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Cite this: DOI: 10.1039/c4cc09251a Received 21st November 2014, Accepted 9th January 2015

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A metal-based tumour necrosis factor-alpha converting enzyme inhibitor† Chung-Hang Leung,*a Li-Juan Liu,a Lihua Lu,b Bingyong He,b Daniel W. J. Kwong,b Chun-Yuen Wong*c and Dik-Lung Ma*b

DOI: 10.1039/c4cc09251a www.rsc.org/chemcomm

We report herein a novel iridium(III) complex 1 as an antitumour necrosis factor agent and the first metal-based inhibitor of TACE enzymatic activity. Complex 1 inhibited TNF-a secretion and p38 phosphorylation in human monocytic THP-1 cells.

Tumour necrosis factor-alpha (TNF-a) is a pro-inflammatory cytokine whose dysfunction plays a key role in the development of human inflammatory diseases.1 In particular, TNF-a overexpression has been implicated in cases of inflammatory bowel disease (IBD), which includes Crohn’s disease and ulcerative colitis, a chronic recurrent systemic disease that affects more than 4 million people worldwide.2 Different classes of inhibitors that directly target TNF-a have been developed for the treatment of inflammatory diseases, including monoclonal antibodies,3 small molecules4 and metal complexes.5 In the clinic, infliximab, a mouse-human chimeric antibody targeting TNF-a,6 has been used for the treatment of Crohn’s disease.7 However, biologics have the potential to cause anti-antibody immune responses and weaken the immune system to opportunistic infections.8 An alternative strategy to repressing TNF-a activity is to inhibit TNF-a converting enzyme (TACE), a metalloprotease that cleaves the 26 kDa membrane-bound TNF-a precursor protein (pro-TNF-a) to form the biologically active 17 kDa soluble TNF-a protein.9 Therefore, TACE has also been considered as a potential target for the treatment of inflammatory diseases.10 TACE, also known as disintegrin and metalloprotease 17 (ADAM17), belongs to the metzincin class of metalloproteinases that have the ability to separate membrane-anchored proteins from the cell surface in organisms.11 Besides cleaving pro-TNF-a, a

State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China. E-mail: [email protected] b Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China. E-mail: [email protected] c Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, China. E-mail: [email protected] † Electronic supplementary information (ESI) available: Experimental details. See DOI: 10.1039/c4cc09251a

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a wide range of other membrane-bound proteins have been recognized as the substrates for TACE, including the ligands for epidermal growth factor receptor (EGFR),12 cellular adhesion molecule L-selectin,13 and amyloid precursor protein (APP).14 To date, a number of compounds belonging to different chemical classes have been reported as TACE inhibitors.15 However, no TACE inhibitor has passed phase II clinical trials yet, with some compounds exhibiting lack of efficacy, high hepatotoxicity, doselimiting musculoskeletal side effects or failure of very potent low nanomolar inhibitors in cells and human whole blood.16 Metal complexes have emerged as a promising class of modulators of enzyme activities, protein–protein interactions and transcriptional activities.17 In particular, kinetically-inert transition metal complexes have been developed that display high degrees of selectivity for biomolecular targets due to their unique geometry and steric and electronic properties.18 To our knowledge, no metal-based inhibitors of TACE have yet been reported in the literature. We report herein a novel iridium(III) complex 1 as the first metal-based inhibitor of TACE enzymatic activity. In order to identify promising metal-based scaffolds as TACE inhibitors, we first examined the ability of an in-house panel of structurally diverse complexes containing various C^N and N^N ligands 1–4 (Fig. 1) to inhibit TACE enzymatic activity in a cell-free system. In this assay, the purified human recombinant TACE protein was incubated with the 5-FAM fluorescent substrate Abz-LAQAVRSSSR-Dpa and complexes 1–4. Cleavage of the substrate by TACE results in the restoration of fluorescence of 5-FAM. The results showed that complex 1 exhibited a significant inhibition of the interaction between TACE and the substrate (Fig. 2). Given the promising activity exhibited by complex 1, we decided to synthesize the rhodium(III) congener 5 in order to investigate the effect of metal ion substitution on TACE activity for this type of complex. Interestingly, we observed that the rhodium(III) complex 5, which differs from the iridium(III) complex 1 only in the nature of the metal center, showed reduced potency. DFT calculations conducted on the cations for complexes 1 and 5 showed that their molecular

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Fig. 3 Complex 1 inhibits TACE enzymatic activity as determined by a fluorimetric assay. Human purified TACE protein (1 mg mL 1) was preincubated with complex 1 at the indicated concentrations for 10 min, and the reaction was initiated by the addition of the 5-FAM fluorescent substrate (100 nM). After 30 min, the reaction was stopped and the fluorescence intensity of the wells was monitored at an excitation of 490 nm and an emission of 520 nm using a microplate reader. IC50 value: 28 mM. Error bars represent the standard deviations of the results from three independent experiments.

Fig. 1 Chemical structures of cyclometalated rhodium(III) and iridium(III) complexes 1–5.

Fig. 2 Complexes 1–5 inhibit TACE enzymatic activity as determined by a fluorimetric assay. Human purified TACE protein (1 mg mL 1) was preincubated with complexes 1–5 (50 mM) for 10 min, and the reaction was initiated by the addition of 5-FAM fluorescent substrate (100 nM). After 30 min, the reaction was stopped and the fluorescence intensity of the wells was monitored at an excitation of 490 nm and an emission of 520 nm using a microplate reader. TAPI-0 (1 mM) was used as a positive control. Error bars represent the standard deviations of the results from three independent experiments.

structures were very similar, but that the charge distribution throughout the complexes was quite different (Fig. S1 in the ESI†). More specifically, the metal center in complex 1 (Ir, ¨wdin atomic charge = 1.67) carried a higher negative charge Lo than in complex 5 (Rh, charge = 0.43). These data suggest that

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the charge localization within a metal complex may have a significant influence on the ability of the complexes to bind to and inhibit TACE enzymatic activity. The synthesis and characterization of complexes 1–5 (as PF6 salts) are given in the ESI.† The spectroscopic data of the complexes are presented in Table S1 (ESI†). The complexes were stable in [d6]DMSO/D2O (2 : 1) solution at 298 K and 310 K for at least seven days as revealed by 1H NMR spectroscopy and UV-Vis spectroscopy (Fig. S2 in the ESI† shows representative spectra for complex 1) and in DMSO/serum (2 : 1) solution at 298 K for at least seven days as verified by UV-Vis spectroscopy (Fig. S3 in ESI† shows representative spectra for complex 1). Complex 1 was then subjected to a dose-response assay to evaluate its potency against TACE enzymatic activity. The results revealed that complex 1 exhibited dose-dependent inhibition of TACE activity in vitro, with an IC50 value of 28 mM (Fig. 3). In contrast, the IC50 value of complex 5 against TACE activity was determined to be 4100 mM (Fig. S4 in the ESI†). In addition, a kinetic study was carried out using a fluorimetric assay to understand the nature of inhibition of TACE activity by complex 1 (Fig. S5 in ESI†). The apparent equilibrium dissociation constant (Kd app) for TACE bound to the 5-FAM fluorescent substrate was found to be 0.025 mM, and the Ki app value of complex 1 was determined to be 7.84 mM. The double-reciprocal plot showed that complex 1 decreased Amax but had no effect on Kd app, suggesting that complex 1 was a non-competitive inhibitor of TACE with respect to the 5-FAM fluorescent substrate. TACE catalyses the cleavage and release of soluble TNF-a from the surface of the cell. Therefore, a TNF-a immunoassay was performed to investigate the inhibition of complex 1 on TACE enzymatic activity in cellulo. A model of macrophage-like cells was obtained by treatment of monocytic leukemia THP-1 cells with phorbol 12-myristate 13-acetate (PMA) for three days.

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Fig. 4 Complex 1 inhibits TNF-a secretion from PMA-differentiated THP1 cells stimulated with LPS as determined by ELISA. THP-1 cells were treated with the indicated concentrations of complex 1 and stimulated with LPS (1 mg mL 1). Culture supernatants were transferred to microtitre wells containing immobilized with human TNF-a monoclonal antibody. The binding activity was detected using yellow antibody and HRPconjugated secondary antibody. IC50 value: 11.24 mM. Error bars represent the standard deviations of the results from three independent experiments.

PMA-differentiated THP-1 cells, which display enhanced expression of the receptor for lipopolysaccharides (LPS), adhered and stopped proliferating.19 The differentiated THP-1 cells were then further treated with complex 1 and LPS, and the secretion of TNF-a from the cells into culture supernatant was determined by ELISA. Gratifyingly, the results showed that complex 1 was able to reduce the secretion of TNF-a in PMAdifferentiated THP-1 cells induced by LPS in a dose-dependent manner, with an IC50 value of 11.24 mM (Fig. 4). This result suggests that complex 1 potentially inhibited TACE activity in THP-1 cells, blocking the processing of pro-TNF-a and slowing the release of soluble TNF-a. p38 MAP kinase, an important regulating factor in the response of inflammation or stress signals, directly activates TACE via phosphorylation of the cytoplasmic domain of TACE.20 Immunoblotting studies were conducted to monitor the impact of complex 1 on the expression and phosphorylation of p38 MAP kinase in differentiated THP-1 cells stimulated with

Fig. 5 Complex 1 inhibits the phosphorylation of p38 from PMAdifferentiated THP-1 cells stimulated with LPS. PMA-differentiated THP-1 cells were treated with the indicated concentrations of complex 1 and stimulated with LPS (1 mg mL 1). Protein lysates were analysed by western blotting using p-p38 and p38 antibodies. Equal loading was determined by b-actin levels.

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LPS. We found that complex 1 was able to reduce the phosphorylation of p38 MAP kinase without significant effect on the expression of p38 MAP kinase in LPS-stimulated cells treated with complex 1 compared to control cells (Fig. 5). These results suggest that the inhibition of TACE activity by complex 1 in cells may be partly attributed to the down-regulation of p38 MAP kinase phosphorylation. In summary, we have presented a novel iridium(III) complex 1 as the first metal-based inhibitor of TACE activity. Complex 1 blocked the secretion of TNF-a from PMA-differentiated THP-1 cells stimulated with LPS, presumably through the inhibition of TACE activity and/or p38 MAP kinase phosphorylation. DFT calculations and kinetic experiments were also performed to understand the mechanism of TACE inhibition by complex 1. We envisage that complex 1 may be considered as a useful scaffold for the development of more potent TACE inhibitors for the treatment of inflammatory diseases. This work is supported by Hong Kong Baptist University (FRG2/13-14/008), Centre for Cancer and Inflammation Research, School of Chinese Medicine (CCIR-SCM, HKBU), the Health and Medical Research Fund (HMRF/13121482), the Research Grants Council (HKBU/201811, HKBU/204612, and HKBU/201913), the French National Research Agency/Research Grants Council Joint Research Scheme (A-HKBU201/12), the Science and Technology Development Fund, Macao SAR (103/2012/A3) and the University of Macau (MYRG091(Y3-L2)-ICMS12-LCH, MYRG121(Y3-L2)ICMS12-LCH and MRG023/LCH/2013/ICMS).

Notes and references 1 J. R. Bradley, J. Pathol., 2008, 214, 149. 2 (a) L. Jostins, S. Ripke, R. K. Weersma, R. H. Duerr, D. P. McGovern, K. Y. Hui, J. C. Lee, L. P. Schumm, Y. Sharma and C. A. Anderson, et al., Nature, 2012, 491, 119; (b) M. F. Neurath, Nat. Rev. Immunol., 2014, 14, 329; (c) L. Kaufman, E. Loftus, W. Sandborn, P. Kammer and D. Pardi, Am. J. Gastroenterol., 2010, 105, S476. 3 (a) K. A. Papadakis, O. A. Shaye, E. A. Vasiliauskas, A. Ippoliti, M. C. Dubinsky, J. Birt, J. Paavola, S. K. Lee, J. Price and S. R. Targan, et al., Am. J. Gastroenterol., 2005, 100, 75; (b) A. Nesbitt, G. Fossati, M. Bergin, P. Stephens, S. Stephens, R. Foulkes, D. Brown, M. Robinson and T. Bourne, Inflammatory Bowel Dis., 2007, 13, 1323. 4 (a) S. Zhuang, B. Lu, R. A. Daubert, K. D. Chavin, L. Wang and R. G. Schnellmann, Kidney Int., 2009, 75, 304; (b) C. P. Papaneophytou, A. K. Mettou, V. Rinotas, E. Douni and G. A. Kontopidis, ACS Med. Chem. Lett., 2013, 4, 137; (c) D. S.-H. Chan, H.-M. Lee, F. Yang, C.-M. Che, C. C. L. Wong, R. Abagyan, C.-H. Leung and D.-L. Ma, Angew. Chem., Int. Ed., 2010, 49, 2860. 5 C.-H. Leung, H.-J. Zhong, H. Yang, Z. Cheng, D. S.-H. Chan, V. P.-Y. Ma, R. Abagyan, C.-Y. Wong and D.-L. Ma, Angew. Chem., Int. Ed., 2012, 51, 9010. 6 R. Baldassano, C. P. Braegger, J. C. Escher, K. DeWoody, D. F. Hendricks, G. F. Keenan and H. S. Winter, Am. J. Gastroenterol., 2003, 98, 833. 7 (a) M. C. Stephens, M. A. Shepanski, P. Mamula, J. E. Markowitz, K. A. Brown and R. N. Baldassano, Am. J. Gastroenterol., 2003, 98, 104; (b) H. Wajant, K. Pfizenmaier and P. Scheurich, Cell Death Differ., 2003, 10, 45. 8 M. A. Palladino, F. R. Bahjat, E. A. Theodorakis and L. L. Moldawer, Nat. Rev. Drug Discovery, 2003, 2, 736. 9 (a) R. A. Black, C. T. Rauch, C. J. Kozlosky, J. J. Peschon, J. L. Slack, M. F. Wolfson, B. J. Castner, K. L. Stocking, P. Reddy and S. Srinivasan, et al., Nature, 1997, 385, 729; (b) M. L. Moss, S. L. C. Jin, M. E. Milla, D. M. Bickett, W. Burkhart, H. L. Carter, W. J. Chen, W. C. Clay, J. R. Didsbury and D. Hassler, et al., Nature, 1997, 386, 738; (c) A. J. H. Gearing, P. Beckett, M. Christodoulou,

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Published on 09 January 2015. Downloaded by Selcuk University on 23/01/2015 03:09:30.

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M. Churchill, J. M. Clements, M. Crimmin, A. H. Davidson, A. H. Drummond, W. A. Galloway and R. Gilbert, et al., J. Leukocyte Biol., 1995, 57, 774. (a) R. C. Newton, K. A. Solomon, M. B. Covington, C. P. Decicco, P. J. Haley, S. M. Friedman and K. Vaddi, Ann. Rheum. Dis., 2001, 60, iii25; (b) M. L. Moss, J. M. White, M. H. Lambert and R. C. Andrews, Drug Discovery Today, 2001, 6, 417. (a) C. P. Blobel, Nat. Rev. Mol. Cell Biol., 2005, 6, 32; (b) K. Maskos, C. Fernandez-Catalan, R. Huber, G. P. Bourenkov, H. Bartunik, G. A. Ellestad, P. Reddy, M. F. Wolfson, C. T. Rauch and B. J. Castner, et al., Proc. Natl. Acad. Sci. U. S. A., 1998, 95, 3408. (a) P. A. Kenny and M. J. Bissell, J. Clin. Invest., 2007, 117, 337; (b) A. Franovic, I. Robert, K. Smith, G. Kurban, A. Pause, L. Gunaratnam and S. Lee, Cancer Res., 2006, 66, 8083; (c) M. Borrell-Pages, F. Rojo, J. Albanell, J. Baselga and J. Arribas, EMBO J., 2003, 22, 1114; (d) N. Normanno, C. Bianco, A. De Luca and D. S. Salomon, Front. Biosci., 2001, 6, D685. (a) B. Ponnuchamy and R. A. Khalil, Circ. Res., 2008, 102, 1139; (b) D. M. Smalley and K. Ley, J. Cell. Mol. Med., 2005, 9, 255. (a) M. L. Kim, B. Zhang, I. P. Mills, M. E. Milla, K. R. Brunden and V. M. Lee, J. Neurosci., 2008, 28, 12052; (b) T. M. J. Allinson, E. T. Parkin, A. J. Turner and N. M. Hooper, J. Neurosci. Res., 2003, 74, 342. S. DasGupta, P. R. Murumkar, R. Giridhar and M. R. Yadav, Bioorg. Med. Chem., 2009, 17, 444. (a) S. Elliott and T. Cawston, Drugs Aging, 2001, 18, 87; (b) M. L. Moss, L. Sklair-Tavron and R. Nudelman, Nat. Clin. Pract. Rheumatol., 2008, 4, 300; (c) P. R. Murumkar, S. DasGupta, S. R. Chandani, R. Giridhar and M. R. Yadav, Expert Opin. Ther. Pat., 2010, 20, 31.

Chem. Commun.

ChemComm 17 (a) P. C. Bruijnincx and P. J. Sadler, Curr. Opin. Chem. Biol., 2008, 12, 197; (b) H. Huang, P. Zhang, B. Yu, Y. Chen, J. Wang, L. Ji and H. Chao, J. Med. Chem., 2014, 57, 8971; (c) E. Meggers, Angew. Chem. Int. Ed., 2011, 50, 2442; (d) C.-M. Che and F. M. Siu, Curr. Opin. Chem. Biol., 2010, 14, 255; (e) C. G. Hartinger, M. Groessl, S. M. Meier, A. Casini and P. J. Dyson, Chem. Soc. Rev., 2013, 42, 6186; ( f ) A. Meyer, C. P. Bagowski, M. Kokoschka, M. Stefanopoulou, H. Alborzinia, S. Can, D. H. Vlecken, W. S. Sheldrick, S. Wolfl and I. Ott, Angew. Chem., Int. Ed., 2012, 51, 8895; ( g) A. Leonidova, V. Pierroz, L. A. Adams, N. Barlow, S. Ferrari, B. Graham and G. Gasser, ACS Med. Chem. Lett., 2014, 5, 809; (h) A. J. McConnell, M. H. Lim, E. D. Olmon, H. Song, E. E. Dervan and J. K. Barton, Inorg. Chem., 2012, 51, 12511; (i) S. Lee, X. Y. Zheng, J. Krishnamoorthy, M. G. Savelieff, H. M. Park, J. R. Brender, J. H. Kim, J. S. Derrick, A. Kochi and H. J. Lee, et al., J. Am. Chem. Soc., 2014, 136, 299; ( j) D.-L. Ma, H.-Z. He, K.-H. Leung, D. S.-H. Chan and C.-H. Leung, Angew. Chem., Int. Ed., 2013, 52, 7666; (k) C.-H. Leung, S. Lin, H.-J. Zhong and D.-L. Ma, Chem. Sci., 2014; (l) C.-H. Leung, H.-Z. He, L.-J. Liu, M. Wang, D. S.-H. Chan and D.-L. Ma, Coord. Chem. Rev., 2013, 257, 3139; (m) D.-L. Ma, D. S.-H. Chan and C.-H. Leung, Acc. Chem. Res., 2014, 47, 3614; (n) B. Y.-W. Man, H.-M. Chan, C.-H. Leung, D. S.-H. Chan, L.-P. Bai, Z.-H. Jiang, H.-W. Li and D.-L. Ma, Chem. Sci., 2011, 2, 917. 18 K. J. Kilpin and P. J. Dyson, Chem. Sci., 2013, 4, 1410. 19 S. C. Dreskin, G. W. Thomas, S. N. Dale and L. E. Heasley, J. Immunol., 2001, 166, 5646. 20 (a) P. L. Xu and R. Derynck, Mol. Cell, 2010, 37, 551; (b) S. Kumar, J. Boehm and J. C. Lee, Nat. Rev. Drug Discovery, 2003, 2, 717.

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A metal-based tumour necrosis factor-alpha converting enzyme inhibitor.

We report herein a novel iridium(III) complex 1 as an antitumour necrosis factor agent and the first metal-based inhibitor of TACE enzymatic activity...
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