TNF-α Converting Enzyme Inhibitors Research Articles

A functional role for eicosanoid-lysophospholipids in activating monocyte signaling

Recently, eicosanoid-lysophospholipids were identified as novel metabolites generated from the direct cyclooxygenase- or lipoxygenase-catalyzed oxidation of 2-arachidonoyl-lysophospholipids produced from either phospholipase A1-mediated hydrolysis of diacyl arachidonoyl-phospholipids or through the cytochrome c-catalyzed oxidative hydrolysis of the vinyl ether linkage of arachidonoyl-plasmalogens. Although the metabolic pathways generating eicosanoid-lysophospholipids have been increasingly appreciated, the signaling functions of eicosanoid-lysophospholipids remain largely unknown. Herein, we demonstrate that 2-12(S)-HETE-lysophospholipids as well as nonesterified 12(S)-HETE are potent lipid mediators that activate THP-1 human monocytic cells to generate tumor necrosis factor α (TNFα) and interleukin 8 (IL8). Remarkably, low nanomolar concentrations of 12(S)-HETE-lysophospholipids, but not other oxidized signaling lipids examined activated THP-1 cells resulting in the production of large amounts of TNFα. Moreover, TNFα release induced by 12(S)-HETE-lysophospholipids was inhibited by the TNFα converting enzyme inhibitor TAPI-0 indicating normal processing of TNFα in THP-1 cells stimulated with these agonists. Western blotting analyses revealed that 12(S)-HETE-lysophospholipids activated the phosphorylation of NFκB p65, suggesting activation of the canonical NFκB signaling pathway. Importantly, activation of THP-1 cells to release TNFα was stereoselective with 12(S)-HETE favored over 12(R)-HETE. Furthermore, the EC50 of 2-12(S)-HETE-lysophosphatidylcholine in activating THP-1 cells was 2.1 nm, whereas the EC50 of free 12(S)-HETE was 23 nm Additionally, lipid extracts of activated platelets were separated by RP-HPLC demonstrating the coelution of 12(S)-HETE with fractions initiating TNFα release. Collectively, these results demonstrate the potent signaling properties of 2-12(S)-HETE-lysophospholipids and 12(S)-HETE by their ability to release TNFα and activate NFκB signaling thereby revealing a previously unknown role of 2-12(S)-HETE-lysophospholipids in mediating inflammatory responses.

The TNFR1/mTNFα signaling axis initiates tumor-promoting inflammation and is associated with poor outcome in neuroblastoma

Abstract Neuroblastoma (NB) is a poorly differentiated, aggressive pediatric solid tumor for which half of high-risk cases have no identified genetic alteration. While such high-risk tumors are often infiltrated by macrophages that generate an inflammatory gene signature predictive of poor outcome, the initiation mechanism of this pro-tumorigenic inflammation remains unknown. In our analysis of clinical outcome-linked NB gene expression data, high tumor expression of TNFR1 was strongly correlated with poor event-free survival (5-year EFS 15% vs 70%, bonf p=9.3e-07), highlighting the potential importance of TNFα signaling in NB. Using TNFα knockout human NB cell lines, we determined that monocytes, but not NB cells, produce TNFα during in vitro co-culture. Furthermore, using a TNFα converting enzyme inhibitor to prevent secretion of soluble TNFα, we found that the membrane-bound form of TNFα (mTNFα) expressed on monocytes was critical for NF-κB pathway activation and downstream production of IL-6, a known NB growth factor. Neutralization of TNFα or its receptor TNFR1, but not TNFR2, in a monocyte-NB co-culture prevented NF-κB activation in monocytes, reducing IL-6 production by 50% compared to controls (p <0.01). Our results demonstrate that TNFR1-expressing NB cells activate monocytes via contact-dependent mTNFα signaling, leading to NF-κB activation and production of IL-6, a known driver of NB progression and therapy resistance. These findings reveal a novel, druggable mechanism of tumor-promoting inflammation in NB.

TNF-α converting enzyme (TACE) inhibitor prevents insulin resistance and diabetic peripheral neuropathy

TNF-α converting enzyme (TACE) inhibitor prevents insulin resistance and diabetic peripheral neuropathy

Effects of Soluble Tumor Necrosis Factor (TNF) on Antibody-Dependent Cellular Cytotoxicity of Therapeutic anti-TNF-α Antibody

ABSTRACTAnti-TNF antibodies are major therapeutics for rheumatoid arthritis and have been approved for marketing in many countries. Antibody-dependent cellular cytotoxicity (ADCC) is considered to be a potential mechanism of action of anti-TNF antibodies, since some anti-TNF antibodies have been confirmed to induce cytotoxic effects on TNF-producing cells via ADCC and complement-dependent cytotoxicity (CDC) in in vitro experiments. In this study, we established a new stable effector cell line expressing human FcγRIIIa, CD16:KHYG-1, and compared the performance of this cell line with that of peripheral blood mononuclear cells (PBMCs) in ADCC assays against CHO-derived target cells expressing protease-sensitive pro-TNF. Although an inhibitory effect of soluble TNF released from pro-TNF expressing cells on ADCC activity was seen, clear dose-responsive ADCC activities were observed even in the presence or absence of TNF-α converting enzyme (TACE) inhibitor. However, significant differences in the ADCC activities in the presence or absence of TACE inhibitor were only noted when CD16:KHYG-1 cells were used as the effector cells. Our findings indicate that soluble TNF may influence ADCC activity of anti-TNF antibody. Moreover, the fact that the influence was able to be detected only in the case using stable effector cell also suggests that the stable effector cell established this time enable highly accurate ADCC measurement.

Development of a prodrug of hydantoin based TACE inhibitor

Our research on hydantoin based TNF-α converting enzyme (TACE) inhibitors led to fused bi-heteroaryl hydantoin series that demonstrate sub-nanomolar potency (Ki) as well as excellent activity in human whole blood (hWBA). However, lead compound 2 posed some formulation challenges which prevented it for further development. A prodrug approach was investigated to address this issue. The pivalate prodrug 3 can be formulated as stable neutral form and demonstrated improved DMPK properties when compared with parent compound.

The activation of EGFR promotes myocardial tumor necrosis factor-α production and cardiac failure in endotoxemia.

To study the effect of EGFR activation on the generation of TNF-α and the occurrence of cardiac dysfuncetion during sepsis, PD168393 and erlotinib (both are EGFR inhibitors) were applied to decreased the production of TNF-α and phosphrylation of ERK1/2 and p38 induced by LPS in cardiomyocytes. These results were further proved by specifically knocked down the expression of EGFR in vitro. Both TAPI-1, a TNF-α converting enzyme (TACE) inhibitor, and TGF-α neutralizing antibody could inhibit the activation of EGFR and the generation of TNF-α mRNA after LPS treatment. The increase of TGF-α in response to LPS could also be suppressed by TAPI-1. On the other hand, exogenous TGF-α increased the expression of TNF-α mRNA and partially reversed the inhibitory effect of TAPI-1 on expression of TNF-α mRNA in response to LPS indicating that the transactivation of EGFR by LPS in cardiomyocytes needs the help of TACE and TGF-α. In endotoxemic mice, inhibition the activation of EGFR not only decreased TNF-α production in the myocardium but also improved left ventricular pump function and ameliorated cardiac dysfunction and ultimately improved survival rate. All these results provided a new insight of how EGFR regulation the production of TNF-α in cardiomyocytes and a potential new target for the treatment of cardiac dysfunction in sepsis.

GM6001 Increases Anastomotic Leakage following Colonic Obstruction Possibly by Impeding Epithelialization.

Emergency operations performed on an obstructed colon are accompanied by an increased risk of anastomotic insufficiency. Tissue-destructive matrix metalloproteinase (MMP) activity is elevated in the obstructed colon and contributes to a loss of suture-holding submucosal collagen, which may be mediated by tumor necrosis factor (TNF)-α. Our aim was to study the effect of the non-selective MMP and TNF-α converting enzyme (TACE) inhibitor GM6001 (30 mg/kg) on anastomosis repair in obstructed left colon. GM6001 has been proved to be highly efficacious in elective anastomosis rodent models. A partial obstruction of the distal colon was induced in male Sprague-Dawley rats. After 4 d the obstructed colonic segment was resected, and an end-to-end anastomosis was constructed. Seven days later, the anastomoses were evaluated for clinical leakage. Histopathological and immunohistochemical assessments were also performed. Finally, the direct effect of GM6001 on epithelialization was studied in cultured colonic epithelial cells. Unlike the robust beneficial effect on anastomosis under uncomplicated conditions, here GM6001 had a negative impact on anastomotic wound healing following colonic obstruction and substantially (p=0.004) more rats in the GM6001 group (75%) than in the control group (11%) had developed anastomotic leakage. In the anastomotic wounds, the myofibroblast abundance and cell proliferation were similar in the two groups. Histologically, GM6001 treatment resulted in wider and minimally epithelialized wounds that were commonly necrotic on the luminal side and infiltrated with numerous granulocytes. In vitro, GM6001 also delayed (p=0.026) epithelialization of denuded intestinal epithelium grown on type I collagen. Non-selective MMP/TACE inhibition with GM6001 increased the anastomotic complications following colon obstruction. Inhibition of epithelialization is one possible mechanism responsible for the increased leakage following GM6001 treatment.

A new technique of bronchial microsampling and proteomic analysis of epithelial lining fluid in a rat model of acute lung injury

The standard technique to evaluate the proteins present in epithelial lining fluid (ELF) is bronchoalveolar lavage (BAL). Bronchoscopic microsampling (BMS) method has been developed for humans as a less invasive alternative. We establish the usefulness of a rat bronchial microsampling (rBMS) to evaluate various proteins in ELF in lipopolysaccharide (LPS)-induced lung injury models in rats. In the first experiment of this study, we validate that whether the rBMS can obtain information from ELF in place of BAL. Tumor necrosis factor (TNF)-α concentrations were increased in the rBMS samples similar to BAL 1 and 3h after LPS instillation. In the second part of this study, a proteomic analysis of the rBMS, using the Protein ChipR system, revealed the presence of proteins whose molecular weights corresponded to TNF-related proteins in the LPS-treated rats. In rats treated with a TNF-α converting enzyme inhibitor, the concentrations of these proteins in rBMS decreased or disappeared. In the third experiment, rBMS was performed without tracheostomy at 6 and 24h after instillation of LPS, and a rat multiple cytokines assay system detected heterogeneous variations in the concentrations of interleukin (IL)-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, TNF-αand interferon (IFN)-γ in ELF. The cytokine profile was significantly modified by pre-treatment with dexamethasone. This new rBMS technique could be used to measure TNF-α in LPS-induced acute lung injury (ALI) as well as for proteomic analysis, without sacrificing the rats. Furthermore, this procedure enables the serial collection of ELF, which would allow the examination of time-dependent cytokine variations in rat ALI model.

2-(2-Aminothiazol-4-yl)pyrrolidine-based tartrate diamides as potent, selective and orally bioavailable TACE inhibitors

TNF-α converting enzyme (TACE) inhibitors are promising agents to treat inflammatory disorders and cancer. We have investigated novel tartrate diamide TACE inhibitors where the tartrate core binds to zinc in a unique tridentate fashion. Incorporating (R)-2-(2-N-alkylaminothiazol-4-yl)pyrrolidines into the left hand side amide of the tartrate scaffold led to the discovery of potent and selective TACE inhibitors, some of which exhibited good rat oral bioavailability.

Novel TNF-α converting enzyme (TACE) inhibitors as potential treatment for inflammatory diseases

Our research on hydantoin based TNF-α converting enzyme (TACE) inhibitors has led to an acetylene containing series that demonstrates sub-nanomolar potency (K(i)) as well as excellent activity in human whole blood. These studies led to the discovery of highly potent TACE inhibitors with good DMPK profiles.

Structure based optimization of chromen-based TNF-α converting enzyme (TACE) inhibitors on S1′ pocket and their quantitative structure–activity relationship (QSAR) study

A series of coumarin based TACE inhibitors were designed to bind in S1′ pocket of TACE enzyme based on their docking study. Twelve analogues were synthesized and most of compounds were active in vitro TACE enzyme inhibition as well as cellular TNF-α inhibition. Among these, 15l effectively inhibited the production of serum TNF-α by oral administration at a dose of 30 mg/kg. Compound 15l also showed a good oral bioavailability at 42% and effectively inhibited paw edema in rat carrageenan model. Quantitative structure–activity relationship (QSAR) study using genetic function approximation technique (GFA) and docking study were performed to confirm the series of coumarin core TACE inhibitors. QSAR model have been evaluated internally and externally using test set prediction. Through docking study of each molecule, it is validated that the electrostatic descriptors from the QSAR equation could explain the importance of S1′ pocket and the TACE inhibitory activity well.

Lymphotoxin-αβ heterotrimers are cleaved by metalloproteinases and contribute to synovitis in rheumatoid arthritis

Tumor necrosis factor-superfamily (TNF-SF) members, lymphotoxin (LT)-α and LTβ, are proinflammatory cytokines associated with pathology in rheumatoid arthritis. LTα3 homotrimers are secreted, whereas LTα 1β 2 heterotrimers are expressed on the surface of activated lymphocytes. As many TNF-SF members are actively cleaved from cell membranes, we determined whether LTαβ heterotrimers are also cleaved, and are biologically active in rheumatoid arthritis (RA) patients. LTαβ heterotrimers were detected in culture supernatants from activated human T-helper (Th) 0, Th1, and Th17 cells, together with LTα3 and TNFα. The heterotimers were actively cleaved from the cell surface by ADAM17 metalloproteinase (MMP) and MMP-8, and cleavage was inhibited by TAPI-1, a TNF-α converting enzyme (TACE) inhibitor. Soluble LTαβ was detected in serum from both normal donors and RA patients, and was elevated in synovial fluid from RA patients compared to osteoarthritis (OA) patients. Levels of LTαβ in RA patient synovial fluid correlated with increased TNFα, IL-8, IL-12, IL-1β, IFN-γ, and IL-6 cytokines. Moreover, recombinant LTα1β2-induced CXCL1, CXCL2, IL-6, IL-8, VCAM-1, and ICAM-1 from primary synovial fibroblasts isolated from RA patients. Therefore, soluble LTαβ in synovial fluid is associated with a proinflammatory cytokine milieu that contributes to synovitis in RA.

The discovery of novel tartrate-based TNF-α converting enzyme (TACE) inhibitors

A novel series of TNF-α convertase (TACE) inhibitors which are non-hydroxamate have been discovered. These compounds are bis-amides of l-tartaric acid (tartrate) and coordinate to the active site zinc in a tridentate manner. They are selective for TACE over other MMP’s. We report the first X-ray crystal structure for a tartrate-based TACE inhibitor.

Synthesis and activity of tryptophan sulfonamide derivatives as novel non-hydroxamate TNF-α converting enzyme (TACE) inhibitors

A novel series of non-hydroxamate tryptophan sulfonamide derivatives containing a butynyloxy P1′ moiety was identified as inhibitors of TNF-α converting enzyme (TACE). The structure–activity relationship of the series was examined via substitution on the tryptophan indole ring. Of the compounds investigated, 2-(4-(but-2-ynyloxy)phenylsulfonamido)-3-(1-(4-methoxybenzyl)-1 H-indol-3-yl)propanoic acid ( 12p) has the best in vitro potency against isolated TACE enzyme with an IC 50 of 80 nM. Compound 12p also shows good selectivity over MMP-1, -13, -14.

Lung microvascular mitochondria regulate TNFα‐induced TNFR1 shedding

Ligation of endothelial (EC) TNFα receptor 1 (TNFR1) by circulating tumor necrosis factor alpha (TNFα) promotes lung inflammation. TNFR1 shedding by inflammatory cells limits inflammation. However, it is not known whether TNFR1 shedding occurs in lung microvessels. To determine shedding mechanisms, we viewed ECs of single microvessels in inflated, isolated, blood‐perfused rat lungs by live confocal microscopy. To quantify cytosolic (Ca2+cyt) and mitochondrial (Ca2+mit) calcium levels, we co‐infused dyes fluo 4 AM and rhod 2 AM respectively. To determine TNFR1 EC expression, we infused vehicle or TNFα then microinjected a fluorophore‐conjugated TNFR1mAb followed with a buffer wash. After infusion of vehicle alone, Ca2+cyt and Ca2+mit oscillation amplitudes and EC TNFR1 expression were stable. By contrast, after TNFα infusion (1 ng/ml, 10 min) oscillation amplitudes increased two fold (p<0.05, n=3) and TNFR1 fluorescence decreased by 68±7% (p<0.05, n=4), indicating TNFR1 shedding. Pre‐infusion of TNFα converting enzyme (TACE) inhibitor TAPI‐1, or mitochondrial SOD mimetic, MnTBAP blocked TNFα‐mediated TNFR1 decrease. These findings imply novel signaling in which TNFR1 ligation enhances Ca2+mit oscillations, leading to increased mitROS triggering TNFR1 shedding. We provide the first evidence that TNFα induces TNFR1 shedding in lung EC in situ. We propose TNFR1 shedding limits lung inflammation.

Development of a Suitable Process for the Preparation of a TNF-α Converting Enzyme Inhibitor, WAY-281418

A suitable process for the preparation of kilogram quantities of a TNF-α converting enzyme (TACE) inhibitor (WAY-281418) was developed using isatin 13 as starting material and an efficient coupling step for the formation of sulfonamide 8 in a 15% overall yield. Process preparation of (+)-(1S,2R)-2-aminocyclopentane-1-carboxylic acid (7, (+)-cispentacin), a chiral component for WAY-281418, was successfully scaled up via an asymmetric hydrogenation reaction. Crystallization allowed the isolation of all intermediates and the final product 9.

Discovery of Novel Hydroxamates as Highly Potent Tumor Necrosis Factor-α Converting Enzyme Inhibitors: Part I—Discovery of Two Binding Modes

Through a de novo design approach, hydroxamates derived from trans-cyclopropyl dicarboxylate were examined as potential TNF-alpha converting enzyme (TACE) inhibitors. Two distinctive series of inhibitors (A and B) were identified and shown to have different structure-activity relationship trends and selectivity profiles against other matrix metalloproteases despite their close structural similarities. X-ray crystallography of the inhibitors binding to the TACE enzyme demonstrates that each series derives its activity from the opposite enantiomer of the cyclopropyl scaffolds, which display almost superimposable hydroxamate groups that coordinate to the zinc at the catalytic site. Mode A inhibitors occupy the S1'-S3' binding pockets, whereas mode B resides in the nonprime binding sites.

Chromen-based TNF-α converting enzyme (TACE) inhibitors: Design, synthesis, and biological evaluation

A series of coumarin types MMP inhibitors were designed based on gelastatin hydroxamates ( 1) and evaluated for TACE, cellular TNF-α, and NO inhibitory activities. Among them, compounds 9b had potent inhibitory activities in enzymatic and cellular assays and good selectivity to MMP-2 and MMP-9. Further investigation of 9b will be carried out for its efficacy in RA animal model system.

Synthesis and structure–activity relationship of a novel, non-hydroxamate series of TNF-α converting enzyme inhibitors

A novel series of TNF-α converting enzyme (TACE) inhibitors which are non-hydroxamate have been discovered. These compounds use a triazolethione moiety as the zinc binding ligand and exhibit IC50 values from 1.5 to 100nM in a porcine TACE assay. They also have excellent selectivities over other MMPs.

A rat pharmacokinetic/pharmacodynamic model for assessment of lipopolysaccharide-induced tumor necrosis factor-alpha production

Introduction Tumor necrosis factor-alpha (TNFα) participates in many inflammatory processes. TNFα modulators show beneficial effects for the treatment of many diseases including rheumatoid arthritis. The purpose of this study was to validate a rat pharmacokinetic/pharmacodynamic (PK/PD) model for rapid assessment of drug candidates that intended to interrupt TNFα synthesis or release. Methods Rats received intravenous (IV) or oral administrations of test article or dose vehicle, followed by LPS challenge. Plasma levels of test article and TNFα were determined. The areas under the concentration–time curves (AUC drug and AUC TNFα) were calculated. The overall percentage of inhibition on TNFα release in vivo was calculated by comparing AUC TNFα of the test article treated group against that for the vehicle control group. Results The dosing vehicles tested in this study did not increase plasma TNFα level. At IV dose of up to 100 μg/kg, LPS did not alter the pharmacokinetics of the compound tested. Using a selective TNFα converting enzyme (TACE) inhibitor as model compound, this PK/PD model demonstrated its ability to correlate plasma test article concentration with its biological activity of lowering the LPS-induced TNFα plasma levels in vivo. Discussion A rat PK/PD model for evaluation of the effect of drug candidates on LPS-induced TNFα synthesis and/or release has been investigated. This model provides integrated information on pharmacokinetics and in vivo potency of the test articles.