The molecular pharmacology of AMD11070: An orally bioavailable CXCR4 HIV entry inhibitor Renee M. Mosi a , Virginia Anastassova a , Jennifer Cox a , Marilyn C. Darkes a , Stefan R. Idzan a , Jean Labrecque a , Gloria Lau c , Kim L. Nelson a , Ketan Patel a , Zefferino Santucci a , Rebecca S.Y. Wong a , Renato T. Skerlj c , Gary J. Bridger a , Dana Huskens b , Dominique Schols b , Simon P. Fricker c, * a Formally of AnorMED Inc., #200 – 20353 64th Avenue, Langley, BC V2Y 1N5, Canada b Rega Institute for Medical Research, Leuven, Belgium c Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701, USA 1. Introduction The introduction of highly active antiretroviral therapy (HAART) has dramatically changed the outcome for HIV-1 infected persons with improvements in morbidity and mortality. However, there are challenges associated with chronic use of reverse transcriptase inhibitors and protease inhibitors, the components of HAART, including emergence of resistance and chronic toxicities which limit patient compliance such as diarrhea, anemia and lipodystrophy [1]. There is therefore a need for new agents with different molecular targets. The inhibition of viral entry has emerged as a validated target for HIV infection and the first approved entry inhibitor was enfuvirtide which acts by blocking gp41-mediated fusion [2]. The discovery that HIV requires both host CD4 and a chemokine receptor as a co-receptor for cell entry stimulated research into chemokine receptor inhibitors as a new class of HIV drugs [3,4]. Chemokines are 8–10 kDa proteins defined by the number and relative spacing of cysteine residues at the N-terminal end of the protein. The two major families are CC and CXC in which there are two cysteine residues that are either adjacent (CC) or separated by one amino acid residue (CXC). They are mediators of hematopoiesis and inflammation, regulating lymphocyte development, homing and trafficking. Their receptors belong to the class A family of seven transmembrane (7 TM) G- protein coupled receptors (GPCR) [5,6]. HIV uses two chemokine receptors, CCR5 and CXCR4, proven targets for HIV drugs [3,7]. CCR5 is the principle co-receptor for viral transmission in the early, clinically latent stage of the disease, whereas CXCR4 usage emerges in the later stages and is associated with a decrease in CD4 cell count and accelerated disease progression [4]. Virus that uses CCR5 as a co-receptor is characterized as M (macrophage)-tropic, CCR5-using or R5, and virus that uses CXCR4 as T (T lymphocyte)-tropic, CXCR4-using or X4. The CCR5 inhibitor Selzentry 1 (maraviroc) was approved by the FDA for use in treatment experienced patients in August 2007 [8–10], and in treatment naı ¨ve patients in November 2009 [11,12]. The bicyclam Mozobil 1 (plerixafor) is a selective antagonist of CXCR4 [13,14] and is an inhibitor of T-tropic, X4 virus [15–17], and was demonstrated to reduce the X4 viral load in HIV-1 infected subjects [18]. Inhibition of CXCR4 by plerixafor also leads to leukocytosis and mobilization of hematopoietic stem cells (HSC) from the bone marrow in both healthy volunteers and in patients Biochemical Pharmacology 83 (2012) 472–479 A R T I C L E I N F O Article history: Received 19 August 2011 Accepted 21 November 2011 Available online 28 November 2011 Keywords: AMD11070 HIV Chemokine receptor CXCR4 Allosteric A B S T R A C T In order to enter and infect human cells HIV must bind to CD4 in addition to either the CXCR4 or the CCR5 chemokine receptor. AMD11070 was the first orally available small molecule antagonist of CXCR4 to enter the clinic. Herein we report the molecular pharmacology of AMD11070 which is a potent inhibitor of X4 HIV-1 replication and the gp120/CXCR4 interaction. Using the CCRF-CEM T cell line that endogenously expresses CXCR4 we have demonstrated that AMD11070 is an antagonist of SDF-1a ligand binding (IC 50 = 12.5 Æ 1.3 nM), inhibits SDF-1 mediated calcium flux (IC 50 = 9.0 Æ 2.0 nM) and SDF- 1a mediated activation of the CXCR4 receptor as measured by a Eu-GTP binding assay (IC 50 = 39.8 Æ 2.5 nM) or a [ 35 S]-GTPgS binding assay (IC 50 = 19.0 Æ 4.1 nM), and inhibits SDF-1a stimulated chemotaxis (IC 50 = 19.0 Æ 4.0 nM). AMD11070 does not inhibit calcium flux of cells expressing CXCR3, CCR1, CCR2b, CCR4, CCR5 or CCR7, or ligand binding to CXCR7 and BLT 1 , demonstrating selectivity for CXCR4. In addition AMD11070 is able to inhibit the SDF-1b isoform interactions with CXCR4; and N-terminal truncated variants of CXCR4 with equal potency to wild type receptor. Further mechanistic studies indicate that AMD11070 is an allosteric inhibitor of CXCR4. ß 2011 Elsevier Inc. All rights reserved. * Corresponding author. Tel.: +1 508 271 4598; fax: +1 508 661 8791. E-mail address: simon.fricker@genzyme.com (S.P. Fricker). Contents lists available at SciVerse ScienceDirect Biochemical Pharmacology jo u rn al h om epag e: ww w.els evier.c o m/lo cat e/bio c hem p har m 0006-2952/$ – see front matter ß 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bcp.2011.11.020