Inhibitors of Chemokine receptors as Anti-inflammatory Reagents in the Eye – I-CKAIRE
Inhibitors of cell migration as Anti-Inflammatory Reagents in eye inflammation
Iinflammation is now considered as an important feature in many pathological conditions. Among immune mediators, chemokines control leukocyte trafficking. Chemokines and their receptors are thus central to the inflammatory process and are attractive targets for the pharmaceutical industry.
To develop chemokine-based therapeutic strategies for the treatment of AMD.
We identified the first short peptide called ECL1i with strong CCR2 antagonist activity. ECL1i blocks CCR2 activity without interfering with the natural ligand binding site, a major advantage compared to conventional non-peptide molecules and antibodies against CCR2 because ECL-1 antagonizes CCR2 independently of the concentration of the natural agonist. Therefore, the level of the drug concentration needed to achieve therapeutic activity should be easier to predict.<br />Our specific aims are thus to <br />- identify key amino acids involved in CCR2 blockade for the first-in-class peptide, <br />- optimize length and stability for the follow-up CCR2 blocking peptides,<br />- determine in vivo fate of the lead compound in order to define therapeutic dosage,<br />- test the lead compound as drug in AMD and choroïdal neovascularization models.
The specific tasks of I-CKAIRE can be summarized as follows:
1) Characterization of the peptide antagonism
a) Identification of the minimal peptide in vitro
b) Full characterization of the antagonist peptide CCR2 interaction
2) Directed molecular evolution of the antagonist
a) Identification of the optimal peptide regarding charge, extension and hydrophobicity
b) In vitro toxicity characterization
3) In vivo survey of the fate of the selected peptide
a) Peptide fate after intraperitoneal, intravenous, intravitreous and topical delivery
4) Preclinical evaluation in murine models of eye inflammation
a) Light-induced subretinal inflammation/degeneration models
b) Laser-induced choroidal neovascularization
c) Newborn hyperglycemia induced retinopathy model
Exploitation, Dissemination and Management of intellectual properties
We developed the first small allosteric peptidic modulator of CCR2 that we calledECL1i. We showed that this modulator displays functional selectivity in vitro by inhibiting CCR2-induced migration without affecting other functions classically associated to CCR2 signaling. Moreover, a treatment with clinically feasible doses of ECL1i reduces inflammatory monocytes recruitment and disease progression in two in vivo models, an acute inflammatory model, i.e peritonitis and a chronic inflammatory model, i.e a mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis.
Proof of concept of chemokine-based anti-inflammatory reagents
1. Sennlaub F., Auvynet C., Calippe B., Hu S.H., Lavalette S., Dominguez E., Camelo S. Poupel L., Levy O., Guyon E., Saederup N., Charo I.F., Van Rooijen N., Nandrot E., Bourges J.-L., Behar-Cohen F., Sahel J.-A., Guillonneau X., Raoul W. and Combadiere C. CCR2+ monocytes infiltrate atrophic lesions in age-related macular disease and mediate photoreceptor degeneration in a model of experimental subretinal inflammation. EMBO Mol.Med 2013 Nov;5(11):1775-93
Age-related macular degeneration (AMD) is the leading cause of vision loss among older adults in the European community. Actual therapies do not treat the neurodegenerative aspect of AMD but only its angiogenic symptoms without tackling the cause of the disease.
Recent evidences suggest an implication of inflammatory mediators in the pathogenesis of AMD and we focused our interest on the potential role of chemotactic cytokines or chemokines in the redistribution of the immune cells in AMD. We showed that, both in human and mice, the chemokine receptor CX3CR1 is a key mediator in AMD etiology (Combadiere et al, JCI. 2007). Unpublished works funded by the ANR “Maladies Neurologiques et Psychiatriques” program (CHARMD, 2008-2012 to Dr. C. Combadiere) and by the European Community (ERC Starting Grant, 2007-2012 to Dr. F. Sennlaub) indicate that 1) in human, AMD is associated with increased intraocular levels of CCL2, another chemokine that specifically interacts with the receptor CCR2, 2) CCR2-positive “inflammatory” monocytes infiltrate the subretinal space, 3) genetic deletion of CCL2 or CCR2 prevents inflammatory monocyte recruitment, microglial cell accumulation, and photoreceptor degeneration in vivo in the Cx3cr1-/- mouse model of AMD and finally 4) pharmacological inhibition of CCR2 using commercial reagents protects from cardinal features of AMD in mice. Our data suggests that CCL2/CCR2 inhibition represents a powerful tool for controlling inflammation and neurodegeneration in AMD.
Based on an original hypothesis-driven model of allosteric receptor inhibition, we designed series of small peptides and identified a 7 amino acid-long peptide termed ECL-1 with strong CCR2 antagonist activity (European Patent; EP11305816.8). Indeed, ECL-1 inhibited CCR2-triggered-proximal (calcium release) and -distal (kinase activation) intracellular signaling, ultimately blocking cellular function like cell migration. Importantly, this antagonist peptide did not modify ortholog ligand binding indicating an allosteric interaction with its receptor. In vivo, ECL-1 blocked peritoneal infiltration by monocyte in a murine model of peritonitis and more recent data showed that ECL-1 protects from retinal degeneration in a murine model of AMD. This set of data indicates that we identify the first CCR2 antagonist peptide with strong in vivo activity.
Commercial interest in therapeutic peptides has soared due in part to advances in synthesis, delivery and formulation techniques. The specific aims of our project are thus to (i) identify key amino acids involved in CCR2 blockade for the first-in-class peptide, (ii) optimize length and stability for the follow-up CCR2 blocking peptides, (iii) determine in vivo fate of the lead compound in order to define therapeutic dosage and (iv) test the lead compound as drug in atrophic AMD and choroidal neovascularization models.
The project gathers three academic groups with complementary education training, skills and fields of research. The range of activities cover extends from fundamental structure/function studies on chemokines/chemokine receptors, the clinical and translational research on ocular pathologies to the protection and valorization of the intellectual properties. The capacity of the groups to generate and diffuse jointly the results of its research is already proved by the development of a number of reagents and animal models, as well as publications of high standard and patent applications. In conclusion, our joint efforts have already demonstrated the feasibility of our research plan and the positive momentum gained through our collaboration.
The aim and the ambition of this work are to develop novel chemokine-based therapeutic strategies for the treatment of atrophic and wet AMD and possibly extended them to additional inflammatory eye diseases. The project should lead to strong reinforcement of the actual ECL-1 patent family that may lead to transfer by license to a pharmaceutical company.
Monsieur Christophe COMBADIÈRE (Laboratoire Immunité et Infection UMR-S945 UPMC INSERM) – firstname.lastname@example.org
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
UMR-S 945 Laboratoire Immunité et Infection UMR-S945 UPMC INSERM
IDV Institut de la Vision_UMR S 968 Inserm_UMR CNRS 7210 UPMC
DGRTT UPMC Bureau Entreprise et Transfert de Technologie DGRTT UPMC
Help of the ANR 249,600 euros
Beginning and duration of the scientific project: December 2012 - 24 Months