Synthesis and evaluation of thiazepines as interleukin-1beta converting enzyme (ICE) inhibitors

A series of monocyclic thiazepine inhibitors of interleukin-1beta converting enzyme (ICE) were synthesized in eight steps from commercially available intermediates. In vitro biological evaluation showed the thiazepines to be moderately potent ICE inhibitors, with the most active compound exhibiting an IC50 value of 30 nM in an enzyme inhibition assay. Compounds of this class possessed good selectivity against the related enzymes caspase-3 and caspase-8.     

Synthesis and evaluation of unsaturated caprolactams as interleukin-1beta converting enzyme (ICE) inhibitors

Peptidomimetic compounds possessing a caprolactam ring constraint were prepared and evaluated as interleukin-1beta converting enzyme (ICE) inhibitors. The caprolactam ring was used to constrain the P3 region of our inhibitors. This strategy proved to be effective for the synthesis of ICE inhibitors, maintaining key hydrogen bond interactions with the enzyme and invoking a preferred conformation for binding. Several compounds exhibited IC(50) values less than 10nM in a caspase-1 enzyme assay and less than 100nM in a THP-1 whole cell assay measuring IL-1beta production. Two compounds, 13c and 13j, were found to have good oral bioavailability (>50%) in rats when administered as prodrugs.     

Structure-based design of nonpeptide inhibitors of interleukin-1beta converting enzyme (ICE, caspase-1).

A novel class of reversible inhibitors of Interleukin-1beta-converting enzyme (ICE, caspase-1) were discovered by iterative structure-based design. Guided by the X-ray crystal structure of analogues 1, 7 and 10 bound to ICE, we have designed a nonpeptide series of small molecule inhibitors. These compounds incorporate an arylsulfonamide moiety which replaces Val-His unit (P3-P2 residues) amino acids of the native substrate. The synthesis of the core structure, structure-activity relationships (SARs), and proposed binding orientation based on molecular modeling studies for this series of ICE inhibitors are described.     

Synthesis and evaluation of novel 1-(2-acylhydrazinocarbonyl)-cycloalkyl carboxamides as interleukin-1beta converting enzyme (ICE) inhibitors

Novel 1-(2-acylhydrazinocarbonyl)cycloalkyl carboxamides were designed as peptidomimetic inhibitors of interleukin-1beta converting enzyme (ICE). A short synthesis was developed and moderately potent ICE inhibitors were identified (IC(50) values <100 nM). Most of the synthesized examples were selective for ICE versus the related cysteine proteases caspase-3 and caspase-8, although several dual-acting inhibitors of ICE and caspase-8 were identified. Several of the more potent ICE inhibitors were also shown to inhibit IL-1beta production in a whole cell assay (IC(50) < 500 nM).     

Synthesis and evaluation of novel 8,5-fused bicyclic peptidomimetic compounds as interleukin-1beta converting enzyme (ICE) inhibitors

An 8,5-fused bicyclic peptidomimetic ring system generated by a stereoselective ring metathesis reaction was elaborated into potent inhibitors of interleukin-1beta converting enzyme (ICE, caspase-1). Multiple compounds were found that exhibited ICE IC50 values < 10 nM and were selective over caspase-3 and caspase-8. These active analogs generally possessed good activity (IC50 values < 100 nM) in a whole cell assay measuring IL-1beta production. Pharmacokinetic analysis of the ethyl acetal prodrug form of a selected active lead revealed a compound with a reasonable plasma half-life (1.1 h) and good oral bioavailability (30%).     

Inhibition of interleukin-1beta (IL-1beta) production in human cells by ribozymes against IL-1beta and IL-1beta converting enzyme (ICE)

We and others have shown previously that hairpin ribozyme genes, when stably expressed in cells, can reduce the steady-state levels of target mRNA and their cognate proteins. Despite this capability, ribozymes have not been as widely used in knockdown experiments as one might expect, probably because specific rules governing the selection of ribozymes that will have high activity have not been described. In this report, we show that parallel screening of less than 10 ribozyme expression constructs, with no advanced knowledge of cleavage activity or preselection, can efficiently identify knockdown ribozymes. This empirical selection study, which used interleukin-1beta (IL-1beta) and IL-1beta converting enzyme (ICE) as example targets, resulted in (1) the rapid identification of ribozymes that can reduce the production of IL-1beta in THP-1 cultures by 10-fold and (2) the consequent direct generation of stable knockdown cell lines. We conclude, based on these and similar studies, that parallel screening of ribozyme constructs could be used in high throughput gene functional analysis programs as a means of rapidly generating specific knockdown cell lines.     

Gingival and dermal fibroblasts produce interleukin-1 beta converting enzyme and interleukin-1 beta but not interleukin-18 even after stimulation with lipopolysaccharide

Epithelial cells play a critical role in periodontal disease through the secretion of pro-inflammatory cytokines such as interleukin-1 beta (IL-1 beta) and interleukin-18 (IL-18). However, the role played by fibroblasts is still unclear. The rationale of this study was to throw light on the role of gingival fibroblasts in periodontal disease. We thus investigated the expression of IL-1 beta, IL-18, and ICE mRNA and the secretion of the corresponding proteins by human normal gingival fibroblasts before and after stimulation with lipopolysaccharide (LPS) from Porphyromonas gingivalis and Escherichia coli. IL-1 beta, IL-18, and ICE mRNA expression was evaluated by RT-PCR. Proteins were analyzed by Western blot and ELISA. We demonstrated that gingival fibroblasts expressed ICE mRNA. Basal expression of ICE was modulated following cell stimulation with lipopolysaccharide (5 mug/ml). However, gingival fibroblasts expressed low levels of IL-1 beta mRNA. The expression was potentiated by LPS. The expression of IL-1 beta mRNA was followed by the secretion of IL-1 beta but not IL-18 protein. Our study suggests that fibroblasts may be involved in the defense against infections via an IL-1 beta-mediated but not an IL-18-mediated mechanism.     

Purification of interleukin-1 beta converting enzyme, the protease that cleaves the interleukin-1 beta precursor.

We have purified the IL-1 beta converting enzyme from the THP-1 cell line using standard chromatographic techniques and obtained the N-terminal amino acid sequence of this novel protein. After stimulation of THP-1 cells with lipopolysaccharide, hydroxyurea, and silica, the protease was solubilized by multiple freeze/thawing. The protein was purified by ion-exchange chromatography, affinity chromatography on blue agarose, gel filtration, and chromatofocusing. The molecular weight of the protein is approximately 22,000 Da and the pI is between 7.1 and 6.8. The overall yield for this procedure was 16% of the activity found in the initial cell lysates. An antiserum raised against a peptide based on the N-terminus was used to precipitate the protease, confirming our identification of the 22,000-Da protein as the IL-1 beta converting enzyme.     

Viral and cellular interleukin-10 (IL-10)-related cytokines: from structures to functions

The anti-inflammatory and immunosuppressive activities of IL-10 have been extensively studied during the last 10 years. More recently a series of new cytokines, structurally related to IL-10, were described. This family includes mda-7, IL-19, IL-20, IL-TIF/IL-22, and AK155. Most of the biological functions of these cytokines remain to be unraveled but new data are coming out steadily. Although none of these "IL-10 homologs" mimics IL-10 activities, they are likely to be involved in inflammatory processes as well. mda-7, IL-19 and IL-20 form a subfamily within IL-10 homologs, based on conserved amino acid sequences, and on the use of shared receptor complexes. Functional studies have stressed the potential suppressing activity of mda-7 on tumor growth. As for IL-20, its overexpression in transgenic mice led to skin abnormalities, reminiscent of psoriatic lesions in humans. IL-TIF/IL-22 is a Th1 cytokine, and was shown to upregulate the acute phase reactant production by liver cells. Finally, for AK155, originally described as a gene induced upon T cell transformation by Herpes-virus saimiri, functional data are still lacking to determine its biological activities.     

Characterization of mice deficient in interleukin-1β converting enzyme

Interleukin-1β converting enzyme (ICE) processes the inactive proIL-1β to the proinflammatory mature IL-1β. ICE belongs to a family of cysteine proteases that have been implicated in apoptosis. To address the biological functions of ICE, we generated ICE-deficient mice through gene targeting technology. ICE-deficient mice developed normally, appeared healthy, and were fertile. Peritoneal macrophages from ICE-deficient mice underwent apoptosis normally upon ATP treatment. Thymocytes from young ICE-deficient mice also underwent apoptosis when triggered by dexamethasone, gamma irradiation, or aging. ICE-deficient mice had a major defect in the production of mature IL-1β and had impaired IL-1α production on LPS stimulation in vitro and in vivo. ICE-deficient mice were resistant to LPS-induced endotoxic shock. J. Cell. Biochem. 64:27–32. © 1997 Wiley-Liss, Inc.     

Genetic variation in pro-inflammatory cytokines (interleukin-1beta, interleukin-1alpha and interleukin-6) associated with the aggressive forms, survival, and relapse prediction of breast carcinoma

OBJECTIVES: Interleukin-1 (IL-1) and interleukin-6 (IL-6) are determining factors in the immune and inflammatory responses to tumors cells. Experimental data suggest that interleukin-1 and interleukin-6 play important roles in the development and progression of breast cancer. We designed a broad study to investigate the susceptibility and prognostic implications of the genetic variation in IL-1alpha, IL-1beta and IL-6 in breast carcinoma. EXPERIMENTAL DESIGN: We used the polymerase chain reaction and restriction enzyme digestion to characterize the genetic variation of IL-1alpha, IL-1beta and IL-6 in 305, unrelated Tunisian patients with breast carcinoma and 200 healthy control subjects. Associations between the genetic markers and the clinicopathological parameters, the specific overall survival rate (OVS) of breast carcinoma and the disease free-survival rate (DFS) were assessed using univariate and multivariate analyses. RESULTS: Both IL-6 (-597) GA and IL-6 (-174) GC heterozygous genotypes were found to be significantly associated with breast carcinoma (OR = 1.59, p = 0.024 and OR = 1.61, p = 0.022 respectively). A highly significant association was found between the (+3954) T allele of IL1-B gene and the aggressive phenotype of breast carcinoma as defined by the high histological grade, axillary lymph node metastasis and large tumor size. The IL-1alpha (-889) TT homozygous genotype showed a significant association with reduced disease-free survival and/or overall survival rate. The IL-1beta (+3954) TT, IL-6 (-597) GG and IL-6 (-174) GG homozygous genotypes were found to be associated with reduced DFS but not with overall survival. CONCLUSIONS: The polymorphisms in the promoter region of the IL-6 gene may represent a marker for the increased risk of breast carcinoma. Genetic variations in IL-1alpha, IL-1beta and IL-6 may predict the clinical outcome of breast carcinoma.     

The interleukin-1β converting enzyme family of cysteine proteases

Interleukin-1β converting enzyme (ICE) is the first enzyme of a new family of cysteine endoproteinases to be isolated and characterized. An overview of the structure and activity of ICE is outlined together with highlights of salient features common to members of each of the family members. J. Cell. Biochem. 64:2–10. © 1997 Wiley-Liss, Inc.     

Mechanical stretch induced interleukin-18 (IL-18) expression through Angiotensin subtype 1 receptor (AT1R) and endothelin-1 in cardiomyocytes

Interleukin-18 (IL-18) is a proinflammatory cytokine with multiple biological functions. We and others have demonstrated that an increased level of circulating IL-18 is one of the risk factors for cardiovascular diseases. Endothelin-1 (ET-1) has been reported to be a potent hypertrophy-promoting factor through RhoA and Rho-Kinase. Mechanical stretch induces a hypertrophic response, partly through the production of ET-1 through Endothelin A receptor (ETAR). Moreover, it has also been reported that mechanical stretch induces cardiac hypertrophy through Angiotensin subtype 1 receptor (AT1R). However, the mechanism by which the IL-18 gene expression is regulated in cardiomyocytes has not yet been fully understood. This study was designed to elucidate the functional significance of IL-18 gene expression in response to mechanical stretch. Neonatal rat cardiomyocytes cultured on silicone dishes were subjected to stretch. The moderate 20% mechanical stretch resulted in the elevation of IL-18 expression in a time-dependent manner with the maximal level achieved 36 hours after the stretch. Olmesartan, AT1R antagonist inhibited stretch-induced IL-18 expression. ETAR blockade BQ123 inhibited stretch-induced IL-18 expression. However, the Endothelin B receptor (ETBR) receptor blockade BQ788 did not inhibit this reaction. ET-1 induced IL-18 expression, with a peak induction after 4 hours of incubation. These results might suggest that stretch stimulation of cardiomyocytes induced ET-1 and, subsequently, ET-1 up-regulated the IL-18 expression. Furthermore, Fasudil, a Rho-Kinase inhibitor, and Simvastatin, a HMG-CoA reductase inhibitor, led to a significant reduction in mechanical stretch-induced IL-18 expression. These results indicated, for the first time, that IL-18 expression is induced by mechanical stretch in cardiomyocytes via the ETAR, AT1R, and the Rho/Rho-K pathways. The induction of IL-18 from cardiomyocytes by mechanical stress might cause the deterioration of cardiac functions in autocrine and paracrine fashion. The inhibition of IL-18 expression induced by mechanical stress might be one of the mechanisms that account for the beneficial cardiovascular effects of AT1R antagonist, ETAR blockade, Statin, and Rho-Kinase inhibitor.     

Synthesis and evaluation of novel 8,6-fused bicyclic peptidomimetic compounds as interleukin-1beta converting enzyme inhibitors

Two novel 8,6-fused bicyclic peptidomimetic ring systems were synthesized utilizing olefin metathesis as the key reaction for the formation of the eight-membered ring. Both peptidomimetic scaffolds were further elaborated into potent ICE inhibitors, with numerous compounds exhibiting caspase-1 IC₅₀s less than 10 nM.     

Phenylalkyl ketones as potent reversible inhibitors of interleukin-1β converting enzyme

Phenylalkyl ketones are potent reversible inhibitors of interleukin-1β converting enzyme (ICE). The extended alkyl chain ketones AcTyrValAlaAspCO(CH₂)_nPh display increased potency over the simple aryl ketone. In particular, the tetrapeptide AcTyrValAlaAspCOPh has a K_i of 10μM while AcTyrValAlaAspCO(CH₂)₅Ph has a K_i of 18.5nM.     

Dose-effect of interleukin-10 and its immunoregulatory role in Crohn's disease

BACKGROUND: Interleukin-10 (IL-10) is currently being extensively studied in clinical trials for the treatment of Crohn's disease (CD). Only marginal effects have, however, been reported, and the dose-response curve was bell-shaped contrasting with the reported data from in vitro experiments. AIM: To use another in vitro model to analyze the effect of rhIL-10 and rhIL-4 on the spontaneous mucosal TNF-alpha secretion in patients with CD, and to characterize the phenotype of the cells targeted by rhIL-10. METHODS: Non-inflamed colon biopsies from CD patients were cultured for 16 hours in presence of different concentrations of rhIL-10 or rhIL-4. The numbers of TNF-alpha-secreting cells among isolated lamina propria mononuclear cells (LPMNC) were estimated by Elispot. RESULTS: Both rhIL-10 and rhIL-4 down-regulate TNF-alpha secretion by LPMNC from CD patients, with a more pronounced effect with rhIL-10. These effects were closely linked to the cytokine concentrations used, with a bell-shaped dose-response curve. Residual TNF-alpha secretion, in the presence of optimal rhIL-10 concentration was mainly attributable to CD3+ T cells. In contrast, at higher rhIL-10 concentrations, CD3- cells contributed significantly to the TNF-alpha secretion. CONCLUSIONS: The in vitro model we used, demonstrates that IL-4, but mostly IL-10, efficiently suppresses TNF-alpha secretion in LPMNC from CD patients, with a dose-response curve similar to results obtained in vivo. Resistance at high rhIL-10 concentrations was associated with a change in the phenotype of TNF-alpha-secreting cells.