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

A novel series of TNF-alpha 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 100 nM in a porcine TACE assay. They also have excellent selectivities over other MMPs.     

Rational design, synthesis and structure-activity relationships of a cyclic succinate series of TNF-alpha converting enzyme inhibitors. Part 2: lead optimization

Modifications of the lead TACE inhibitor 1 (N-hydroxy-trans-2-[[4-(4-quinolinyloxymethyl)anilinyl]carbonyl]-1-cyclohexanecarboxamide) at the cyclohexyl ring and the quinoline moiety led to the identification of a series of piperidine containing TACE inhibitors with potent activity in the inhibition of TNF-alpha release in the whole blood assay (WBA). The most potent analogue IM491 [N-hydroxy-(5S,6S)-1-methyl-6-[[4-(2-methyl-4-quinolinylmethoxy)anilinyl]carbonyl]-5-piperidinecarboxamide] exhibited an IC(50) value of 20 nM in WBA with excellent selectivity over MMP-1, -2 and -9 and is orally bioavailable with an F value of 43% in beagle dogs.     

Alpha,beta-cyclic-beta-benzamido hydroxamic acids: novel templates for the design, synthesis, and evaluation of selective inhibitors of TNF-alpha converting enzyme (TACE)

Selective inhibitors of TNF-alpha Converting Enzyme (TACE) based on (1R,2S)-cyclopentyl, (3S,4S)-pyrrolidinyl, and (3R,4S)-tetrahydrofuranyl beta-benzamido hydroxamic acids have been synthesized and evaluated. This study has led to the discovery of novel inhibitors whose profiles include activity against TACE in an enzyme assay, potency in the suppression of LPS-stimulated TNF-alpha in human whole blood, selectivity against a panel of MMPs and oral bioavailability.     

Processing of tumor necrosis factor by the membrane-bound TNF-alpha-converting enzyme, but not its truncated soluble form.

Tumour necrosis factor (TNF)-alpha-converting enzyme (TACE) is a membrane protein belonging to the ADAM (a disintegrin and metalloproteinase) family that cleaves various membrane proteins, including the proform of TNF-alpha. In this study, we constructed expression vectors for the membrane-bound full-length TACE (mTACE) and its truncated soluble form (sTACE). When a human TNF-alpha expression vector was introduced into human 293 cells, processing of TNF-alpha to its mature form was enhanced by coexpressing mTACE, and this processing was inhibited by a metalloproteinase inhibitor. On the other hand, coexpression of sTACE had no effect on the processing of TNF-alpha, although the culture medium of sTACE-transfected cells could cleave a peptide containing the TNF-alpha cleavage site. Fas ligand (FasL)-transfected 293 cells released a considerable amount of soluble FasL, and coexpression of neither mTACE nor sTACE enhanced this shedding. Immunoprecipitation and Western blotting analysis with cells that were cotransfected with TACE and TNF-alpha indicated that both mTACE and sTACE could interact with the proform of TNF-alpha. In the same assay, neither mTACE nor sTACE interacted with FasL. The catalytic domain-lacking TACE mutant, which could also interact TNF-alpha, showed a dominant negative effect on not only TNF-alpha secretion but also FasL secretion. These results suggest that binding of the membrane-anchored but not the soluble form of TACE to TNF-alpha results in efficient ectodomain shedding, and that FasL secretase is a metalloproteinase similar, but not identical, to TACE.     

Potent, selective, orally bioavailable inhibitors of tumor necrosis factor-alpha 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.     

Synthesis and structure-activity relationship of a novel sulfone series of TNF-alpha converting enzyme inhibitors

Replacement of the amide functionality in IM491 (N-hydroxy-(5S,6S)-1-methyl-6-[[4-(2-methyl-4-quinolinylmethoxy)anilinyl]carbonyl]5-piperidinecarboxamide) with a sulfonyl group led to a new series of alpha,beta-cyclic and beta,beta-cyclic gamma-sulfonyl hydroxamic acids, which were potent TNF-alpha converting enzyme (TACE) inhibitors. Among them, inhibitor 4b (N-hydroxy-(4S,5S)-1-methyl-5-[[4-(2-methyl-4-quinolinylmethoxy)phenyl]sulfonylmethyl]-4-pyrrolidinecarboxamide) exhibited IC50 values of < 1 nM and 180 nM in porcine TACE (pTACE) and cell assays, respectively, with excellent selectivity over MMP-1, -2, -9 and -13 and was orally bioavailable with an F value of 46% in mice.     

Mapping and characterization of the functional epitopes of tissue inhibitor of metalloproteinases (TIMP)-3 using TIMP-1 as the scaffold: a new frontier in TIMP engineering

Tumor necrosis factor-alpha (TNF-alpha) converting enzyme (TACE/ADAM-17) is responsible for the release of TNF-alpha, a potent proinflammatory cytokine associated with many chronic debilitating diseases such as rheumatoid arthritis. Among the four variants of mammalian tissue inhibitor of metalloproteinases (TIMP-1 to -4), TACE is specifically inhibited by TIMP-3. We set out to delineate the basis for this specificity by examining the solvent accessibility of every epitope on the surface of a model of the truncated N-terminal domain form of TIMP-3 (N-TIMP-3) in a hypothetical complex with the crystal structure of TACE. The epitopes suspected of interacting with TACE were systematically transplanted onto N-TIMP-1. We succeeded in transforming N-TIMP-1 into an active inhibitor for TACE (K(i)(app) 15 nM) with the incorporation of Ser4, Leu67, Arg84, and the TIMP-3 AB-loop. The combined effects of these epitopes are additive. Unexpectedly, introduction of "super-N-TIMP-3" epitopes, defined in our previous work, only impaired the affinity of N-TIMP-1 for TACE. Our mutagenesis results indicate that TIMP-3-TACE interaction is a delicate process that requires highly refined surface topography and flexibility from both parties. Most importantly, our findings confirm that the individual characteristics of TIMP could be transplanted from one variant to another.     

Non-hydroxamate 5-phenylpyrimidine-2,4,6-trione derivatives as selective inhibitors of tumor necrosis factor-alpha converting enzyme

New inhibitors of tumor necrosis factor-alpha converting enzyme (TACE) were discovered with a pyrimidine-2,4,6-trione in place of the commonly used hydroxamic acid. These non-hydroxamate TACE inhibitors were developed by incorporating a 4-(2-methyl-4-quinolinylmethoxy)phenyl group, an optimized TACE selective P1' group. Several leads were identified with IC50 values around 100 nM in a porcine TACE assay and selective over MMP-1, -2, -9, -13, and aggrecanase.     

Discovery of low nanomolar non-hydroxamate inhibitors of tumor necrosis factor-alpha converting enzyme (TACE)

Using a pyrimidine-2,4,6-trione motif as a zinc-binding group, a series of selective inhibitors of tumor necrosis factor-alpha converting enzyme (TACE) was discovered. Optimization of initial lead 1 resulted in a potent inhibitor (51), with an IC(50) of 2 nM in a porcine TACE assay. To the best of our knowledge, compound 51 and related analogues represent first examples of non-hydroxamate-based inhibitors of TACE with single digit nanomolar potency.     

Gelastatins and their hydroxamates as dual functional inhibitors for TNF-alpha converting enzyme and matrix metalloproteinases: synthesis, biological evaluation, and mechanism studies

The hydroxamic acid analogues (2) of the natural product gelastatins (1) were prepared by 1 step conversion reaction. The synthetic analogues (2) showed potent enzymatic inhibitory activities against MMP-2, MMP-9, and TACE IC50's of 6, 23, and 28 nM, respectively. In addition, 2 were able to inhibit TNF-alpha production effectively in mice as well as in a macrophage cell line, RAW 264.7. The protective effect of 2 also was examined on LPS-induced acute septic shock model. The mechanism of TNF-alpha inhibition was examined by RT-PCR and Western blot analyses. The relation of TACE and alpha-secretase was examined using cellular alpha-secretase assays on IMR-32 and SH-SY5Y cell lines. The docking mode of 2 with the catalytic domain of TACE was illustrated to analyze the binding mode for the further analogue design.     

Implication of TNF-alpha convertase (TACE/ADAM17) in inducible nitric oxide synthase expression and inflammation in an experimental model of colitis

Tumour necrosis factor-alpha (TNF-alpha) is a pro-inflammatory cytokine which is shed in its soluble form by a disintegrin and metalloproteinase (ADAM) called TNF-alpha convertase (TACE; ADAM17). TNF-alpha plays a role in inflammatory bowel disease (IBD) and is involved in the expression of inducible nitric oxide synthase (iNOS) which has also been implicated in IBD. The study was designed to investigate whether colitis induced by trinitrobenzene sulphonic acid (TNBS) in rats produces an increase in TACE activity and/or expression and whether its pharmacological inhibition reduces TNF-alpha levels, iNOS expression and colonic damage in this model. TNBS (30 mg in 0.4 ml of 50% ethanol) was instilled into the colon of female Wistar rats. Saline or TACE inhibitor BB1101 (10 mg/kg/day) was administered intraperitoneally 5 days after TNBS instillation. On day 10, colons were removed and assessed for pathological score, myeloperoxidase (MPO), NO synthase (NOS), TACE enzymatic activity and protein levels, colonic TNF-alpha and NOx- levels. Instillation of TNBS caused an increase in TACE activity and expression and the release of TNF-alpha. TNBS also resulted in iNOS expression and colonic damage. BB1101 blocked TNBS-induced increase in TACE activity, TNF-alpha release and iNOS expression. Concomitantly, BB1101 ameliorated TNBS-induced colonic damage and inflammation. TNBS causes TNF-alpha release by an increase in TACE activity and expression and this results in the expression of iNOS and subsequent inflammation, suggesting that TACE inhibition may prove useful as a therapeutic means in IBD.     

Discovery of N-hydroxy-2-(2-oxo-3-pyrrolidinyl)acetamides as potent and selective inhibitors of tumor necrosis factor-alpha converting enzyme (TACE)

New inhibitors of tumor necrosis factor-alpha converting enzyme (TACE) were discovered using an N-hydroxy-2-(2-oxo-3-pyrrolidinyl)acetamide scaffold. The series was found to be potent in a porcine TACE (pTACE) assay with IC(50)s typically below 5 nM. For most compounds, selectivity for pTACE relative to MMP-1,-2, and -9 is at least 300-fold. Compound 2o was potent in inhibition of TNFalpha production in a human whole blood assay (WBA) with an IC(50) of 0.42 micro M.     

Discovery of selective hydroxamic acid inhibitors of tumor necrosis factor-alpha converting enzyme.

Modification of the P(1)' substituent of macrocyclic matrix metalloproteinase (MMP) inhibitors provided compounds that are selective for inhibition of tumor necrosis factor-alpha converting enzyme (TACE) over MMP-1 and MMP-2. Several analogues potently inhibited the release of TNF-alpha in a THP-1 cellular assay. Compounds containing a trimethoxyphenyl group in the P(1)' substituent demonstrated TACE selectivity across several series of hydroxamate-based inhibitors.     

Reactive oxygen species mediate tumor necrosis factor alpha-converting, enzyme-dependent ectodomain shedding induced by phorbol myristate acetate.

Ectodomain shedding of cell surface membrane-anchoring proteins is an important process in a wide variety of physiological events(1, 2). Tumor necrosis factor alpha (TNF-alpha) converting enzyme (TACE) is the first discovered mammalian sheddase responsible for cleavage of several important surface proteins, including TNF-alpha, TNF p75 receptor, L-selectin, and transforming growth factor-a. Phorbol myristate acetate (PMA) has long been known as a potent agent to enhance ectodomain shedding. However, it is not fully understood how PMA activates TACE and induces ectodomain shedding. Here, we demonstrate that PMA induces both reactive oxygen species (ROS) generation and TNF p75 receptor shedding in Mono Mac 6 cells, a human monocytic cell line, and l-selectin shedding in Jurkat T-cells. ROS scavengers significantly attenuated PMA-induced TNF p75 receptor shedding. Exogenous H2O2 mimicked PMA-induced enhancement of ectodomain shedding, and H2O2-induced shedding was blocked by TAPI, a TACE inhibitor. Furthermore, both PMA and H2O2 failed to cause ectodomain shedding in a cell line that lacks TACE activity. By use of an in vitro TACE cleavage assay, H2O2 activated TACE that had been rendered inactive by the addition of the TACE inhibitory pro-domain sequence. We presume that the mechanism of TACE activation by H2O2 is due to an oxidative attack of the pro-domain thiol group and disruption of its inhibitory coordination with the Zn++ in the catalytic domain of TACE. These results demonstrate that ROS production is involved in PMA-induced ectodomain shedding and implicate a role for ROS in other shedding processes.     

Rational design, synthesis and structure-activity relationships of a cyclic succinate series of TNF-alpha converting enzyme inhibitors. Part 1: lead identification

Rational design based on the broad spectrum MMP inhibitor CGS 27023A led to the identification of a novel series of cyclic succinate TACE inhibitors. As a mixture of two enantiomers, the lead compound 17b exhibited potent enzyme activity (IC(50)=8 nM) in the inhibition of porcine TNF-alpha converting enzyme (pTACE) and excellent selectivity over aggrecanase and MMP-1, -2 and -9.     

以重组TACE胞外功能域从噬菌体随机肽库中筛选TACE的抑制肽

肿瘤坏死因子转换酶 (TACE)是加工裂解TNF-α前体的关键酶 ,参与了许多炎症的发生发展过程。为通过肽库筛选得到TACE的抑制肽 ,首先制备筛选靶分子 ,用RT PCR从人外周血单核细胞中分别扩增出TACE的催化区 (T800 )和整个胞外区 (T1300 ) ,然后分别克隆至pET-28a和pET-28c中 ,转化大肠杆菌BL2-1(DE3) ,经IPTG诱导表达出带有His-tag的目的蛋白 ,两者均为包涵体 ,变性复性后过Ni²⁺-NTA亲和层析柱得到纯度达90%的重组蛋白。以纯化的重组T800和T1300分别筛选噬菌体展示随机 15肽库 ,对筛选克隆进行ELISA检测、竞争抑制实验和序列分析。从两个独立的筛选过程中得到一个相同的阳性克隆序列“TRWLVYFSRPYLVAT” ,固相Fmoc法合成该短肽 ,观察其在LPS诱导人单核细胞产生sTNF-α中的作用。结果表明 ,筛选到的短肽可显著抑制TACE的活性 ,减少TNF-α的分泌 ,抑制率可达 60.3% ,为抗炎小分子药物的设计和改造提供线索和依据。     

Expression and function of tumour necrosis factor-alpha-converting enzyme in the central nervous system

Tumour necrosis factor-alpha (TNF-alpha)-converting enzyme (TACE/ADAM17) is a membrane protein belonging to the ADAM (a disintegrin and a metalloprotease) family able to cleave various membrane proteins, including the transmembrane form of TNF-alpha at its physiological processing site. Being an ADAM, TACE may mediate not only proteolysis but also adhesive interactions; however, the role of the disintegrin domain of TACE has not been studied. In the central nervous system (CNS), little is known about the physiological role of TACE, but some important pathophysiological functions have been reported recently, with both neurotoxic and neuroprotective repercussions. This article discusses and reviews the main contributions to this field of investigation addressing the expression and function of TACE in the CNS.     

Multiple metalloproteinases process protransforming growth factor-alpha (proTGF-alpha)

Shedding of TNF-alpha requires a single cleavage event, whereas the ectodomain of proTGF-alpha is cleaved at N-proximal (N-terminal) and membrane proximal (C-terminal) sites to release mature TGF-alpha. Tumor necrosis factor-alpha converting enzyme (TACE) was shown to have a central role in the shedding of both factors. Here we show that cleavage of the proTGF-alpha C-terminal site, required for release of mature growth factor, is less sensitive to a panel of hydroxamates than TNF-alpha processing. Recombinant TACE cleaves TNF-alpha and N-terminal TGF-alpha peptides 50-fold more efficiently than the C-terminal TGF-alpha peptide. Moreover, fractionation of rat liver epithelial cell membranes yields two populations: one contains TACE and cleaves peptides corresponding to TNF-alpha and both proTGF-alpha processing sites, while the other lacks detectable TACE and cleaves only the C-terminal proTGF-alpha processing site. Activities in both fractions are inhibited by hydroxamates and EDTA but not by cysteine, aspartate, or serine protease inhibitors. Both membrane fractions also contain ADAM 10. ADAM 10 correctly cleaves peptides and a soluble form of precursor TGF-alpha (proTGFecto) at the N-terminal site but not the C-terminal site. However, the kinetics of N-terminal peptide cleavage by ADAM 10 are 90-fold less efficient than TACE. Our findings indicate that while TACE is an efficient proTGF-alpha N-terminal convertase, a different activity, distinguishable from TACE, exists that can process proTGF-alpha at the C-terminal site. A model that accounts for these findings and the requirement for TACE in TGF-alpha shedding is proposed.     

Tumor necrosis factor-alpha stimulates proliferation of rat ovarian theca-interstitial cells.

Tumor necrosis factor-alpha (TNF-alpha) is a potent modulator of ovarian function, affecting steroidogenesis of both granulosa and theca-interstitial (T-I) cells. Women with polycystic ovary syndrome (PCOS) have increased levels of serum TNF-alpha. The present study evaluated the effects of TNF-alpha on T-I cell proliferation. Purified rat T-I cells were cultured for 48 h with or without TNF-alpha (0.001-1 nM), insulin-like growth factor I (IGF-I; 10 nM), and/or insulin (10 nM). Proliferation was measured by [(3)H]thymidine incorporation assay and by counting the steroidogenically active (stained positive for 3beta-hydroxysteroid dehydrogenase; 3beta-HSD) and inactive (3beta-HSD negative) cells. TNF-alpha stimulated thymidine incorporation in a dose-dependent fashion (up to 3.2-fold; P < 0.01). Insulin and IGF-I stimulated T-I proliferation (respectively, by up to 2.4- and 3.1-fold; P < 0.001). TNF-alpha potentiated effects of insulin and IGF-I in a dose-dependent and additive fashion (up to 6.7-fold; P < 0.001). TNF-alpha (1 nM) increased total cell count (by 80%, P < 0.05) and the proportion of 3beta-HSD-positive cells (by 19%, P < 0.05). Flow cytometry DNA analysis revealed that TNF-alpha (1 nM) increased the proliferative index by up to 16% (P = 0.05). The present findings demonstrate that TNF-alpha stimulates mitotic activity of T-I cells by increasing the proportion of actively dividing cells and preferentially increasing the number of steroidogenically active cells. The effects of TNF-alpha appear to be independent of those induced by insulin and IGF-I. We postulate that TNF-alpha may play a pathophysiologic role in disorders of the T-I compartment, such as PCOS.