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Why and how to find neutraligands targeting chemokines?
Drug Discovery Today: Technologies
Vol. 9, No. 4 2012
Editors-in-Chief Kelvin Lam – Harvard University, USA Henk Timmerman – Vrije Universiteit, The Netherlands DRUG DISCOVERY
TODAY
TECHNOLOGIES
Chemokine receptors
Why and how to find neutraligands targeting chemokines? Jean-Luc Galzi1,*, Muriel Haas1, Nelly Frossard2, Marcel Hibert2 1
Biotechnology and Cellular Signalling, UMR 7242 CNRS-Universite´ de Strasbourg, School of Biotechnology, Bd Se´bastien Brant, BP 10413, 67412 Illkirch Cedex, France 2 Therapeutic Innovation, UMR 7200 CNRS-Universite´ de Strasbourg, Faculte´ de Pharmacie, 74 route du Rhin, 67401 Illkirch, France
Inspired by viruses and parasites that protect themselves from host immune system with the help of
Section Editor: Martine Smit – Vrije Universiteit, Amsterdam
neutralizing substances that block the action of chemokines, an emerging class of new compounds with anti-inflammatory and anticancer properties can be developed. These small chemicals interact with the chemokine to prevent its actions on the subset of its natural target chemokine receptors. A proof of concept, given by a small molecule that neutralize CXCL12, and act in vivo as anti-inflammatory in a mouse model of airway hypereosinophilia will be illustrated. Strategies to identify and characterize such ‘neutraligands’ are presented, as well as their potential interest in the therapeutic armamentarium targeting chemokine signaling is discussed. Introduction Chemokines are small, secreted chemotactic cytokines that ensure the key function of attracting leukocytes during inflammatory processes. Chemokines, produced by specific cell types on the site of inflammation, cross the endothelial cell wall and remain immobilized on the luminal surface of the endothelium to activate leukocytes and trigger their egress from the blood circulation to the inflamed tissue. In addition to attracting immune and inflammatory cells, chemokines contribute to brain, cardiac and vascular tissue
*Corresponding author.: J.-L. Galzi ([email protected]) 1740-6749/$ ß 2012 Elsevier Ltd. All rights reserved.
patterning during development, as well as to the regulation of gene expression to control cell proliferation and apoptosis. There are nowadays 45 different chemokines that all bind and activate ca. 25 receptors from the family of G protein-coupled receptors linked to Gi-mediated intracellular signaling. The inflammatory response is meant to the repair of damaged tissue after wound, allergen stimulation, or infection by microorganisms and parasites. Chemokines and their receptors, together with other mediators of inflammation, contribute to the onset of the inflammatory response, to its maintenance, as well as to its resolution. Dysfunction of the chemokine system, like maintenance of chemokine expression over time or decreased function of clearing processes, will result in impairment of the resolution phase. Chronic inflammatory processes [1] such as those occurring in asthma, chronic obstructive pulmonary disease, inflammatory bowel disease, transplant rejection or neuropathies, as well as autoimmune diseases like multiple sclerosis, rheumatoid arthritis, psoriasis or lupus erythematosus, will then take place.
Chemokines retraction in inflammation and infection: state of the art Decoy receptors Resolution is a multistep process that is regulated throughout the inflammatory response. Regarding chemokine signaling systems, this process includes termination of chemokine expression, degradation of the chemokine, induction of
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decoy receptor protein expression, desensitization of chemokine receptors, or down regulation of chemokine receptor [2]. Of particular interest are the systems involved in chemokine trapping, also called decoys, which are used under normal and pathological conditions. Human beings as well as different categories of pathogens naturally express decoy proteins that intercept chemokines and lower their circulating levels. In humans, decoy proteins specialized in chemokine clearance are G protein coupled receptor-like transmembrane proteins known as D6, DARC, CRAM-B, CCX-CKR. These proteins bind chemokines, without activating intracellular responses, and undergo co-internalization with the bound chemokine [3]. DARC, which binds 12 different chemokines, is expressed at high levels on red blood cells where it functions in clearing the blood from circulating chemokines. DARC is also expressed on endothelial cells from post capillary venules under normal conditions, and becomes expressed in arteries and capillaries during infection, inflammation or graft rejection [4]. The absence of DARC expression in certain human populations [5] correlates with aggravated inflammatory reactions. The D6 protein binds 12 different chemokines, mostly proinflammatory CC chemokines. Its expression is strongly regulated, and significant expression is detected on leukocytes that invade inflamed tissues [6]. D6 is a constitutively recycling protein predominantly located on endosomes where the bound chemokine is directed for degradation. Its most likely function thus is the clearing of tissues from remaining chemokine, as part of the resolution phase [7]. When heterologously expressed, the CCX-CKR decoy protein promotes continuous chemokine internalization and scavenges large amounts of the homeostatic CCR7-selective chemokines CCL19 and CCL21, controlling naı¨ve T cell and dendritic cell trafficking [8,9]. In vivo, CCX-CKR is expressed in lymphoid organs, stromal and epidermal cells. It controls homeostasis of chemokines, regulates immune response kinetic, and influences CD4+ T cell differentiation. CCXCKR mRNA levels are reduced in response to proinflammatory cytokines such as TNFa, interferon g or interleukin-1b, supporting participation of this decoy protein in inflammation. In addition to being cleared by CCX-CKR, CCL19 is also scavenged by another chemokine decoy receptor, CRAM-B, which is expressed on B cells in a maturation stage-dependent manner, and modulates T- and B-lymphocytes and dendritic cell trafficking [10]. Although still under investigation to firmly establish its physiological function(s) the second CXCL12 receptor, CXCR7, is endowed with a decoy function [11,12]. This receptor signals neither through G proteins, nor through MAP-kinase or PI3 kinase pathways. It however recruits beta-arrestin in response to CXCL12 and CXCL11 stimulation and internalizes these two chemokines. In an elegant study [13], CXCR7 was shown to control directionality of e246
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migrating primordium germ cells during fish lateral line development. In all circumstances where expression of any of the decoy receptor protein is lowered, the inflammatory responses is more severe [5], and cancer tumor development is enhanced [14,15].
Receptor mediated internalization of chemokine Besides this set of decoy receptors ‘professionalized’ in chemokine clearing, the conventional chemokine receptors also contribute to the regulation of circulating chemokine concentrations, by scavenging them in an inflammation-controlled manner or in homeostatic conditions. The initial demonstration was made with CCR5, the expression of which is upregulated in apoptotic neutrophils and T cells to sequester CCL3, CCL4, CCL5 chemokines, and thus contribute to the resolution of the immune/inflammatory response [16]. Similarly, CCR2/ mice have extremely high levels of CCL2 at sites of allo-induced inflammation, and mice deleted from CX3CR1, CXCR2 or CXCR3 exhibit significantly increased levels of the chemokines CX3CL1, CXCL1 or CXCL10 in serum and tissues [17]. This shows that chemokine receptors not only mediate immune/inflammatory responses, but also partly define response thresholds, and contribute to the resolution phase of the inflammation process.
Why should one find neutraligands? The principle of removing chemokines or blocking their action to lower immune response, by means of proteins and molecules that intercept normal chemokine signaling, is largely exploited by pathogens and parasites that infect mammals including humans [18,19]. The topic has been studied since the beginning of the 1980s and several major avoidance strategies were identified (Fig. 1): (i) production of soluble or membrane-bound chemokine binding proteins that neutralize and/or internalize chemokines, (ii) production of soluble proteins that inhibit chemokine binding to the extracellular matrix and thus prevent chemoattractant gradient formation, and (iii) production of chemokine-like proteins that antagonize chemokine receptors; in addition, pathogens also target the chemokine receptors by (iv) producing functional chemokine receptors on the pathogen (schistosoma) to help it finding the target cells, or (v) producing chemokine receptors in the infected cell (viral infections) to promote cell growth and survival. In all cases, the efficiency of the immune/inflammatory system is significantly lowered and pathogens thrive in host tissues [20,3,19]. In a drug discovery strategy, chemokine receptor blockade may be hazardous as it may (i) directly affect chemokine levels and body responsiveness to exogenous aggression like allergens and/or tumor growth, or (ii) selectively affect only a subset of responses linked to a given receptor [21,22]. Therefore, attention turned toward evaluating the potential of
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Drug Discovery Today: Technologies | Chemokine receptors
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Figure 1. The different modes of chemokine retraction. There are several types of chemokine interceptors, or scavengers. (a) The membrane bound interceptors mimic the natural chemokine receptor, but are devoid of signaling properties. They can be of endogenous or exogenous origin, (b) the soluble interceptors, or neutraligands, such as viral soluble proteins, evasins or the small molecules described in the text, bind to the chemokine and prevent its interaction with the chemokine receptor and (c) the second category of soluble interceptors that bind to the domain that recognizes extracellular matrix components such as GAGs. These molecules abolish gradient formation and render chemokines available to the interaction with the membrane bound interceptors for internalization in cells.
molecules that abrogate chemokine signaling by interacting with the chemokine. This investigation started by testing pathogen decoy proteins in disease models. The soluble protein M-T7 from myxoma virus has been evaluated as a potential therapeutic tool. M-T7 reduces post-operative responses in murine models of tissue engraftment [23,24] in a spectacular manner. The more recently identified evasin proteins from blood sucking ticks also open promising therapeutic routes [25]. Evasins form a group of four small immuno-modulating proteins that bind chemokines with moderate to high selectivity and neutralize them [26]. Evasin-1 efficiently chelates CCL3 and reduces inflammatory responses, that is neutrophil influx, to intraperitoneal injection of CCL3 in mice [26]. New drug discovery programs, as a consequence, focus on the identification of small molecules that bind to chemokines, rather than to their receptors, with the rationale that inhibition of chemokines will have fewer side effects on tissue physiology, as compared to inhibition of the receptor itself as discussed above.
How to find neutraligands? A general approach to identify small neutralizing molecules targeting chemokines, hereafter referred to as neutraligands, addresses the issue of possible interactions between the chemokine and a drug, preferably with the possibility to carry out
time resolved recordings. This approach can be declined in several ways, that combine direct binding monitoring together or not with virtual screening. One example is that of chemokine–chemokine receptor monitoring through fluorescence resonance energy transfer (FRET) as shown in Fig. 2 [27–29]. In this assay, the chemokine receptor is fused with Green Fluorescent Protein (GFP) at its amino terminal end, and the chemokine is chemically labeled with a fluorophore that behaves as an acceptor of excited GFP, typically Texas Red, lissamine, or derivatives in the Bodipy and Alexa series. Binding of the fluorescent chemokine to its receptor is detected as a reversible reduction of GFP fluorescence emission. Among variants of this assay, readers can refer to homogeneous time resolved fluorescence (HTRF) techniques [30]. Detection of chemokine neutraligands relies on the principle that the binding process can be biased to detect binding to a receptor or to the chemokine, depending on the sequence of addition of molecules (Fig. 3A), and provided that binding can be monitored in real time. Specifically, the compound to be tested is preincubated (from minutes up to hours) with the cells expressing the chemokine receptor or with the chemokine before FRET recording. The specific protocol adaptation consists in preparing daughter test compound plates in which the tracer ligand (fluorescent chemokine) is added to the wells to achieve preincubation of the chemokine with the compounds. Real time binding www.drugdiscoverytoday.com
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Figure 2. Fluorescence resonance energy transfer as a mean to find new molecules. Left panel: The chemokine receptor CXCR4 (green) is expressed as a functional fusion protein with EGFP in mammalian cells. The chemokine CXCL12 (red) is produced by total chemical synthesis and chemically linked to the fluorescent group Texas Red [28]. Right panel: Upon addition of Texas Red-labeled CXCL12 to cells expressing GFP-CXCR4, the intensity of GFP emission (510 nm) declines in a time- and dose-dependent manner. Emission reduction is prevented by previous addition – or reversed by subsequent addition – of a competitor. Quantification of the interaction parameters for both the fluorescent chemokine and for the competitor is easily carried out by analyzing signals obtained at variable compound doses. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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Figure 3. How to find and characterize chemokine neutraligand binding. Identification of a neutralizing compound relies on a kinetic-based binding determination according to the following principle. Preincubation of a molecule with its ‘receptor’ prior to detect ligand binding will bias ligand interaction kinetics. Accordingly, the chemical library must be preincubated with the chemokine (A) before being added to cells expressing the chemokine receptor. In the example shown in (B) the amplitude of CXCL12 binding to CXCR4 (similar to association in Fig. 2 right panel) is much smaller when the neutraligand is preincubated with the chemokine than when the neutraligand is preincubated with the receptor. The signal can be monitored on commercial calcium response detectors. N.B. the same experiment carried out with a classical competitor (i.e. T134 or AMD3100) results in mirror image. (C) Characterization of direct binding of neutraligand to a chemokine. The example reports on the intrinsic fluorescence of the unique tryptophan residue from CXCL12. The active neutraligand (triangles) extinguishes Trp fluorescence in a dose dependent manner while an inactive analog does not. Other protocols of direct binding investigations are reported in [29].
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observation that CXCR4 binds sulphated CXCL12 with high affinity, rational drug design was carried out. The structure of CXCR4 peptides bound to sulphated CXCL12 was solved by NMR, and the resulting structural models used for in silico ligand design. A compound, 3-(naphthalene-2-carbonylthiocarbamoylamino) benzoic acid (ZINC ID 310454), which binds the chemokine and competes with CXCR4 [31], was identified. Another, more generic approach, uses immobilized chemokines, and detects interactions with peptides from libraries or fluids through surface plasmon resonance. This is a powerful approach to discover chemokine binders, among which some will interfere with chemokine–chemokine receptor complex formation. Other compounds, such as those that prevent interactions with glycosaminoglycans (GAGs) may also be detected. The major hurdle with SPR detection still remains the size of small molecule ligands, which cause small mass variation when they bind to the chemokine. Better success rate are therefore obtained with peptides larger than ten amino acids, that is with compounds of 1200 Da or more molecular weight. Regarding functional characterization of newly identified neutraligands, specific response protocols can be adapted from those developed for binding monitoring. It can indeed be demonstrated that preincubation of a neutraligand with a chemokine will affect functional response properties in a manner that differs from those obtained after preincubation of the neutraligand with cells expressing the receptor. This is generally easily detected as responses such as chemotaxis, calcium elevation or cyclic AMP regulation are under kinetic control. Fig. 4A illustrates how experimental conditions can
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kinetic is necessarily recorded because at equilibrium, the level of chemokine binding will not depend on the preincubation process. In the example shown in Fig. 3B, the amplitude of FRET recording (receptor sites occupancy) is low when the neutraligand is preincubated with the chemokine, and much larger when the neutraligand is preincubated with the receptor (to which it does not bind). Chalcone 4 or ((E)-1-(40 chlorophenyl)-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1one) which neutralizes CXCL12 was discovered that way, as a compound that inhibits binding of CXCL12 to CXCR4 [29]. Noteworthy, the assay can be conducted in a high throughput format on calcium-type plate readers on which GFP fluorescence can be monitored in real time to follow ligand binding kinetic. Reading the same plate before and after chemical library plus chemokine addition further allows parallelization of data analyses to improve assay robustness. Confirmation of the neutralizing properties is carried out using biophysical methods and functional assays. In the instance of CXCL12 neutralization by chalcones, the active chalcone molecule, chalcone 4 [29] quenches intrinsic fluorescence of the unique tryptophan residue present in CXCL12, not the inactive analogs. Also, CXCL12 increases active chalcone molecule solubility in a highly significant manner and finally, microcalorimetry allows the interaction between chalcone and chemokine to be quantified. Other biophysical methods include surface plasmon resonance (SPR) [26] (see below), NMR heteronuclear shift perturbation determinations [31] or co-crystal structure solution. Some of the above mentioned approaches have led to the identification of neutraligands [31,32]. Based on the
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Figure 4. How to find and characterize chemokine neutraligand function. The neutraligand can be characterized in vitro and in vivo. (A) Human peripheral blood CD4+ lymphocytes (PBLs) migrate toward CXCL12 at a 30 nM concentration in the lower compartment of a Boyden chamber. If the neutraligand is preincubated with PBLs, chemotaxis is not different from control experiment with CXCL12 alone. By contrast, if neutraligand is preincubated with chemokine, chemotaxis is inhibited in a dose dependent manner. (B) The panel reports cell type counting in bronchoalveolar lavages from mice that have developed an allergic response to ovalbumin. As was reported for anti-CXCR4 or anti-CXCL12 antibodies [39], anti-CXCL12 neutraligand inhibits recruitment of eosinophils (eosino) and lymphocytes (lympho) in the airway, as previously described for Th2 mediated inflammatory responses. Macrophages (macro); neutrophils (neutro).
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be set up to discriminate chemotactic inhibition resulting from receptor inhibition or chemokine neutralization. In the example shown, chalcone 4 displays efficient inhibition of CD4+ peripheral blood lymphocyte chemotaxis when preequilibrated with CXCL12, not with the CXCR4-positive cells. Similar results are obtained in calcium concentration elevation.
Functional explorations with neutraligands targeting chemokines When two receptors bind the chemokine, as is the case in HPV immortalized keratinocytes that express CXCR4 and CXCR7, functional responses to neutraligand and to CXCR4 antagonist differ quantitatively and qualitatively. In these cells that overexpress CXCL12 and its two receptors, inhibition of cell proliferation is better achieved with chalcone 4 than with blockade of any of the two receptors using specific antibodies or ligands [33]. Inhibition of chemokine signaling by neutraligands is functionally confirmed at the whole animal level, on disease models (Haas 2008, Vieira 2009, Montecucco 2010, Russo 2011). Anti-CXCL12 neutraligand for instance can be injected intraperitoneally for weeks to mice without any sign of toxic effect. CXCL12 neutralization by chalcone 4 reduces eosinophilic infiltration into the inflamed airways in a mouse model of allergic hypereosinophilia [29], Fig. 4B. Whereas neutralization by evasin-3 blocks eosinophilia into the gut in a model of experimental colitis [34], or neutrophilia during myocardial injury [35]. Neutralization by evasin-1 reduces leukocyte infiltration in the lung in a model of pulmonary fibrosis [36].
Concluding remarks Altogether, there are accumulating evidence that neutralization of chemokines with small chemical compounds is realistic, and that it represents a potent chemical biology – as well as therapeutic – approach to study chemokine signaling and to treat inflammatory disorders. Regarding inflammation, one important issue will be to determine whether the inhibition of chemokine signaling will allow the resolution strategy to occur. This will have to be studied in physiological and pathophysiological instances. It is now known for several receptors from the CXC, CC or decoy groups whether and how they participate in the onset or the resolution of inflammation [16,6,3,37,38], and thus how they might be targeted for anti-inflammatory and resolution therapeutic indications. Similarly, neutralization of chemokines should be considered with regard to chemokine gradient disruption, chemokine sequestering and/or draining activity to optimally envision therapeutic development.
References 1 Koelink, P.J. et al. (2011) Targeting chemokine receptors in chronic inflammatory diseases: an extensive review. Pharmacol. Ther. 133, 1–18
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2 Bennett, L.D. et al. (2011) Mechanisms regulating chemokine receptor activity. Immunology 134, 246–256 3 Mantovani, A. et al. (2006) Tuning inflammation and immunity by chemokine sequestration: decoys and more. Nat. Rev. Immunol. 6, 907–918 4 Gardner, L. et al. (2006) Temporal expression pattern of Duffy antigen in rheumatoid arthritis: up-regulation in early disease. Arthritis Rheum. 54, 2022–2026 5 Vergara, C. et al. (2008) Gene encoding Duffy antigen/receptor for chemokines is associated with asthma and IgE in three populations. Am. J. Respir. Crit. Care Med. 178, 1017–1022 6 Graham, G.J. and McKimmie, C.S. (2006) Chemokine scavenging by D6: a movable feast? Trends Immunol. 27, 381–386 7 Jamieson, T. et al. (2005) The chemokine receptor D6 limits the inflammatory response in vivo. Nat. Immunol. 6, 403–411 8 Comerford, I. et al. (2006) The chemokine receptor CCX-CKR mediates effective scavenging of CCL19 in vitro. Eur. J. Immunol. 36, 1904–1916 9 Comerford, I. et al. (2011) The atypical chemokine receptor CCX-CKR scavenges homeostatic chemokines in circulation and tissues and suppresses Th17 responses. Blood 116, 4130–4140 10 Catusse, J. et al. (2010) Role of the atypical chemoattractant receptor CRAM in regulating CCL19 induced CCR7 responses in B-cell chronic lymphocytic leukemia. Mol. Cancer 9, 297 11 Thelen, M. and Thelen, S. (2008) CXCR7, CXCR4 and CXCL12: an eccentric trio? J. Neuroimmunol. 198, 9–13 12 Levoye, A. et al. (2009) CXCR7 heterodimerizes with CXCR4 and regulates CXCL12-mediated G protein signaling. Blood 113, 6085–6093 13 Dambly-Chaudiere, C. et al. (2007) Control of cell migration in the development of the posterior lateral line: antagonistic interactions between the chemokine receptors CXCR4 and CXCR7/RDC1. BMC Dev. Biol. 7, 23 14 Wang, J. et al. (2006) Enhanced expression of Duffy antigen receptor for chemokines by breast cancer cells attenuates growth and metastasis potential. Oncogene 25, 7201–7211 15 Wang, J. et al. (2008) The role of CXCR7/RDC1 as a chemokine receptor for CXCL12/SDF-1 in prostate cancer. J. Biol. Chem. 283, 4283–4294 16 Ariel, A. et al. (2006) Apoptotic neutrophils and T cells sequester chemokines during immune response resolution through modulation of CCR5 expression. Nat. Immunol. 7, 1209–1216 17 Cardona, A.E. et al. (2008) Scavenging roles of chemokine receptors: chemokine receptor deficiency is associated with increased levels of ligand in circulation and tissues. Blood 112, 256–263 18 Alcami, A. (2003) Viral mimicry of cytokines, chemokines and their receptors. Nat. Rev. Immunol. 3, 36–50 19 Galzi, J.L. et al. (2010) Neutralizing endogenous chemokines with small molecules. Principles and potential therapeutic applications. Pharmacol. Ther. 126, 39–55 20 Murphy, P.M. (2000) Viral antichemokines: from pathogenesis to drug discovery. J. Clin. Invest. 105, 1515–1517 21 Kiss, D.L. et al. (2009) The functional antagonist Met-RANTES: a modified agonist that induces differential CCR5 trafficking. Cell. Mol. Biol. Lett. 14, 537–547 22 Kim, H.Y. et al. (2010) The CXCR4 antagonist AMD3100 has dual effects on survival and proliferation of myeloma cells in vitro. Cancer Res. Treat. 42, 225–234 23 Bedard, E.L. et al. (2003) Chemokine-binding viral protein M-T7 prevents chronic rejection in rat renal allografts. Transplantation 76, 249–252 24 Liu, L. et al. (2004) Viral chemokine-binding proteins inhibit inflammatory responses and aortic allograft transplant vasculopathy in rat models. Transplantation 77, 1652–1660 25 Frauenschuh, A. et al. (2007) Molecular cloning and characterization of a highly selective chemokine-binding protein from the tick Rhipicephalus sanguineus. J. Biol. Chem. 282, 27250–27258 26 Deruaz, M. et al. (2008) Ticks produce highly selective chemokine binding proteins with antiinflammatory activity. J. Exp. Med. 205, 2019–2031 27 Vollmer, J.Y. et al. (1999) Subcellular compartmentalization of activation and desensitization of responses mediated by NK2 neurokinin receptors. J. Biol. Chem. 274, 37915–37922 28 Valenzuela-Fernandez, A. et al. (2001) Optimal inhibition of X4 HIV isolates by the CXC chemokine stromal cell-derived factor 1 alpha requires
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interaction with cell surface heparan sulfate proteoglycans. J. Biol. Chem. 276, 26550–26558 Hachet-Haas, M. et al. (2008) Small neutralizing molecules to inhibit actions of the chemokine CXCL12. J. Biol. Chem. 283, 23189–23199 Hermand, P. et al. (2008) Functional adhesiveness of the CX3CL1 chemokine requires its aggregation. Role of the transmembrane domain. J. Biol. Chem. 283, 30225–30234 Veldkamp, C.T. et al. (2010) Targeting SDF-1/CXCL12 with a ligand that prevents activation of CXCR4 through structure-based drug design. J. Am. Chem. Soc. 132, 7242–7243 Peled, A. et al. (2009) Methods of inhibiting chemokine binding to chemokine receptors. USP7488717 Chow, K.Y. et al. (2010) A pivotal role for CXCL12 signaling in HPVmediated transformation of keratinocytes: clues to understanding HPVpathogenesis in WHIM syndrome. Cell Host Microbe 8, 523–533
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Vieira, A.T. et al. (2009) Treatment with a novel chemokine-binding protein or eosinophil lineage-ablation protects mice from experimental colitis. Am. J. Pathol. 175, 2382–2391 Montecucco, F. et al. (2010) Single administration of the CXC chemokinebinding protein Evasin-3 during ischemia prevents myocardial reperfusion injury in mice. Arterioscler. Thromb. Vasc. Biol. 30, 1371–1377 Russo, R.C. et al. (2011) Therapeutic effects of evasin-1, a chemokine binding protein, in bleomycin-induced pulmonary fibrosis. Am. J. Respir. Cell Mol. Biol. 45, 72–80 Hansell, C. and Nibbs, R. (2007) Professional and part-time chemokine decoys in the resolution of inflammation. Sci. STKE 2007, pe18 Soehnlein, O. and Lindbom, L. (2010) Phagocyte partnership during the onset and resolution of inflammation. Nat. Rev. Immunol. 10, 427–439 Gonzalo, J.A. et al. (2000) Critical involvement of the chemotactic axis CXCR4/stromal cell-derived factor-1 alpha in the inflammatory component of allergic airway disease. J. Immunol. 165, 499–508
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Editors-in-Chief Kelvin Lam – Harvard University, USA Henk Timmerman – Vrije Universiteit, The Netherlands DRUG DISCOVERY
TODAY
TECHNOLOGIES
Chemokine receptors
Why and how to find neutraligands targeting chemokines? Jean-Luc Galzi1,*, Muriel Haas1, Nelly Frossard2, Marcel Hibert2 1
Biotechnology and Cellular Signalling, UMR 7242 CNRS-Universite´ de Strasbourg, School of Biotechnology, Bd Se´bastien Brant, BP 10413, 67412 Illkirch Cedex, France 2 Therapeutic Innovation, UMR 7200 CNRS-Universite´ de Strasbourg, Faculte´ de Pharmacie, 74 route du Rhin, 67401 Illkirch, France
Inspired by viruses and parasites that protect themselves from host immune system with the help of
Section Editor: Martine Smit – Vrije Universiteit, Amsterdam
neutralizing substances that block the action of chemokines, an emerging class of new compounds with anti-inflammatory and anticancer properties can be developed. These small chemicals interact with the chemokine to prevent its actions on the subset of its natural target chemokine receptors. A proof of concept, given by a small molecule that neutralize CXCL12, and act in vivo as anti-inflammatory in a mouse model of airway hypereosinophilia will be illustrated. Strategies to identify and characterize such ‘neutraligands’ are presented, as well as their potential interest in the therapeutic armamentarium targeting chemokine signaling is discussed. Introduction Chemokines are small, secreted chemotactic cytokines that ensure the key function of attracting leukocytes during inflammatory processes. Chemokines, produced by specific cell types on the site of inflammation, cross the endothelial cell wall and remain immobilized on the luminal surface of the endothelium to activate leukocytes and trigger their egress from the blood circulation to the inflamed tissue. In addition to attracting immune and inflammatory cells, chemokines contribute to brain, cardiac and vascular tissue
*Corresponding author.: J.-L. Galzi ([email protected]) 1740-6749/$ ß 2012 Elsevier Ltd. All rights reserved.
patterning during development, as well as to the regulation of gene expression to control cell proliferation and apoptosis. There are nowadays 45 different chemokines that all bind and activate ca. 25 receptors from the family of G protein-coupled receptors linked to Gi-mediated intracellular signaling. The inflammatory response is meant to the repair of damaged tissue after wound, allergen stimulation, or infection by microorganisms and parasites. Chemokines and their receptors, together with other mediators of inflammation, contribute to the onset of the inflammatory response, to its maintenance, as well as to its resolution. Dysfunction of the chemokine system, like maintenance of chemokine expression over time or decreased function of clearing processes, will result in impairment of the resolution phase. Chronic inflammatory processes [1] such as those occurring in asthma, chronic obstructive pulmonary disease, inflammatory bowel disease, transplant rejection or neuropathies, as well as autoimmune diseases like multiple sclerosis, rheumatoid arthritis, psoriasis or lupus erythematosus, will then take place.
Chemokines retraction in inflammation and infection: state of the art Decoy receptors Resolution is a multistep process that is regulated throughout the inflammatory response. Regarding chemokine signaling systems, this process includes termination of chemokine expression, degradation of the chemokine, induction of
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decoy receptor protein expression, desensitization of chemokine receptors, or down regulation of chemokine receptor [2]. Of particular interest are the systems involved in chemokine trapping, also called decoys, which are used under normal and pathological conditions. Human beings as well as different categories of pathogens naturally express decoy proteins that intercept chemokines and lower their circulating levels. In humans, decoy proteins specialized in chemokine clearance are G protein coupled receptor-like transmembrane proteins known as D6, DARC, CRAM-B, CCX-CKR. These proteins bind chemokines, without activating intracellular responses, and undergo co-internalization with the bound chemokine [3]. DARC, which binds 12 different chemokines, is expressed at high levels on red blood cells where it functions in clearing the blood from circulating chemokines. DARC is also expressed on endothelial cells from post capillary venules under normal conditions, and becomes expressed in arteries and capillaries during infection, inflammation or graft rejection [4]. The absence of DARC expression in certain human populations [5] correlates with aggravated inflammatory reactions. The D6 protein binds 12 different chemokines, mostly proinflammatory CC chemokines. Its expression is strongly regulated, and significant expression is detected on leukocytes that invade inflamed tissues [6]. D6 is a constitutively recycling protein predominantly located on endosomes where the bound chemokine is directed for degradation. Its most likely function thus is the clearing of tissues from remaining chemokine, as part of the resolution phase [7]. When heterologously expressed, the CCX-CKR decoy protein promotes continuous chemokine internalization and scavenges large amounts of the homeostatic CCR7-selective chemokines CCL19 and CCL21, controlling naı¨ve T cell and dendritic cell trafficking [8,9]. In vivo, CCX-CKR is expressed in lymphoid organs, stromal and epidermal cells. It controls homeostasis of chemokines, regulates immune response kinetic, and influences CD4+ T cell differentiation. CCXCKR mRNA levels are reduced in response to proinflammatory cytokines such as TNFa, interferon g or interleukin-1b, supporting participation of this decoy protein in inflammation. In addition to being cleared by CCX-CKR, CCL19 is also scavenged by another chemokine decoy receptor, CRAM-B, which is expressed on B cells in a maturation stage-dependent manner, and modulates T- and B-lymphocytes and dendritic cell trafficking [10]. Although still under investigation to firmly establish its physiological function(s) the second CXCL12 receptor, CXCR7, is endowed with a decoy function [11,12]. This receptor signals neither through G proteins, nor through MAP-kinase or PI3 kinase pathways. It however recruits beta-arrestin in response to CXCL12 and CXCL11 stimulation and internalizes these two chemokines. In an elegant study [13], CXCR7 was shown to control directionality of e246
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migrating primordium germ cells during fish lateral line development. In all circumstances where expression of any of the decoy receptor protein is lowered, the inflammatory responses is more severe [5], and cancer tumor development is enhanced [14,15].
Receptor mediated internalization of chemokine Besides this set of decoy receptors ‘professionalized’ in chemokine clearing, the conventional chemokine receptors also contribute to the regulation of circulating chemokine concentrations, by scavenging them in an inflammation-controlled manner or in homeostatic conditions. The initial demonstration was made with CCR5, the expression of which is upregulated in apoptotic neutrophils and T cells to sequester CCL3, CCL4, CCL5 chemokines, and thus contribute to the resolution of the immune/inflammatory response [16]. Similarly, CCR2/ mice have extremely high levels of CCL2 at sites of allo-induced inflammation, and mice deleted from CX3CR1, CXCR2 or CXCR3 exhibit significantly increased levels of the chemokines CX3CL1, CXCL1 or CXCL10 in serum and tissues [17]. This shows that chemokine receptors not only mediate immune/inflammatory responses, but also partly define response thresholds, and contribute to the resolution phase of the inflammation process.
Why should one find neutraligands? The principle of removing chemokines or blocking their action to lower immune response, by means of proteins and molecules that intercept normal chemokine signaling, is largely exploited by pathogens and parasites that infect mammals including humans [18,19]. The topic has been studied since the beginning of the 1980s and several major avoidance strategies were identified (Fig. 1): (i) production of soluble or membrane-bound chemokine binding proteins that neutralize and/or internalize chemokines, (ii) production of soluble proteins that inhibit chemokine binding to the extracellular matrix and thus prevent chemoattractant gradient formation, and (iii) production of chemokine-like proteins that antagonize chemokine receptors; in addition, pathogens also target the chemokine receptors by (iv) producing functional chemokine receptors on the pathogen (schistosoma) to help it finding the target cells, or (v) producing chemokine receptors in the infected cell (viral infections) to promote cell growth and survival. In all cases, the efficiency of the immune/inflammatory system is significantly lowered and pathogens thrive in host tissues [20,3,19]. In a drug discovery strategy, chemokine receptor blockade may be hazardous as it may (i) directly affect chemokine levels and body responsiveness to exogenous aggression like allergens and/or tumor growth, or (ii) selectively affect only a subset of responses linked to a given receptor [21,22]. Therefore, attention turned toward evaluating the potential of
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Figure 1. The different modes of chemokine retraction. There are several types of chemokine interceptors, or scavengers. (a) The membrane bound interceptors mimic the natural chemokine receptor, but are devoid of signaling properties. They can be of endogenous or exogenous origin, (b) the soluble interceptors, or neutraligands, such as viral soluble proteins, evasins or the small molecules described in the text, bind to the chemokine and prevent its interaction with the chemokine receptor and (c) the second category of soluble interceptors that bind to the domain that recognizes extracellular matrix components such as GAGs. These molecules abolish gradient formation and render chemokines available to the interaction with the membrane bound interceptors for internalization in cells.
molecules that abrogate chemokine signaling by interacting with the chemokine. This investigation started by testing pathogen decoy proteins in disease models. The soluble protein M-T7 from myxoma virus has been evaluated as a potential therapeutic tool. M-T7 reduces post-operative responses in murine models of tissue engraftment [23,24] in a spectacular manner. The more recently identified evasin proteins from blood sucking ticks also open promising therapeutic routes [25]. Evasins form a group of four small immuno-modulating proteins that bind chemokines with moderate to high selectivity and neutralize them [26]. Evasin-1 efficiently chelates CCL3 and reduces inflammatory responses, that is neutrophil influx, to intraperitoneal injection of CCL3 in mice [26]. New drug discovery programs, as a consequence, focus on the identification of small molecules that bind to chemokines, rather than to their receptors, with the rationale that inhibition of chemokines will have fewer side effects on tissue physiology, as compared to inhibition of the receptor itself as discussed above.
How to find neutraligands? A general approach to identify small neutralizing molecules targeting chemokines, hereafter referred to as neutraligands, addresses the issue of possible interactions between the chemokine and a drug, preferably with the possibility to carry out
time resolved recordings. This approach can be declined in several ways, that combine direct binding monitoring together or not with virtual screening. One example is that of chemokine–chemokine receptor monitoring through fluorescence resonance energy transfer (FRET) as shown in Fig. 2 [27–29]. In this assay, the chemokine receptor is fused with Green Fluorescent Protein (GFP) at its amino terminal end, and the chemokine is chemically labeled with a fluorophore that behaves as an acceptor of excited GFP, typically Texas Red, lissamine, or derivatives in the Bodipy and Alexa series. Binding of the fluorescent chemokine to its receptor is detected as a reversible reduction of GFP fluorescence emission. Among variants of this assay, readers can refer to homogeneous time resolved fluorescence (HTRF) techniques [30]. Detection of chemokine neutraligands relies on the principle that the binding process can be biased to detect binding to a receptor or to the chemokine, depending on the sequence of addition of molecules (Fig. 3A), and provided that binding can be monitored in real time. Specifically, the compound to be tested is preincubated (from minutes up to hours) with the cells expressing the chemokine receptor or with the chemokine before FRET recording. The specific protocol adaptation consists in preparing daughter test compound plates in which the tracer ligand (fluorescent chemokine) is added to the wells to achieve preincubation of the chemokine with the compounds. Real time binding www.drugdiscoverytoday.com
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Figure 2. Fluorescence resonance energy transfer as a mean to find new molecules. Left panel: The chemokine receptor CXCR4 (green) is expressed as a functional fusion protein with EGFP in mammalian cells. The chemokine CXCL12 (red) is produced by total chemical synthesis and chemically linked to the fluorescent group Texas Red [28]. Right panel: Upon addition of Texas Red-labeled CXCL12 to cells expressing GFP-CXCR4, the intensity of GFP emission (510 nm) declines in a time- and dose-dependent manner. Emission reduction is prevented by previous addition – or reversed by subsequent addition – of a competitor. Quantification of the interaction parameters for both the fluorescent chemokine and for the competitor is easily carried out by analyzing signals obtained at variable compound doses. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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Figure 3. How to find and characterize chemokine neutraligand binding. Identification of a neutralizing compound relies on a kinetic-based binding determination according to the following principle. Preincubation of a molecule with its ‘receptor’ prior to detect ligand binding will bias ligand interaction kinetics. Accordingly, the chemical library must be preincubated with the chemokine (A) before being added to cells expressing the chemokine receptor. In the example shown in (B) the amplitude of CXCL12 binding to CXCR4 (similar to association in Fig. 2 right panel) is much smaller when the neutraligand is preincubated with the chemokine than when the neutraligand is preincubated with the receptor. The signal can be monitored on commercial calcium response detectors. N.B. the same experiment carried out with a classical competitor (i.e. T134 or AMD3100) results in mirror image. (C) Characterization of direct binding of neutraligand to a chemokine. The example reports on the intrinsic fluorescence of the unique tryptophan residue from CXCL12. The active neutraligand (triangles) extinguishes Trp fluorescence in a dose dependent manner while an inactive analog does not. Other protocols of direct binding investigations are reported in [29].
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observation that CXCR4 binds sulphated CXCL12 with high affinity, rational drug design was carried out. The structure of CXCR4 peptides bound to sulphated CXCL12 was solved by NMR, and the resulting structural models used for in silico ligand design. A compound, 3-(naphthalene-2-carbonylthiocarbamoylamino) benzoic acid (ZINC ID 310454), which binds the chemokine and competes with CXCR4 [31], was identified. Another, more generic approach, uses immobilized chemokines, and detects interactions with peptides from libraries or fluids through surface plasmon resonance. This is a powerful approach to discover chemokine binders, among which some will interfere with chemokine–chemokine receptor complex formation. Other compounds, such as those that prevent interactions with glycosaminoglycans (GAGs) may also be detected. The major hurdle with SPR detection still remains the size of small molecule ligands, which cause small mass variation when they bind to the chemokine. Better success rate are therefore obtained with peptides larger than ten amino acids, that is with compounds of 1200 Da or more molecular weight. Regarding functional characterization of newly identified neutraligands, specific response protocols can be adapted from those developed for binding monitoring. It can indeed be demonstrated that preincubation of a neutraligand with a chemokine will affect functional response properties in a manner that differs from those obtained after preincubation of the neutraligand with cells expressing the receptor. This is generally easily detected as responses such as chemotaxis, calcium elevation or cyclic AMP regulation are under kinetic control. Fig. 4A illustrates how experimental conditions can
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kinetic is necessarily recorded because at equilibrium, the level of chemokine binding will not depend on the preincubation process. In the example shown in Fig. 3B, the amplitude of FRET recording (receptor sites occupancy) is low when the neutraligand is preincubated with the chemokine, and much larger when the neutraligand is preincubated with the receptor (to which it does not bind). Chalcone 4 or ((E)-1-(40 chlorophenyl)-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1one) which neutralizes CXCL12 was discovered that way, as a compound that inhibits binding of CXCL12 to CXCR4 [29]. Noteworthy, the assay can be conducted in a high throughput format on calcium-type plate readers on which GFP fluorescence can be monitored in real time to follow ligand binding kinetic. Reading the same plate before and after chemical library plus chemokine addition further allows parallelization of data analyses to improve assay robustness. Confirmation of the neutralizing properties is carried out using biophysical methods and functional assays. In the instance of CXCL12 neutralization by chalcones, the active chalcone molecule, chalcone 4 [29] quenches intrinsic fluorescence of the unique tryptophan residue present in CXCL12, not the inactive analogs. Also, CXCL12 increases active chalcone molecule solubility in a highly significant manner and finally, microcalorimetry allows the interaction between chalcone and chemokine to be quantified. Other biophysical methods include surface plasmon resonance (SPR) [26] (see below), NMR heteronuclear shift perturbation determinations [31] or co-crystal structure solution. Some of the above mentioned approaches have led to the identification of neutraligands [31,32]. Based on the
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Figure 4. How to find and characterize chemokine neutraligand function. The neutraligand can be characterized in vitro and in vivo. (A) Human peripheral blood CD4+ lymphocytes (PBLs) migrate toward CXCL12 at a 30 nM concentration in the lower compartment of a Boyden chamber. If the neutraligand is preincubated with PBLs, chemotaxis is not different from control experiment with CXCL12 alone. By contrast, if neutraligand is preincubated with chemokine, chemotaxis is inhibited in a dose dependent manner. (B) The panel reports cell type counting in bronchoalveolar lavages from mice that have developed an allergic response to ovalbumin. As was reported for anti-CXCR4 or anti-CXCL12 antibodies [39], anti-CXCL12 neutraligand inhibits recruitment of eosinophils (eosino) and lymphocytes (lympho) in the airway, as previously described for Th2 mediated inflammatory responses. Macrophages (macro); neutrophils (neutro).
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be set up to discriminate chemotactic inhibition resulting from receptor inhibition or chemokine neutralization. In the example shown, chalcone 4 displays efficient inhibition of CD4+ peripheral blood lymphocyte chemotaxis when preequilibrated with CXCL12, not with the CXCR4-positive cells. Similar results are obtained in calcium concentration elevation.
Functional explorations with neutraligands targeting chemokines When two receptors bind the chemokine, as is the case in HPV immortalized keratinocytes that express CXCR4 and CXCR7, functional responses to neutraligand and to CXCR4 antagonist differ quantitatively and qualitatively. In these cells that overexpress CXCL12 and its two receptors, inhibition of cell proliferation is better achieved with chalcone 4 than with blockade of any of the two receptors using specific antibodies or ligands [33]. Inhibition of chemokine signaling by neutraligands is functionally confirmed at the whole animal level, on disease models (Haas 2008, Vieira 2009, Montecucco 2010, Russo 2011). Anti-CXCL12 neutraligand for instance can be injected intraperitoneally for weeks to mice without any sign of toxic effect. CXCL12 neutralization by chalcone 4 reduces eosinophilic infiltration into the inflamed airways in a mouse model of allergic hypereosinophilia [29], Fig. 4B. Whereas neutralization by evasin-3 blocks eosinophilia into the gut in a model of experimental colitis [34], or neutrophilia during myocardial injury [35]. Neutralization by evasin-1 reduces leukocyte infiltration in the lung in a model of pulmonary fibrosis [36].
Concluding remarks Altogether, there are accumulating evidence that neutralization of chemokines with small chemical compounds is realistic, and that it represents a potent chemical biology – as well as therapeutic – approach to study chemokine signaling and to treat inflammatory disorders. Regarding inflammation, one important issue will be to determine whether the inhibition of chemokine signaling will allow the resolution strategy to occur. This will have to be studied in physiological and pathophysiological instances. It is now known for several receptors from the CXC, CC or decoy groups whether and how they participate in the onset or the resolution of inflammation [16,6,3,37,38], and thus how they might be targeted for anti-inflammatory and resolution therapeutic indications. Similarly, neutralization of chemokines should be considered with regard to chemokine gradient disruption, chemokine sequestering and/or draining activity to optimally envision therapeutic development.
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2 Bennett, L.D. et al. (2011) Mechanisms regulating chemokine receptor activity. Immunology 134, 246–256 3 Mantovani, A. et al. (2006) Tuning inflammation and immunity by chemokine sequestration: decoys and more. Nat. Rev. Immunol. 6, 907–918 4 Gardner, L. et al. (2006) Temporal expression pattern of Duffy antigen in rheumatoid arthritis: up-regulation in early disease. Arthritis Rheum. 54, 2022–2026 5 Vergara, C. et al. (2008) Gene encoding Duffy antigen/receptor for chemokines is associated with asthma and IgE in three populations. Am. J. Respir. Crit. Care Med. 178, 1017–1022 6 Graham, G.J. and McKimmie, C.S. (2006) Chemokine scavenging by D6: a movable feast? Trends Immunol. 27, 381–386 7 Jamieson, T. et al. (2005) The chemokine receptor D6 limits the inflammatory response in vivo. Nat. Immunol. 6, 403–411 8 Comerford, I. et al. (2006) The chemokine receptor CCX-CKR mediates effective scavenging of CCL19 in vitro. Eur. J. Immunol. 36, 1904–1916 9 Comerford, I. et al. (2011) The atypical chemokine receptor CCX-CKR scavenges homeostatic chemokines in circulation and tissues and suppresses Th17 responses. Blood 116, 4130–4140 10 Catusse, J. et al. (2010) Role of the atypical chemoattractant receptor CRAM in regulating CCL19 induced CCR7 responses in B-cell chronic lymphocytic leukemia. Mol. Cancer 9, 297 11 Thelen, M. and Thelen, S. (2008) CXCR7, CXCR4 and CXCL12: an eccentric trio? J. Neuroimmunol. 198, 9–13 12 Levoye, A. et al. (2009) CXCR7 heterodimerizes with CXCR4 and regulates CXCL12-mediated G protein signaling. Blood 113, 6085–6093 13 Dambly-Chaudiere, C. et al. (2007) Control of cell migration in the development of the posterior lateral line: antagonistic interactions between the chemokine receptors CXCR4 and CXCR7/RDC1. BMC Dev. Biol. 7, 23 14 Wang, J. et al. (2006) Enhanced expression of Duffy antigen receptor for chemokines by breast cancer cells attenuates growth and metastasis potential. Oncogene 25, 7201–7211 15 Wang, J. et al. (2008) The role of CXCR7/RDC1 as a chemokine receptor for CXCL12/SDF-1 in prostate cancer. J. Biol. Chem. 283, 4283–4294 16 Ariel, A. et al. (2006) Apoptotic neutrophils and T cells sequester chemokines during immune response resolution through modulation of CCR5 expression. Nat. Immunol. 7, 1209–1216 17 Cardona, A.E. et al. (2008) Scavenging roles of chemokine receptors: chemokine receptor deficiency is associated with increased levels of ligand in circulation and tissues. Blood 112, 256–263 18 Alcami, A. (2003) Viral mimicry of cytokines, chemokines and their receptors. Nat. Rev. Immunol. 3, 36–50 19 Galzi, J.L. et al. (2010) Neutralizing endogenous chemokines with small molecules. Principles and potential therapeutic applications. Pharmacol. Ther. 126, 39–55 20 Murphy, P.M. (2000) Viral antichemokines: from pathogenesis to drug discovery. J. Clin. Invest. 105, 1515–1517 21 Kiss, D.L. et al. (2009) The functional antagonist Met-RANTES: a modified agonist that induces differential CCR5 trafficking. Cell. Mol. Biol. Lett. 14, 537–547 22 Kim, H.Y. et al. (2010) The CXCR4 antagonist AMD3100 has dual effects on survival and proliferation of myeloma cells in vitro. Cancer Res. Treat. 42, 225–234 23 Bedard, E.L. et al. (2003) Chemokine-binding viral protein M-T7 prevents chronic rejection in rat renal allografts. Transplantation 76, 249–252 24 Liu, L. et al. (2004) Viral chemokine-binding proteins inhibit inflammatory responses and aortic allograft transplant vasculopathy in rat models. Transplantation 77, 1652–1660 25 Frauenschuh, A. et al. (2007) Molecular cloning and characterization of a highly selective chemokine-binding protein from the tick Rhipicephalus sanguineus. J. Biol. Chem. 282, 27250–27258 26 Deruaz, M. et al. (2008) Ticks produce highly selective chemokine binding proteins with antiinflammatory activity. J. Exp. Med. 205, 2019–2031 27 Vollmer, J.Y. et al. (1999) Subcellular compartmentalization of activation and desensitization of responses mediated by NK2 neurokinin receptors. J. Biol. Chem. 274, 37915–37922 28 Valenzuela-Fernandez, A. et al. (2001) Optimal inhibition of X4 HIV isolates by the CXC chemokine stromal cell-derived factor 1 alpha requires
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