Tumor Necrosis Factor-α Converting Enzyme Research Articles

Active-site determinants of substrate recognition by the metalloproteinases TACE and ADAM10

The metalloproteinases TACE [tumour necrosis factor alpha-converting enzyme; also known as ADAM17 (a disintegrin and metalloproteinase 17)] and ADAM10 are the primary enzymes responsible for catalysing release of membrane-anchored proteins from the cell surface in metazoan organisms. Although the repertoire of protein substrates for these two proteases is partially overlapping, each one appears to target a subset of unique proteins in vivo. The mechanisms by which the two proteases achieve specificity for particular substrates are not completely understood. We have used peptide libraries to define the cleavage site selectivity of TACE and ADAM10. The two proteases have distinct primary sequence requirements at multiple positions surrounding the cleavage site in their substrates, which allowed us to generate peptide substrates that are highly specific for each of these proteases. The major difference between the two protease specificities maps to the P1' position (immediately downstream of the cleavage site) of the substrate. At this position, TACE is selective for smaller aliphatic residues, whereas ADAM10 can accommodate aromatic amino acids. Using mutagenesis we identified three residues in the S1' pockets of these enzymes that dramatically influence specificity for both peptide and protein substrates. Our results suggest that substrate selectivity of TACE and ADAM10 can be at least partly rationalized by specific features of their active sites.

Activation of endothelial intrinsic NF-κB pathway impairs protein C anticoagulation mechanism and promotes coagulation in endotoxemic mice

AbstractAlthough the role of systemic activation of the nuclear factor κB (NF-κB) pathway in septic coagulation has been well documented, little is known about the contribution of endothelial-specific NF-κB signaling in this pathologic process. Here, we used transgenic mice that conditionally overexpress a mutant I-κBα, an inhibitor of NF-κB, selectively on endothelium, and their wild-type littermates to define the role of endothelial-specific NF-κB in septic coagulation. In wild-type mice, lipopolysaccharide (LPS) challenge (5 mg/kg intraperitoneally) caused markedly increased plasma markers of coagulation, decreased plasma fibrinogen level, and widespread tissue fibrin deposition, which were abrogated by endothelial NF-κB blockade in transgenic mice. Endothelial NF-κB blockade inhibited tissue factor expression in endothelial cells, but not in leukocytes. Endothelial NF-κB blockade did not inhibit LPS-induced tissue factor expression in heart, kidney, and liver. Endothelial NF-κB blockade prevented LPS down-regulation of endothelial protein C receptor (EPCR) and thrombomodulin protein expressions, inhibited tissue tumor necrosis factor-α converting enzyme activity, reduced EPCR shedding, and restored plasma protein C level. Our data demonstrate that endothelial intrinsic NF-κB signaling plays a pivotal role in septic coagulation and suggests a link between endothelial-specific NF-κB activation and the impairment of the thrombomodulin-protein C-EPCR anticoagulation pathway.

Selective and specific regulation of ectodomain shedding of angiotensin-converting enzyme 2 by tumor necrosis factor α-converting enzyme

Angiotensin-converting enzyme 2 (ACE2) is a newly identified regulator of the renin-angiotensin system. This type I membrane-anchored protein has a catalytically active ectodomain that undergoes shedding. Tumor necrosis factor alpha-converting enzyme (TACE) has been shown to be involved in ACE2 shedding. Although pathophysiological significance of ACE2 shedding has been suggested, regulation of this process by TACE is not clearly defined. We characterized TACE-mediated constitutive ectodomain shedding of ACE2 using wild-type Chinese Hamster Ovary (WT-CHO), the TACE-mutant M2 (M2-CHO) cells, and EC-4 and EC-2 cells that are fibroblasts from wild-type and TACE-null mice, respectively. ACE2 was constitutively cleaved to release two distinct major soluble forms. The deglycosylated molecular masses of the larger (LSF) and smaller soluble form (SSF) were approximately 80 and 70 kDa, respectively. These forms had equivalent enzyme activities. Reduced shedding for the LSF from M2-CHO and EC-2 cells when compared with WT-CHO and EC-4 cells, respectively, was noted. TACE reconstitution in EC-2 cells expressing ACE2 resulted in increase in LSF but not SSF release, demonstrating a main role of TACE in LSF release and distinct regulations of release of the two soluble forms. Deletions of the juxtamembrane region of ACE2 reduced LSF release in CHO cell lines, whereas it abolished TACE-induced shedding in EC-2 cells. Analysis of TACE structural domains confirmed that the active site in the catalytic domain is essential for ACE2 shedding but that noncatalytic domains also play additional roles. These results demonstrate selective and specific regulation of constitutive shedding of ACE2 by TACE.

Preconditioning with Sublethal Ischemia or Intermittent Normobaric Hyperoxia Up-regulates Glutamate Transporters and Tumor Necrosis Factor-α Converting Enzyme in the Rat Brain

Recent studies suggest that sublethal ischemia and intermittent normobaric hyperoxia (InHO) protect the brain from subsequent ischemic injury. In this, changes in the expression of excitatory amino-acid transporters (EAATs) and tumor necrosis factor-alpha converting enzyme (TACE) may play a role. We sought to identify and clarify the nature of any such changes. Rats were divided into 3 experimental groups, each of 15 animals. The first group was exposed to normobaric hyperoxia (fractional inspired oxygen concentration 95%) for 4 hours/day for 6 consecutive days (InHO). The second group acted as controls, and was exposed to 21% oxygen in the same chamber (room air). The third group acted as a model of ischemic preconditioning, and was exposed to 21% oxygen in the same chamber and subjected to 10 minutes of temporary middle cerebral artery (MCA) occlusion (tMCAO). After 24 hours, 9 animals from each group were subjected to 60 minutes of right MCA occlusion (MCAO). After 24 hours of reperfusion, neurologic deficit score and infarct volume were assessed in MCAO-operated subgroups. The remaining 6 animals in each group remained intact and, 48 hours after pretreatment, were killed for assessment of EAATs and TACE expression in the ipsilateral hemisphere. Preconditioning with InHO and tMCAO decreased neurologic deficit score and infarct volume, and increased expression of EAAT1, EAAT2, EAAT3, and TACE. InHO and tMCAO are associated with expression of EAAT1, EAAT2, EAAT3, and TACE, consistent with an active role in the genesis of ischemic protection.

CB-12181, a New Azasugar-Based Matrix Metalloproteinase/Tumor Necrosis Factor-α Converting Enzyme Inhibitor, Inhibits Vascular Endothelial Growth Factor-Induced Angiogenesis in Vitro and Retinal Neovascularization in Vivo

To evaluate the anti-angiogenic efficacy of CB-12181 [an azasugar derivative that has inhibitory actions against matrix metalloproteinases (MMPs) and tumor necrosis factor-alpha (TNF-alpha) converting enzyme (TACE)], we investigated the suppressing ability on in vitro (tube formation by endothelial cells) and in vivo (retinal neovascularization on murine ischemia-induced proliferative retinopathy) models of angiogenesis. For in vitro analysis, a capillary-like tube formation model using human umbilical vein endothelial cells (HUVECs) and fibroblasts co-culture assay was employed. Tube formation of HUVECs was stimulated by vascular endothelial growth factor (VEGF) and incubated with different concentrations of CB-12181 (0.1-100 microM) for 11 days. For in vivo analysis, mice were exposed to 75% oxygen between postnatal days 7 and 12 (P7 to P12). Then, the mice were removed from the oxygen treatment and treated with CB-12181 (1, 15, or 50 mg/kg) by daily subcutaneous injection from the time of reintroduction to room air at P12 until P16. At P17, pathological and physiological angiogenesis was quantified using retinal flat-mounts visualized by fluorescent angiography. In the in vitro angiogenesis model, CB-12181 significantly suppressed VEGF-induced HUVEC tube formation. Furthermore, in the in vivo angiogenesis model, administration of CB-12181 significantly suppressed retinal neovascularization without any apparent side effects on physiological revascularization to the oxygen-induced obliteration area. These results suggest that CB-12181 might be useful in the treatment of various diseases that depend on pathologic angiogenesis, and especially valuable for the treatment of diabetic retinopathy and retinopathy of prematurity.

Tumor Necrosis Factor-α–Converting Enzyme Is a Key Regulator of Agonist-Induced Cardiac Hypertrophy and Fibrosis

Cardiac remodeling is associated with hypertrophy and fibrosis processes, which may depend on the activity of matrix metalloproteinases (MMPs) and "a disintegrin and metalloproteinases" (ADAMs). We investigated whether ADAM-17 (tumor necrosis factor-alpha-converting enzyme [TACE]) plays a role in agonist-induced cardiac remodeling and the relationships established among TACE, MMP-2, and ADAM-12. We targeted TACE in rodent models of spontaneous and agonist-induced hypertension using RNA interference combined with quantitative RT-PCR, activity determinations, and functional studies. Treatment of spontaneously hypertensive rats with previously validated TACE small-interfering RNA for 28 days resulted in systemic knockdown of TACE expression. TACE knockdown effectively stopped the development of cardiac hypertrophy. Mice receiving angiotensin II (1.4 mg/kg per day for 12 days) exhibited cardiac hypertrophy, as well as fibrosis, which was associated with elevated myocardial expression of molecular markers of hypertrophy (alpha-skeletal actin, beta-myosin heavy chain, and brain natriuretic peptide) and fibrosis (collagen types I and III and fibronectin), as well as MMP-2 and ADAM-12. Treatment with TACE small-interfering RNA (but not with PBS or luciferase small-interfering RNA) inhibited TACE expression, thus preventing angiotensin II-induced cardiac hypertrophy and fibrosis. Moreover, knockdown of TACE inhibited angiotensin II-induced overexpression of markers of myocardial hypertrophy and fibrosis, as well as ADAM-12 and MMP-2. These findings provide the first in vivo evidence that agonist-induced cardiac hypertrophy and fibrosis processes are signaled through TACE, which acts through novel pathways involving transcriptional regulation of ADAM-12 and MMP-2. Targeting TACE has potential therapeutic importance for modulating agonist-induced cardiac remodeling.

Release of Soluble Tumor Necrosis Factor Receptor 1 from Corneal Epithelium by TNF-α–Converting Enzyme-Dependent Ectodomain Shedding

An involvement of tumor necrosis factor-alpha-converting enzyme (TACE)-dependent ectodomain shedding in the release of soluble tumor necrosis factor receptor 1 (sTNFR1) from corneal epithelium was evaluated. In vitro experiments were performed using the human SV40-transformed human corneal epithelial cell (HCEC) line. Ectodomain shedding was stimulated by phorbol myristate acetate (PMA, 3 microM) or peptidoglycan (PGN, 100 microg/mL), with or without TACE inhibition, using TNF-alpha processing inhibitor-1 (TAPI-1, 250 microg/mL) or tissue inhibitor of metalloproteinase-3 (TIMP-3, 2 microg/mL) by addition to the HCEC culture medium. The concentrations of sTNFR1 in culture medium were analyzed by enzyme-linked immunosorbent assay. To induce an inflammatory response in the ocular surface, corneal alkali burn of BALB/c mice was made from a filter paper dipped in 1 N NaOH solution. TNFR1 expression in corneal and conjunctival epithelia was evaluated by immunohistochemistry 28 days after wounding. In HCEC culture medium, sTNFR1 release was significantly increased by the addition of PMA (t-test, P < 0.01) or PGN (P < 0.01). The increased release of sTNFR1 was significantly inhibited by the addition of TAPI-1 or TIMP-3, indicating the possibility of TACE-dependent ectodomain shedding of TNFR1. In the corneal alkali burn model, TNFR1 was expressed in corneal and conjunctival epithelia. TACE-dependent ectodomain shedding of sTNFR1 was recognized in corneal epithelium. In the inflamed ocular surface, TNFR1 was expressed in the corneal and conjunctival epithelia after alkali burn treatment. sTNFR1, released from the ocular surface, may play an anti-inflammatory role in the inflammatory condition.

Identification of Ligand-Induced Proteolytic Cleavage and Ectodomain Shedding of VEGFR-1/FLT1 in Leukemic Cancer Cells

Vascular endothelial growth factor receptor-1/fms-related tyrosine kinase 1 (VEGFR-1/FLT1) is expressed as a membrane-bound receptor tyrosine kinase and as an alternatively spliced soluble protein (sVEGFR-1) containing the 1-6 IgG-like domain of its ectodomain. sVEGFR-1 is known as a naturally occurring inhibitor of angiogenesis and as a surrogate marker for cancer progression; it is also linked to pregnancy-induced hypertension called preeclampsia and to avascularity of normal cornea. It remains an open question whether alternative mRNA splicing is the only mechanism by which sVEGFR-1 is generated. In this study, we show that in leukemic cancer cells, PlGF and VEGF-A both induce tyrosine phosphorylation of VEGFR-1 and render it susceptible to ectodomain shedding, resulting in the generation of sVEGFR-1 and an intracellular cytoplasmic fragment. Activation of protein kinase C and tumor necrosis factor-alpha-converting enzyme family metalloproteases are critically required for the occurrence of sVEGFR-1. Following the removal of the ectodomain, the remnant of VEGFR-1 remains attached to the membrane, and the activity of gamma-secretase/presenilin is required for its release from the cell membrane. We propose that sVEGFR-1 produced via ectodomain shedding plays a prominent role in the VEGF receptor system by antagonizing VEGF receptor signaling by acting as a dominant-negative form and/or forming a nonsignaling dimerizing complex with VEGF receptors.

Thrombospondin 2 Potentiates Notch3/Jagged1 Signaling

Extracellular thrombospondins (TSP or THBS) and the Notch family of transmembrane receptors share a role in multiple, overlapping cellular functions and participate in developmental signaling and pathological reactions to tissue injury. We demonstrate that TSP2, but not TSP1, enhances the potency of Notch3 signal transduction. In addition, TSP2 reduces cancer cell proliferation in a Notch-ligand dependent fashion. The loss of TSP2 in knock-out mice reduces Notch target gene expression. TSP2 binds directly to Notch3 and Jagged1. TSP1 also binds to Notch3 and Jagged1; however, only TSP2 augments the interaction between Notch3 and Jagged1. These studies demonstrate that the diverse functions of TSP2 may also include a role as an intermediary protein that facilitates transcellular receptor-ligand interactions.

Niaspan Treatment Increases Tumor Necrosis Factor-α-Converting Enzyme and Promotes Arteriogenesis after Stroke

We tested the hypothesis that Niaspan (a prolonged release formulation of niacin) increases tumor necrosis factor-alpha-converting enzyme (TACE) expression and Notch signaling activity and promotes arteriogenesis after stroke. Rats were subjected to middle cerebral artery occlusion and were treated with or without Niaspan. Niaspan significantly elevated local cerebral blood flow, and increased arteriogenesis as indicated by increased arterial diameter and vascular smooth muscle cell (VSMC) proliferation in the ischemic brain after stroke. The increased arteriogenesis significantly correlated with the functional outcome after stroke. Niaspan treatment of stroke upregulated TACE, Notch1, and Notch intracellular domain expression in the ischemic brain. To further investigate the mechanisms of Niaspan-induced arteriogenesis, a primary brain arterial culture was used. Niacin treatment significantly increased arterial sprouting and VSMC migration compared with control nontreated arterial cells. Inhibition of TACE by the TACE inhibitor or knockdown of TACE gene expression in brain arterial culture significantly attenuated Niacin-induced arterial sprouting and VSMC migration. In addition, TACE treatment of arterial culture significantly increased arterial VSMC migration and arterial sprouting. Knockdown of Notch1 marginally decreased arterial sprouting and VSMC migration compared with scrambled control. Niaspan promotes arteriogenesis, which is mediated, in part, by TACE.

Transforming Growth Factor β Induces Clustering of HER2 and Integrins by Activating Src-Focal Adhesion Kinase and Receptor Association to the Cytoskeleton

It has been proposed that cross talk between integrin and growth factor receptor signaling such as ErbB2 (HER2) is required for activation of downstream effectors and ErbB2-mediated mammary tumorigenesis. Here we show that transforming growth factor beta (TGF-beta) induced focal adhesion kinase (FAK)-dependent clustering of HER2 and integrins alpha(6), beta(1), and beta(4) in HER2-overexpressing mammary epithelial cells without altering the total and surface levels of HER2 receptors. This effect was mediated by ligand-induced epidermal growth factor receptor (EGFR) activation and the subsequent phosphorylation of Src and FAK. We have previously reported that TGF-beta up-regulates EGFR ligand shedding through a mechanism involving the phosphorylation of tumor necrosis factor-alpha-converting enzyme (TACE/ADAM17). Knockdown of TACE, FAK, or integrin alpha(6) by siRNA or inhibition of EGFR or Src by specific inhibitors abrogated TGF-beta-induced receptor clustering and signaling to phosphatidylinositol 3-kinase-Akt. Finally, inhibition of Src-FAK reversed TGF-beta-induced resistance to the therapeutic HER2 inhibitor trastuzumab in HER2-overexpressing breast cancer cells. Taken together, these data suggest that, by activating Src-FAK, TGF-beta integrates ErbB receptor and integrin signaling to induce cell migration and survival during breast cancer progression.

Chlorella powder inhibits the activities of peptidase cathepsin S, PLA2, cyclooxygenase-2, thromboxane synthase, tyrosine phosphatases, tumor necrosis factor-α converting enzyme, calpain and kinases

A Chlorella powder was tested in 118 in vitro enzyme assay systems. The powder showed potent inhibitions of peptidase cathepsin S, thromboxane A2 synthase and cyclooxygenase-2 in a dose-concentration manner with IC50±standard error of the mean values of 3.46±0.93 µg/ml, 3.23±0.69 µg/ml, and 44.26±9.98 µg/ml, respectively. Other activities observed were inhibitions of tumor necrosis factor-α converting enzyme, protein tyrosine phosphatase (SHP-2), calpain, protein kinases and protein tyrosine phosphatases. Chlorella powder had no significant effect on cyclooxygenase-1. These actions to inhibit cyclooxygenase-2 and thromboxane synthase could contribute to the purported anti-inflammatory and anti-thrombotic effects of Chlorella. These results reveal important potential biochemical activities to be developed that, if confirmed by in vivo studies, might be exploited for the prevention or treatment of several serious pathologies, including inflammatory diseases, immune and cancer.

Discovery of novel hydroxamates as highly potent tumor necrosis factor-α converting enzyme inhibitors. Part II: Optimization of the S3′ pocket

A series of cyclopropyl hydroxamic acids were prepared. Many of the compounds displayed picomolar affinity for the TACE enzyme while maintaining good to excellent selectivity profiles versus MMP-1, -2, -3, -7, -14, and ADAM-10. X-ray analysis of an inhibitor in the TACE active site indicated that the molecules bound to the enzyme in the S1′–S3′ pocket.

Production of the Soluble Form of KIT, s-KIT, Abolishes Stem Cell Factor-Induced Melanogenesis in Human Melanocytes

The signaling of stem cell factor (SCF) and its receptor KIT (membrane-bound KIT; m-KIT) plays an important role in melanocyte development, survival, proliferation, and melanogenesis. It has been demonstrated in other systems that a soluble form of m-KIT released from the cell surface (s-KIT) regulates SCF signaling, although there have been no reports pertaining to the existence and the biological role of s-KIT in melanocytes. In this study, we therefore examined the involvement of s-KIT in melanogenesis. Western blotting analysis revealed that treatment with phorbol 12-myristate-13-acetate (PMA) or 4-aminophenylmercuric acetate (APMA) induced s-KIT production in cultured human melanocytes. Inhibitors of tumor necrosis factor-alpha-converting enzyme (TACE) and metalloproteinases (MMPs) muted this release of s-KIT into the media. Human recombinant s-KIT added to melanocytes inhibited SCF-induced phosphorylation of m-KIT, resulting in suppression of SCF-induced melanogenesis. Additionally, APMA-induced s-KIT production abolished SCF-induced melanogenesis as effectively as a KIT-neutralizing antibody. Concomitantly, APMA and TACE inhibitors significantly decreased and increased melanin synthesis, respectively, in an in vitro skin model. Taken together, these findings provided an insight into the elaborate mechanism of SCF/m-KIT signaling in human melanocytes and suggested that production of s-KIT contributes to the regulation of human skin pigmentation. Journal of Investigative Dermatology (2008) 128, 1763-1772; doi:10.1038/jid.2008.9; published online 31 January 2008.

Localization of the Delta-like-1-binding Site in Human Notch-1 and Its Modulation by Calcium Affinity

The Notch signaling pathway plays a key role in a myriad of cellular processes, including cell fate determination. Despite extensive study of the downstream consequences of receptor activation, very little molecular data are available for the initial binding event between the Notch receptor and its ligands. In this study, we have expressed and purified a natively folded wild-type epidermal growth factor-like domain (EGF) 11-14 construct from human Notch-1 and have used flow cytometry and surface plasmon resonance analysis to demonstrate a calcium-dependent interaction with the human ligand Delta-like-1. Site-directed mutagenesis of three of the calcium-binding sites within the Notch-(11-14) fragment indicated that only loss of calcium binding to EGF12, and not EGF11 or EGF13, abrogates ligand binding. Further mapping of the ligand-binding site within this region by limited proteolysis of Notch wild-type and mutant fragments suggested that EGF12 rather than EGF11 contains the major Delta-like-1-binding site. Analysis of an extended fragment EGF-(10-14), where EGF11 is placed in a native context, surprisingly demonstrated a reduction in ligand binding, suggesting that EGF10 modulates binding by limiting access of ligand. This inhibition could be overcome by the introduction of a calcium binding mutation in EGF11, which decouples the EGF-(10-11) module interface. This study therefore demonstrates that long range calcium-dependent structural perturbations can influence the affinity of Notch for its ligand, in the absence of any post-translational modifications.

In vivo preconditioning with normobaric hyperoxia induces ischemic tolerance partly by triggering tumor necrosis factor-α converting enzyme/tumor necrosis factor-α/nuclear factor-κB

Recent studies suggest that intermittent and prolonged normobaric hyperoxia (HO) results in brain ischemic tolerance (BIT), reducing ischemic brain injury. We have attempted to determine the time course of HO-induced BIT, and to explore the putative roles of tumor necrosis factor-α (TNF-α) converting enzyme (TACE), TNF-α, and nuclear factor-κB (NF-κB) activation in mediating this effect. Two core experimental protocols were applied to rats (experiments 1 [E1] and 2 [E2] respectively). E1 rodents comprised six subgroups, breathing room air (RA; O 2=21%), or 95% oxygen (HO) for 4, 8, 16 h (4RA, 8RA, 16RA and 4HO, 8HO, 16HO respectively). E2 rodents were divided into subgroups, exposed to 95% inspired HO for 4 h/day for six consecutive days (intermittent hyperoxia, InHO) or for 24 continuous hours (prolonged hyperoxia, PrHO). Each of these had a control group exposed to 21% oxygen in the same chamber. Twenty-four hours after pretreatment, each group was randomly divided to receive 60 min right middle cerebral artery occlusion (MCAO-operated), sham-operation (without MCAO), or no operation (intact). After 24 h reperfusion, neurologic deficit score (NDS), brain water content, Evans Blue extravasation (as a marker of blood–brain barrier permeability), TACE expression, serum TNF-α, and phosphor- κBα levels were assessed in all animals, and infarct volume in the MCAO-operated subgroups. E1: Compared with the control (RA) group, infarct volume was reduced by 58.6% and 64.4% in 16 h and 24 h respectively. NDS and Evans Blue extravasation was also reduced in 16 h and 24 h. There was no statistical difference among 4 h and 8 h. E2: Preconditioning with prolonged and intermittent HO decreased NDS, infarct volume and upregulated TACE and increased phosphor-κBα and serum TNF-α level significantly. Although further studies are needed to clarify the mechanisms of brain ischemic tolerance, InHO and PrHO may partly exert their effects via triggering TACE/TNF-α/NF-κB.

Tumor Necrosis Factor-α-converting Enzyme (TACE) Levels in Periodontal Diseases

Tumor necrosis factor-alpha-converting enzyme (TACE) is a metalloprotease which can shed several cytokines from the cell membrane, including receptor activator of NF-kappaB ligand (RANKL). This study aimed to investigate the hypothesis that TACE would be elevated in the gingival crevicular fluid (GCF) of persons with periodontitis. Total TACE amounts in GCF were higher in persons with chronic and aggressive periodontitis than in those with gingivitis or in healthy persons. TACE concentrations in GCF were higher in persons with chronic and aggressive periodontitis than in those with gingivitis, although not significantly higher than in healthy persons. Persons with chronic periodontitis receiving immunosuppressive treatment exhibited over 10-fold lower TACE levels than the other periodontitis groups. TACE was positively correlated with probing pocket depth, clinical attachment levels, and RANKL concentrations in GCF. In conclusion, the increased GCF TACE levels in persons with periodontitis and their positive correlation with RANKL may indicate an association of this enzyme with alveolar bone loss, and may warrant special attention in future therapeutic approaches.

Potent, selective, orally bioavailable inhibitors of tumor necrosis factor-α converting enzyme (TACE): Discovery of indole, benzofuran, imidazopyridine and pyrazolopyridine P1′ substituents

Potent and selective inhibitors of tumor necrosis factor-alpha converting enzyme (TACE) were discovered with several new heterocyclic P1' groups in conjunction with cyclic beta-amino hydroxamic acid scaffolds. Among them, the pyrazolopyridine provided the best overall profile when combined with tetrahydropyran beta-amino hydroxamic acid scaffold. Specifically, inhibitor 49 showed IC(50) value of 1 nM against porcine TACE and 170 nM in the suppression of LPS-induced TNF-alpha of human whole blood. Compound 49 also displayed excellent selectivity over a wide panel of MMPs as well as excellent oral bioavailability (F%>90%) in rat n-in-1 PK studies.

α,β-Cyclic-β-benzamido hydroxamic acids: Novel oxaspiro[4.4]nonane templates for the discovery of potent, selective, orally bioavailable inhibitors of tumor necrosis factor-α converting enzyme (TACE)

Two novel oxaspiro[4.4]nonane beta-benzamido hydroxamic scaffolds have been synthesized in enantio- and diasteriomerically pure form. These templates proved to be exceptional platforms that have led to the discovery of potent inhibitors of TACE that are active in a cellular assay measuring suppression of LPS-induced TNF-alpha. Furthermore, these inhibitors are selective against related MMPs, demonstrate permeability in a Caco-2 assay, and display good oral bioavailability.

Regulated Proteolytic Processing of Tie1 Modulates Ligand Responsiveness of the Receptor-tyrosine Kinase Tie2

Regulated ectodomain shedding followed by intramembrane proteolysis has recently been recognized as important in cell signaling and for degradation of several type I transmembrane proteins. The receptor-tyrosine kinase Tie1 is known to undergo ectodomain cleavage generating a membrane-tethered endodomain. Here we show Tie1 is a substrate for regulated intramembrane proteolysis. After Tie1 ectodomain cleavage the newly formed 45-kDa endodomain undergoes additional proteolytic processing mediated by gamma-secretase to generate an amino-terminal-truncated 42-kDa fragment that is subsequently degraded by proteasomal activity. This sequential processing occurs constitutively and is stimulated by phorbol ester and vascular endothelial growth factor. To assess the biological significance of regulated Tie1 processing, we analyzed its effects on angiopoietin signaling. Activation of ectodomain cleavage causes loss of phosphorylated Tie1 holoreceptor and generation of phosphorylated receptor fragments in the presence of cartilage oligomeric protein angiopoietin 1. A key function of gamma-secretase is in preventing accumulation of these phosphorylated fragments. We also find that regulated Tie1 processing modulates ligand responsiveness of the Tie-1-associated receptor Tie2. Activation of Tie1 ectodomain cleavage increases cartilage oligomeric protein angiopoietin 1 activation of Tie2. This correlates with increased ability of Tie2 to bind ligand after shedding of the Tie1 extracellular domain. A similar enhancement of ligand activation of Tie2 is seen when Tie1 expression is suppressed by RNA interference. Together these data indicate that Tie1, via its extracellular domain, limits the ability of ligand to bind and activate Tie2. Furthermore the data suggest that regulated processing of Tie1 may be an important mechanism for controlling signaling by Tie2.