is achievable in SRIF receptor-negative tumors, whose growth is driven by factors (gastrin, insulin-like growth factor-1, etc.) that are downregulated by SRIF. The use of radiolabeled somatostatin analogs represents a new diagnostic approach. [111In-DTPA]octreotide was developed for gamma camera imaging of SRIF receptor-positive malignancies, such as gasteroenteropancreatic tumors. Visualization of SRIF receptor-positive tumors in humans is emerging as an important methodology, both in tumor staging and predicting therapeutic response to octreotide. Recently, five SRIF receptor subtypes (SSTR1-5) have been cloned, all of which bind SRIF with high affinity. In contrast, SRIF receptor subtypes 1-5 have different binding profiles for short SRIF analogs. Octreotide, SSTR5, show moderate affinity for SSTR3 and fail to bind with high affinity to the other subtypes (SSTR1 and 4). Accordingly, the oncological profile of these three analogs is apparently similar. In conclusion, somatostatin analogs are a promising class of compounds for diagnosis and treatment of cancer. Current work is focussed on the identification of further SRIF receptor subtype-selective analogs with potential in oncology.

Binding properties of somatostatin receptor subtypes

In the past few years, five different somatostatin (SRIF) receptor subtypes (sst1.5) have been identified, which form a distinct group in the superfamily of G-protein-coupled receptors. The naturally occurring somatostatins SRIF-28, SRIF-25, and SRIF-14 all reveal high-affinity binding for sst1.5. In contrast, short synthetic analogs that are in clinical use, such as SMS 201-995, RC-160, or BIM 23014, primarily interact with the sst2 subtype. Some SRIF analogs were previously reported to be selective for one SRIF receptor subtype, eg, the sst2 (MK 678), the sst3 (BIM 23056), or the sst5 (BIM 23052, L362-855) subtype. However, when we studied the binding affinities of these SRIF analogs for human (h) sst1.5 expressed in either CHO or COS-1 cells, we were unable to confirm these previously reported selectivities. The absence of sst antagonists is a major drawback for investigating the functional role of each sst subtype. We used site-directed mutagenesis to identify amino acids that determine ligand specificity for sst2. A single Ser305 to Phe mutation in TM VII increased the affinity of hsst1, for SMS 201-995 nearly 100-fold, and when Gln291 was also exchanged to Asn in TM VII of hsst1, almost full sst2-like binding of SMS 201-995 was obtained. These data may aid in the design and synthesis of new selective type sst ligands. We have identified the expression of sst subtypes in nonclassical SRIF target tissue such as the lung. The pKi values for SRIF and various SRIF analogs in rat lung tissue preparations were in close correlation with those obtained for CHO cells expressing the sst4 subtype. Furthermore, reverse transcriptase polymerase chain reaction (RT-PCR) experiments revealed the predominant expression of mRNA specific for sst4 in mouse, rat, and human lung tissue, confirmed by autoradiographies of rat lung. No specific binding for [125I]Tyr3-SMS 201-995 was detected, since SMS 201-995 has low affinity for sst4. In contrast, specific binding of [125I]SRIF-28 to rat lung sections was demonstrated, which could be displaced by unlabelled SRIF-14 and SRIF-28, indicating specific, high affinity binding of this radioligand to sst4 receptors.

Drug design at peptide receptors

Somatostatin (SRIF, somatotropin release inhibiting factor), discovered for its inhibitory action on growth hormone (GH) secretion from pituitary, is an abundant neuropeptide. Two forms, SRIF14 and SRIF28 exist. Recently, a second family of peptides with very similar sequences and features was described; the cortistatins (CST), CST17 and CST29 which are brain selective. The five cloned SRIF receptors (sst1–5) belong to the G-protein coupled/heptathelical receptor family. Structural and operational features distinguish two classes of receptors; SRIF1-sst2/sst3/sst5 (high affinity for octreotide or seglitide) and SRIF2=sst1/sst4 (very low affinity for the aforementioned ligands). The affinity of SRIF receptors for somatostatins and cortistatins is equally high, and it is not clear whether selective receptors do exist for one or the other of the peptides. Several radiologlands label all SRIF receptors, e.g., [125I]LTT-SRIF28, [125I]CGP23996, [125I]Tyr10cortistatin or [125I]Tyr11SRIF14. In contrast, [125I]Tyr3octreotide, [125I]BIM23027, [125I]MK678 or [125I]D-Trp8SRIF14 label predominantly SRIF1 sites, especially sst2 and possibly sst5 receptors. In brain, [125I]Tyr3octreotide binding equates with sst2 receptor mRNA distribution. Native SRIF2 receptors can be labeled with [125I]SRIF14 in the presence of high NaCl in brain (sst1) or lung (sst4) tissue. Short cyclic or linear peptide analogs show selectivity for sst2/sst5 (octreotide, lanreotide, BIM 23027), sst1 (CH-275), sst3 (sst3-ODN-8), or sst5 receptors (BIM 23268); although claims for selectivity have not always been confirmed. Beta peptides with affinity for SRIF receptors are also reported. The general lack of SRIF receptor antagonists is unique for peptide receptors, although CYN 154806 is a selective and potent sst2 antagonist. Nonpeptide ligands are still rare, although a number of molecules have been reported with selectivity and potency for sst1 (L 757,519), sst2 (L 779,976), sst3 (L 796,778), sst4 (NNC 26-9100, L 803,087) or sst1/sst5 receptors (L 817,018). Such molecules are essential to establish the role of SRIF receptors, e.g., sst1 in hypothalamic glutamate currents: sst2 in inhibiting release of GH, glucagon, TSH, gastric acid secretion, pain, seizures and tumor growth, and sst5 in vascular remodeling and inhibition of insulin and GH release.

Discovery of 3-(1H-indol-3-yl)-4-[2-(4-methylpiperazin-1-yl)quinazolin-4-yl]pyrrole-2,5-dione (AEB071), a potent and selective inhibitor of protein kinase C isotypes

A series of novel maleimide-based inhibitors of protein kinase C (PKC) were designed, synthesized, and evaluated. AEB071 (1) was found to be a potent, selective inhibitor of classical and novel PKC isotypes. 1 is a highly efficient immunomodulator, acting via inhibition of early T cell activation. The binding mode of maleimides to PKCs, proposed by molecular modeling, was confirmed by X-ray analysis of 1 bound in the active site of PKCalpha.

1,4-Diazepane-2,5-diones as novel inhibitors of LFA-1

1,4-Diazepane-2,5-diones (2) are found to be a new class of potent LFA-1 inhibitors. The synthesis, structure, and biological evaluation of these 1,4-diazepine-2,5-diones and related derivatives are described.

Inhibition of inducible nitric oxide synthase improves graft function and reduces tubulointerstitial injury in renal allograft rejection.

Increased levels of nitric oxide (NO) are found in rejecting renal allografts. Inducible NO synthase (iNOS) in infiltrating monocytes/macrophages could lead to NO bursts. NO may modulate the inflammatory response of early rejection due to its high reactivity with superoxide to yield peroxynitrite. To define the role of iNOS in acute renal allograft, rejection effects of the specific iNOS blockers iminoethyl-lysine and 7-butylhexahydro-1H-azepin-2-imine, monohydrochloride on renal function and morphology were investigated in renal allografts. Lewis rats received Brown Norway grafts with one kidney left in situ. All recipients were treated with low dose cyclosporine-A (2.5 mg/kg BW/day s.c.) to allow moderate rejection. In addition, one group received iminoethyl-lysine (10 mg/kg BW/day gavage) and one group received butylhexahydro-azepin-imine (3.4 mg/kg BW/day i.p.). Sham operated Brown Norway donor rats served as baseline controls. Compared to controls, low dose cyclosporine-A decreased glomerular filtration rate (P<0.05) and numerically increased renal vascular resistance. Adding iminoethyl-lysine to cyclosporine-A improved renal hemodynamics. Adding butylhexahydro-azepin-imine to cyclosporine-A practically restored glomerular filtration rate and renal vascular resistance (P<0.05) to control levels. Grafts treated with cyclosporine-A alone showed vascular, glomerular and tubulointerstitial lesions. Adding iminoethyl-lysine or butylhexahydro-azepin-imine to cyclosporine-A did not significantly reduce vascular and glomerular injury, but diminished tubulointerstitial injury as well as nitrotyrosine staining in tubular epithelium (P<0.05). Thus, adding the iNOS blockers iminoethyl-lysine or butylhexahydro-azepin-imine to cyclosporine-A improved graft function and reduced tubulointerstitial lesions.

Deficiency of MALT1 Paracaspase Activity Results in Unbalanced Regulatory and Effector T and B Cell Responses Leading to Multiorgan Inflammation

The paracaspase MALT1 plays an important role in immune receptor-driven signaling pathways leading to NF-κB activation. MALT1 promotes signaling by acting as a scaffold, recruiting downstream signaling proteins, as well as by proteolytic cleavage of multiple substrates. However, the relative contributions of these two different activities to T and B cell function are not well understood. To investigate how MALT1 proteolytic activity contributes to overall immune cell regulation, we generated MALT1 protease-deficient mice (Malt1PD/PD) and compared their phenotype with that of MALT1 knockout animals (Malt1−/−). Malt1PD/PD mice displayed defects in multiple cell types including marginal zone B cells, B1 B cells, IL-10–producing B cells, regulatory T cells, and mature T and B cells. In general, immune defects were more pronounced in Malt1−/− animals. Both mouse lines showed abrogated B cell responses upon immunization with T-dependent and T-independent Ags. In vitro, inactivation of MALT1 protease activity caused reduced stimulation-induced T cell proliferation, impaired IL-2 and TNF-α production, as well as defective Th17 differentiation. Consequently, Malt1PD/PD mice were protected in a Th17-dependent experimental autoimmune encephalomyelitis model. Surprisingly, Malt1PD/PD animals developed a multiorgan inflammatory pathology, characterized by Th1 and Th2/0 responses and enhanced IgG1 and IgE levels, which was delayed by wild-type regulatory T cell reconstitution. We therefore propose that the pathology characterizing Malt1PD/PD animals arises from an immune imbalance featuring pathogenic Th1- and Th2/0-skewed effector responses and reduced immunosuppressive compartments. These data uncover a previously unappreciated key function of MALT1 protease activity in immune homeostasis and underline its relevance in human health and disease.

Structural basis of guanine nucleotide exchange mediated by the T-cell essential Vav1.

The guanine nucleotide exchange factor (GEF) Vav1 plays an important role in T-cell activation and tumorigenesis. In the GEF superfamily, Vav1 has the ability to interact with multiple families of Rho GTPases. The structure of the Vav1 DH-PH-CRD/Rac1 complex to 2.6 A resolution reveals a unique intramolecular network of contacts between the Vav1 cysteine-rich domain (CRD) and the C-terminal helix of the Vav1 Dbl homology (DH) domain. These unique interactions stabilize the Vav1 DH domain for its intimate association with the Switch II region of Rac1 that is critical for the displacement of the guanine nucleotide. Small angle x-ray scattering (SAXS) studies support this domain arrangement for the complex in solution. Further, mutational analyses confirms that the atypical CRD is critical for maintaining both optimal guanine nucleotide exchange activity and broader specificity of Vav family GEFs. Taken together, the data outline the detailed nature of Vav1s ability to contact a range of Rho GTPases using a novel protein-protein interaction network.

Effects of the novel protein kinase C inhibitor AEB071 (Sotrastaurin) on rat cardiac allograft survival using single agent treatment or combination therapy with cyclosporine, everolimus or FTY720

NVP‐AEB071 (AEB, sotrastaurin), an oral inhibitor of protein kinase C (PKC), effectively blocks T‐cell activation. The immunosuppressive effects of oral AEB were demonstrated in a rat local graft versus host (GvH) reaction and rat cardiac transplantation models. T‐cell activation was suppressed by 95% in blood from AEB‐treated rats, with a positive correlation between T‐cell inhibition and AEB blood concentration. In GvH studies, AEB inhibited lymph node swelling dose‐dependently (3–30 mg/kg). BN and DA cardiac allografts were acutely rejected within 6–10 days post‐transplantation in untreated LEW rats. AEB at 10 and 30 mg/kg b.i.d. prolonged BN graft survival to a mean survival time of 15 and >28 days, and DA grafts to 6.5 and 17.5 days, respectively. In the DA to LEW model, combining a nonefficacious dose of AEB (10 mg/kg b.i.d.) with a nonefficacious dose of cyclosporine, everolimus or FTY720 led to prolonged median survival times (26 days, >68 days and >68 days, respectively). Pharmacokinetic monitoring excluded drug–drug interactions, suggesting synergy. In conclusion, these studies are the first to demonstrate that AEB prolongs rat heart allograft survival safely as monotherapy and in combination with nonefficacious doses of cyclosporine, everolimus or FTY720. Thus, AEB may have the potential to offer an alternative to calcineurin inhibitor‐based therapies.