Identification of novel allosteric nonpeptidergic inhibitors of the human cytomegalovirus-encoded chemokine receptor US28 Henry F. Vischer  , * , Janneke W. Hulshof   , Saskia Hulscher, Silvina A. Fratantoni, Mark H. P. Verheij, Jane Victorina, Martine J. Smit, Iwan J. P. de Esch, Rob Leurs Leiden/Amsterdam Center for Drug Research, Division of Medicinal Chemistry, Faculty of Sciences, VU University Amsterdam, De Boelelaan 1083, 1081 HV, Amsterdam, The Netherlands article info Article history: Received 21 September 2009 Revised 25 November 2009 Accepted 28 November 2009 Available online 6 December 2009 Keywords: HCMV Viral GPCR Nonpeptidergic Constitutive activity Chemokine Inverse agonist Herpesvirus abstract Human cytomegalovirus (HCMV) is a widespread human pathogen, possessing onco-modulatory proper- ties. Constitutive signaling of the HCMV-encoded chemokine receptor US28 and its ability to bind a broad spectrum of chemokines might facilitate HCMV-associated tumor progression. Novel nonpeptidergic chemotypes were identified as neutral antagonists or inverse agonists on US28, that allosterically inhibit chemokine binding to US28. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Human cytomegalovirus (HCMV) is an infectious b-herpesvirus that establishes a lifelong latent infection in the majority of the human population and (ab)uses human cells for survival and prop- agation. Occasional reactivation and shedding of new infectious virions allow viral dissemination to other individuals. In order to establish viral latency and widespread propagation, HCMV has to hijack infected host cells and evade anti-viral immune responses. To this end, HCMV encodes important regulatory proteins that are expressed by infected host cells. One of these virally-encoded genes is the G protein-coupled receptor US28. US28 binds a broad variety of inflammatory chemokines allowing cell migration along chemokine gradients. In addition, recruitment of leukocytes to the microenvironment of HCMV-infected cells is impaired as inflam- matory chemokines are depleted by constitutive and rapid US28- mediated internalization. 1–3 Although latent infection is generally asymptomatic, HCMV has been associated with several chronic inflammatory diseases and cancer. 4 Importantly, US28 activates in a ligand-independent manner proliferative and inflammatory signal transduction pathways. 5–7 This US28-mediated constitutive signaling induces a transformed and pro-angiogenic phenotype in NIH-3T3 cells, and promotes tumorigenesis in a nude mice model. 8 Identification of small nonpeptidergic ligands is essential to investigate the role of US28 in evasion of the immune system and HCMV-associated cancers. Moreover, such molecules might be con- sidered as promising therapeutics for anti-viral intervention. Previ- ously, we identified VUF2274 (1) [5-(4-(4-chlorophenyl)-4- hydroxy-piperidin-1-yl)-2,2-diphenylpentanenitrile] as a nonpept- idergic allosteric inverse agonist for US28 (Fig. 1a). 9 Compound 1 inhibited US28-mediated constitutive signaling, chemokine binding, and US28-mediated human immunodeficiency virus (HIV) co-recep- tor activity with a low lM potency (EC 50 and IC 50 , respectively). 9 In subsequent lead optimization programs, approximately 100 new analogues were synthesized and pharmacologically character- ized. 10,11 These structure–activity relationship (SAR) studies re- vealed the importance of the 4-phenylpiperidine moiety. However, the potency of this class of compounds could not be improved beyond the low lM range. In addition to these 4-substituted piperidine der- ivates (e.g., 1 and 2), series of piperazinyldibenzothiepine (e.g., 3, 4), cinchonidine derivates (e.g., 5) and benzamides like 6 have been de- scribed in the patent literature as inhibitors of chemokine binding to US28 with IC 50 values in the 0.3–1 lM range (Fig. 1a). 12–14 In order to identify novel chemotypes that can inhibit constitu- tive US28-mediated signaling and/or chemokine binding that might be used as new starting point for lead optimization, we screened a selection of compounds from our in-house collection. 0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2009.11.060 * Corresponding author. Tel.: +31 0 205987588; fax: +31 0 205987610. E-mail address: vischer@few.vu.nl (H.F. Vischer).   Both authors contributed equally to this work. Bioorganic & Medicinal Chemistry 18 (2010) 675–688 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc