Synthesis and in vitro evaluation of 2-amino-4-N-piperazinyl-6-(3,4-dimethoxyphenyl)-pteridines as dual immunosuppressive and anti-inflammatory agents.

Screening of a pteridine-based compound library led to the identification of compounds exhibiting immunosuppressive as well as anti-inflammatory activity. Optimization afforded a series of 2-amino-4-N-piperazinyl-6-(3,4-dimethoxyphenyl)pteridine analogues. The most potent congeners in this series displayed low nM IC(50) values in the Mixed Lymphocyte Reaction (MLR) assay. In addition, these compounds also have potent anti-inflammatory activity as measured in the Tumor Necrosis Factor (TNF) assay.

Immunosuppressive activity of a new pteridine derivative (4AZA1378) alleviates severity of TNBS-induced colitis in mice.

Besides TNF, activated T cells play a central role in the pathogenesis of inflammatory bowel diseases such as Crohn's disease. New therapies are still awaited to cure these often debilitating diseases. Natural occurring pteridines such as tetrahydrobiopterin (BH4) and neopterin have been reported to have immune modulating activities. Starting from a pteridine scaffold library, we intended to select compounds with potent in vitro inhibitory effects on T cells and to evaluate in vivo efficacy of selected compounds on trinitrobenzenesulphonate (TNBS) colitis in mice. Compound 4AZA1378 was selected because it potently inhibits human T cell proliferation at low nM concentrations (IC50 4 nM) while an almost 50-fold higher concentration was needed to inhibit LPS-induced TNF production. Mice treated with 4AZA1378 had less severe signs of colitis after TNBS rectal administration, with a more rapid weight recovery. Myeloperoxidase (MPO) activity and intralesional cytokine production were lower in mice of the treated groups. Furthermore anti-TNBS antibody responses were completely inhibited by treatment with 4AZA1378. In conclusion, we identified a pteridine analogue 4AZA1378 with immunosuppressive activity and a strong remission-inducing effect in TNBS colitis, supporting further pre-clinical and clinical development of this novel molecule for treatment of inflammatory diseases.

Anti-inflammatory activity of a pteridine derivative (4AZA2096) alleviates TNBS-induced colitis in mice.

Elevated production of tumor necrosis factor (TNF) plays a central role in the pathogenesis of many inflammatory diseases, such as rheumatoid arthritis and Crohn's disease. Naturally occurring pteridine analogs have been reported to have potent immunomodulatory activity, especially on TNF production. The aim of this study is to identify small molecule TNF inhibitiors derived from pteridine and to prove their in vivo efficacy in an inflammatory model. A focused chemical library based on the pteridine scaffold was screened in vitro on lipopolysaccharide (LPS)-induced TNF production in peripheral blood mononuclear cells (PBMC). One synthetic pteridine analog (4AZA2096), shown to have strong inhibitory activity, was selected and tested for its efficacy to treat trinitrobenzenesulfonate (TNBS)-induced colitis in mice, a model of Crohn's disease. Colitis was induced by rectal administration of 1 mg TNBS in 50% ethanol after presensitization via the skin. The synthetic pteridine analog 4AZA2096 was shown to potently inhibit LPS-induced TNF production in vitro. Colitic mice treated with 4AZA2096 orally (20 mg/kg/day) recovered more rapidly and, histologically, had a reduction of inflammatory lesions, less edema, a reduction of goblet cell loss, and reduced wall thickness. Cell infiltration in the colon, especially infiltration of neutrophils, as shown by myeloperoxidase (MPO) activity, was reduced in 4AZA2096-treated animals. Intralesional TNF production was lower in mice of the treated groups, whereas interleukin-18 (IL-18) and interferon-gamma (IFN-gamma) mRNA were not affected. Treatment had no effect on anti-TNBS antibody production, arguing against generalized immunosuppression. In conclusion, we identified a pteridine derivative, 4AZA2096, with strong inhibitory activity on TNF production and a remission- inducing effect in TNBS colitis, supporting further preclinical and clinical development of this novel TNF inhibitor for treatment of inflammatory diseases.

A novel Ca2+-induced Ca2+ release mechanism in A7r5 cells regulated by calmodulin-like proteins.

Intracellular Ca2+ release is involved in setting up Ca2+ signals in all eukaryotic cells. Here we report that an increase in free Ca2+ concentration triggered the release of up to 41 +/- 3% of the intracellular Ca2+ stores in permeabilized A7r5 (embryonic rat aorta) cells with an EC50 of 700 nm. This type of Ca2+-induced Ca2+ release (CICR) was neither mediated by inositol 1,4,5-trisphosphate receptors nor by ryanodine receptors, because it was not blocked by heparin, 2-aminoethoxydiphenyl borate, xestospongin C, ruthenium red, or ryanodine. ATP dose-dependently stimulated the CICR mechanism, whereas 10 mm MgCl2 abolished it. CICR was not affected by exogenously added calmodulin (CaM), but CaM1234, a Ca2+-insensitive CaM mutant, strongly inhibited the CICR mechanism. Other proteins of the CaM-like neuronal Ca2+-sensor protein family such as Ca2+-binding protein 1 and neuronal Ca2+ sensor-1 were equally potent for inhibiting the CICR. Removal of endogenous CaM, using a CaM-binding peptide derived from the ryanodine receptor type-1 (amino acids 3614-3643) prevented subsequent activation of the CICR mechanism. A similar CICR mechanism was also found in 16HBE14o-(human bronchial mucosa) cells. We conclude that A7r5 and 16HBE14o-cells express a novel type of CICR mechanism that is silent in normal resting conditions due to inhibition by CaM but becomes activated by a Ca2+-dependent dissociation of CaM. This CICR mechanism, which may be regulated by members of the family of neuronal Ca2+-sensor proteins, may provide an additional route for Ca2+ release that could allow amplification of small Ca2+ signals.

The role of calmodulin for inositol 1,4,5-trisphosphate receptor function.

Intracellular calcium release is a fundamental signaling mechanism in all eukaryotic cells. The ryanodine receptor (RyR) and inositol 1,4,5-trisphosphate receptor (IP(3)R) are intracellular calcium release channels. Both channels can be regulated by calcium and calmodulin (CaM). In this review we will first discuss the role of calcium as an activator and inactivator of the IP(3)R, concluding that calcium is the most important regulator of the IP(3)R. In the second part we will further focus on the role of CaM as modulator of the IP(3)R, using results of the voltage-dependent Ca(2+) channels and the RyR as reference material. Here we conclude that despite the fact that different CaM-binding sites have been characterized, their function for the IP(3)R remains elusive. In the third part we will discuss the possible functional role of CaM in IP(3)-induced Ca(2+) release (IICR) by direct and indirect mechanisms. Special attention will be given to the Ca(2+)-binding proteins (CaBPs) that were shown to activate the IP(3)R in the absence of IP(3).

Inhibition of the inositol trisphosphate receptor of mouse eggs and A7r5 cells by KN-93 via a mechanism unrelated to Ca2+/calmodulin-dependent protein kinase II antagonism.

KN-93, a Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitor, concentration-dependently and reversibly inhibited inositol 1,4,5-trisphosphate receptor (IP(3)R)-mediated [Ca(2+)](i) signaling in mouse eggs and permeabilized A7r5 smooth muscle cells, two cell types predominantly expressing type-1 IP(3)R (IP(3)R-1). KN-92, an inactive analog, was ineffective. The inhibitory action of KN-93 on Ca(2+) signaling depended neither on effects on IP(3) metabolism nor on the filling grade of Ca(2+) stores, suggesting a direct action on the IP(3)R. Inhibition was independent of CaMKII, since in identical conditions other CaMKII inhibitors (KN-62, peptide 281-309, and autocamtide-related inhibitory peptide) were ineffective and since CaMKII activation was precluded in permeabilized cells. Moreover, KN-93 was most effective in the absence of Ca(2+). Analysis of Ca(2+) release in A7r5 cells at varying [IP(3)], of IP(3)R-1 degradation in eggs, and of [(3)H]IP(3) binding in Sf9 microsomes all indicated that KN-93 did not affect IP(3) binding. Comparison of the inhibition of Ca(2+) release and of [(3)H]IP(3) binding by KN-93 and calmodulin (CaM), either separately or combined, was compatible with a specific interaction of KN-93 with a CaM-binding site on IP(3)R-1. This was also consistent with the much smaller effect of KN-93 in permeabilized 16HBE14o(-) cells that predominantly express type 3 IP(3)R, which lacks the high affinity CaM-binding site. These findings indicate that KN-93 inhibits IP(3)R-1 directly and may therefore be a useful tool in the study of IP(3)R functional regulation.

Localization and function of a calmodulin-apocalmodulin-binding domain in the N-terminal part of the type 1 inositol 1,4,5-trisphosphate receptor.

Calmodulin (CaM) is a ubiquitous protein that plays a critical role in regulating cellular functions by altering the activity of a large number of proteins, including the d-myo-inositol 1,4,5-trisphosphate (IP3) receptor (IP3R). CaM inhibits IP3 binding in both the presence and absence of Ca2+ and IP3-induced Ca2+ release in the presence of Ca2+. We have now mapped and characterized a Ca2+-independent CaM-binding site in the N-terminal part of the type 1 IP3R (IP3R1). This site could be responsible for the inhibitory effects of CaM on IP3 binding. We therefore expressed the N-terminal 581 amino acids of IP3R1 as a His-tagged recombinant protein, containing the functional IP3-binding pocket. We showed that CaM, both in the presence and absence of Ca2+, inhibited IP3 binding to this recombinant protein with an IC50 of approx. 2 microM. Deletion of the N-terminal 225 amino acids completely abolished the effects of both Ca2+ and CaM on IP3 binding. We mapped the Ca2+-independent CaM-binding site to a recombinant glutathione S-transferase fusion protein containing the first 159 amino acids of IP3R1 and then made different synthetic peptides overlapping this region. We demonstrated that two synthetic peptides matching amino acids 49-81 and 106-128 bound CaM independently of Ca2+ and could reverse the inhibition of IP3 binding caused by CaM. This suggests that these sequences are components of a discontinuous Ca2+-independent CaM-binding domain, which is probably involved in the inhibition of IP3 binding by CaM.