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Molecular Mechanism of AMD3100 Antagonism in the CXCR4 Receptor

2004; Elsevier BV; Volume: 279; Issue: 4 Linguagem: Inglês

10.1074/jbc.m309546200

1083-351X

Mette M. Rosenkilde, Lars‐Ole Gerlach, Janus S. Jakobsen, Renato T. Skerlj, Gary Bridger, Thue W. Schwartz,

interferon and immune responses

AMD3100 is a symmetric bicyclam, prototype non-peptide antagonist of the CXCR4 chemokine receptor. Mutational substitutions at 16 positions located in TM-III, -IV, -V, -VI, and -VII lining the main ligand-binding pocket of the CXCR4 receptor identified three acid residues: Asp171 (AspIV:20), Asp262 (AspVI:23), and Glu288 (GluVII:06) as the main interaction points for AMD3100. Molecular modeling suggests that one cyclam ring of AMD3100 interacts with Asp171 in TM-IV, whereas the other ring is sandwiched between the carboxylic acid groups of Asp262 and Glu288 from TM-VI and -VII, respectively. Metal ion binding in the cyclam rings of AMD3100 increased its dependence on Asp262 and provided a tighter molecular map of the binding site, where borderline mutational hits became clear hits for the Zn(II)-loaded analog. The proposed binding site for AMD3100 was confirmed by a gradual build-up in the rather distinct CXCR3 receptor, for which the compound normally had no effect. Introduction of only a Glu at position VII:06 and the removal of a neutralizing Lys residue at position VII:02 resulted in a 1000-fold increase in affinity of AMD3100 to within 10-fold of its affinity in CXCR4. We conclude that AMD3100 binds through interactions with essentially only three acidic anchor-point residues, two of which are located at one end and the third at the opposite end of the main ligand-binding pocket of the CXCR4 receptor. We suggest that non-peptide antagonists with, for example, improved oral bioavailability can be designed to mimic this interaction and thereby efficiently and selectively block the CXCR4 receptor. AMD3100 is a symmetric bicyclam, prototype non-peptide antagonist of the CXCR4 chemokine receptor. Mutational substitutions at 16 positions located in TM-III, -IV, -V, -VI, and -VII lining the main ligand-binding pocket of the CXCR4 receptor identified three acid residues: Asp171 (AspIV:20), Asp262 (AspVI:23), and Glu288 (GluVII:06) as the main interaction points for AMD3100. Molecular modeling suggests that one cyclam ring of AMD3100 interacts with Asp171 in TM-IV, whereas the other ring is sandwiched between the carboxylic acid groups of Asp262 and Glu288 from TM-VI and -VII, respectively. Metal ion binding in the cyclam rings of AMD3100 increased its dependence on Asp262 and provided a tighter molecular map of the binding site, where borderline mutational hits became clear hits for the Zn(II)-loaded analog. The proposed binding site for AMD3100 was confirmed by a gradual build-up in the rather distinct CXCR3 receptor, for which the compound normally had no effect. Introduction of only a Glu at position VII:06 and the removal of a neutralizing Lys residue at position VII:02 resulted in a 1000-fold increase in affinity of AMD3100 to within 10-fold of its affinity in CXCR4. We conclude that AMD3100 binds through interactions with essentially only three acidic anchor-point residues, two of which are located at one end and the third at the opposite end of the main ligand-binding pocket of the CXCR4 receptor. We suggest that non-peptide antagonists with, for example, improved oral bioavailability can be designed to mimic this interaction and thereby efficiently and selectively block the CXCR4 receptor. The CXCR4 receptor is expressed much more broadly than chemokine receptors in general, i.e. not only on a wide variety of leukocytes but also on cells outside the immune system; for example, in the central nervous system (1.Unutmaz D. Littman D.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1615-1618Crossref PubMed Scopus (43) Google Scholar, 2.Cho C. Miller R.J. J. Neurovirol. 2002; 8: 573-584Crossref PubMed Scopus (52) Google Scholar). In contrast to many chemokine receptors, the CXCR4 receptor is only activated by a single chemokine ligand, stromal cell-derived factor (SDF-1α, 1The abbreviations used are: SDF-1, stromal cell-derived factor; 7TM, seven transmembrane domain; cyclam, 1,4,8,11-tetraazacyclotet-radecane moiety; HIV, human immunodeficiency virus; G-CSF, granulocyte colony-stimulating factor; PI, phosphatidyl-inositol; IP10, inter-feron-γ-inducible protein; ITAC, interferon-inducible T cell α-chemoattractant.1The abbreviations used are: SDF-1, stromal cell-derived factor; 7TM, seven transmembrane domain; cyclam, 1,4,8,11-tetraazacyclotet-radecane moiety; HIV, human immunodeficiency virus; G-CSF, granulocyte colony-stimulating factor; PI, phosphatidyl-inositol; IP10, inter-feron-γ-inducible protein; ITAC, interferon-inducible T cell α-chemoattractant. also called CXCL12). Like many chemokine receptors, the CXCR4 receptor is involved in the control of migration and tissue targeting, i.e. homing of leukocytes. Importantly, SDF-1α and the CXCR4 receptor play a central role for the anchorage of CD34+ stem cells in the bone marrow. The importance of the receptor is emphasized by the fact that targeted deletion of either the gene for CXCR4 or for its ligand in both cases leads to embryologic lethality (3.Zou Y.-R. Kottmann A.H. Kuroda M. Taniuchi I. Littman D.R. Nature. 1998; 393: 595-599Crossref PubMed Scopus (2113) Google Scholar, 4.Tachibana K. Hirota S. Iizasa H. Yoshida H. Kawabata K. Kataoka Y. Kitamura Y. Matsushima K. Yoshida N. Hishikawa S. Kishimoto T. Nagasawa T. Nature. 1998; 393: 591-595Crossref PubMed Scopus (1314) Google Scholar, 5.Nagasawa T. Hirota S. Tachibana K. Takukura N. Nishikawa S.-I. Kitamura Y. Yoshida N. Kikutani H. Kishimoto T. Nature. 1996; 382: 635-637Crossref PubMed Scopus (1999) Google Scholar). This is only rarely observed in knockouts of genes for chemokine receptors and, in fact, also rarely for 7TM G protein-coupled receptors in general. The CXCR4 receptor is also expressed on many different types of cancer cells, and it seems to function both as a survival factor and to be responsible for the "chemotactic" spread of cancer cells as metastasis, for example, to the bone marrow where the SDF-1α ligand for the receptor is produced in large quantities. AMD3100, which is composed of two 1,4,8,11-tetraazacy-clotetradecane (cyclam) moieties connected by a conformationally constraining linker, is a prototype non-peptide antagonist of the CXCR4 receptor. The compound was discovered as an anti-HIV agent long before it was realized that it functioned through specific blockade of the CXCR4 receptor, which is used as a co-receptor for cell entry by so-called X4 strains of the AIDS virus. AMD3100 is a specific CXCR4 antagonist that inhibits the binding and function of SDF-1α with high affinity and potency (6.Tasker P.A. Sklar L. J. Cryst. Mol. Struct. 1975; 5: 329-344Crossref Scopus (130) Google Scholar, 7.Choi H.J. Suh M.P. J. Am. Chem. Soc. 1998; 120: 10622-10628Crossref Scopus (373) Google Scholar). AMD3100 has been shown to block the outgrowth of all X4 as well as dual-tropic (T cell- and macrophage-tropic) HIV variants in vitro. During the development of AMD3100, it was discovered that the compound increases white blood cell counts in the blood and, importantly, that the compound is able to mobilize stem cells from the bone marrow. Thus, currently the compound, in combination with G-CSF, is in clinical trials for stem cell mobilization for auto-transplantations in, for example, multiple myeloma patients. In fact, strong evidence has been presented which indicates that many other stem cell mobilizing regimes, such as cyclophosphamide and G-CSF, act through disruption of the SDF-CXCR4 system, thus emphasizing the crucial role of this system in the control of stem cell homing and mobilization (8.Levesque J.P. Hendy J. Takamatsu Y. Simmons P.J. Bendall L.J. J. Clin. Invest. 2003; 111: 187-196Crossref PubMed Scopus (637) Google Scholar). In an initial study with just a few targeted mutations, we identified two acidic residues, Asp171 (AspIV:20) and Asp262 (AspVI:23), that are located in the main ligand-binding pocket of the CXCR4 receptor as being important for the binding of AMD3100 (9.Gerlach L.O. Skerlj R.T. Bridger G.J. Schwartz T.W. J. Biol. Chem. 2001; 276: 14153-14160Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). That study was based on the knowledge of the strong preference of the cyclam moiety for interactions with carboxylic acid groups (21.Bridger G.J. Skerlj R.T. Adv. Antiviral Drug Des. 1999; 3: 161-229Crossref Scopus (25) Google Scholar). Asp171 and Asp262 were also found to be essential for the function of the CXCR4 receptor as a co-receptor for HIV (10.Donzella G.A. Schols D. Lin S.W. Este J.A. Nagashima K.A. Maddon P.J. Allaway G.P. Sakmar T.P. Henson G. De Clercq E. Moore J.P. Nat. Med. 1998; 4: 72-77Crossref PubMed Scopus (681) Google Scholar). The present study was aimed at performing a rather exhaustive mutational analysis (Fig. 1) of the molecular mechanism of action of AMD3100 using the monoclonal antibody 12G5 and not SDF-1α as the radioligand. The reason for this is that the binding of the endogenous chemokine ligand was affected by certain mutations down in the main ligand-binding pocket, which limited the number of residues that could be addressed with that ligand as a probe. The 12G5 antibody is a relevant probe to use because the interactions of bicyclams with CXCR4 monitored by the inhibition of 12G5 binding follows a similar structure-activity relationship for the inhibition of HIV-replication (9.Gerlach L.O. Skerlj R.T. Bridger G.J. Schwartz T.W. J. Biol. Chem. 2001; 276: 14153-14160Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, 11.Este J.A. Cabrera C. De Clercq E. Struyf S. Van Damme J. Bridger G. Skerlj R.T. Abrams M.J. Henson G. Gutierrez A. Clotet B. Schols D. Mol. Pharmacol. 1999; 55: 67-73Crossref PubMed Scopus (113) Google Scholar, 12.Bridger G.J. Skerlj R.T. Thornton D. Padmanabhan S. Martellucci S.A. Henson G.W. Abrams M.J. Yamamoto N. De Vreese K. Pauwels R. J. Med. Chem. 1995; 38: 366-378Crossref PubMed Scopus (226) Google Scholar, 13.Bridger G.J. Skerlj R.T. Padmanabhan S. Martellucci S.A. Henson G.W. Abrams M.J. Joao H.C. Witvrouw M. De Vreese K. Pauwels R. De Clercq E. J. Med. Chem. 1996; 39: 109-119Crossref PubMed Scopus (61) Google Scholar). Importantly, the rather simple binding mode of the prototype CXCR4 non-peptide antagonist AMD3100, which was found through mutational disruption of the binding of the non-peptide compound in the CXCR4 receptor, was proven through the transfer of the essential components of this binding site to the binding pocket of the otherwise rather distinct CXCR3 receptor. Materials—The human chemokines CXCL12/SDF-1, CXCL10/IP10, and CXCL11/ITAC were purchased from Peprotech. The CXCR4-specific monoclonal antibody 12G5 was kindly provided by Jim Hoxie (University of Pennsylvania, Philadelphia, PA). Human CXCR4 was kindly provided by Timothy N. C. Wells (Serono Pharmaceutical Research Institute, Geneva, Switzerland), and human CXCR3 was kindly provided by Kuldeep Neote (Pfizer Inc., Groton, CT). The promiscuous chimeric G-protein GαΔ6qi4myr was kindly provided by Evi Kostenis (7TM-Pharma, Denmark). [3H]myo-inositol (PT6–271) and Bolton-Hunter reagent for iodination of 12G5 were purchased from Amersham Pharmacia Biotech (Uppsala, Sweden). AG 1-X8 anion-exchange resin was obtained from Bio-Rad. Site-directed Mutagenesis—Point mutations were introduced in the receptors by the polymerase chain reaction overlap extension technique (14.Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene. 1989; 77: 51-59Crossref PubMed Scopus (6808) Google Scholar) using the wild-type CXCR4 or the wild-type CXCR3 receptor as template. All reactions were carried out using the Pfu polymerase (Stratagene) under conditions recommended by the manufacturer. The generated mutations were cloned into the eukaryotic expression vector pcDNA3+. The mutations were verified by restriction endonuclease digestion and DNA sequencing (ABI 310, PerkinElmer). Iodination of 12G5—The Bolton-Hunter reagent was dried by a gentle stream of nitrogen for 30–60 min. 250 pmol of 12G5 was incubated on ice with 1 mCi Bolton-Hunter reagent in a total volume of 50 μlof0.1 mm borate buffer, pH 8.5, for 1 h. The reaction was terminated by the addition of 0.25 ml of the borate buffer supplemented with 0.2 m glycine and the Bolton-Hunter-labeled 12G5 separated from free Bolton-Hunter reagent by column chromatography (Econo-Pac DC10, Bio-Rad; Ref. 15.Signoret N. Rosenkilde M.M. Klasse P.J. Schwartz T.W. Malim M.H. Hoxie J.A. Marsh M. J. Cell. Science. 1998; 111: 2819-2830PubMed Google Scholar). Transfections and Tissue Culture—COS-7 cells were grown at 10% CO2 and 37 °C in Dulbecco's modified Eagle's medium with glutamax (21885–025, Invitrogen) adjusted with 10% fetal bovine serum, 180 units/ml penicillin, and 45 μg/ml streptomycin (PenStrep). Transfection of the COS-7 cells was performed by the calcium phosphate precipitation method (16.Rosenkilde M.M. Kledal T.N. Bräuner-Osborne H. Schwartz T.W. J. Biol. Chem. 1999; 274: 956-961Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Binding Experiments—COS-7 cells were transferred to culture plates 1 day after transfection. The number of cells seeded per well was determined by the apparent expression efficiency of the receptors and was aimed at obtaining 5–10% specific binding of the added radioactive ligand (2 × 104 to 1 × 105 cells/well for the different CXCR4 constructs). Two days after transfection, cells were assayed by competition binding for 3 h at 4 °C using 32 pm [125I]12G5 plus unlabeled ligand in 0.5 ml of a 50 mm Hepes buffer, pH 7.4, supplemented with 1 mm CaCl2, 5 mm MgCl2, and 0.5% (w/v) bovine serum albumin. After incubation, cells were washed quickly 2× in 4 °C binding buffer supplemented with 0.5 m NaCl. Nonspecific binding was determined as the binding in the presence of 0.1 μm unlabeled 12G5. Determinations were made in duplicates. Phosphatidyl-inositol Assay (PI-turnover)—COS-7 cells were transfected according to the procedure mentioned above. Briefly, 6 × 106 cells were transfected with 20 μg of receptor cDNA in addition to 30 μgofthe promiscuous chimeric G-protein, GαΔ6qi4myr, which turns the Gαi-coupled signal (the most common pathway for endogenous chemokine receptors) into the Gαq pathway (phospholipase C activation measured as PI-turnover; Ref. 17.Kostenis E. Trends Pharmacol. Sci. 2001; 22: 560-564Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). One day after transfection, COS-7 cells (2.5 × 104 cells/well) were incubated for 24 h with 2 μCi of [3H]myo-inositol in 0.4 ml of growth medium per well. Cells were washed twice in 20 mm Hepes, pH 7.4, supplemented with 140 mm NaCl, 5 mm KCl, 1 mm MgSO4, 1 mm CaCl2, 10 mm glucose, and 0.05% (w/v) bovine serum albumin, and were incubated in 0.4 ml of buffer supplemented with 10 mm LiCl at 37 °C for 90 min in the presence of various concentrations of chemokines or AMD analogs together with a constant concentration of chemokine corresponding to 80% of maximal stimulation. Cells were extracted by the addition of 1 ml of 10 mm formic acid to each well, followed by incubation on ice for 30–60 min. The generated [3H]inositol phosphates were purified on AG 1-X8 anion-exchange resin (18.Berridge M.J. Dawson M.C. Downes C.P. Heslop J.P. Irvin R.F. Biochem. J. 1983; 212: 473-482Crossref PubMed Scopus (1537) Google Scholar). Determinations were made in duplicates. Calculations—IC50 and EC50 values were determined by nonlinear regression, and Bmax values were calculated by using Prism version 3.0 software (GraphPad Software, San Diego). Mapping of the Binding Site for AMD3100 in the CXCR4 Receptor—Based on knowledge of cyclam-carboxylic acid interactions, two acid residues located at each end of the main ligand-binding pocket of the CXCR4 receptor, Asp171 (AspIV: 20) and Asp262 (AspVI:23), have previously been identified as key interaction points for AMD3100 (9.Gerlach L.O. Skerlj R.T. Bridger G.J. Schwartz T.W. J. Biol. Chem. 2001; 276: 14153-14160Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, 19.Labrosse B. Brelot A. Heveker N. Sol N. Schols D. De Clercq E. Alizon M. J. Virol. 1998; 72: 6381-6388Crossref PubMed Google Scholar). As shown in Table I and Fig. 2, these 2 positions plus 15 other positions in TM-III, -IV, -V, -VI, and -VII of the CXCR4 receptor were probed by mutational substitutions using the radio-labeled monoclonal antibody, 12G5, as a radioligand. In most cases, the side chain was substituted with the small methyl group of Ala. However, in some cases, a structurally similar but uncharged Asn residue was introduced instead of a charged Asp; in some cases, steric-hindrance mutagenesis was performed through introduction of larger side chains such as Phe or Trp for Ala, Gly, or Ile (20.Holst B. Zoffmann S. Elling C.E. Hjorth S.A. Schwartz T.W. Mol. Pharmacol. 1998; 53: 166-175Crossref PubMed Scopus (64) Google Scholar). None of the substitutions impaired 12G5 binding to the CXCR4 receptor as reflected in the Kd and Bmax values presented in Table I, indicating that the overall structure as well as the cell surface expression of the receptor was unaffected by the mutations. However, five of the substitutions impaired the binding of AMD3100 more than 10-fold. Substitution of As-pIV:20 (Asp171) to Asn has previously been shown to affect AMD3100 38-fold in competition against the natural chemokine ligand SDF-1α (9.Gerlach L.O. Skerlj R.T. Bridger G.J. Schwartz T.W. J. Biol. Chem. 2001; 276: 14153-14160Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). However, when using 12G5 as a radioligand, this substitution only resulted in a 15-fold impairment of AMD3100 binding. In contrast, substitution of AlaIV:24 (Ala175, located four residues after AspIV:20) with a Phe residue affected AMD3100 binding 40-fold. In the x-ray crystal structure of rhodopsin, the residue which corresponds to Ala175 is located at the start of extracellular loop 2 facing down into the main ligand-binding pocket. The relatively large effect of the steric-hindrance substitution of Ala175 with Phe is in good agreement with the assumption that one of the cyclam rings of AMD3100 should be located in the pocket between TM-III, -IV, and -V, i.e. in front of Asp171, which is where the large aromatic side chain of the introduced Phe residue would be expected to be located in the (A175F) mutant.Table IAffinity of 12G5, AMD3100, and AMD3100 (Zn2) for the wild-type CXCR4 and large amount of mutations located in the main binding pocket The data were obtained from competition binding with 125I (Bolton-Hunter)-labeled 12G5 antibody as radioligand on transiently transfected COS-7 cells. Values in parentheses indicate the number of experiments. Maximum specific binding for each mutant is given as Bmax (fmol/105 cell). Fmut indicates the effect of the mutant on the affinity of each ligand compared to wildtype affinity.ResidueBmax ± SEM12G5AMD3100AMD3479NumberPositionlog Kd ± SEMKd(N)fmutlog Ki ± SEMKi(N)fmutlog Ki ± SEMKi(N)fmutfmol/105 cellnmumumwtCXCR4137 ± 37–8.80 ± 0.091.57(19)1.0–6.05 ± 0.110.89(17)1.0–7.61 ± 0.100.02(9)1.0TM-IIIH113AIII:0349 ± 14–8.79 ± 0.231.63(4)1.0–5.75 ± 0.191.8(7)2.0–7.00 ± 0.220.10(4)4.1T117AIII:0761 ± 28–9.21 ± 0.150.62(5)0.4–6.34 ± 0.090.46(5)0.5–7.86 ± 0.090.01(4)0.6TM-IVT168AV:1785 ± 49–9.20 ± 0.190.63(6)0.4–6.13 ± 0.120.74(6)0.8–7.84 ± 0.260.01(4)0.6D171NIV:2070 ± 11–9.06 ± 0.090.87(14)0.6–4.87 ± 0.1313(13)15–6.55 ± 0.230.28(11)12F174AIV:2344 ± 15–9.29 ± 0.240.51(5)0.3–5.50 ± 0.163.16(5)3.5–6.17 ± 0.310.68(4)28A175FIV:2442 ± 21–8.81 ± 0.121.55(4)1.0–4.45 ± 0.2436(4)40–5.07 ± 0.348.6(3)350TM-VV196AV:0127 ± 6–9.05 ± 0.100.89(4)0.6–5.33 ± 0.134.6(4)5.2–5.78 ± 0.171.7(3)68Q200AV:05139 ± 15–9.31 ± 0.140.49(3)0.3–6.25 ± 0.100.56(3)0.6–7.75 ± 0.070.02(3)0.7G207WV:12117 ± 76–9.03 ± 0.320.93(4)0.6–5.52 ± 0.333.02(4)3.4–6.60 ± 0.050.25(2)10TM-VIY255AVI:16235 ± 101–8.87 ± 0.131.36(4)0.9–5.01 ± 0.279.7(4)11–6.28 ± 0.090.53(3)22Y256AV:17172 ± 73–8.87 ± 0.161.36(4)0.9–5.40 ± 0.334.0(3)5–6.73 ± 0.230.19(4)7.6I259AVI:2040 ± 8–9.52 ± 0.120.30(3)0.2–5.81 ± 0.051.5(3)1.7–7.56 ± 0.060.03(3)1.1I259WVI:2071 ± 6–8.99 ± 0.161.03(3)0.7–5.34 ± 0.334.6(3)5.2–6.08 ± 0.360.84(2)34D262NVI:23162 ± 59–8.94 ± 0.131.15(13)0.7–4.34 ± 0.1546(13)52–4.89 ± 0.5813(3)522S263AV:24101 ± 44–9.44 ± 0.200.36(3)0.2–5.27 ± 0.145.4(3)6.1–7.10 ± 0.170.08(3)3.2TM-VIIH281AVII:-01116 ± 35–9.08 ± 0.160.83(8)0.5–6.80 ± 0.210.16(11)0.2–7.54 ± 0.140.03(5)1.2I284AVII:02116 ± 16–8.83 ± 0.141.47(5)0.9–5.96 ± 0.361.1(4)1.2–6.64 ± 0.070.23(3)9.3E288AVII:06140 ± 46–9.02 ± 0.060.96(16)0.6–4.20 ± 0.1763(15)71–5.64 ± 0.322.3(3)93 Open table in a new tab None of the substitutions in TM-V affected AMD3100 more than 5-fold, whereas in TM-VI, the mutational analysis (as expected) pointed to AspVI:23 (Asp262) but also to TyrVI:16 (Tyr255) as being interaction points for AMD3100. Surprisingly, substitution of IleVI:20 (Ile259, located in between these two residues and facing right into the middle of the main ligand-binding pocket) had only minimal effect on AMD3100 binding (Table I and Fig. 2). Even the introduction of a large Trp residue in position VI:20 only gave a 5-fold effect on AMD3100 binding. In TM-VII, substitution of IleVII:02 (Ile284) had no effect on AMD3100 binding, despite the fact that this residue (like IleVI: 20) is facing directly into the main pocket and at a location close to AspVI:23. In contrast, substitution of GluVII:06 (Glu288) to Ala affected AMD3100 around 70-fold. Thus, besides the previously described Asp171 and Asp262, the major result of the mutational analysis using 12G5 as a radioligand was the identification of an additional acid residue Glu288 as a potential interaction point for AMD3100. Effect of Zn(II) on the Mutational Mapping of AMD3100 Binding—Binding of Zn(II) in the cyclam rings of AMD3100 is known to increase its affinity for the CXCR4 receptor around 35-fold (Table I; Refs. 21.Bridger G.J. Skerlj R.T. Adv. Antiviral Drug Des. 1999; 3: 161-229Crossref Scopus (25) Google Scholar and 22.Gerlach L.O. Jakobsen J.S. Jensen K.P. Rosenkilde M.R. Skerlj R.T. Ryde U. Bridger G.J. Schwartz T.W. Biochemistry. 2003; 42: 710-717Crossref PubMed Scopus (129) Google Scholar). Previously, we have found that this increased affinity in fact is determined by only one of the Zn(II) ions in one of the cyclam rings and that the effect is achieved through interaction with the carboxylic group of AspVI:23 (Asp262) (22.Gerlach L.O. Jakobsen J.S. Jensen K.P. Rosenkilde M.R. Skerlj R.T. Ryde U. Bridger G.J. Schwartz T.W. Biochemistry. 2003; 42: 710-717Crossref PubMed Scopus (129) Google Scholar). When the Zn(II)-loaded AMD3100 (also called AMD3479) was probed in the library of mutated CXCR4 receptors, an interesting picture emerged. The four to five residues that were identified as interaction points for AMD3100 itself were all positive, but the effects of the mutations were in all cases (except for Asp171) larger for the Zn(II)-loaded version than for AMD3100 alone (Table I and Fig. 2). Moreover, a number of substitutions which had given small, single-digit effects for AMD3100 now gave clear, i.e. > 10-fold, effects for the Zn(II)-loaded version: 28-versus 3.5-fold (PheIV:23 or Phe174), 68-versus 5.2-fold (ValV:01 or Val196), and 34-versus 5.2-fold (IleVI:20 or Ile259) (Table I and Fig. 2). Important Acidic Residue in TM-VII for the Binding of AMD3100 —GluVII:06 (Glu288) is clearly the most interesting among the novel potential interaction sites for AMD3100, because the only other mutation which has a relatively large effect on the binding of the antagonist (Ala175 to Phe) is considered to be a reflection of steric hindrance. As shown in Fig. 3, by using [125I]12G5 as a radioligand, the Glu288 to Ala substitution shifted the competition binding curve for AMD3100 with or without Zn(II) 70- to 90-fold to the right. We were unable to use [125I]SDF-1α as radioligand in binding experiments in this construct because of a lack of specific binding (data not shown). This is very likely a reflection of a decreased affinity of SDF-1α on the Glu288 to Ala mutant receptor, as the potency of SDF-1α in signaling was impaired 87-fold in this construct (Fig. 4A). Thus, Glu288 seems to be a residue that is critical for the function of SDF-1α as an agonist on the CXCR4 receptor. By using appropriate sub-maximal doses of SDF-1α on the wild-type and on the Glu288 to Ala mutant form of CXCR4, respectively, it was possible to demonstrate that, not only the affinity (Table I and Fig. 3), but also the potency of AMD3100 (with or without Zn(II)) were highly dependent on the presence of a Glu in position VII:06. Thus, in the wild-type CXCR4, receptor potencies of 79 nm and 17 nm were observed for AMD3100 and the Zn(II)-loaded version, respectively, whereas right shifts of 23- to 116-fold of the bicyclam inhibition curves were observed in the Glu288 to Ala mutant form of CXCR4 (Fig. 4B).Fig. 4Effect of Glu to Ala substitution at position VII:06 (Glu288) in CXCR4 on SDF-1-induced PI-turnover and inhibition by AMD3100. Whole-cell phospholipase C activity (PI-turnover) was measured in COS-7 cells after co-transfection of receptor cDNA with a promiscuous, chimeric G protein Gqi4myr (as described in detail under "Experimental Procedures"). Dose-response curves for SDF-1 are shown in A: wild-type receptor (▵) and E288A-CXCR4 (▴). Inhibition of SDF-1-induced PLC activity by AMD3100 are shown in B: wild-type receptor (○), E288A-CXCR4 (•), AMD3100(Zn2), wild-type receptor (□), and E288A-CXCR4 (▪). Data are shown as means ± S.E. (n = 3–5).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Thus, the overall picture for the AMD3100 binding mode is that it is critically dependent on two acid residues at the extracellular ends of transmembrane segments VI and VII, AspVI:23 and GluVII:06, respectively, plus one Asp residue located at the opposite end of the main ligand-binding pocket, i.e. in position IV:20. Surprisingly, few other residues that are known to be lining each end of the pocket as well as the space in between seem to be directly involved in the recognition of the relatively large bicyclam compound. For the Zn(II)-loaded version of AMD3100, a similar picture is observed; however, the dependence on Asp262 is increased 10-fold, and a number of other residues on the inner face of TM-IV, -V, and -VI are picked up in the mutational analysis. Transfer of the Binding Site for AMD3100 to the CXCR3 Receptor—A survey of the main ligand-binding pocket of all human 7TM receptors revealed that the combination of AspIV: 20, AspVI:23, and GluVII:06 is unique to the CXCR4 receptor, which is in agreement with the fact that AMD3100 is known to be a highly selective antagonist for the CXCR4 receptor (23.Hatse S. Princen K. Bridger G. De Clercq E. Schols D. FEBS Lett. 2002; 527: 255-262Crossref PubMed Scopus (372) Google Scholar). However, among the chemokine receptors, we verified that the CXCR3 receptor had two of the three residues, i.e. AspIV:20 and AspVI:23, but that it had a Ser residue in position VII:06 instead of a Glu as in CXCR4 (Fig. 5). Interestingly, at position VII:02, a Lys residue is located in the CXCR3 receptor (Lys300), which very likely will form a neutralizing salt bridge with AspVI:23 (Asp278) because of the close proximity of the extracellular ends of TM-VI and VII (Fig. 5). Thus, in the CXCR3 receptor, it is very likely that of the three proposed key residues, only one, AspIV:20 (Asp186), is available for interaction with AMD3100. To try to verify the proposed binding site for AMD3100, which in the CXCR4 receptor is based on mutational "destruction" of the binding and function of the non-peptide antagonist, we attempted to use the binding pocket of the CXCR3 receptor as a scaffold to gradually build up the binding site for the antagonist. As expected, AMD3100 had no effect on the inositol phosphate accumulation induced by the agonist chemokines ITAC or IP10 in the CXCR3 receptor. Moreover, neither introduction of Glu at position VII:06 nor substitution of LysVII:02 with a non-neutralizing Ala residue changed this (Fig. 6). However, in the CXCR3 construct, where these two substitutions were combined to ensure the presence and exposure of all three proposed key interacting residues for AMD3100, the bicyclam antagonist inhibited in a dose-dependent manner both the ITAC and the IP10-induced signaling, with IC50 values of 1.1 and 9.3 μm, respectively (Fig. 6). However, only a partial inhibition down to 35–40% of the maximal stimulation was observed with AMD3100. As shown in Fig. 7, this was not the case for the Zn(II)- or Ni(II)-loaded analogs of AMD3100 which acted as full antagonists.Fig. 7Improvement of the inhibitory potency and efficacy of AMD3100 in inhibiting agonist-induced IP turnover in the K300A,S304E-CXCR3 receptor. Dose-dependent inhibition of 10 nm ITAC (A and B) or 10 nm IP10 (C and D) induced IP turnover by AMD3100 (▪) and by the Zn(II)-loaded version (▴) (A and C) or the Ni(II)-loaded version (⋄) (B and D). Data are shown as means ± S.E. (n = 3–5).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Metal Ion-loaded Versions of AMD3100 Are More Potent and Efficacious Antagonists—Besides the fact that the metal ion-loaded versions of AMD3100 completely inhibited the agonist activity in the "CXCR4-mutated" CXCR3 receptor (Fig. 7), a surprisingly high potency of these compounds was observed. Thus, for the Zn(II)-loaded AMD3100, left-shifts of 19- to 69-fold were observed in the inhibition of the ITAC- and IP10-induced acti