Inhibition of Tumor Necrosis Factor- –Converting Enzyme by a Selective Antagonist Protects Brain from Focal Ischemic Injury in Rats

Tumor necrosis factor (TNF ) is an immunomodulatory and proinflammatory cytokine implicated in neuroinflammation and neuronal damage in response to cerebral ischemia. Tumor necrosis factorconverting enzyme (TACE or ADAM17) is a key sheddase that releases TNF from its inactive cell-bound precursor. Using a selective small molecule inhibitor of TACE, DPH-067517, we tested the hypothesis that inhibition of TNF formation might have a salutary effect in ischemic stroke induced by embolic occlusion of the middle cerebral artery (MCAO). DPH-067517 selectively inhibited TACE enzyme activity in vitro (Ki 2.8 nM), and effectively suppressed ischemia-induced increase in soluble TNF in brain tissue after systemic administration. DPH-067517 (3 and 30 mg/kg, i.p. administered 15 min before MCAO) produced 43% (n 8, p 0.16) and 58% (n 8, p 0.05) reduction in infarct size and 36% (p 0.05) and 23% (p 0.05) reduction in neurological deficits, respectively. The salutary effect of DPH-067517 in ischemic brain injury was also observed when the first dose was administrated 60 min after the onset of ischemia. Inhibition of TACE had no effect on apoptosis measured by levels of active caspase-3 expression and DNA fragmentation. Our data suggest that inhibition of TACE might be a potential therapeutic strategy for neuroprotection after focal ischemic stroke. Tumor necrosis factor-converting enzyme (TACE, or ADAM17) is a member of the ADAM (A Disintegrin and Metalloproteinase) family of proteases containing both a disintegrin and a metalloproteinase domain (Doedens and Black, 2000). ADAMs form a large group of cell surface proteins that combine features of both cell surface adhesion molecules and proteases. So far, 31 ADAMs have been identified in the public database, 17 of which contain the sequences consistent with metalloproteinase activity. TACE is a membrane-anchored, zinc-dependent metalloproteinase that was originally identified for its ability to release soluble TNF from transmembrane pro-TNF (Black et al., 1997; Reddy et al., 2000). Focal stroke is a pathophysiological condition caused by decreased blood supply to the brain. The deprivation of oxygen and glucose in the ischemic brain eventually leads to cell death (necrosis and apoptosis), inflammation, and tissue repair (del Zoppo et al., 2000; Wang and Feuerstein, 2000). TNF is a key inflammatory mediator that has been demonstrated to be upregulated in brain ischemia (Liu et al., 1994; Wang et al., 1994) and to play a detrimental role in neuronal survival. Administration of TNF during an ischemic brain insult has been shown to augment the injury, as evidenced by increased tissue damage and neurological deficits (Barone et al., 1997). Correspondingly, experiments with neutralizing anti-TNF antibodies or sTNF-R1, administered directly into the cerebroventricular system, reduced ischemic damage and improved functional outcome (Dawson et al., 1996; Barone et al., 1997; Nawashiro et al., 1997a). However, several other studies suggested a protective role of the TNF signaling pathway in tolerance to ischemic or traumatic brain injuries (Bruce et al., 1996; Nawashiro et al., 1997b; Liu et al., 2000). TNF null mutations in mice result in exacerbation of lesions and functional deficits after cerebral ischemia (Bruce et al., 1996). Studies conducted with animals exposed to TNF before ischemic injuries showed remarkable tolerance to the ischemic insult, as ABBREVIATIONS: TACE, tumor necrosis factor-converting enzyme; ADAM, a disintegrin and metalloproteinase; ADAMTS, ADAM family proteins with thrombospondin type I motifs; TNF , tumor necrosis factor ; DPH-067517, N-{4 -[2-(hydroxylamino)-2-oxoethyl]-2 ,6 -dimethyl-4piperidinyl}-4-[(2-methyl-4-quinolinyl)methoxy]benzamide; MMP, matrix metalloproteinase; ELISA, enzyme-linked immunosorbent assay; MCAO, occlusion of the middle cerebral artery; MCA, middle cerebral artery; CBF, cerebral blood flow; TTC, 2,3,5-triphenyltetrazolium chloride; IL, interleukin; BB-1101, 2S-allyl-N-hydroxy-3R-isobutyl-N-(1S-methylcarbamoyl-2-phenylethyl)-succinamide. 0026-895X/04/6504-890–896$20.00 MOLECULAR PHARMACOLOGY Vol. 65, No. 4 Copyright © 2004 The American Society for Pharmacology and Experimental Therapeutics 3008/1141033 Mol Pharmacol 65:890–896, 2004 Printed in U.S.A. 890 at A PE T Jornals on M ay 0, 2017 m oharm .aspeurnals.org D ow nladed from evidenced by better functional outcome and lesion reduction (Nawashiro et al., 1997b; Liu et al., 2000). Although there is considerable evidence for the generation of TNF after cerebral ischemia, the regulatory mechanisms of postischemic TNF synthesis remain poorly understood. It is likely that mRNA transcription is important in TNF generation, yet for TNF to become functionally active, cleavage from its membrane bound form is necessary. Thus, TACE activity is central to TNF production and, therefore, of potential regulatory importance. To explore the role of TACE and TNF in ischemic injury, we have investigated the effect of a novel, potent, and selective TACE inhibitor, DPH-067517, on brain damage and neurobehavioral consequence in a rat model of thromboembolic focal stroke. Materials and Methods TACE Activity Assay. DPH-067517 (Fig. 1) was synthesized by Bristol-Myers Squibb (Princeton, NJ). The affinity of DPH-067517 for TACE was determined using partially purified porcine enzyme and a synthetic fluorogenic substrate, (7-methoxy coumarin-4 acetyl)-PLAQAV-(3-[2,4-dinitrophenyl])-2,3-diaminopropanolRSSSR-NH2 (Quality Controlled Biochemicals, Hopkinton, MA). Partially purified TACE enzyme was obtained from porcine spleen after a previously described procedure (Moss et al., 1997). The peptide substrate was diluted to a final concentration of 10 M in a buffer containing 50 mM Tricine pH 7.5, 100 mM NaCl, 10 mM CaCl2, and 1 mM ZnCl2. The enzyme reaction contained 25 l of 2 nM partially purified TACE plus the diluted peptide in 200l final volume in the presence or absence of inhibitors. Reaction mixtures were incubated for 1 h on an orbital shaker at 27°C. Reactions were quenched by the addition of 20 l of 500 M EDTA. Fluorescence measurements were performed in a CytoFluor Multi-Well Plate Reader series 4000. Cleavage of internal quenched substrate liberated emission active 7-methoxy coumarin-4 acetyl product causing an increase in fluorescence emission at 395 nM (excitation wavelength was 330 nM). The rate of emission change was proportional to enzyme activity. The data were analyzed as described previously (Xue et al., 1998). The potency of DPH-067517 for ADAM-10 (R&D System, Minneapolis, MN) was determined by using the same assay protocol as the one used for TACE except for changes of enzyme concentration (100 nM) and incubation time (16 h). MMP Activity Assay. For MMP activity assays, a fluorogenic peptide substrate, (7-methoxy coumarin-4-yl)acetyl-Pro-Leu-Gly-Leu(3-[2,4-dinitrophenyl])-L-2,3-diaminopropanol-Ala-Arg-NH2 (Biochem), was used for MMP-1, -2, -3, -8, -9, -13, -14, and -15 reactions. A different fluorogenic peptide substrate, (7-metthoxycoumarin-4-yl)acetylArg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys (2,4-dinitrophenyl)-NH2 (R&D Systems), was used for the MMP-10 reaction. MMP enzymes were purchased from Chemicon International (Temecula, CA), InVitek, (Berlin, Germany), or R&D Systems Inc. (Minneapolis, MN). The appropriate peptide substrate was diluted to a final concentration of 10 M in a buffer containing 50 mM Tricine, pH 7.5, 100 mM NaCl, 10 mM CaCl2, and 1 mM ZnCl2, and an appropriate amount of MMP (between 0.2 and 10 nM depending on the enzyme) plus the diluted peptide in 200l final volume in the presence or absence of the inhibitors. This was incubated for 1 h on orbital shaker at 27°C. Reactions were quenched by adding 20 l of EDTA (500 mM), and activity was determined by measuring the fluorescence at 330 nm excitation and 395 nm emission in a CytoFluor 96-Well plate reader. The data were analyzed as described previously (Xue et al., 1998). ADAM-TS Activity Assay. ADAMTS enzymes are ADAM family proteins with thrombospondin type I motifs. ADAMTSs are active metalloproteinases associated with the extracellular matrix. The inhibition of selected ADAMTS family members was determined by using an enzyme-linked immunosorbent assay (ELISA)-like assay format. A biotinylated 41-amino acid peptide substrate was immobilized onto streptavidin-coated 96-well microtiter plates. Proteolysis of this substrate at the correct amide bond was then detected using an antibody specific for the neoepitope generated upon hydrolysis (Miller et al., 2003). Focal Brain Ischemia. Rats were housed and cared for in accordance with the National Insititutes of Health’s Guide for the Care and Use of Laboratory Animals. Procedures using lab animals were approved by our Institutional Animal Care and Use Committee. Male Sprague-Dawley rats at 20 weeks of age weighing 340 to 450 g were used for these experiments. Rats were anesthetized with gas inhalation composed of a 30% oxygen (0.3 liter/min) and 70% nitrous oxide (0.7 liter/min) mixture. The gas was passed through an isoflurane vaporizer set to deliver 3 to 4% isoflurane during initial induction and 1.5 to 2% during surgery. Details for animal care and monitoring during operation have been described previously (Wang