A long-lived peptide-conjugated iridium(iii) complex as a luminescent probe and inhibitor of the cell migration mediator, formyl peptide receptor 2.

Chemical Science View Article Online Open Access Article. Published on 01 October 2018. Downloaded on 11/22/2018 9:46:57 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. EDGE ARTICLE Cite this: Chem. Sci., 2018, 9, 8171 All publication charges for this article have been paid for by the Royal Society of Chemistry View Journal | View Issue A long-lived peptide-conjugated iridium(III) complex as a luminescent probe and inhibitor of the cell migration mediator, formyl peptide receptor 2† Kasipandi Vellaisamy,‡a Guodong Li,‡b Wanhe Wang, and Dik-Lung Ma *a ‡a Chung-Hang Leung *b Formyl peptide receptors play important biological and therapeutic roles in wound repair and inflammatory diseases. In this work, we present a luminescent iridium(III) complex (6) conjugated with the peptide agonist WKYMVm as a luminescent formyl peptide receptor 2 (FPR2) imaging probe in living cells. Complex 6 displayed ideal cell imaging characteristics, high photostability and low cytotoxicity. Competition assays with a known FPR2 antagonist, WRW4, and siRNA knockdown experiments both revealed that complex 6 selectively targeted FPR2 in living HUVEC cells. Moreover, complex 6 regulated FPR2 signalling in HUVEC Received 21st June 2018 Accepted 29th September 2018 cells as shown using a mechanical scratch assay. Finally, complex 6 reduced epithelial cell migration capacity and inhibited lipoxin A4 (LXA4)-triggered cell migration in HUVEC cells, demonstrating the DOI: 10.1039/c8sc02733a ability of this complex to inhibit FPR2 in living cells. To our knowledge, this is the first long-lived probe rsc.li/chemical-science for imaging FPR2 in living cells. Introduction Leukocyte responses to chemotactic factors or chemoattractants during inammation are dependent on specic G protein-coupled receptors.1 One class of these receptors is formyl-peptide receptors (FPRs), which include formyl peptide receptor 1 (FPR1), formyl peptide receptor 2 (ALX/FPR2 or FPR2), and formyl peptide receptor 3 (FPR3).2 FPRs facilitate the trafficking of phagocytes to the site of infection, and modulate the survival and the phagocytic activity of inltrating cells. Moreover, FPRs play an important role in inammatory angiogenesis.3 A cathelicidin peptide, LL-37, promoted angiogenesis in human umbilical vein endothelial cells (HUVECs) through an FPR2 signalling pathway.4 In liver epithelial cells, FPRs are capable of responding to mitochondrial antigens to enhance wound healing caused by linear scratch injury.5 In another study, annexin A1 (ANXA1) activated FPRs to repair intestinal wounds by a redox process.6 However, the understanding of FPR functions in wound healing is limited. Moreover, FPRs are emerging as therapeutic targets for a range of diseases, including cancer, inammation, amyloidosis and a Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China. E-mail: b State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China. E-mail: † Electronic supplementary 10.1039/c8sc02733a information (ESI) ‡ These authors contributed equally to this work. This journal is © The Royal Society of Chemistry 2018 available. See DOI: neurodegenerative diseases.7 Therefore, the development of highly sensitive and selective probes for FPRs is required for further elucidating the biological and therapeutic roles of FPRs in wound repair and disease. Noninvasive molecular-imaging techniques are powerful tools for studying biological processes in living systems.8,9 Peptide-based imaging probes have been increasingly studied due to their compatibility with living systems, high binding affinity, and adequate permeability.10,11 For FPRs, an FPR1 targetable cFLFLF peptide complexed with various radioactive metals (e.g. Ga(III), In(III), Gd(III), 99Tc and 64Cu) has been widely employed for imaging of inammation in various animal models.12–16 Peptide-based uorescent probes have received considerable recent attention due to their high sensitivity, selectivity, and safe imaging performance.17 For example, peptide-based uorescent probes have been reported for imaging matrix metalloproteinase (MMP)18,19 and cysteine protease.20–22 However, only a few peptide-based uorescent probes have been reported for FPR1 16,23 and FPR2.24 Transition metal complexes are emerging as a promising class of luminophores for studying biological processes in living cells,25–28 due to their long emission lifetime, large Stokes shi, and high photostability. However, the poor cell permeability of transition metal complex-based probes still remains a challenge for their further bioimaging application. One effective strategy to overcome this bottleneck is the conjugation of a transition metal complex with a specically targetable peptide.29,30 In recent years, considerable effort has been made to develop Chem. Sci., 2018, 9, 8171–8177 | 8171 View Article Online Open Access Article. Published on 01 October 2018. Downloaded on 11/22/2018 9:46:57 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Chemical Science transition metal complex–peptide conjugates as imaging probes,29,31 such as cobaltocenium–peptide conjugates for studying cellular uptake,32 ruthenium-short peptide conjugates for nuclear staining,33 and ruthenium-cyclic RGD peptide conjugates for targeting integrin receptors.34 Considering the additional favorable characteristics possessed by iridium(III) complexes including high quantum yields and tunable luminescence,35,36 the combination of an iridium(III) complex and a peptide may provide additional synergism for bioimaging applications. However, peptide-functionalized iridium(III) complexes as bioimaging probes have not been widely explored.37–42 The hexapeptide WKYMVm is a selective FPR2 agonist with high affinity, which was identied through screening of a synthetic peptide library.43,44 WKYMVm promoted recovery from tissue damage via an FPR2-dependent pathway in various disease models,44 such as cutaneous wound healing of diabetic mice,45 and a hindlimb ischemia model.46 In this study, we conjugated WKYMVm to a cyclometalated iridium(III) complex to develop a new luminescent probe for FPR2 (Fig. 1). This peptide-conjugated cyclometalated iridium(III) complex 6 visualized FPR2 in living cells. As complex 6 bears the peptide ligand (WKYMVm) agonist, we believe that the peptideconjugated complex would not only be able to effectively recognize FPRs, but also regulate FPR2-mediated functions (Scheme 1).47 To our knowledge, this is the rst report of an iridium(III) complex for visualizing FPR2 in living cells. Edge Article Results and discussion Synthesis of peptide-conjugated iridium(III) complex 6 The peptide-conjugated iridium(III) complex 6 has the general structure of [Ir(C^N)2(N^N)](PF6) (where C^N ¼ 2-phenylpyridine (ppy), and N^N ¼ 1,10-phenanthrolin-5-am (...truncated)