SEARCH

SEARCH BY CITATION

Keywords:

  • aminoglycoside–polyarginine conjugates;
  • chemokines;
  • enhanced mobilization of hematopoietic progenitor cells;
  • G-protein-coupled receptors;
  • molecular docking

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Conclusions
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Mobilization of hematopoietic stem and progenitor cells (HSPCs) from the bone marrow to the peripheral blood is utilized in clinical HSPC transplantation protocols. Retention of HSPCs in the bone marrow is determined by relationships between the chemokine chemokine (C-X-C motif) ligand 12 (CXCL12) and its major receptor C-X-C chemokine receptor type 4 (CXCR4), and disruption of this retention by CXCR4 antagonists such as AMD3100 induces rapid HSPC mobilization. Here, we report that aminoglycoside–polyarginine conjugates (APACs) and N-α-acetyl-nona-d-arginine (r9) induce mobilization of white blood cells and, preferentially, immature hematopoietic progenitor cells (HPCs) in mice, similarly to AMD3100. Remarkably, administration of AMD3100 with each one of the APACs or r9 caused additional HPC mobilization. The mobilizing activity of APACs and r9 was accompanied by a significant elevation in plasma CXCL12 levels. To further understand how APACs, r9 and their combinations with AMD3100 compete with CXCL12 binding to CXCR4, as well with antibody against CXCR4 for CXCR4 binding, we have undertaken an approach combining experimental validation and docking to determine plausible binding modes for these ligands. On the basis of our biological and docking findings, and recently published NMR data, we suggest that combination of pairs of compounds such as APACs (or r9) with AMD3100 induces more efficient disruption of the CXCL12–CXCR4 interaction than AMD3100 alone, resulting in enhanced HPC mobilization.


Abbreviations
ALL

acute lymphoblastic leukemia

ALX40-4C

N-α-acetyl-nona-d-arginine amide

APAC

aminoglycoside–polyarginine conjugate

BM

bone marrow

CFU

colony-forming unit

CXCL12

chemokine (C-X-C motif) ligand 12

CXCR4

C-X-C chemokine receptor type 4

EL

extracellular loop

GAG

glycosaminoglycan

G-CSF

granulocyte colony-stimulating factor

HPC

hematopoietic progenitor cell

HSPC

hematopoietic stem and progenitor cell

IL

interleukin

Neam-r9

nona-d-arginine–neamine conjugate

Neo-r9

nona-d-arginine–neomycin conjugate

PB

peripheral blood

r9

N-α-acetyl-nona-d-arginine

PDB

Protein Data Bank

SDM

site-directed mutagenesis

TM

transmembrane

WBC

white blood cell

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Conclusions
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Transplantation of stem cells is a preferred strategy in the treatment of a variety of hematological malignancies and disorders. In recent years, the use of peripheral blood (PB) as a source of hematopoietic stem and progenitor cells (HSPCs) for transplantation after high-dose chemotherapy has emerged as a common clinical practice [1]. Although the majority of HPSCs reside within the bone marrow (BM), a very small subset of immature cells are also found in the PB as part of steady-state homeostasis [2]. However, a process termed HSPC mobilization can significantly amplify these low levels [3]. Clinical HSPC mobilization regimens, including repeated daily stimulation with the cytokine granulocyte colony-stimulating factor (G-CSF), induce this process, leading to enhanced proliferation, differentiation and recruitment of HSPCs to the circulation, allowing their harvesting for stem cell transplantation protocols.

Interactions between the chemokine chemokine (C-X-C motif) ligand 12 (CXCL12) (also named stromal-derived factor-1α) and its major receptor C-X-C chemokine receptor type 4 (CXCR4) play a crucial role in HSPC retention within the BM [4]. Thus, disruption of the CXCL12–CXCR4 interaction may result in HSPC egress from the BM to the circulation. Indeed, a single administration of the CXCR4 antagonist AMD3100 (also termed plerixafor or Mozobil) results in rapid (within 1 h) mobilization of immature hematopoietic progenitor cells (HPCs) from the BM into the PB in mice [5], non-human primates [6], and humans [7,8]. Similar mobilization was demonstrated in the murine system by use of another CXCR4 antagonist, 4F-benzoyl-TN14003 [9]. However, the mechanisms mediating rapid mobilization of HSPCs from the BM following administration of CXCR4 antagonists are still poorly understood. We have recently demonstrated that AMD3100-induced HPC mobilization is dependent on CXCR4-mediated enhanced release of CXCL12 from activated BM stromal cells to the murine circulation [10].

Previously, we designed and synthesized a new set of aminoglycoside–polyarginine conjugates (APACs) (Fig. 1A), and reported the structure–function relationships of these novel X4 HIV-1 entry inhibitors [11]. We found that APACs inhibit a wide range of T-tropic HIV-1 isolates, interacting with CXCR4. Our previous in vitro studies demonstrated that APACs compete with antibodies against CXCR4 and inhibit CXCL12-induced cell migration in a dose-dependent manner [11,12]. Both the length of the polyarginine (6-mer versus 9-mer) and the aminoglycoside core of the APACs exhibited differential effects on the capacity of the APACs to affect CXCL12-induced cell migration, supporting the notion that the core and the length of the arginine chain affect the specificity of the interaction of APACs with CXCR4 [11,12]. We have also shown that fluorescein isothiocyanate derivatives of nona-d-arginine–neomycin conjugate (Neo-r9), nona-d-arginine–neamine conjugate (Neam-r9) and N-α-acetyl-nona-d-arginine (r9) display efficient cell uptake and accumulate intracellularly [11]. Here, we show that 9-mer APACS or r9 induce mobilization of mature blood leukocytes and, preferentially, immature HPCs in mice, accompanied by an elevation in functional CXCL12 plasma levels. Moreover, enhanced mobilizing and CXCL12-releasing effects were demonstrated upon administration of APACs or r9 in combination with AMD3100.

image

Figure 1.  Schematic representation of (A) APACs and (B) AMD3100.

Download figure to PowerPoint

Crystal structures of CXCR4 bound to small-molecule (IT1t) and cyclic peptide (CVX15) CXCR4 antagonists were recently determined [13]. However, the N-terminal residues (1–24 or 1–26) did not have interpretable electron density, and they are probably disordered in these structures [13]. As the CXCR4 N-terminus is critical for CXCL12 binding [14], we modeled the missing N-terminal part of CXCR4. To further understand how APACs, r9 and their combinations with AMD3100 compete with CXCL12 binding to CXCR4, we have undertaken an approach combining experimental validation and docking to determine plausible binding modes for these ligands, and also determined the common CXCR4-binding sites of APACs, r9, AMD3100, and CXCL12. A two-step binding model for the CXCL12–CXCR4 interaction was originally proposed by Clark-Lewis et al. [15], on the basis of extensive structure–function studies of CXCL12, and was recently validated on the basis of NMR studies [16]. The authors showed that both AMD3100 and CXCL12 could simultaneously bind to CXCR4 without release of CXCL12 from CXCR4 [16]. On the basis of these results and our findings described below, that administration of combinations of APACs (or r9) with AMD3100 enhanced the rapid mobilization of HPCs from the BM to blood and the release of CXCL12, we propose that combination of pairs of compounds such as APACs (or r9) with AMD3100 induces more efficient disruption of the CXCL12–CXCR4 interaction than AMD3100 alone. To understand the mechanism of the synergistic effect of combination of two CXCR4 inhibitors, we applied a multistep docking approach (Fig. S1). Taken together, our results clearly show that APACs and r9, as well as AMD3100, are able to interfere with the CXCL12–CXCR4 interaction. Predicted binding sites of APACs, r9, and AMD3100, mapped to the CXCR4 ligand-binding pocket, are also presented. Notably, predicted new putative binding sites for APACs and r9 mapped onto the CXCR4 N-terminus/extracellular loop (EL) 2, when these compounds bind to CXCR4 simultaneously with AMD3100, are identified. Our structural and functional results support the notion that two different CXCR4 antagonists simultaneously inhibit the CXCL12–CXCR4 interaction much more strongly than each one alone.

Results and Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Conclusions
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

APACs and r9 induce rapid preferential mobilization of HPCs and provide an additive mobilizing effect in combination with AMD3100 accompanied by elevated plasma levels of functional CXCL12

So far, G-CSF is the major mobilizing agent known to induce a dramatic elevation in the number of HSPCs in the PB. However, a significant number of patients fail to mobilize the required HSPC dose for successful transplantation [1]. Recently, AMD3100 has been approved for clinical mobilization in combination with G-CSF in lymphoma and multiple myeloma patients undergoing autologous transplantation [7]. When combined with G-CSF, AMD3100 synergistically augments mobilization of HPCs capable of increased in vitro migration to a gradient of CXCL12 and repopulation of transplanted immune-deficient NOD/SCID mice [5,17]. G-CSF activity facilitates a reduction in the amount of BM CXCL12 by means of proteolytic enzymes, leading to disruption of the CXCR4–CXCL12 interaction [3]. However, G-CSF-induced mobilization, in addition to showing broad interindividual variations in HSPC mobilization, requires 5-day repeated dosing and is frequently associated with various side effects [18]. Hence, improved methods of HSPC mobilization for hematopoietic rescue are warranted. One such approach may be a search for substitution of G-CSF in combination with AMD3100 aiming to more efficient CXCR4 binding while avoiding the proinflammatory signals exerted by G-CSF. In order to achieve this goal, we examined the capacity of APACs and r9 to mobilize HPCs alone and in combination with AMD3100.

To assess the mobilizing potential of APACs and r9, we used the protocol applied for AMD3100 that demonstrated the maximal HPC-mobilizing effect upon single subcutaneous injection at 5 mg·kg−1 (125.9 nm), with mice being killed 1 h later [5,10]. By using this protocol, we found that Neo-r9, Neam-r9 and r9 had significant HPC-mobilizing activity upon administration at 1, 5 and 10 mg·kg−1, and a slightly (although not statistically different) more pronounced effect upon application at 1 mg·kg−1 (Table S1). At 0.5 mg·kg−1, these compounds were ineffective (data not shown). On the basis of these findings, we used Neo-r9, Neam-r9 and r9 in all experiments at 1 mg·kg−1 (6.9, 8.1, and 10 nm, respectively). As shown in Fig. 2A, Neo-r9, Neam-r9 and r9 induced significant white blood cell (WBC) mobilization (1.82, 2.56 and 1.8-fold stimulation, respectively, as compared with an NaCl/Pi control), with no significant difference between these compounds. It is of note that AMD3100 applied as a standard had a similar effect, namely, 1.88-fold stimulation. Remarkably, however, according to molar calculations, the efficacy of Neo-r9, Neam-r9 and r9 was 12.5–18-fold stronger than that of AMD3100.

image

Figure 2.  Administration of APACs and r9 to mice leads to mobilization of WBCs and HPCs and increases in CXCL12 plasma levels, which all are enhanced in the presence of AMD3100. Mice were injected with NaCl/Pi (control), Neo-r9 (Neo), Neam-r9 (Nm) or r9 alone (all at 1 mg·kg−1) and in combination with AMD3100 (AMD, 5 mg·kg−1), as described in Experimental procedures. (A) Number of WBCs·mL−1 PB. (B) Number of CFUs·mL−1 PB, reflecting HPC number. (C) CXCL12 plasma levels detected by ELISA. Means ± standard deviations of three mice per group from a total of three separate experiments are shown. NS, not statistically different (P > 0.05) from mice injected with AMD3100 alone (AMD).

Download figure to PowerPoint

In combination experiments, AMD3100, Neo-r9, Neam-r9 and r9 were injected separately but simultaneously. In these experiments, r9 showed a significant additive WBC-mobilizing effect (1.79-fold stimulation as compared to the effect of AMD3100 alone), whereas Neo-9 and Neam-9 did not augment the WBC-mobilizing activity of AMD3100 (Fig. 2A).

Upon examining HPC mobilization, we found that Neo-r9, Neam-r9 and r9 injected alone induced robust HPC mobilization (Fig. 2B). Interestingly, the mobilizing activity of Neam-r9 was similar to that of AMD3100 (5.95 and 6.33-fold stimulation, respectively, as compared with the NaCl/Pi control). Although the effects of Neo-r9 and r9 were weaker (4.15 and 3.87-fold stimulation, respectively, as compared with the NaCl/Pi control), the overall HPC-mobilizing activity of APACs was much stronger than their WBC-mobilizing activity (Fig. 2A), implying a preferential HPC-mobilizing potential, as compared with mature blood leukocytes. Similar favored HPC mobilization was demonstrated recently by the examination of 4F-benzoyl-14003 [9] and AMD3100 [10]. Importantly, short (6-mer) APACs as well as neomycin were unable to induce WBC and HPC mobilization (data not shown).

In combination with AMD3100, Neo-r9, Neam-r9 and r9 had significant additive HPC-mobilizing effects (2.03, 1.62 and 1.91-fold stimulation, respectively, as compared with the effect of AMD3100 alone) (Fig. 2B). These data suggest that combination of APACs with AMD3100 induces a preferential additive HPC-mobilizing effect as compared with WBC mobilization, suggesting the potential application of such combinations in clinical stem cell transplantation protocols.

Little is known about the mechanisms governing rapid HSPC mobilization. We have recently shown that AM3100-induced HPC mobilization is dependent on enhanced release of functional CXCL12 to the murine circulation from activated BM stromal cells, thus creating a CXCL12 gradient that is capable of chemoattracting CXCR4+ HPCs [10]. In order to examine whether such a mechanism is involved in APAC-induced mobilization, we determined the plasma levels of noncleaved functional CXCL12 in mice injected with APACs alone and in combination with AMD3100. In these experiments, we found that injection of Neo-r9, Neam-r9, r9 and AMD3100 was accompanied by similar (2.67, 2.28, 1.57 and 2.17-fold stimulation, respectively) increases in CXCL12 plasma levels. These observations suggest that APACs and r9, like AMD3100, are able to enhance the release of CXCL12 from the BM to the circulation. It is noteworthy that the combination of Neo-r9, Neam-r9 and r9 with AMD3100 resulted in a significant, ∼ 1.5-fold, additional elevation in CXCL12 plasma levels (Fig. 2C).

Altogether, these observations show that 9-mer APACs rapidly, strongly and preferentially mobilize HPCs. This activity is significantly enhanced in the presence of AMD3100. The mechanism of APAC-induced mobilization may involve enhanced secretion of CXCL12 by BM stromal cells, creating a gradient of functional CXCL12 that facilitates egress of HPCs from the BM to the circulation.

Putative CXCR4-binding sites of each ligand

It is well known that the CXCR4 natural ligand CXCL12 has predominantly positive potential, which permits it to bind to the highly negatively charged extracellular domains of CXCR4. According to site-directed mutagenesis (SDM) data [14,19,20] and NMR studies [16,21], CXCL12 binds to the N-terminus and the ligand-binding pocket of CXCR4. Neo-r9, Neam-r9, r9, AMD3100 and CVX15 also possess strong positive potentials (Fig. S2) and can compete with CXCL12 for binding to CXCR4.

Experimentally, the capability of AMD31000 and some other anti-CXCR4 agents to bind CXCR4 was demonstrated in competition assays by using labeled CXCL12 or mAbs against CXCR4 [20,22–26]. In one of these studies, AMD3100 and the CXCR4 antagonists T140, T140012 and T134 efficiently blocked binding of an antibody against CXCR4 (12G5) to membrane CXCR4 on the human pre-B acute lymphoblastic leukemia (ALL) cell line NALM-6 and primary human ALL cells [26]. By using another pre-B-ALL cell line, G2, which is known to express CXCR4 at a high level [27], we found that AMD3100 and r9 significantly reduced binding of 12G5 to CXCR4 (Fig. 3). Most importantly, combination of these compounds led to an additional dramatic reduction in CXCR4 binding, comparable to that obtained when the cells were pretreated with CXCL12 alone (Fig. 3).

image

Figure 3.  Inhibition of binding of antibody against CXCR4 CXCR4 on G2 cells by AMD3100 and r9. G2 cells were incubated untreated (UT), with CXCL12, AMD3100 (AMD) r9 or their combination, and then stained with 12G5, as described in Experimental procedures. Representative dot blot analysis is presented from two separate experiments.

Download figure to PowerPoint

To test the ability of the docking program molfit to reassemble the crystal structure of CXCR4 in complex with CVX15 [Protein Data Bank (PDB) entry 3OE0] [13], we performed a full geometric–electrostatic scan of CVX15 bound to CXCR4 (Fig. S3A). The first top-ranked molfit solution was very similar to the crystal structure (Fig. S3B). A cluster consisting of 27 top-ranking solutions was located in the ligand-binding pocket (Fig. S3C); the solutions ranked 2–8 were also similar to the crystal structure. The geometric–electrostatic docking of Neo-r9 revealed its strong preference for binding CXCR4 in the ligand-binding pocket (Fig. 4A), which was determined in the crystal structures of ligand–CXCR4 complexes. The geometric–electrostatic docking results for Neam-r9 and r9 were similar to those for Neo-r9. It is of note that all of our tested ligands were in the unbound state only at the step of their construction and accompany energy minimizations in water.

image

Figure 4.  (A–C) Results of ligand–CXCR4 geometric–electrostatic scans of molfit. Each solution is the plausible position of the ligand on CXCR4, represented by its center of mass. The 1000 top-ranked molfit solutions are shown as points. (A) molfit scan of Neo-r9 alone (colored blue). The first top-ranked molfit solution is shown as green sticks. (B) molfit scan of AMD3100 alone (colored magenta). The first top-ranked molfit solution is shown as sticks. (C) molfit scan of Neo-r9 in the presence of AMD3100. (D–F) Electrostatic potential maps calculated by delphi. The equipotential contours at −3 kT/e and +3 kT/e are shown. Red indicates −3 kT/e and blue +3 kT/e. (D) Unbound CXCR4. (E) AMD3100 bound to CXCR4. (F) Both AMD3100 and Neo-r9 bound to CXCR4.

Download figure to PowerPoint

The interaction energy ranges for the ligand–CXCR4 complexes in which the ligands docked to the experimentally determined binding surface were significantly lower than the energies calculated for complexes with the ligands bound in other regions (Table 1). All ligand–CXCR4 complexes (Neo-r9, Neam-r9, r9, and AMD3100) with the lowest interaction energies were obtained by refinement of the corresponding top-ranking molfit solution. The nine arginines of Neo-r9 formed salt bridges with CXCR4 residues in the ligand-binding pocket and the N-terminus (Glu2, Glu15, Asp22, Glu32, Glu179, Asp181, Asp193, Asp262, and Glu288) (Fig. 5A; Table 2). The neomycin core of Neo-r9 formed hydrogen bonds with Asp171 and Asp187. According to our docking results, several CXCR4 residues (Glu15, Tyr21, Asp22, Pro27, Tyr116, Asp171, Asp187, Asp262, and Glu288) are common to the CXCR4 binding surfaces for Neo-r9 and CXCL12. This result could explain their competitive binding.

Table 1.   Interaction energies for the ligand–CXCR4 complexes.
CompoundEnergy (kcal·mol−1)
Final complexesComplexes in the other regions (control)
  1. a In the presence of AMD3100. b In the presence of CVX15.

Neo-r9 alone−3163 to −3027−2566 to −2145
Neam-r9 alone−3153 to −2987−2512 to −2147
r9 alone−3099 to −2898−2443 to −2187
AMD3100−1198 to −1126−767 to −605
Neo-r9a−3068 to −2856−2533 to −2187
Neam-r9a−2926 to −2788−2458 to −2097
r9a−2867 to −2687−2409 to −2059
Neo-r9b−3076 to −2992−2501 to −2093
image

Figure 5.  CXCR4 ligand-binding modes. (A) Neo-r9 (cyan) alone, (B) Neam-r9 (pink), (C) r9 (gray), and (D) AMD3100 (magenta) alone. (E) Comparison of CXCR4 ligand-binding modes of docked ligands: Neo-r9, Neam-r9, r9, and AMD3100, with ligands CVX15 (PDB entry 3OE0 [13], red) and IT1t (PDB entry 3ODU [13], blue) from crystal structures. CXCR4 is shown as a yellow transparent ribbon. The CXCR4 residues that participate in Neo-r9, Neam-r9, r9 and AMD3100 binding are shown as sticks. Oxygen atoms are red, and nitrogen atoms are blue. The CXCR4 residues that participate in CXCL12 binding are in red. Ligands are shown in stick representation. Salt bridges are shown as red dashed lines, and hydrogen bonds as blue dashed lines. CXCR4 residues that participate in the formation of salt bridges and hydrogen bonds are shown in italics and underlined.

Download figure to PowerPoint

Table 2.    CXCR4 ligand-binding sites. EL and TM residue distribution is according to [13].
(A) N-terminus
LigandResidues
Met1Glu2Ile4Ile6Tyr7Thr8Ser9Asp10Asn11Tyr12Glu14Glu15Met16
CXCL12a   ++b+++++b++b+b
12G5c  +d++d+++++d++d+d
Neo-r9 alone +         +b,d 
Neam-r9 alone +         +b,d+b,d
r9 alone +         +b,d 
Neo-r9e+++d +b,d    +b,d   
Neam-r9e+++d +b,d        
r9e+++d +b,d        
(B) N-terminus (continued)
LigandResidues
Gly17Ser18Gly19Asp20Tyr21Asp22Ser23Met24Glu26Pro27Arg30Glu31Glu32
CXCL12a+b++b+b+b+b+++b+b+b+b+b
12G5c+d++d+d+d+d+++d+d+d+d+d
Neo-r9 alone    +b+b,d   +b,d  +b,d
Neam-r9 alone+b,d    +b,d   +b,d  +b,d
r9 alone     +b,d   +b,d  +b,d
Neo-r9e  +b,d+b,d+b,d   +b,d+b,d+b,d+b,d+b,d
Neam-r9e  +b,d+b,d+b,d   +b,d +b,d+b,d+b,d
r9e  +b,d+b,d+b,d   +b,d+b,d +b,d+b,d
CVX15f         +b,d   
(C) TM1, TM2, TM3, TM4
LigandResidues
TM1TM2TM3TM4
Leu41Trp94Asp97His113Tyr116Thr117Leu120Asp171
CXCL12g ++b +b  +b
12G5c  +d     
Neo-r9 alone+  ++b  +b
AMD3100h  +b,d++b+++b
CVX15f   ++b+ +b
(D) EL2
LigandResidues
Asn176Ser178Glu179Asp181Asp182Ile185Cys186Asp187Arg188Phe189Tyr190Pro191Asn192
CXCL12g       +b     
12G5c+ +d+d+d     +d  
Neo-r9 alone ++d+d + +b+++d  
Neam-r9 alone  +d+d   +b++   
r9 alone  +d+d+d        
AMD3100h       +b+    
CVX15f +    ++b+++d++
(E) TM5, TM6
LigandResidues
TM5TM6
Asp193Val196Phe199Gln200His203Tyr255Ile259Asp262Leu266
CXCL12g+b     ++b 
Neo-r9 alone+b+ +   +b+
Neam-r9 alone+b      +b 
r9 alone+b      +b+
AMD3100h    ++   
CVX15f+b+++ + +b+
(F) TM7
LigandResidues
Glu277His281Ile284Ser285Glu288
  1. a According to NMR data [21]. b Common residues for CXCL12 and AMD3100, Neo-r9, Neam-r9 or r9 binding. c [14]. d Common residues for 12G5 and AMD3100, Neo-r9, Neam-r9 or r9 binding. e In the presence of AMD3100. f [13]. g According to SDM data [14,19,20,25,31]. h Same data for AMD3100 alone and in combination with Neo-r9, Neam-r9, or r9.

CXCL12g    +b
Neo-r9 alone + ++b
Neam-r9 alone + ++b
r9 alone++ ++b
AMD3100g   ++b
CVX15f+++++b

The final Neam-r9–CXCR4 complex with the lowest interaction energy (−3153 kcal·mol−1) generally resembled the Neo-r9–CXCR4 complex (Fig. 5B; Table 2). Neam-r9 also formed nine salt bridges and two hydrogen bonds with CXCR4 residues. Neam-r9 also had multiple common residues with CXCL12, suggesting strong competition with CXCL12–CXCR4 binding. The final r9–CXCR4 complex with the lowest interaction energy (−3099 kcal·mol−1; Table 2; Fig. 5C) was also generally similar to the Neo-r9–CXCR4 and Neam-r9–CXCR4 complexes. This compound also formed nine salt bridges, but only one hydrogen bond.

The geometric–electrostatic docking scan for AMD3100 bound to CXCR4 revealed a cluster of solutions, located at the bottom of the ligand-binding pocket, between the transmembrane (TM) helices (Fig. 4B). The range of interaction energies for the AMD3100–CXCR4 complexes was −1198 to −1126 kcal·mol−1 (Table 1). The AMD3100–CXCR4 complex with the lowest energy is presented in Fig. 5D, and the contacts between AMD3100 and CXCR4 are listed in Table 2. The nitrogens (two on the first cyclam ring and two on the second one of AMD3100) undergo strong electrostatic interactions with Asp97, Asp187, Asp171, and Glu288. The CXCR4 residues His113, Ty116, Thr117, Leu120, Arg188, His203, Tyr255 and Ser285 also contribute to AMD3100 binding. The SDM results showed that Tyr45, Trp94, Asp97, Ty116, Asp171, Tyr190, Asp262 and Glu288 are important for AMD3100 binding [25]. In general, our docking results are in agreement with previously published docking data for AMD3100–CXCR4 homology models [28,29]. The AMD3100–CXCR4 complexes in this region are energetically more favorable (−1198 to −1126 kcal·mol−1) than the AMD3100–CXCR4 complexes obtained in other regions (−767 to −605 kcal·mol−1). All five AMD3100–CXCR4 complexes obtained between the TM helices resemble each other, and differ only in hydrophobic interactions with the ligand. Hence, the full geometric–electrostatic scan, final refinement, SDM data [20,22,25] and docking results of other groups [28–30] strongly suggest that AMD3100 binding to CXCR4 is very specific and is restricted to the bottom of the ligand-binding pocket between the TM helices. The binding site of AMD3100 overlaps with the CXCR4 residues that, according to SDM results, are important for CXCL12 binding [14,19,20,25], suggesting strong competition between these ligands (Table 2; Fig. 5D).

Neo-r9, Neam-r9 and r9 are comparable in size to the 16-residue ligand CVX15. CVX15 occupies most of the volume of the CXCR4 ligand-binding pocket (PDB entry 3OE0 [13]). The binding modes of Neo-r9, Neam-r9 and r9 are similar to that of CVX15 (Fig. 5E). In contrast, the binding mode of AMD3100 is similar to that of the small-molecule ligand IT1t (PDB entry 3ODU [13]). AMD3100 and IT1t contact only the CXCR4 residues at the bottom of the ligand-binding pocket, and do not undergo interactions with N-terminal residues.

The effect of combinations of APACs (or r9) with AMD3100 on the release of CXCL12 from CXCR4

On the basis of extensive structure–function studies of CXCL12 and on NMR studies, a two-step (also called two-site) binding model for the CXCL12–CXCR4 interaction was proposed [15,16]. According to this binding model, in the first step the β-sheet, 50-s loop, and N-loop of CXCL12 interact with the CXCR4 extracellular region and facilitate the rapid binding and efficient anchoring of CXCL12 on the extracellular side of CXCR4 [16]. In this step, the highly dynamic CXCL12 N-terminus searches for the binding cavities buried within the TM helices. The NMR structure of CXCL12 in complex with the CXCR4 N-terminal fragment (PDB entry 2K05 [21]) probably represents at least part of the ‘first step’ complex, and shows important interactions between CXCL12 and the CXCR4 N-terminal residues that are missing in the crystal structures of ligand–CXCR4 complexes [13] (Fig. 6). In the second binding step, the CXCL12 N-terminus interacts with the CXCR4 TM region. This interaction triggers conformational changes in the CXCR4 TM that induce G-protein signaling. Moreover, the NMR data reveal that the CXCL12 N-terminus is not responsible for CXCR4 binding in the presence of AMD3100, and that the binding of the CXCL12 β-sheet and 50-s loop to CXCR4 is unaffected by AMD3100 [16]. The authors suggested that when the stable interaction between CXCR4 and CXCL12 occurs (excluding the N-terminus), the AMD3100–CXCR4 complex still exists [16]. Taking into consideration that AMD3100 binds CXCR4 in the bottom of the ligand-binding pocket between the TM helices [22,25], the NMR experiments demonstrated that the CXCL12 N-terminus binds CXCR4 in the TM region, whereas the other CXCL12 regions should bind CXCR4 extracellular regions [16]. These two interactions should occur independently, as AMD3100 could block only the interaction of the CXCL12 N-terminus and the CXCR4 TM region [16]. In other words, AMD3100 binding could block G-protein signaling, but still permit the CXCL12–CXCR4 interaction, and both AMD3100 and CXCL12 could simultaneously bind to CXCR4. Therefore, CXCL12 may not be released from CXCR4 in the presence of AMD3100 only [16]. These data may explain our findings that AMD3100 alone has weaker effects on cell mobilization and CXCL12 release than it does in combination with one of the other inhibitors: Neo-r9, Neam-r9, or r9. Thus, a question arises: can Neo-r9, Neam-r9 or r9 bind CXCR4 even though the region at the bottom of the ligand-binding pocket between the TM helices of CXCR4 is already occupied by the specific binder AMD3100?

image

Figure 6.  Schematic diagram of the two-step mechanism for the CXCL12–CXCR4 interaction. The NMR structure of CXCL12 in complex with the 38-residue sulfotyrosine-containing peptide derived from the CXCR4 N-terminus (PDB entry 2K05 [21]) is thought to represent at least part of the ‘first step’ complex (presented in the upper part of the figure). Note that the conformations and contacts of CXCL12 and the CXCR4 N-terminus within the whole receptor are somewhat different from those of CXCL12 and the 38-residue sulfotyrosine-containing peptide. The CXCR4 N-terminal peptide is colored yellow. The CXCR4 residues that participate in CXCL12 binding are shown as sticks. Oxygen atoms are red, nitrogen atoms are blue, and sulfur atoms of sulfotyrosines are orange. Salt bridges are shown as red dashed lines, and hydrogen bonds as blue dashed lines. The CXCR4 residues that participate in the formation of salt bridges and hydrogen bonds are shown in italics and underlined. CXCL12 is shown as a purple ribbon, and the CXCL12 residues that participate in the formation of salt bridges and hydrogen bonds with the CXCR4 N-terminus are shown as sticks. CXCL12 arginines and lysines are colored cyan. For details about CXCR4 residues that participate in CXCL12 binding, see also Table 2. SB salt bridge.

Download figure to PowerPoint

The electrostatic potential of the AMD3100–CXCR4 complex with the lowest energy was calculated, revealing that AMD3100 binding to CXCR4 significantly changed the electrostatic potential from negative to positive in the TM region (Fig. 4E). The geometric–electrostatic molfit docking scans of Neo-r9, Neam-r9 and r9 bound to the AMD3100–CXCR4 complex revealed a single cluster for each ligand containing the 1000 top-ranked molfit solutions, located at the CXCR4 N-terminus and at the top of the ligand-binding pocket (only the results for Neo-r9 are presented in Fig. 4C).

The refined CXCR4–AMD3100 complexes with Neo-r9, Neam-r9 and r9 with the lowest interaction energies were obtained from the first top-ranked molfit solution for each ligand. The Neo-r9–CXCR4–AMD3100 complex with the lowest interaction energy (−3068 kcal·mol−1) is shown (Fig. 7A; Table 2). The arginines of Neo-r9 formed eight salt bridges with the CXCR4 N-terminal and EL2 residues Glu2, Asp20, Glu26, Glu31, Glu32, Glu179, Asp181, and Asp182, and two hydrogen bonds with Met1 and Tyr7. Neo-r9 also formed two additional hydrogen bonds with Tyr12 and Tyr21 via the amino groups of its neomycin core. According to our docking results, several CXCR4 residues participating in CXCL12 binding [14,19,20,25] also participated in Neo-r9 binding (Table 2), suggesting strong competition between these two ligands at the CXCR4 N-terminus.

image

Figure 7.  CXCR4 ligand-binding modes (continuation). (A) Combination of Neo-r9 and AMD3100. (B) Combination of Neam-r9 and AMD3100. (C) Combination of r9 and AMD3100. (D) Combination of Neo-r9 and CVX15. CXCR4 is shown as a yellow ribbon. The colors, representations and labels are as in Fig. 5.

Download figure to PowerPoint

The refined Neam-r9–CXCR4–AMD3100 (−2926 kc al·mol−1; Fig. 7B; Table 2) and r9–CXCR4–AMD3100 (−2867 kcal·mol−1; Fig. 7C; Table 2) complexes with the lowest interaction energies were very similar to the Neo-r9–CXCR4–AMD3100 complex. The interaction energy of r9 was comparable to those of Neo-r9 and Neam-r9, and it had multiple contacts with CXCR4 N-terminal and EL2, including common with CXCL12, suggesting relatively strong competition with the natural ligand.

Although the ranges of interaction energies of CXCR4–AMD3100 complexes for Neo-r9, Neam-r9, and r9, i.e. at the N-terminus/EL2, were higher than those for complexes of these ligands alone with CXCR4, the absolute values of interaction energies of ligand–CXCR4–AMD3100 complexes were also sufficient (Table 1). These data suggest that these ligands preferably bind CXCR4 in the ligand-binding pocket. However, when AMD3100 blocks CXCR4 in the ligand-binding pocket between the TM helices, r9, Neo-r9 and Neam-r9 can form energetically favorable complexes with CXCR4 at the upper part of this ligand-binding pocket and at its N-terminus.

To further understand how N-α-acetyl-nona-d-arginine amide (ALX40-4C) (which is similar to r9) interacts with CXCR4 and to demonstrate the specificity of its interaction, Doranz et al. [31] used a series of previously described CXCR4 chimeras and mutants. Substitution of the distal N-terminus of CXCR4 had little impact on the ability of ALX40-4C to block HIV-1 infection. ALX40-4C was completely incapable of inhibiting infection mediated by CXCR4-D193K [31] (this residue is situated in the ligand-binding pocket).It is of note that these studies were performed with ALX40-4C alone. We also propose that, when Neor-r9, Neam-r9 and r9 are administered alone, they preferably bind CXCR4 in the ligand-binding pocket and only partially at the N-terminus. Probably, only when the bottom of the ligand-binding pocket between the TM helices of CXCR4 is already occupied by AMD3100 can these ligands bind to the N-terminus/EL2. According to the NMR structure of CXCL12 complexed with a 38-residue sulfotyrosine-containing peptide derived from the CXCR4 N-terminus [21], CXCL12 arginines and lysines form four salt bridges (Arg8–Asp10, Arg41–Glu14, Arg47–Asp20, and Lys56–Glu32) and hydrogen bonds with sulfotyrosines Tyr12 and Tyr21 (Fig. 6). Our docking results show that Neor-r9, Neam-r9 and r9 bind almost all of these CXCR4 N-terminal residues (except for Glu14).

The electrostatic potential of the CVX15–CXCR4 complex, based on the crystal structure [13] with the modeled N-terminus (presented here), was calculated, and docking of Neo-r9 to this complex was performed according to our docking procedure. The position of Neo-r9 complexed with CXCR4–CVX15 (Fig. 7D) was similar to that in the Neo-r9–CXCR4–AMD3100 complex. The Neo-r9–CXCR4–CVX15 interaction was also found to be energetically favorable – the interaction energy of Neo-r9 of this complex was even lower than that of Neo-r9 in the Neo-r9–CXCR4–AMD3100 complex. Thus, it can be suggested that Neo-r9 and CVX15 also simultaneously bind CXCR4, completely preventing its binding to CXCL12.

To illustrate the effect of simultaneous binding of Neo-r9 and AMD3100, we calculated their electrostatic potential under conditions when both of these ligands are bound to CXCR4 (Fig. 4F). The simultaneous binding dramatically changed the electrostatic potential of CXCR4, completely preventing the highly positively charged CXCL12 from binding to CXCR4.

Interestingly, complexes of CXCR4 with Neo-r9, Neam-r9 and r9 alone with the lowest interaction energies obtained in the ligand-binding pocket, as well as CXCR4–AMD3100 complexes with these ligands, reveal strong overlap with 12G5 (Table 2). These results could explain competitive binding between r9 and 12G5. Notably, combination of r9 with AMD3100 led to an additional dramatic reduction in CXCR4 binding, similar to that obtained when the cells were pretreated with CXCL12 (Fig. 3). Previously, we demonstrated the competition of Neo-r9, Neam-r9 and r9 alone with 12G5 [11].

Our experimental and modeling findings demonstrate a major role of CXCR4 binding in rapid HPC mobilization by AMD3100 and APACs. However, additional mechanisms, such as interaction of glycosaminoglycans (GAGs) with CXCL12, might also be involved. It has been found that a single injection of fucoidan at 50–100 mg·kg−1 in mice results in strong and rapid (within 30 min to 3 h) mobilization of HPCs accompanied by a significant elevation in CXCL12 plasma levels [32,33]. More recently, Albanese et al. [34] demonstrated the ability of two GAG mimetics to induce rapid mobilization of HPCs, also followed by an elevation of CXCL12 plasma levels; these effects of GAG mimetics were diminished by application of antibodies against CXCL12. A proposed mechanism includes the ability of GAGs to bind CXCL12 at its heparin-binding domain, thus displacing sequestered CXCL12 from heparan sulfate GAGs, and consequently increasing the circulating levels of CXCL12 [32]. These observations suggest that, in addition to their direct CXCR4-blocking activity, our polyarginine-containing compounds may also compete with the CXCL12–GAG associations that are needed for optimal CXCL12 binding to CXCR4. This interesting problem could be the subject of a further independent study.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Conclusions
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Altogether, our modeling data, which are strongly supported by the experimental results, suggest that the AMD3100–CXCR4 complex still permits the CXCL12–CXCR4 interaction and only partially prevents CXCL12 release from CXCR4. When Neo-r9, Neam-r9 and r9 alone bind to CXCR4 in the ligand-binding pocket, they probably also do not completely release CXCL12 from CXCR4, although they are more potent competitors of CXCL12 than AMD3100, owing to their partial interaction with the N-terminal region. It is of note that administration of only 1 mg·kg−1 each of Neo-r9, Neam-r9 and r9 (corresponding to 6.9–10 nm) to mice induces similar mobilization of HPCs and increases in functional CXCL12 plasma levels as 5 mg·kg−1 AMD3100 (corresponding to 125.9 nm). Combination of one of these ligands with AMD3100 can more efficiently prevent the CXCL12–CXCR4 interaction. These results support a significant enhancement of HPC mobilization as and CXCL12-releasing effects upon application of the combination of two different CXCR4 ligands, namely AMD3100 and one of the APACs (or r9). On the basis of our docking results, we predict that combination of each one of the APACs or r9 with CVX15 could also affect the CXCL12–CXCR4 interaction more efficiently than CVX15 alone. Our compounds may utilize the extracellular part of CXCR4, in addition to the well-known ligand-binding pocket within the TM helices. These data could be important for the development of HPC-mobilizing drugs.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Conclusions
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

The synthesis, purification and analysis of Neo-r9, Neam-r9, and r9 were performed as previously described [11]. AMD3100 was purchased from Sigma-Aldrich (Rehovot, Israel).

HPC mobilization protocol

The Weizmann Institute Animal Care and Use Committee approved all animal experiments. B6.SJL mice (8–10 weeks old), bred in the Weizmann Institute, were injected subcutaneously with NaCl/Pi (control), Neo-r9, Neam-r9 and r9 (all at 1 mg·kg−1; 6.9, 8.1, and 10 nm, respectively) or AMD3100 (5 mg·kg−1; 125.9 nm). In the preliminary experiments, these doses of APACs and r9 revealed the maximal mobilizing effect (Table S1). In combination experiments, mice were subcutaneously injected with AMD3100 together with Neo-r9, Neam-r9 or r9 at the doses indicated above. In both groups, mice were killed 1–1.5 h after injection of the compounds, the PB samples were collected in heparinized tubes by cardiac aspiration of CO2-asphyxiated mice, and the number of WBCs was calculated. After centrifugation at 400 g for 5 min, plasma was collected and used for the determination of CXCL12 protein levels by ELISA. For evaluation of the number of HPCs, PB mononuclear cells were isolated with Lymphoprep (Fresenius Kabi Norge AS), and 2 × 105 per mL collected cells were seeded for the HPC colony assay as previously described [35], with 50 ng·mL−1 murine stem cell factor, 5 ng·mL−1 murine interleukin (IL)-3, 5 ng·mL−1 murine granulocyte–macrophage colony-stimulating factor, and 2 U·mL−1 human erythropoietin. Colonies, reflecting colony-forming progenitors, were scored 7 days after plating and presented as colony-forming progenitor number·mL−1 blood.

CXCL12 ELISA

CXCL12 levels in tested plasma samples were determined by ELISA as described previously [36]. The K15C mAb (INRA, Paris, France) recognizing the first three amino acids of CXCL12 was utilized as a capture antibody (10 μg·mL−1) in order to measure the levels of noncleaved, functional CXCL12 [10].

Flow cytometry

The abilities of r9, AMD3100 and their combination to compete with CXCR4 were studied by flow cytometry with the strongly CXCR4-expressing pre-B-ALL cell line G2 [27] and the protocol excluding CXCR4 internalization [26]. Cells (5 × 104 cells in 50 μL of FACS buffer [NaCl/Pi containing 1% fetal bovine serum (Invitrogen, Grand Island, NY, USA) and 0.2% sodium azide, pH 7.4) were first incubated untreated, with CXCL12 (2 μm; PeproTech, Rocky Hill, NJ, USA), AMD3100 (100 nm), r9 (1 μm) or AMD3100 and r9 at 4 °C for 30 min, and then with 12G5 (R&D Systems, Minneapolis, MN, USA) at 4 °C for an additional 30 min. The cells were then washed in FACS buffer at 4 °C, and stained with Alexa Fluor 488 (Invitrogen, Molecular Probes, Eugene, USA) at 4 °C for 30 min and analyzed by flow cytometry on a FACSCalibur (Becton Dickinson, San Jose, CA, USA) with cellquest software.

Statistical analysis

Data were analyzed with a two-tailed Student’s t-test by Excel 2004. Values with P < 0.05 were considered to be statistically significant. Error bars represent standard errors.

CXCR4 and ligands

The crystal structures of CXCR4 with its ligands IT1t and CVX15 were recently determined [13]. N-terminal residues of CXCR4 (1–24 or 1–26, depending on structure) are absent in these structures. We modeled the missing N-terminal part of CXCR4 with the homology module of insightII (Accelrys, San Diego, CA, USA). NMR data for the structure of the CXCR4 N-terminus in complex with CXCL12 (PDB entry 2K05 [21]) were applied for N-terminus modeling. The modeled N-terminus was added to the crystal structure of CXCR4 (PDB entry 3OE0 [13]) and energy minimized, and the following conditions were fulfilled: the modeled N-terminus had no steric clashes with the ligand CVX15, and did not interfere with CXCR4 homodimer formation.

AMD3100 and r9 were constructed by using the biopolymer module of insightII. Modeling of Neo-r9 and Neam-r9 has been described previously [37,38]. These modeled compounds were solvated in water and energy minimized. The ligands were docked to the CXCR4 crystal structure complemented with the modeled N-terminus (Fig. S1).

Docking of individual ligands

In the first step, each ligand was individually docked to CXCR4 with the geometric–electrostatic version of the rigid-body docking program molfit [39,40]. The electrostatic potential of each molecule was calculated with delphi as implemented in the insightII package [41]. The final refinement of the top-scoring models from the geometric–electrostatic molfit full scan was performed by energy minimization with the discover3 module of insightII, with default settings. We refined the five top-ranked molfit solutions for each ligand; five high-scoring molfit solutions in the other regions were used as a negative control. The backbone of CXCR4 was fixed, except for the N-terminal residues 1–28. The energies of the refined complexes were estimated with the docking module of insightII, according to maximal lengths of ligands (15–31 Å).

Docking of combinations of two ligands

The second ligand was docked to the final ligand–CXCR4 complex obtained in the previous step. Four pairs of ligands were tested: Neo-r9/AMD3100, Neam-r9/AMD3100, r9/AMD3100, and Neo-r9/CVX15. The electrostatic potential of the ligand–CXCR4 complex was recalculated by delphi. Thereafter, a new, full geometric–electrostatic molfit scan was performed for the second ligand (e.g. Neo-r9) and CXCR4 complexed with the first ligand (e.g. AMD3100). The same molfit parameters were employed in all steps: grid interval of 1.0 Å and rotational interval of 12°. Next, energy minimization with discover3 was performed to refine the five top-scored solutions from the molfit scan for the second ligand.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Conclusions
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank M. Eisenstein for critical remarks, discussions, and providing the docking program molfit, and G. Borkow, O. Kollet, R. Alon, A. Dar and K. Lapid for discussions and critical reading of the manuscript. The docking program molfit was downloaded from: http://www.weizmann.ac.il/Chemical_Research_Support/molfit.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Conclusions
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
  • 1
    Pelus LM & Fukuda S (2008) Chemokine-mobilized adult stem cells; defining a better hematopoietic graft. Leukemia 22, 466473.
  • 2
    Wright DE, Wagers AJ, Gulati AP, Johnson FL & Weissman IL (2001) Physiological migration of hematopoietic stem and progenitor cells. Science 294, 19331936.
  • 3
    Lapidot T & Petit I (2002) Current understanding of stem cell mobilization: the roles of chemokines, proteolytic enzymes, adhesion molecules, cytokines, and stromal cells. Exp Hematol 30, 973981.
  • 4
    Dar A, Kollet O & Lapidot T (2006) Mutual, reciprocal SDF-1/CXCR4 interactions between hematopoietic and bone marrow stromal cells regulate human stem cell migration and development in NOD/SCID chimeric mice. Exp Hematol 34, 967975.
  • 5
    Broxmeyer HE, Orschell CM, Clapp DW, Hangoc G, Cooper S, Plett PA, Liles WC, Li X, Graham-Evans B, Campbell TB et al. (2005) Rapid mobilization of murine and human hematopoietic stem and progenitor cells with AMD3100, a CXCR4 antagonist. J Exp Med 201, 13071318.
  • 6
    Larochelle A, Krouse A, Metzger M, Orlic D, Donahue RE, Fricker S, Bridger G, Dunbar CE & Hematti P (2006) AMD3100 mobilizes hematopoietic stem cells with long-term repopulating capacity in nonhuman primates. Blood 107, 37723778.
  • 7
    Devine SM, Vij R, Rettig M, Todt L, McGlauchlen K, Fisher N, Devine H, Link DC, Calandra G, Bridger G et al. (2008) Rapid mobilization of functional donor hematopoietic cells without G-CSF using AMD3100, an antagonist of the CXCR4/SDF-1 interaction. Blood 112, 990998.
  • 8
    Liles WC, Rodger E, Broxmeyer HE, Dehner C, Badel K, Calandra G, Christensen J, Wood B, Price TH & Dale DC (2005) Augmented mobilization and collection of CD34+ hematopoietic cells from normal human volunteers stimulated with granulocyte-colony-stimulating factor by single-dose administration of AMD3100, a CXCR4 antagonist. Transfusion 45, 295300.
  • 9
    Abraham M, Biyder K, Begin M, Wald H, Weiss ID, Galun E, Nagler A & Peled A (2007) Enhanced unique pattern of hematopoietic cell mobilization induced by the CXCR4 antagonist 4F-benzoyl-TN14003. Stem Cells 25, 21582166.
  • 10
    Dar A, Schajnovitz A, Lapid K, Kalinkovich A, Itkin T, Ludin A, Kao WM, Battista M, Tesio M, Kollet O et al. (2011) Rapid mobilization of hematopoietic progenitors by AMD3100 and catecholamines is mediated by CXCR4-dependent SDF-1 release from bone marrow stromal cells. Leukemia 25, 12861296.
  • 11
    Hegde R, Borkow G, Berchanski A & Lapidot A (2007) Structure–function relationship of novel X4 HIV-1 entry inhibitors – l- and d-arginine peptide–aminoglycoside conjugates. FEBS J 274, 65236536.
  • 12
    Lapidot A, Berchanski A & Borkow G (2008) Insight into the mechanisms of aminoglycoside derivatives interaction with HIV-1 entry steps and viral gene transcription. FEBS J 275, 52365257.
  • 13
    Wu B, Chien EY, Mol CD, Fenalti G, Liu W, Katritch V, Abagyan R, Brooun A, Wells P, Bi FC et al. (2010) Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists. Science 330, 10661071.
  • 14
    Brelot A, Heveker N, Montes M and Alizon M (2000) Identification of residues of CXCR4 critical for human immunodeficiency virus coreceptor and chemokine receptor activities. J Biol Chem 275, 2373623744.
  • 15
    Crump MP, Gong JH, Loetscher P, Rajarathnam K, Amara A, Arenzana-Seisdedos F, Virelizier JL, Baggiolini M, Sykes BD & Clark-Lewis I (1997) Solution structure and basis for functional activity of stromal cell-derived factor-1; dissociation of CXCR4 activation from binding and inhibition of HIV-1. EMBO J 16, 69967007.
  • 16
    Kofuku Y, Yoshiura C, Ueda T, Terasawa H, Hirai T, Tominaga S, Hirose M, Maeda Y, Takahashi H, Terashima Y et al. (2009) Structural basis of the interaction between chemokine stromal cell-derived factor-1/CXCL12 and its G-protein-coupled receptor CXCR4. J Biol Chem 284, 3524035250.
  • 17
    Cashen A, Lopez S, Gao F, Calandra G, MacFarland R, Badel K & DiPersio J (2008) A phase II study of plerixafor (AMD3100) plus G-CSF for autologous hematopoietic progenitor cell mobilization in patients with Hodgkin lymphoma. Biol Blood Marrow Transplant 14, 12531261.
  • 18
    Tigue CC, McKo JM, Evens AM, Trifilio SM, Tallman MS & Bennett CL (2007) Granulocyte-colony stimulating factor administration to healthy individuals and persons with chronic neutropenia or cancer: an overview of safety considerations from the Research on Adverse Drug Events and Reports project. Bone Marrow Transplant 40, 185192.
  • 19
    Tian S, Choi WT, Liu D, Pesavento J, Wang Y, An J, Sodroski JG & Huang Z (2005) Distinct functional sites for human immunodeficiency virus type 1 and stromal cell-derived factor 1alpha on CXCR4 transmembrane helical domains. J Virol 79, 1266712673.
  • 20
    Rosenkilde MM, Gerlach LO, Hatse S, Skerlj RT, Schols D, Bridger GJ & Schwartz TW (2007) Molecular mechanism of action of monocyclam versus bicyclam non-peptide antagonists in the CXCR4 chemokine receptor. J Biol Chem 282, 2735427365.
  • 21
    Veldkamp CT, Seibert C, Peterson FC, De la Cruz NB, Haugner JC III, Basnet H, Sakmar TP & Volkman BF (2008) Structural basis of CXCR4 sulfotyrosine recognition by the chemokine SDF-1/CXCL12. Sci Signal 1, ra4, 1–9.
  • 22
    Gerlach LO, Skerlj RT, Bridger GJ & Schwartz TW (2001) Molecular interactions of cyclam and bicyclam non-peptide antagonists with the CXCR4 chemokine receptor. J Biol Chem 276, 1415314160.
  • 23
    Hatse S, Princen K, Bridger G, De Clercq E & Schols D (2002) Chemokine receptor inhibition by AMD3100 is strictly confined to CXCR4. FEBS Lett 527, 255262.
  • 24
    Fricker SP, Anastassov V, Cox J, Darkes MC, Grujic O, Idzan SR, Labrecque J, Lau G, Mosi RM, Nelson KL et al. (2006) Characterization of the molecular pharmacology of AMD3100: a specific antagonist of the G-protein coupled chemokine receptor CXCR4. Biochem Pharmacol 72, 588596.
  • 25
    Wong RS, Bodart V, Metz M, Labrecque J, Bridger G & Fricker SP (2008) Comparison of the potential multiple binding modes of bicyclam, monocylam, and noncyclam small-molecule CXC chemokine receptor 4 inhibitors. Mol Pharmacol 74, 14851495.
  • 26
    Juarez J, Bradstock KF, Gottlieb DJ & Bendall LJ (2003) Effects of inhibitors of the chemokine receptor CXCR4 on acute lymphoblastic leukemia cells in vitro. Leukemia 17, 12941300.
  • 27
    Spiegel A, Kollet O, Peled A, Abel L, Nagler A, Bielorai B, Rechavi G, Vormoor J & Lapidot T (2004) Unique SDF-1-induced activation of human precursor-B ALL cells as a result of altered CXCR4 expression and signaling. Blood 103, 29002907.
  • 28
    Pettersson S, Perez-Nueno VI, Mena MP, Clotet B, Este JA, Borrel JI & Teixido J (2010) Novel monocyclam derivatives as HIV entry inhibitors: design, synthesis, anti-HIV evaluation, and their interaction with the CXCR4 co-receptor. ChemMedChem 5, 12721281.
  • 29
    Lam AR, Bhattacharya S, Patel K, Hall SE, Mao A & Vaidehi N (2011) Importance of receptor flexibility in binding of cyclam compounds to the chemokine receptor CXCR4. J Chem Inf Model 51, 139147.
  • 30
    Trent JO, Wang ZX, Murray JL, Shao W, Tamamura H, Fujii N & Peiper SC (2003) Lipid bilayer simulations of CXCR4 with inverse agonists and weak partial agonists. J Biol Chem 278, 4713647144.
  • 31
    Doranz BJ, Filion LG, Diaz-Mitoma F, Sitar DS, Sahai J, Baribaud F, Orsini MJ, Benovic JL, Cameron W & Doms RW (2001) Safe use of the CXCR4 inhibitor ALX40-4C in humans. AIDS Res Hum Retroviruses 17, 475486.
  • 32
    Sweeney EA, Lortat-Jacob H, Priestley GV, Nakamoto B & Papayannopoulou T (2002) Sulfated polysaccharides increase plasma levels of SDF-1 in monkeys and mice: involvement in mobilization of stem/progenitor cells. Blood 99, 4451.
  • 33
    Hidalgo A, Peired AJ, Weiss LA, Katayama Y & Frenette PS (2004) The integrin alphaMbeta2 anchors hematopoietic progenitors in the bone marrow during enforced mobilization. Blood 104, 9931001.
  • 34
    Albanese P, Caruelle D, Frescaline G, Delbe J, Petit-Cocault L, Huet E, Charnaux N, Uzan G, Papy-Garcia D & Courty J (2009) Glycosaminoglycan mimetics-induced mobilization of hematopoietic progenitors and stem cells into mouse peripheral blood: structure/function insights. Exp Hematol 37, 10721083.
  • 35
    Petit I, Szyper-Kravitz M, Nagler A, Lahav M, Peled A, Habler L, Ponomaryov T, Taichman RS, Arenzana-Seisdedos F, Fujii N et al. (2002) G-CSF induces stem cell mobilization by decreasing bone marrow SDF-1 and up-regulating CXCR4. Nat Immunol 3, 687694.
  • 36
    Kalinkovich A, Tavor S, Avigdor A, Kahn J, Brill A, Petit I, Goichberg P, Tesio M, Netzer N, Naparstek E et al. (2006) Functional CXCR4-expressing microparticles and SDF-1 correlate with circulating acute myelogenous leukemia cells. Cancer Res 66, 1101311020.
  • 37
    Berchanski A & Lapidot A (2007) Prediction of HIV-1 entry inhibitors neomycin–arginine conjugates interaction with the CD4-gp120 binding site by molecular modeling and multistep docking procedure. Biochim Biophys Acta 1768, 21072119.
  • 38
    Berchanski A & Lapidot A (2009) Computer-based design of novel HIV-1 entry inhibitors: neomycin conjugated to arginine peptides at two specific sites. J Mol Model 15, 281294.
  • 39
    Heifetz A, Katchalski-Katzir E & Eisenstein M (2002) Electrostatics in protein–protein docking. Protein Sci 11, 571587.
  • 40
    Berchanski A, Shapira B & Eisenstein M (2004) Hydrophobic complementarity in protein–protein docking. Proteins 56, 130142.
  • 41
    Honig B & Nicholls A (1995) Classical electrostatics in biology and chemistry. Science 268, 11441149.

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Conclusions
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Fig. S1. Multistep docking procedure.

Fig. S2. Electrostatic potential maps calculated by delphi as implemented in the insightII package.

Fig. S3. Results of CVX15–CXCR4 geometric–electrostatic scan of molfit.

Table S1. Dose–response effects of studied compounds as mobilizers of HPCs.

Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

FilenameFormatSizeDescription
FEBS_8348_sm_FigS1-3-and-TableS1.zip688KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.