TACE Inhibitors Research Articles

IK682, a tight binding inhibitor of TACE

TNFα converting enzyme (TACE) is the major metalloproteinase for the processing of TNFα, a key inflammatory cytokine. IK682, a hydroxamate compound, was reported to be a potent and specific TACE inhibitor [J.J. Duan, L. Chen, Z.R. Wasserman, Z. Lu, R.Q. Liu, M.B. Covington, M. Qian, K.D. Hardman, R.L. Magolda, R.C. Newton, D.D. Christ, R.R. Wexler, C.P. Decicco, J. Med. Chem. 45 (2002) 4954–4957]. The binding kinetics of IK682 and the ectodomain of human TACE was examined. The k on of IK682 was determined as 1.1 ± 0.3 × 10 8 M −1 min −1. No detectable dissociation of IK682 from TACE was observed following dialysis, dilution, and extensive washing over a maximum of 72 h. This was in contrast to the rapid dissociation of IK682 from ADAM10. LC/MS analysis of the TACE–IK682 complex after dissociation under denaturing conditions indicated that the tight binding is not due to covalent interaction. The X-ray crystal structure of TACE–IK682 complex revealed multiple binding points at the S1′ and S3′ sites and the movement of a loop (from Ala349 to Gly442) to accommodate the binding of the quinolinyl group of IK682 at the S3′ pocket. The conformational changes of TACE may contribute significantly to the high affinity binding as a result of a more stable TACE–inhibitor complex.

Conversion of Potent MMP Inhibitors into Selective TACE Inhibitors.

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TACE-dependent EGF receptor activation in angiotensin-II-induced kidney disease

Angiotensin II (Ang II) has been implicated in the development of cardiovascular disorders and chronic kidney disease (CKD). Ang II causes renal lesions through the activation of tumor necrosis factor (TNF)-alpha-converting enzyme (TACE, also called a disintegrin and a metalloproteinase domain 17) and the release of transforming growth factor (TGF)-alpha, which binds to and activates the epidermal growth factor receptor. Renal lesions such as glomerulosclerosis, tubular atrophy, fibrosis, mononuclear cell infiltration and proteinuria following chronic Ang II infusion are substantially reduced in mice lacking TGF-alpha and those given a specific TACE inhibitor. These findings indicate that the selective inhibition of renal TACE could have therapeutic potential in the treatment of CKD.

Dialogue entre l’angiotensine et le récepteur du facteur de croissance épidermique dans les maladies rénales chroniques : vers une nouvelle approche thérapeutique

Angiotensin plays a major role in renal deterioration, and inhibition of the renin-angiotensin system slows the progression of renal lesions after nephron reduction both in animal models and in humans with chronic kidney diseases. The EGF receptor (EGFR) has recently been recognized as a key molecule in the progression of chronic renal failure, but the interaction between angiotensin and EGF is poorly understood. We show that transgenic mice harboring a dominant negative form of EGFR are resistant to the progression of renal lesions induced by nephron reduction or angiotensin infusion. TGF-alpha, an EGFR ligand, and its sheddase TACE, are overexpressed after angiotensin infusion, and angiotensin-induced renal lesions are blunted in TGF-alpha knockout mice and by pharmacological TACE blockade. After nephron reduction, angiotensin-converting-enzyme inhibitors and angiotensin receptor antagonists prevent TGF-alpha and TACE accumulation. These results indicate that EGFR transactivation by angiotensin plays a crucial role in renal deterioration and that pharmacological inhibitors of TACE might be useful for preventing the progression of chronic kidney diseases.

Acetylenic TACE inhibitors. Part 3: Thiomorpholine sulfonamide hydroxamates

A series of thiomorpholine sulfonamide hydroxamate TACE inhibitors, all bearing propargylic ether P1′ groups, was explored. In particular, compound 5h has excellent in vitro potency against isolated TACE enzyme and in cells, oral activity in a model of TNF-α production and a collagen-induced arthritis model, was selected as a clinical candidate for the treatment of RA.

Blockade of tumor necrosis factor-α-converting enzyme improves experimental small intestinal damage by decreasing matrix metalloproteinase-3 production in rats

Objective. Tumor necrosis factor (TNF)-α-converting enzyme (TACE), which has been purified, regulates maturity of TNF-α. Matrix metalloproteinases (MMPs) play a key role in various inflammatory conditions. The incidence of intestinal damage has increased, but the mechanism and treatment have not been well understood. The purpose of this study was to investigate the roles of TACE and MMP in indomethacin (Indo)-induced intestinal damage as well as the therapeutic effects of TACE inhibitor and selective MMP inhibitor (sMMPi) on this intestinal damage in rats.Material and methods. In the first experiment, serial changes in intestinal ulcers and the production of MMP were investigated. In the second experiment, we assessed the effect of three TACE and/or MMP inhibitors and the production of TNF-α, TACE, MMP-3, -9 and tissue inhibitor of MMP (TIMP)-1. The rats were divided into five groups: a control group, and four groups that received Indo alone, Indo plus TACE inhibitor (GM6001), Indo plus a selective MMP-3 inhibitor and Indo plus an MMP-9/13 inhibitor, respectively.Results. MMP-3 was overexpressed at 24 h after Indo administration, when intestinal injury was most prominent macroscopically and microscopically. GM6001 significantly decreased ulcer severity and suppressed MMP-3 in a dose-dependent fashion. The selective MMP-3 inhibitor dose-dependently ameliorated intestinal damage to the same degree as GM6001, but the MMP-9 inhibitor had no effect on the injury.Conclusions. MMP-3 inhibition ameliorates intestinal damage without apparently affecting either TNF-α or TACE production and the dose–response curve suggests that the beneficial effect of the so-called TACE inhibitor is actually mainly mediated via MMP-3 inhibition rather than TNF-α inhibition.

Screening of Peptide Inhibitors of TACE from a Phage Display Random 15-Peptide Library by Recombinant TACE Ectodomain

Tumor necrosis factor (TNF)-α-converting enzyme (TACE) is the major protease responsible for processing pro-TNF-α from membrane-anchored precursors to secreted TNF-α. In the present study, a 15-peptide library was used to identify potential TACE antagonists. To obtain the recombinant TACE ectodomain and to use it as a selective molecule for the screening of peptide inhibitors of TACE, cDNA coding for the catalytic domain (T800) and full-length ectodomain (T1300) of TACE were amplified by reverse transcription-polymerase chain reaction. The expression plasmid were constructed by inserting T800/T1300 into plasmid pET-28a/c respectively and were transformed into Escherichia coli BL21 (DE3). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis revealed that T800/T1300 were highly expressed in the form of an inclusion body induced by isopropylthiogalactoside. After Ni2+-NTA resin affinity chromatography, the purity of the recombinant T800/T1300 protein was more than 90%. T800 and T1300 proteins were used in the screening of T800/T1300-binding peptides from a phage display random 15-peptide library. After four rounds of biopanning, the positive phage clones were analyzed by enzyme-linked immunosorbent assay, competitive inhibition assay (ELESA), and DNA sequencing. A common amino acid sequence (TRWLVYFSRPYLVAT) was confirmed and synthesized. A synthetic peptide was shown to bind to TACE and to inhibit TNF-α release from lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (PBMC) by up to 60.3%. Fluorescence-activated cell sorter (FACS) analysis revealed that the peptide mediated the accumulation of TNF-α on an LPS-stimulated PBMC surface. These results demonstrate that the TACE-binding peptide is an effective antagonist of TACE and that the deduced motif might be applied to the molecular design of anti-inflammatory drugs.

Heterocyclic Inhibitors of Tumor Necrosis Factor-α Converting Enzyme (TACE)

A variety of heterocyclic ring systems have been prepared as scaffolds for butynyloxyphenyl sulfonamide and sulfone hydroxamic acid inhibitors of TACE enzyme. All scaffolds provided highly active TACE inhibitors, but selectivity, and cellular activity was highly scaffold dependent.

Acetylenic TACE Inhibitors. Part 2. SAR of Six‐Membered Cyclic Sulfonamide Hydroxamates.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Conversion of potent MMP inhibitors into selective TACE inhibitors

Novel sultam hydroxamates with potent MMP activity were transformed into potent TACE inhibitors, lacking MMP activity. To accomplish this we relied on structural differences between the MMP and TACE S1′ pockets and the known advantageous fit of a 2-methyl-4-quinolinylmethoxyphenyl group into this region. From this approach, compound 7d was identified as a potent TACE inhibitor (IC 50 = 3.7 nM) that lacked MMP-1, -2, -9, and -13 activity.

Neutrophil Elastase Induces MUC5AC Mucin Production in Human Airway Epithelial Cells via a Cascade Involving Protein Kinase C, Reactive Oxygen Species, and TNF-α-Converting Enzyme

Mucus hypersecretion is a prominent manifestation in patients with chronic inflammatory airway diseases and contributes to their morbidity and mortality by plugging airways and causing recurrent infections. Human neutrophil elastase (HNE) exists in high concentrations (1-20 microM) in airway secretions of these patients and induces overproduction of MUC5AC mucin, a major component of airway mucus. Previous studies showed that HNE induces MUC5AC mucin production involving reactive oxygen species (ROS) generation and TGF-alpha-dependent epidermal growth factor receptor (EGFR) activation in human airway epithelial cells. However, the molecular mechanisms involved in these responses are not defined. TNF-alpha-converting enzyme (TACE) cleaves pro-TGF-alpha into soluble TGF-alpha and can be activated by ROS. We hypothesize that HNE activates TACE via ROS generation, resulting in cleavage of pro-TGF-alpha, EGFR activation, and MUC5AC mucin expression in airway epithelial cells. Here we show that in human airway epithelial cells HNE increases TGF-alpha release, EGFR phosphorylation, and MUC5AC mucin expression, effects that were attenuated by TACE inhibitor TAPI-1 and by specific knockdown of TACE expression with small interfering RNA, implicating TACE in HNE-induced responses. These responses to HNE were also reduced by pretreatment with ROS scavengers, implicating ROS. Furthermore, we show that HNE causes protein kinase C (PKC) activation and translocation from cytosol to plasma membrane; blockade of this effect by PKC inhibitors reduced HNE-induced ROS generation and other responses, implicating PKC. We conclude that HNE induces MUC5AC mucin expression via a cascade involving PKC-ROS-TACE in human airway epithelial cells.

Acetylenic TACE inhibitors. Part 2: SAR of six-membered cyclic sulfonamide hydroxamates

The SAR of a series of potent sulfonamide hydroxamate TACE inhibitors bearing a butynyloxy P1′ group was explored. In particular, compound 5k has excellent in vitro potency against TACE enzyme and in cells, and oral activity in an in vivo model of TNF-α production and a collagen-induced arthritis model.

Angiotensin II and EGF receptor cross-talk in chronic kidney diseases: a new therapeutic approach

Mechanisms of progression of chronic renal diseases, a major healthcare burden, are poorly understood. Angiotensin II (AngII), the major renin-angiotensin system effector, is known to be involved in renal deterioration, but the molecular pathways are still unknown. Here, we show that mice overexpressing a dominant negative isoform of epidermal growth factor receptor (EGFR) were protected from renal lesions during chronic AngII infusion. Transforming growth factor-alpha (TGF-alpha) and its sheddase, TACE (also known as ADAM17), were induced by AngII treatment, TACE was redistributed to apical membranes and EGFR was phosphorylated. AngII-induced lesions were substantially reduced in mice lacking TGF-alpha or in mice given a specific TACE inhibitor. Pharmacologic inhibition of AngII prevented TGF-alpha and TACE accumulation as well as renal lesions after nephron reduction. These findings indicate a crucial role for AngII-dependent EGFR transactivation in renal deterioration and identify in TACE inhibitors a new therapeutic strategy for preventing progression of chronic renal diseases.

Synthesis and SAR of Diazepine and Thiazepine TACE and MMP Inhibitors.

AbstractFor Abstract see ChemInform Abstract in Full Text.

The Design and Synthesis of Aryl Hydroxamic Acid Inhibitors of MMPs and TACE

AbstractFor Abstract see ChemInform Abstract in Full Text.

Involvement of EGF receptor and c-Src in the survival signals induced by TGF-β1 in hepatocytes

Transforming growth factor beta1 (TGF-beta1) belongs to a family of polypeptide factors, whose cytostatic and apoptotic functions help restrain the growth of mammalian cells. Although solid data established the role of TGF-beta's as suppressor factors in tumorigenic processes, in the context of an advanced stage of disease, TGF-beta's could also play a pro-oncogenic role. We have previously shown that TGF-beta1 induces both pro- and anti-apoptotic signals in foetal rat hepatocytes. In this work, we have focused on its anti-apoptotic mechanism. We show that TGF-beta1 activates the epidermal growth factor receptor (EGFR) and phosphorylates c-Src. EGFR is required for Akt activation. Blocking EGFR signalling amplifies the apoptotic response to TGF-beta1. TGF-beta1 induced a rapid activation of the tumour necrosis factor-alpha-converting enzyme (TACE/ADAM (a disintegrin and metalloprotease) 17). Inhibitors of TACE considerably attenuated Akt activation, which suggests that TGF-beta1 activates EGF signalling in hepatocytes by promoting shedding of EGF-like ligands. The activation of c-Src by TGF-beta1 is EGFR dependent and is required for full Akt phosphorylation and cell survival. Inhibition of EGFR does not block the epithelial-mesenchymal transition (EMT) induced by TGF-beta1 in hepatocytes, which indicates that activation of EGFR plays an essential role in impairing apoptosis, but it is dispensable for the EMT process.

A Comparison of the Binding Sites of Matrix Metalloproteinases and Tumor Necrosis Factor-α Converting Enzyme: Implications for Selectivity

MMPs and TACE (ADAM-17) assume independent, parallel, or opposite pathological roles in cancer, arthritis, and several other diseases. For therapeutic purposes, selective inhibition of individual MMPs and TACE is required in most cases due to distinct roles in diseases and the need to preserve activities in normal states. Toward this goal, we compared force-field interaction energies of five ubiquitous inhibitor atoms with flexible binding sites of 24 known human MMPs and TACE. The results indicate that MMPs 1-3, 10, 11, 13, 16, and 17 have at least one subsite very similar to TACE. S3 subsite is the best target for development of specific TACE inhibitors. Specific binding to TACE compared to most MMPs is promoted by placing a negatively charged ligand part at the bottom of S2 subsite, at the entrance of S1' subsite, or the part of S3' subsite that is close to catalytic zinc. Numerous other clues, consistent with available experimental data, are provided for design of selective inhibitors.

Synthesis and SAR of diazepine and thiazepine TACE and MMP inhibitors

Potent and selective TACE and MMP inhibitors utilizing the diazepine and thiazepine ring systems were synthesized and evaluated for biological activity in in vitro and in vivo models of TNF-α release. Oral activity in the mouse LPS model of TNF-α release was seen. Efficacy in the mouse collagen induced arthritis model was achieved with diazepine 20.

TNFR1 Upregulation Mediates Tolerance after Brain Ischemic Preconditioning

A short ischemic event (ischemic preconditioning (IPC)) can result in subsequent resistance to severe ischemic injury (ischemic tolerance (IT)). The expression and neuroprotective role of tumor necrosis factor (TNF-alpha) have been described in models of IPC and we have showed the participation of its processing enzyme, the TNF-alpha convertase enzyme (TACE) in this process. We have now decided to explore the expression and localization of TNF receptors (TNFR) as well as other signalling mechanisms involved in IT. A period of 10 mins of temporary middle cerebral artery occlusion (tMCAO) was used for focal IPC. To evaluate the ability of IPC to produce IT, permanent MCAO was performed 48 hours after IPC. Ischemic preconditioning produced a reduction in infarct volume, as we showed previously. Ischemic preconditioning caused upregulation of neuronal TNFR1 that was reduced by the selective TACE inhibitor BB1101. Intracerebral administration of TNFR1 antisense oligodeoxynucleotide, which caused a reduction in TNFR1 expression, inhibited the IPC-induced protective effect, showing that TNFR1 upregulation is implicated in IT. Moreover, treatment with BB1101, TNFR1 antisense and lactacystin-a specific proteasome inhibitor-blocked IPC-induced NF-kappaB. Immunohistochemical studies showed the expression of TACE and TNFR1 in neurons. In summary, these data show that IPC produces neuronal upregulation of TACE and TNFR1, and that the pathway TACE/TNF-alpha/TNFR1/NF-kappaB is involved in IT.

Small molecular anti-cytokine agents

The recent successful introduction of the anti-cytokine biologicals Etanercept, Infliximab, Adalimumab, and Anakinra has stimulated the search for anti-cytokine small-molecules. A number of molecular targets have been identified for the development of such small molecular anti-cytokine agents. The focus of this review will be on those inhibitors of cytokine production, which target either p38 mitogen activated protein (MAP) kinase, TNF-alpha converting enzyme (TACE), or IL-1beta converting enzyme (ICE). P38 MAP kinase occupies a central role in the signaling network responsible for the upregulation of proinflammatory cytokines like interleukin 1beta (IL-1beta) and TNF-alpha, and regulates their biosynthesis at both the transcriptional and translational level. TACE and ICE are two proteases required for the processing of proTNF-alpha and proIL-1beta, respectively into their mature, proinflammatory form. Since the mid-1990s, a plethora of inhibitors of p38 MAP kinase, TACE, and ICE has been characterized in vitro, and individual representatives from all three inhibitor classes have in the meantime been advanced into clinical trials. This review will highlight the correlation between effective inhibition at the molecular target and cellular activity in functional assays of cytokine, particularly TNF-alpha and IL-1beta, production. Structure-activity relationships (SAR) will be discussed regarding activity at the respective enzyme target, but also with regard to properties required for efficient in vitro and in vivo cellular activity (e.g., oral availability, solubility, cell penetration, etc.).