TNF-alpha Converting Enzyme Inhibitors Research Articles

The discovery of potential tumor necrosis factor alpha converting enzyme inhibitors via implementation of K Nearest Neighbor QSAR analysis

Tumor necrosis factor-alpha converting enzyme (TACE) is considered as a pro-inflammatory cytokine which catalyzes the formation of TNF-α from membrane bound TNF-α precursor protein. It has important role in various pathological diseases such as inflammation, anorexia, rheumatoid arthritis, cancer and viral replication. Despite the reporting of a variety of TACE inhibitors of diverse chemical scaffolds whether peptide, peptide-like compounds or others, the bioavailability and pharmacokinetic problems are reflected strongly on its clinical effectiveness. In this effort we employed a novel combination of k-nearest neighbor QSAR modeling to select the best critical ligand-TACE contacts capable of elucidation of TACE inhibitory bioactivity among a long list of inhibitors. The recurrence of one valid pharmacophore hypothesis among the successful models emerged the pharmacophore to be used as 3D search queries to screen the National Cancer Institute’s structural database for new TACE inhibitors. Eight potent TACE inhibitors were discovered with inhibition percentage exceeding 50% at 100 μM inhibitor concentration.

Probing the bioactive compounds of Kigelia africana as novel inhibitors of TNF-α converting enzyme using HPLC/GCMS analysis, FTIR and molecular modelling

Tumor Necrosis Factor Alpha Converting Enzyme (TACE) mediates inflammatory disorder and contributes to the pathophysiology of a variety of illnesses, such as chronic inflammation and cancer. This study identified metabolites in solvent extracts of Kigelia africana as putative TACE inhibitors due to the plant’s known anti-inflammatory properties. HPLC-MS/GCMS analysis was used to characterize tentative phytochemicals from K. africana. The identified metabolites (n = 123) were docked with TACE to reveal the lead compounds. Binding free energy, ADMET prediction, molecular dynamics simulation at 100 ns, and DFT calculation were further conducted. The results revealed that K. africana contains sterol, phenols, alkaloids, terpenes and flavonoids. The FTIR shows that the extracts had peaks that correspond to the presence of different functional groups. The quantum polarized ligand docking (QPLD) analysis identified compound (n = 3) with binding affinity higher than standard compound IK-682. The hits also had modest ADMET profiles, interacted with essential residues within TACE binding pockets, and formed stable complexes with the protein. The 100 ns MD simulation shows that the compounds formed fairly stable interactions and complex with the protein as evidenced through RMSF, RMSD and MM-GBA results. The HOMO/LUMO, global descriptive molecular electrostatic potential Fukui function aid in the identification of the compounds’ atomic sites prone to electrophilic/neutrophilic attacks, and non-covalent interactions. This study suggests that K. africana’s bioactive compounds are capable of mitigating inflammation by inhibiting TACE. Communicated by Ramaswamy H. Sarma

Simultaneous monitoring assay for T-cell receptor stimulation-dependent activation of CD4 and CD8 T cells using inducible markers on the cell surface

Isolation of antigen (Ag)-specific T cells is an important step in the investigation of T-cell immunity. Activation-induced markers (AIMs), such as CD154/tumor necrosis factor (TNF)/CD107A/CD134/CD137 enable the sorting of Ag-specific T cells without using human leukocyte antigen (HLA)-multimers. However, optimal conditions suitable for simultaneous detection of both Ag-specific CD4 and CD8 T cells have not been investigated. Here, conditions were optimized to simultaneously detect the maximum number of activated CD4 and CD8 T cells in a TCR-dependent manner. First, the frequency of total pools of AIM-positive cells induced by superantigen, staphylococcal enterotoxin B (SEB), stimulation in various culture conditions was monitored and compared side-by-side. The total amount of AIM-positive CD4 T cells, but not CD8 T cells, was significantly abrogated by addition of brefeldin A. TNF-alpha converting enzyme inhibitor treatment effectively increased the TNF-positive population, without affecting other markers’ positivity. AIM-positive CD4 T cells and CD8 T cells were detected at least 3 h after stimulation. Furthermore, examination of the multiple combination of each marker revealed that minimum contribution of CD134 on the total pool of AIM-positive cells at this setting, suggesting the essential and non-essential AIMs to maximize the detected number of AIM-positive cells.Taken together, this optimized method will be a useful tool for the simultaneous monitoring the T-cell receptor stimulation-dependent activation of CD4 and CD8 T cells using inducible markers on the cell surface including Ag-specific T cells.

Enhanced expression of TACE contributes to elevated levels of sVCAM-1 in endometriosis.

Are increased sVCAM-1 and sICAM-1 levels associated with tumor necrosis factor-alpha-converting enzyme (TACE) activity in endometriosis? Here we provide the first functional evidence that induced TACE activity in human endometriotic epithelial cells is at least in part responsible for the enhanced release of sVCAM-1 from these cells. We and others have shown that serum-soluble (s)VCAM-1 levels are significantly higher in women with endometriosis, compared to disease-free controls. Experimental evidence exists suggesting a role of sICAM-1 and sVCAM-1 in the pathogenesis of endometriosis. TACE was identified as the protease responsible for phorbol 12-myristate 13-acetate (PMA)-induced VCAM-1 release in murine endothelial cells. Additionally, it has recently been shown that TACE is upregulated in the endometrial luminal epithelium of the mid-secretory phase in infertile women. This study was conducted at the Tertiary Endometriosis Referral Center of the Medical University of Vienna. Samples from a total number of 97 women were collected between July 2013 and September 2014. After complete surgical exploration of the abdominopelvic cavity, 49 women with histologically proven endometriosis and 48 endometriosis-free control women were enrolled. Each participating woman contributed only one sample of eutopic endometrium and normal peritoneum, and some of the women with endometriosis contributed samples of diverse types of endometriotic lesions (in total 52 ectopic samples). Among the 49 women with endometriosis, 36 matched samples of endometriotic lesions and corresponding eutopic endometrium were collected. In order to detect sVCAM-1 and TACE protein by ELISA, peritoneal fluid (PF) samples were collected from 44 cases and 32 controls during surgery. Expression of TACE mRNA was analyzed by qRT-PCR in 111 endometrium tissue samples (28 eutopic control samples, 33 eutopic samples from women with endometriosis, 50 ectopic samples from lesions) and 37 healthy peritoneum samples. Immunohistochemistry was performed in 123 tissue samples (39 eutopic control samples, 42 eutopic samples from women with endometriosis, 42 ectopic samples from lesions) and the relation between tissue TACE protein levels and sVCAM-1 secretion was examined. PMA-induced sVCAM-1 release, and TACE- and VCAM-1-transcripts or proteins were measured in an immortalized endometriotic epithelial cell line (11Z) pre-incubated either with TACE inhibitors or following TACE siRNA knockdown. Here, we demonstrate that TACE protein is overexpressed in epithelium of tissue samples of both eutopic endometrium and ectopic lesions of women with endometriosis compared to disease-free controls (P < 0.001 both) and that the overexpression of the protein in the lesions is due to activation of TACE gene transcription (P < 0.001). Moreover, epithelial TACE protein was significantly higher in ectopic samples than in corresponding eutopic tissue of women with the disease (P < 0.001). High endometrial tissue TACE protein expression correlated with higher serum sVCAM-1 levels (P < 0.05) but not with sICAM-1 levels. Inhibition of TACE either by TACE inhibitors or by TACE siRNA knockdown resulted in decreased PMA-induced shedding of sVCAM-1 in vitro (P < 0.005 or P < 0.01, respectively), but the TACE inhibitors did not affect transcription of TACE or VCAM-1. Additionally, we observed an upregulation of TACE in proliferative endometrial epithelium of infertile (P < 0.005), compared to fertile women. TACE was increased in infertile women with endometriosis (P = 0.051) but not in infertile women without endometriosis. Albeit well characterized, our control population included women with other gynecologic diseases, which may have impacted the levels of sVCAM-1 and tissue TACE expression levels, e.g. benign ovarian cysts or uterine fibroids. Thus, the results of our analysis have to be interpreted carefully and in the context of the current experimental settings. The dysregulation of TACE substrate shedding represents a promising yet relatively unexplored area of endometriosis progression and could serve as a basis for the development of new treatments of the disease. This work was supported by the Ingrid Flick Foundation. The authors have no competing interests to declare.

A Computational Model for Predicting ADAM17 Substrate Specificity

Tumor necrosis factor-alpha converting enzyme (TACE) is a membrane-anchored protein that releases the soluble forms of many proteins by a process called ectodomain shedding. TACE has been considered as a potential target in a lot of diseases in autoimmune diseases and in cancers recently. In spite a lot of protein substrates have been found these years for TACE, the substrate selection of TACE is still not known. In this paper, a TACE-peptide complex was constructed, and used for the prediction of substrate sequences and cleavage sites. The result could be useful for understanding the substrate specificity of TACE, and designing better TACE inhibitors in future.

Investigation of the Role of TNF-α Converting Enzyme (TACE) in the Inhibition of Cell Surface and Soluble TNF-α Production by Acute Ethanol Exposure

Toll-like receptors (TLRs) play a fundamental role in the immune system by detecting pathogen associated molecular patterns (PAMPs) to sense host infection. Ethanol at doses relevant for humans inhibits the pathogen induced cytokine response mediated through TLRs. The current study was designed to investigate the mechanisms of this effect by determining whether ethanol inhibits TLR3 and TLR4 mediated TNF-α secretion through inhibition of transcription factor activation or post-transcriptional effects. In NF-κB reporter mice, activation of NF-κB in vivo by LPS was inhibited by ethanol (LPS alone yielded 170,000±35,300 arbitrary units of light emission; LPS plus ethanol yielded 56,120±16880, p = 0.04). Inhibition of protein synthesis by cycloheximide revealed that poly I:C- or LPS-induced secreted TNF-α is synthesized de novo, not released from cellular stores. Using real time RT-PCR, we found inhibition of LPS and poly I:C induced TNF-α gene transcription by ethanol. Using an inhibitor of tumor necrosis factor alpha converting enzyme (TACE), we found that shedding caused by TACE is a prerequisite for TNF-α release after pathogen challenge. Flow cytometry was used to investigate if ethanol decreases TNF-α secretion by inhibition of TACE. In cells treated with LPS, ethanol decreased both TNF-α cell surface expression and secretion. For example, 4.69±0.60% of untreated cells were positive for cell surface TNF-α, LPS increased this to 25.18±0.85%, which was inhibited by ethanol (86.8 mM) to 14.29±0.39% and increased by a TACE inhibitor to 57.88±0.62%. In contrast, cells treated with poly I:C had decreased secretion of TNF-α but not cell surface expression. There was some evidence for inhibition of TACE by ethanol in the case of LPS, but decreased TNF-α gene expression seems to be the major mechanism of ethanol action in this system.

Development of predictive pharmacophore model for in silico screening, and 3D QSAR CoMFA and CoMSIA studies for lead optimization, for designing of potent tumor necrosis factor alpha converting enzyme inhibitors

A chemical feature-based pharmacophore model was developed for Tumor Necrosis Factor-alpha converting enzyme (TACE) inhibitors. A five point pharmacophore model having two hydrogen bond acceptors (A), one hydrogen bond donor (D) and two aromatic rings (R) with discrete geometries as pharmacophoric features was developed. The pharmacophore model so generated was then utilized for in silico screening of a database. The pharmacophore model so developed was validated by using four compounds having proven TACE inhibitory activity which were grafted into the database. These compounds mapped well onto the five listed pharmacophoric features. This validated pharmacophore model was also used for alignment of molecules in CoMFA and CoMSIA analysis. The contour maps of the CoMFA/CoMSIA models were utilized to provide structural insight for activity improvement of potential novel TACE inhibitors. The pharmacophore model so developed could be used for in silico screening of any commercial/in house database for identification of TACE inhibiting lead compounds, and the leads so identified could be optimized using the developed CoMSIA model. The present work highlights the tremendous potential of the two mutually complementary ligand-based drug designing techniques (i.e. pharmacophore mapping and 3D-QSAR analysis) using TACE inhibitors as prototype biologically active molecules.

TACE antagonists blocking ACE2 shedding caused by the spike protein of SARS-CoV are candidate antiviral compounds

Because outbreaks of severe acute respiratory syndrome coronavirus (SARS-CoV) might reemerge, identifying antiviral compounds is of key importance. Previously, we showed that the cellular factor TNF-α converting enzyme (TACE), activated by the spike protein of SARS-CoV (SARS-S protein), was positively involved in viral entry, implying that TACE is a possible target for developing antiviral compounds. To demonstrate this possibility, we here tested the effects of TACE inhibitors on viral entry. In vitro and in vivo data revealed that the TACE inhibitor TAPI-2 attenuated entry of both pseudotyped virus expressing the SARS-S protein in a lentiviral vector backbone and infectious SARS-CoV. TAPI-2 blocked both the SARS-S protein-induced shedding of angiotensin-converting enzyme 2 (ACE2), a receptor of SARS-CoV, and TNF-α production in lung tissues. Since the downregulation of ACE2 by SARS-S protein was proposed as an etiological event in the severe clinical manifestations, our data suggest that TACE antagonists block SARS-CoV infection and also attenuate its severe clinical outcome.

Prediction of novel and selective TNF-alpha converting enzyme (TACE) inhibitors and characterization of correlative molecular descriptors by machine learning approaches

The inhibition of TNF-α converting enzyme (TACE) has been explored as a feasible therapy for the treatment of rheumatoid arthritis (RA) and Crohn's disease (CD). Recently, large numbers of novel and selective TACE inhibitors have been reported. It is desirable to develop machine learning (ML) models for identifying the inhibitors of TACE in the early drug design phase and test the prediction capabilities of these ML models. This work evaluated four ML methods, support vector machine (SVM), k-nearest neighbor ( k-NN), back-propagation neural network (BPNN) and C4.5 decision tree (C4.5 DT), which were trained and tested by using a diverse set of 443 TACE inhibitors and 759 non-inhibitors. A well-established feature selection method, the recursive feature elimination (RFE) method, was used to select the most appropriate descriptors for classification from a large pool of descriptors, and two evaluation methods, 5-fold cross-validation and independent evaluation, were used to assess the performances of these developed models. In this study, all these ML models have already achieved promising prediction accuracies. By using the RFE method, the prediction accuracies are further improved. In k-NN, the model gives the best prediction for TACE inhibitors (98.32%), and the SVM bears the best prediction for non-inhibitors (99.51%). Both the k-NN and SVM model give the best overall prediction accuracy (98.45%). To the best of our knowledge, the SVM model developed in this work is the first one for the classification prediction of TACE inhibitors with a broad applicability domain. Our study suggests that ML methods, particularly SVM, are potentially useful for facilitating the discovery of TACE inhibitors and for exhibiting the molecular descriptors associated with TACE inhibitors.

Interleukin‐10 regulates TNF‐α−converting enzyme (TACE/ADAM‐17) involving a TIMP‐3 dependent and independent mechanism

IL-10 is a potent anti-inflammatory molecule, which regulates TNF-alpha at multiple levels. We investigated whether IL-10 also modulated the activity of the TNF-alpha-converting enzyme (TACE). Using an ex vivo fluorogenic assay we observed that LPS rapidly induced TACE activity in monocytes coinciding with release of soluble TNF-alpha. In the presence of IL-10, TNF-alpha production and activation of surface TACE was significantly inhibited. Paradoxically, both LPS with or without IL-10 led to accumulation of surface TACE (albeit catalytically inactive) over a 24 h period. We investigated whether this was mediated through induction of endogenous tissue inhibitor metalloproteinase-3 (TIMP-3). We found that the inhibition of TACE activity at 2 h by IL-10 was not TIMP-3 dependent but that the late accumulation of surface TACE was prevented with TIMP-3 antibodies. Furthermore, induction of endogenous TIMP-3 was observed by western blotting in both LPS- and in LPS with IL-10-treated monocytes from 6 to 8 h of culture. These results indicate that IL-10 further regulates TNF-alpha by modulating TACE activation at early time points and by contributing to the induction of TIMP-3, the natural inhibitor of active TACE, at later time points. These observations add to our understanding of inflammation and the importance of homeostatic regulators of these events.

Tumor Necrosis Factor—α Converting Enzyme in the Human Placenta Throughout Gestation

Ectodomain shedding of epidermal growth factor receptor ligands such as transforming growth factor- alpha (TGF-alpha), heparin-binding epidermal growth factor-like growth factor (HBEGF), and amphiregulin (AREG) is considered to be important during implantation. Tumor necrosis factor-alpha converting enzyme (TACE) has been suggested as the major sheddase for these molecules. The objectives of this study are (1) to characterize the expression of TACE in the human placenta throughout gestation; (2) to determine the association between the expression of TACE with TGF-alpha, HBEGF, and AREG; (3) to ascertain whether TACE mediates TGF-alpha, HBEGF, and AREG shedding; and (4) to examine the effect of hypoxia on the expression of TACE. By analyzing a total of 55 villous samples representing different gestational ages, the authors found that TACE was continuously expressed in the placentas throughout gestation and that the levels of TACE were positively correlated with the levels of TGF-alpha, HBEGF, and AREG. Preadministration of a TACE inhibitor in villous explant cultures or transfection of cytotrophoblastic cells with TACE-specific small interference RNA decreased the shedding of HBEGF and AREG. Moreover, hypoxia (2% O(2)) caused an increase in the levels of TACE mRNA and protein in villous explants and primary cytotrophoblastic cells in vitro. These results indicate that oxygen regulates the expression of TACE and that TACE may be important for placental development during human pregnancy.

Inhibition of TACE Activity Enhances the Susceptibility of Myeloma Cells to TRAIL.

TNF-related apoptosis-inducing ligand/Apo2 ligand (TRAIL/Apo2L) selectively induces apoptosis in various cancer cells including myeloma (MM) cells. Although TRAIL-mediated immunotherapy is as attractive maneuver against MM because of its tumor-specific cytotoxic activity, the susceptibility of MM cells to TRAIL has been demonstrated to vary among MM cells, which limits clinical applications of TRAIL-mediated immunotherapy. TRAIL is known to bind to 2 different proapoptotic receptors, death receptor 4 (DR4) and DR5. The membrane-bound TRAIL receptors have been reported to be subjected to proteolytic cleavage in melanoma cells in a manner inhibitable by tissue inhibitor of metalloproteinases-3 (TIMP-3), an endogenous inhibitor for TNF-alpha converting enzyme (TACE) (Ahonen M, Oncogene. 2003). Although TACE is regarded as a sheddase for TNF-like ligands/receptors, the role for TACE in TRAIL-mediated cytotoxicity in MM cells is largely unknown. In the present study, we therefore investigated the role for TACE in TRAIL and its receptor editing as well as the effect of TACE inhibition on TRAIL-triggered cytotoxicity in MM cells. MM cells constitutively expressed DR4 on their surface as determined in flow cytometry, while the expression of DR5 as well as TRAIL was negligible. TACE mRNA was constitutively overexpressed in all MM cell lines including RPMI8226, MM.1S, KMS12 and U266, whereas its endogenous inhibitor, TIMP-3, was not detected, suggesting enhanced TACE activity along with down-regulation of TIMP-3 in MM cells. TAPI-0, a TACE inhibitor, up-regulated the cell-surface expression of DR4 about 2-fold in RPMI8226 and U266 cells, while DR5 and TRAIL expression remained undetectable. Immunoblot analyses for DR4 revealed that conditioned media from RPMI8226 and U266 cells contained DR4 protein with a molecular size smaller than that observed in their cell lysates. Interestingly, TAPI-0 treatment down-regulated DR4 levels in culture supernatants of these MM cells, while increasing DR4 levels in their cell lysates as well as on their surface, suggesting ectodomain shedding of DR4 by TACE in MM cells. Importantly, TAPI-0 treatment potentiated TRAIL-triggered cytotoxicity against MM cells including RPMI8226 and MM.1S, which was almost completely antagonized by addition of osteoprotegerin, a soluble inhibitor for TRAIL, indicating a TRAIL-dependent mechanism. Taken together, the present observations demonstrate that cell-surface TRAIL receptor expression is post-transcriptionally down-regulated by endogenous TACE activity enhanced in MM cells, which may protect MM cells from TRAIL-mediated apoptosis by surrounding immune cells expressing TRAIL, and that up-regulation of TRAIL receptor expression by inhibiting TACE activity is able to restore the susceptibility of MM cells to TRAIL. Therefore, these preclinical data provide that TACE inhibitors in combination with TRAIL-based immunotherapy may be a novel therapeutic approach against MM which can not be cured by the present chemotherapeutic modalities.

Pharmacokinetics and Pharmacodynamics of DPC 333 ((2R)-2-((3R)-3-Amino-3{4-[2-methyl-4-quinolinyl) methoxy] phenyl}-2-oxopyrrolidinyl)-N-hydroxy-4-methylpentanamide)), a Potent and Selective Inhibitor of Tumor Necrosis Factor α-Converting Enzyme in Rodents, Dogs, Chimpanzees, and Humans

DPC 333 ((2R)-2-((3R)-3-amino-3{4-[2-methyl-4-quinolinyl) methoxy] phenyl}-2-oxopyrrolidinyl)-N-hydroxy-4-methylpentanamide)) is a potent and selective inhibitor of tumor necrosis factor (TNF)-alpha-converting enzyme (TACE). It significantly inhibits lipopolysaccharide-induced soluble TNF-alpha production in blood from rodents, chimpanzee, and human, with IC(50) values ranging from 17 to 100 nM. In rodent models of endotoxemia, DPC 333 inhibited the production of TNF-alpha in a dose-dependent manner, with an oral ED(50) ranging from 1.1 to 6.1 mg/kg. Oral dosing of DPC 333 at 5.5 mg/kg daily for 2 weeks in a rat collagen antibody-induced arthritis model suppressed the maximal response by approximately 50%. DPC 333 was distributed widely to tissues including the synovium, the site of action for antiarthritic drugs. Pharmacokinetic and pharmacodynamic studies in chimpanzee revealed a systemic clearance of 0.4 l/h/kg, a V(ss) of 0.6 l/kg, an oral bioavailability of 17%, and an ex vivo IC(50) for the suppression of TNF-alpha production of 55 nM (n = 1). In a phase I clinical trial with male volunteers after single escalating doses of oral DPC 333, the terminal half-life was between 3 and 6 h and the ex vivo IC(50) for suppressing TNF-alpha production was 113 nM. Measurement of the suppression of TNF-alpha production ex vivo may serve as a good biomarker in evaluating the therapeutic efficacy of TACE inhibitors. Overall, the pharmacological profiles of DPC 333 support the notion that suppression of TNF-alpha with TACE inhibitors like DPC 333 may provide a novel approach in the treatment of various inflammatory diseases including rheumatoid arthritis, via control of excessive TNF-alpha production.

Effects of PKF242-484 and PKF241-466, novel dual inhibitors of TNF-alpha converting enzyme and matrix metalloproteinases, in a model of intestinal reperfusion injury in mice

Tumor necrosis factor (TNF)-alpha plays an important role in the mediation of reperfusion-induced tissue injury and lethality. Here, we assessed the effects of PKF242-484 and PKF241-466, two dual inhibitors of TNF-alpha converting enzyme (TACE) and matrix metalloproteinases (MMPs), in a model of ischemia and reperfusion injury in mice. Reperfused animals that received PKF242-484 or PKF241-466 treatment had a dose-dependent reduction of TNF-alpha concentrations in serum. Both drugs delayed and partially inhibited the reperfusion-associated lethality. Maximal inhibition occurred at 10 mg/kg. At this dose, both inhibitors reduced reperfusion-associated local and remote tissue injury, as assessed by changes in vascular permeability, neutrophil recruitment and hemorrhage. In addition, the compounds markedly reduced production of TNF-alpha, CXCL1 (keratinocyte-derived chemokine, KC) and CCL2 (monocyte chemoattractant protein-1, MCP-1) in intestine and lungs of animals which underwent reperfusion. FN-439, an inhibitor of MMPs which possesses no effect on TACE, decreased MMP-2 and MMP-3 activity, but failed to affect tissue injury, TNF-alpha production or lethality. Thus, combined TACE and MMP inhibitors might be effective co-adjuvants in treatments of injuries that follow reperfusion of an ischemic vascular territory. The effects of these drugs on TNF-alpha production appear to be more relevant than their effects on MMP inhibition.

Adjuvant TACE inhibitor treatment improves the outcome of TLR2-/-mice with experimental pneumococcal meningitis

BackgroundStreptococcus (S.) pneumoniae meningitis has a high lethality despite antibiotic treatment. Inflammation is a major pathogenetic factor, which is unresponsive to antibiotics. Therefore adjunctive therapies with antiinflammatory compounds have been developed. TNF484 is a TNF-alpha converting enzyme (TACE) inhibitor and has been found efficacious in experimental meningitis. Toll-like receptor 2 (TLR2) contributes to host response in pneumococcal meningitis by enhancing bacterial clearing and downmodulating inflammation. In this study, TNF484 was applied in mice, which lacked TLR2 and exhibited a strong meningeal inflammation.Methods103 CFU S. pneumoniae serotype 3 was inoculated subarachnoidally into C57BL/6 wild type (wt) mice or TLR2-/-, CD14-/- and CD14-/-/TLR2-/- mice. Severity of disease and survival was followed over 9 days. Response to antibiotics (80 mg/kg ceftriaxone i.p. for 5 days) and/or TACE inhibitor treatment (1 mg/kg s.c. twice daily for 4 days) was evaluated. Animals were sacrificed after 12, 24, and 48 h for analysis of bacterial load in cerebrospinal fluid (CSF) and brain and for TNF and leukocyte measurements in CSF.ResultsTLR2-/- mice were significantly sicker than the other mouse strains 24 h after infection. All knockout mice showed higher disease severity after 48 h and died earlier than wt mice. TNF release into CSF was significantly more elevated in TLR2-/- than in the other strains after 24 h. Brain bacterial numbers were significantly higher in all knockout than wt mice after 24 h. Modulation of outcome by antibiotic and TACE inhibitor treatment was evaluated. With antibiotic therapy all wt, CD14-/- and TLR2-/-/CD14-/- mice, but only 79% of TLR2-/- mice, were rescued. TACE inhibitor treatment alone did not rescue, but prolonged survival in wt mice, and in TLR2-/- and CD14-/- mice to the values observed in untreated wt mice. By combined antibiotic and TACE inhibitor treatment 95% of TLR2-/- mice were rescued.ConclusionDuring pneumococcal meningitis strong inflammation in TLR2-deficiency was associated with incomplete responsiveness to antibiotics and complete response to combined antibiotic and TACE inhibitor treatment. TACE inhibitor treatment offers a promising adjuvant therapeutic strategy in pneumococcal meningitis.

NONISCHEMIC LUNG INJURY BY MEDIATORS FROM UNILATERAL ISCHEMIC REPERFUSED LUNG

We hypothesized that the ischemic reperfused (I/R) lung expresses and liberates tumor necrosis factor-alpha (TNF-alpha) to injure the nonischemic lung, and that a TNF-alpha-converting enzyme inhibitor (TACEI) prevents injury of the nonischemic lung by blocking TNF-alpha liberation from the I/R lung. In isolated ventilated rat lungs in which differential perfusion to the right (RL) or left (LL) lung was feasible, LLs were selectively made ischemic (60 min) while maintaining perfusion to RLs, then reperfused (30 min) in a nonrecirculating manner with buffer solution (non-R; n = 18) or in a recirculating manner with buffer containing TACEI (TACEI[+]; n = 18) or without TACEI (TACEI[-]; n = 18). Ischemia reperfusion induced TNF-alpha messenger RNA expression in the ischemic LLs; the expression was highest in TACEI(+) group (P < 0.01). The expression of TNF-alpha, which was detected as immunofluorescence signals on CD34-positive endothelial cells, was observed in ischemic LLs; the highest expression being that in the TACEI(+) group. Wet/dry ratio and protein content in bronchoalveolar lavage fluid were higher in LLs than in RLs, and among the RLs, these 2 parameters were significantly increased in the TACEI(-) group (P < 0.01) in which the RLs were exposed to the TNF-alpha-rich perfusate. On the other hand, protein content in bronchoalveolar lavage fluid of the TACEI(+) group in which RLs were exposed to recirculating perfusate containing little TNF-alpha was decreased to a level close to but still higher than that in the non-R group (P < 0.05). The unilateral I/R lung affected the permeability of the nonischemic lung by liberating mainly TNF-alpha and induced TNF-alpha, interleukin (IL)-1beta, IL-6, and IL-10 messenger RNA expression in the nonischemic lung. These findings support the idea of organ-organ interaction in which an injured organ affects a remote organ by liberating humoral mediators.

Small Molecular anti‐Cytokine Agents

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The TACE Zymogen: Re-Examining the Role of the Cysteine Switch

The tumor necrosis factor-alpha-converting enzyme (TACE) is a member of the disintegrin family of metalloproteinases (ADAMs) that plays a central role in the regulated shedding of a host of cell surface proteins. TACE is biosynthesized as a precursor protein with latent proteolytic activity (zymogen). TACE's zymogen inhibition is mediated by its Pro domain, a 197-amino acid region that serves this function as well as aiding in the secretion of this enzyme through the secretory pathway. We have discovered that a conserved "cysteine switch" consensus motif within TACE's Pro domain is, contrary to expectations, not required for maintenance of the inactive precursor state or for the secretion of this metalloproteinase in its functional form. The only role for this motif seems to be in decreasing TACE's susceptibility to proteolytic degradation during its biogenesis and maturation within the secretory pathway. Interestingly, the Pro domain of TACE seems to carry both its inhibitory and secretory functions through the same mechanism: it seems to prevent the Catalytic domain from accessing its native, functional state, resembling the function of true molecular chaperones. Recent evidence suggests that TACE may also be switched out of the active conformation even by small, drug-like molecules such as the synthetic compound SB-3CT. These findings point at the possibility of developing, in the near future, a new generation of antiinflammatory, noncompetitive TACE inhibitors that would exert negative allosteric modulation over the activity of this key enzyme, mediating several inflammatory diseases and certain cancers.

Substrate Specificity and Novel Selective Inhibitors of TNF-&amp;#945; Converting Enzyme (TACE) from Two-Dimensional Substrate Mapping

We report a systematic analysis of the P1' and P2' substrate specificity of TNF-alpha converting enzyme (TACE) using a peptide library and a novel analytical method, and we use the substrate specificity information to design novel reverse hydroxamate inhibitors. Initial truncation studies, using the amino acid sequence around the cleavage site in precursor-TNF-alpha, showed that good turnover was obtained with the peptide DNP-LAQAVRSS-NH2. Based on this result, 1000 different peptide substrates of the form Biotin-LAQA-P1'-P2'-SSK(DNP)-NH2 were prepared, with 50 different natural and unnatural amino acids at P1' in combination with 20 different amino acids at P2'. The peptides were pooled, treated with purified microsomal TACE, and the reaction mixtures were passed over a streptavidin affinity column to remove unreacted substrate and the N-terminal biotinylated product. C-terminal cleavage products not binding to streptavidin were subjected to liquid chromatography/mass spectrometry analysis where individual products were identified and semiquantitated. 25 of the substrates were resynthesized as discrete peptides and assayed with recombinant TACE. The experiments show that recombinant TACE prefers lipophilic amino acids at the P1' position, such as phenylglycine, homophenylalanine, leucine and valine. At the P2' position, TACE can accommodate basic amino acids, such as arginine and lysine, as well as certain non-basic amino acids such as citrulline, methionine sulfoxide and threonine. These substrate preferences were used in the design of novel reverse hydroxamate TACE inhibitors with phenethyl and 5-methyl-thiophene-methyl side-chains at P1', and threonine and nitro-arginine at P2'.

Therapeutic Potential of TACE Inhibitors in Stroke

Stroke is the third leading cause of death and the leading cause of permanent disability in western countries and the incidence of stroke is expected to increase in the foreseeable future due to the ageing population. The effective treatment of stroke remains challenging due to the complexity and heterogenicity of the disease. Recombinant tissue plasminogen activator (rt-PA) is the only FDA-approved therapy for stroke during the first 3 hr after the disease onset. However the risk of hemorrhage and its narrow therapeutic window has limited its use in clinic. Inflammation has been known to play a crucial role in the induction and development of stroke and tumor necrosis factor-alpha (TNF-alpha) is a central player in the initiation of multiple inflammatory cascades. The recent success of three anti-TNF biologics in the clinic for the treatment of rheumatoid arthritis as well as other inflammatory diseases has further validated TNF159nflammation. TNF-alpha has also been shown to be associated with ischemic stroke. Anti-TNF biologics have been shown to be effective in reducing the disease symptoms in various pre-clinical stroke models. Small molecule TNF inhibitors are highly desirable due to the limitations of protein therapeutics. Tumor necrosis factor-alpha-converting enzyme (TACE) is the major sheddase of TNF-alpha and is essential for the generation of soluble, mature TNF-alpha. Thus TACE appears to be an attractive target for development of oral small molecule TNF-alpha inhibitors. This review summarizes the role of TNF-alpha in stroke and the effect of several TACE/MMP inhibitors in pre-clinical stroke models. The data strongly suggest that TACE/MMP inhibitors have great therapeutic potential and may be valuable alternatives in treating stroke in the clinic.