Backbone cyclic helix mimetic of chemokine (C–C motif) receptor 2: A rational approach for inhibiting dimerization of G protein-coupled receptors Mattan Hurevich a,  , Maya Ratner-Hurevich b,  , Yftah Tal-Gan a , Deborah E. Shalev c , Shlomo Z. Ben-Sasson b , Chaim Gilon a,⇑ a Institute of Chemistry, The Hebrew University of Jerusalem, Safra Campus, Givat Ram, Jerusalem 91904, Israel b Lautenberg Center for General and Tumor Immunology, Hadassah-Hebrew University Medical School, Jerusalem 91120, Israel c The Wolfson Centre for Applied Structural Biology, The Hebrew University of Jerusalem, Safra Campus, Givat Ram, Jerusalem 91904, Israel article info Article history: Received 27 December 2012 Revised 28 February 2013 Accepted 1 March 2013 Available online 23 March 2013 Keywords: G protein-coupled receptors Backbone cyclization Helix mimetics Urea cyclization GPCR dimerization Multiple Sclerosis abstract The transmembrane helical bundle of G protein-coupled receptors (GPCRs) dimerize through helix–helix interactions in response to inflammatory stimulation. A strategy was developed to target the helical dimerization site of GPCRs by peptidomimetics with drug like properties. The concept was demonstrated by selecting a potent backbone cyclic helix mimetic from a library that derived from the dimerization region of chemokine (C–C motif) receptor 2 (CCR2) that is a key player in Multiple Sclerosis. We showed that CCR2 based backbone cyclic peptide having a stable helix structure inhibits specific CCR2-mediated chemotactic migration Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Protein–protein interactions (PPIs) control the function of living cells. Signal transduction, cell cycle, proliferation and metabolism are just examples of the fundamental processes governed by PPI. 1,2 a-Helical sequences constitute the largest class of secondary structures in proteins and are estimated to account for 30% of pro- tein structures. They are not only abundant in PPIs, but also in interactions with DNA or RNA. 3 a-Helical interactions (AHI) are key processes in many diseases and are therefore targeted for drug development. Understanding the correlation between the struc- ture and the specific biological function of the helical regions in proteins is crucial for studying the mechanism of action of these macromolecules. A vast number of drug targets are related to rhodopsin-like G protein-coupled receptors (GPCRs). 4 These proteins contain major helical segments that construct their transmembrane region. At- tempts to structurally characterize these proteins are still ongoing using elaborate NMR techniques. 5 However, the helical segments of GPCRs are buried inside the cell membrane and elucidating their unique structure is not straightforward. These limitations hamper efforts to design GPCR-based drugs. GPCRs that interact with chemokines are generally referred to as chemokine receptors. Chemokines are a large family of proteins that regulate recruitment of leukocytes to sites of inflammation and coordinate their trafficking. 6 Chemokines control leukocyte function by binding and activating specific chemokine receptors 0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmc.2013.03.019 Abbreviations: ACN, acetonitrile allocallyloxycarbonyl; BBB, blood brain barrier; Boc, t-butyloxycarbonyl; BTC, bis(trichloromethyl)carbonate; CCR2, chemokine (C– C motif) receptor 2; CCR5, chemokine (C–C motif) receptor 5; CNS, central nervous system; DIPEA, diisopropylethylamine; DMF,N, N-dimethylformamide; ESI, elec- trospray ionization; EtOAc, ethyl acetate; Fmoc, 9-fluorenylmethyloxycarbonyl; GPCR, G protein-coupled receptor; HATU, [2-(7-aza-1H-benzotriazole-1-yl)-1,1,3, 3-tetramethyluronium hexafluorophosphate]; HBTU, [2-(1H-benzotriazole-1-yl)- 1,1,3,3-tetramethyluronium hexafluorophosphate]; HOAt, 1-hydroxy-7-aza-benzo- triazole; HOBt, 1-hydroxybenzotriazole; HRMS, high resolution mass spectrometry; MALDI, matrix assisted laser desorption ionization; MAPS, microwave assisted peptide synthesis; MBHA, methylbenzhydrylamine; NMP, N-methyl-2-pyrollidone; NMR, nuclear magnetic resonance; NOESY, nuclear overhauser effect spectroscopy; OSu, N-hydroxysuccinimide; Pbf, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sul- fonyl; PFPC, pentafluorophenyl carbonate; ROESY, rotating-frame overhauser effect spectroscopy; RP-HPLC, reverse phase high pressure liquid chromatography; SPPS, solid phase peptide synthesis; tBu, tert-butyl; TDW, tri-distilled water; TFA, trifluoroacetic acid; TFE, 2,2,2-trifluorethanol; TLC, thin layer chromatography; TM-1, transmembrane region-1; TOCSY, totally correlation spectroscopy; TOF, time of flight.. ⇑ Corresponding author. Tel.: +972 2 6585276; fax: +972 2 6585345. E-mail address: chaimgilon@gmail.com (C. Gilon).   Both authors contribute equally to this work. Bioorganic & Medicinal Chemistry 21 (2013) 3958–3966 Contents lists available at SciVerse ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc