This study was undertaken to demonstrate the unique speci¢city of the chemokine receptor CXCR4 antagonist AMD3100. Calcium £ux assays with selected chemokine/cell combinations, a¡ording distinct chemokine receptor speci¢cities, revealed no interaction of AMD3100 with any of the chemokine receptors CXCR1 through CXCR3, or CCR1 through CCR9. In contrast, AMD3100 potently inhibited CXCR4-mediated calcium signaling and chemotaxis in a concentration-dependent manner in di¡erent cell types. Also, AMD3100 inhibited stromal cell-derived factor (SDF)-1-induced endocytosis of CXCR4, but did not a¡ect phorbol ester-induced receptor internalization. Importantly, AMD3100 by itself was unable to elicit intracellular calcium £uxes, to induce chemotaxis, or to trigger CXCR4 internalization, indicating that the compound does not act as a CXCR4 agonist. Speci¢c small-molecule CXCR4 antagonists such as AMD3100 may play an important role in the treatment of human immunode¢ciency virus infections and many other pathological processes that are dependent on SDF-1/CXCR4 interactions (e.g. rheumatoid arthritis, atherosclerosis, asthma and breast cancer metastasis). ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

The plant lectins derived from Galanthus nivalis (Snowdrop) (GNA) and Hippeastrum hybrid (Amaryllis) (HHA) selectively inhibited a wide variety of human immunodeficiency virus type 1 (HIV-1) and HIV-2 strains and clinical (CXCR4-and CCR5-using) isolates in different cell types. They also efficiently inhibited infection of T lymphocytes by a variety of mutant virus strains. GNA and HHA markedly prevented syncytium formation between persistently infected HUT-78/HIV cells and uninfected T lymphocytes. The plant lectins did not measurably affect the antiviral activity of other clinically approved anti-HIV drugs used in the clinic when combined with these drugs. Short exposure of the lectins to cell-free virus particles or persistently HIV-infected HUT-78 cells markedly decreased HIV infectivity and increased the protective (microbicidal) activity of the plant lectins. Flow cytometric analysis and monoclonal antibody binding studies and a PCR-based assay revealed that GNA and HHA do not interfere with CD4, CXCR4, CCR5, and DC-SIGN and do not specifically bind with the membrane of uninfected cells. Instead, GNA and HHA likely interrupt the virus entry process by interfering with the virus envelope glycoprotein. HHA and GNA are odorless, colorless, and tasteless, and they are not cytotoxic, antimetabolically active, or mitogenic to human primary T lymphocytes at concentrations that exceed their antivirally active concentrations by 2 to 3 orders of magnitude. GNA and HHA proved stable at high temperature (50°C) and low pH (5.0) for prolonged time periods and can be easily formulated in gel preparations for microbicidal use; they did not agglutinate human erythrocytes and were not toxic to mice when administered intravenously.

The bicyclam AMD3100 is a highly potent and selective CXCR4 antagonist with strong antiviral activity against human immunodeficiency virus (HIV)-1 and HIV-2, which use CXCR4 as coreceptor for host cell entry. Here, we investigated the interaction of AMD3100 with CXCR4 at the molecular level by mutational analysis. We established a set of stably transfected U87.CD4 cell lines expressing different mutant forms of CXCR4 (i.e., CXCR4[WT], CXCR4[D171N], CXCR4[D262N], CXCR4[D171N,D262N], and CXCR4[H281A]), to compare the activity of the compound against mutated versus wild-type CXCR4. We found that the antagonistic action of AMD3100 against CXCR4--as assessed by the inhibitory effects of the compound on stromal cell-derived factor (SDF-1) binding to its receptor and on SDF-1-induced intracellular calcium signaling, and by displacement of the CXCR4-specific antibody, clone 12G5--was greatly reduced by substitution of Asp(171) and/or Asp(262) by neutral asparagine residue(s). Both aspartates, but most particularly Asp(262), also proved essential for the anti-HIV-1 activity of AMD3100 against the viruses NL4.3, IIIB, and HE. In contrast, substitution of His(281) by a neutral alanine potentiated the antagonistic and antiviral effects of the compound in the different assay systems. Importantly, compared with the wild-type receptor, CXCR4[D262N] was much less effective, whereas CXCR4[D171N,D262N] completely failed as a coreceptor for infection by HIV-1 NL4.3. Thus, the negatively charged aspartate residues at positions 171 and 262, located in transmembrane domains 4 and 6 of the 7-transmembrane receptor, respectively, may represent crucial sites for electrostatic interaction of the positive charges of the bicyclams, as well as for the highly basic V3 loop of the gp120 envelope protein of certain HIV-1 strains.

The chemokine receptors CCR5 and CXCR4 function as coreceptors for human immunodeficiency virus (HIV) and are attractive targets for the development of anti-HIV drugs. The most potent CXCR4 antagonists described until today are the bicyclams. The prototype compound, AMD3100, exhibits potent and selective anti-HIV activity against CXCR4-using (X4) viruses and showed antiviral efficacy in X4 HIV-1-infected persons in a phase II clinical trial. However, AMD3100 lacks oral bioavailability due to its high overall positive charge. Initial structure-activity relationship studies with bicyclam analogues suggested that the bis-macrocyclic structure was a prerequisite for anti-HIV activity. Now, we report that the N-pyridinylmethylene cyclam AMD3465, which lacks the structural constraints mentioned above, fully conserves all the biological properties of AMD3100. Like AMD3100, AMD3465 blocked the cell surface binding of both CXCL12 (the natural CXCR4 ligand), and the specific anti-CXCR4 monoclonal antibody 12G5. AMD3465 dose-dependently inhibited intracellular calcium signaling, chemotaxis, CXCR4 endocytosis and mitogen-activated protein kinase phosphorylation induced by CXCL12. Compared to the bicyclam AMD3100, AMD3465 was even 10-fold more effective as a CXCR4 antagonist, while showing no interaction whatsoever with CCR5. As expected, AMD3465 proved highly potent against X4 HIV strains (IC50: 1-10 nM), but completely failed to inhibit the replication of CCR5-using (R5) viruses. In conclusion, AMD3465 is a novel, monomacrocyclic anti-HIV agent that specifically blocks the interaction of HIV gp120 with CXCR4. Although oral bioavailability is not yet achieved, the monocyclams, with their decreased molecular charge as compared to the bicyclams, embody an important step forward in the design of oral CXCR4 antagonists that can be clinically used as anti-HIV drugs.