Thank you for believing in me and for giving me many practical opportunities to further my scientific development. Paul Nelson, thanks for the many hours of sleep and Quake (but not math) you gave up for me. Allen Hong and Matt Winston, thank you for being my confidants since day one of graduate school.
Landry Fokoua, Chris Marotta, Hanqing Li and Ryan Henning, thanks for the many pleasant conversations;
2 Further modifications are then made at distinct sites along the polysaccharide chain, which include
3 Thus, heparin exhibits higher overall levels of N-sulfation and has greater conversion of GlcA to
4 HS proteoglycans participate in multiple biological processes through modulating the activities of
This research has proven to be technically demanding, given the heterogeneity of the macromolecular organization (sulfated and non-sulfated domains, chain length) and fine structure (sulfation motifs, monosaccharide identity, glycosidic linkage type) of HS polysaccharides /heparin. Representation of the lowest energy models for the molecular binding of HS/heparin interactions with IL-8 and PF4 oligomers. Energy-minimized structures of HS/heparin polysaccharide chains containing the IdoA conformer 1C4 (chair) or 2S0 (tilting boat).
Within the HS/heparin chains, IdoA typically exists in equilibrium in either the 1C4 (chair) or 2S0 (tilted boat) conformation (Figure 4).
8 Heparin-Based Therapeutics in Inflammatory Disease
9 at this specific position also removes uronyl 2-O sulfates. Simultaneous loss of both modifications
Another strategy for obtaining non-anticoagulant anti-inflammatory mimetics is to perform high-throughput screening of a large compound library using these defined criteria. For example, Progen Pharmaceuticals recently developed a new series of homogenously sulfated sugars with a single entity attached to an aglycone lipophilic moiety (PG500) based on the chemical structure of the original drug PI-88.[47] The new series of compounds were first screened for potential anticoagulant activity in human plasma, and any compounds that altered the clotting time of activated partial thromboplastin (PT) were eliminated from further testing. The anti-angiogenic and anti-metastatic activities of the remaining compounds were then evaluated using in vivo tumor growth and proliferation assays.
11 anti-inflammatory activity, it is reasonable to predict that some of the compounds with low anti-
11 anti-inflammatory activity, it is reasonable to predict that several of the compounds with low anti-
12 REFERENCES
L -idopyranosyluronate (9). Compound 9 was prepared in six steps from the commercially available diacetone glucose (Sigma Aldrich) using previously reported
The reaction was quenched with ice-cold H2O (2.0 x 500 mL), filtered through celite, and concentrated under reduced pressure. The reaction mixture was then diluted with CH 2 Cl 2 (500 mL), washed with aqueous NaHCO 3 and H 2 O (200 mL each), dried over MgSO 4 , and concentrated under reduced pressure. After confirming that the reaction was complete by TLC, the reaction was quenched with triethylamine (2.0 mL) and concentrated under reduced pressure.
The reaction was stirred at -10 °C for 10 min, slowly warmed to room temperature over 15 min, quenched with Et 3 N (0.50 mL), filtered through silica, and concentrated under reduced pressure. The reaction was stirred at 0 °C for 12 h, quenched with Et 3 N (1.0 mL) and concentrated under reduced pressure. After confirming the partial disappearance of 5 by TLC, flame-dried K2CO3 (2.4 mg, 20 µmol) was added and the reaction mixture was stirred for 24 h at room temperature.
The reaction was quenched with Dowex 5W-X8 (H+ form), filtered through a Celite pad and concentrated under reduced pressure. The reaction mixture was stirred at room temperature for 24 hours, refluxed at 50°C for 24 hours, quenched with MeOH (1.0 mL) and concentrated to yield a gold syrup. The reaction mixture was loaded directly onto a Sephadex LH-20 gel filtration column and eluted with 1:1 CH2Cl2:MeOH.
The reaction mixture was stirred at room temperature for 24 hours, refluxed at 50°C for 24 hours, quenched with MeOH (5.0 mL) and concentrated to yield a gold syrup. The reaction mixture was stirred at room temperature for 24 hours and refluxed at 50°C for 24 hours, quenched with MeOH (1.0 mL) and concentrated to yield a gold syrup. The reaction mixture was stirred at room temperature for 1 hour, quenched with ethyl vinyl ether (0.10 ml) and diluted with diethyl ether (1.0 ml) and hexanes (0.50 ml) to obtain a white precipitate.
The reaction mixture was filtered using a vacuum filtration system (0.45 µm PES membrane, VWR) and desalted on a Sephadex G-25 column in 100% H2O to obtain the desired product 60%.
71 established that RANTES preferentially recognizes heparin (or the trisulfated motif found in HS)
By fitting the relative values of RANTES binding to a Hill equation using KaleidaGraph software, we determined the EC50 to be 12.2 nM (Figure 8). We used this concentration of RANTES to perform all competitive ELISA experiments with the synthetic glycopolymers. We followed the same experimental format except that RANTES was first preincubated with either heparin or glycopolymer, which allowed us to determine the individual ability of each compound to inhibit RANTES binding to the heparin-bound plates.
However, glycopolymer 1 was a more efficient competitor for RANTES at its concentration of maximum inhibition: while heparin showed a maximum inhibition of 58.4%, glycopolymer 1 inhibited RANTES binding by up to 90.8% below the same.
73 assay conditions. Maximum inhibition values were also determined from the Michaelis-Menten
74 values. We then used the molecular mass of the differentially sulfated disaccharide units to
75 heparin binds to ATIII through a high-affinity glucosaminyl 3-O sulfated pentasaccharide that is
76 complex with ATIII. [16] In agreement with this molecular mechanism, we found that only heparin
77 context of glycosaminoglycan anticoagulant activity. Altogether, our results demonstrate that
79 reported binding sites for HS and RANTES exhibit considerable overlap on CCR3, whereas the
80 important chemokine, without affecting key components of the blood coagulation cascade. We
L1.2 cells (mouse pre-B lymphocytes) stably transfected with CCR3, CCR5 or vector alone were kindly provided by Drs. Cells were maintained in RPMI 1640 (Invitrogen) supplemented with 10% FBS, 100 µg/mL penicillin/streptomycin (Invitrogen), and 50 µM 2-mercaptoethanol (Sigma Aldrich). Cells were routinely analyzed by flow cytometry (FACSCalibur, Beckman Dickenson) to verify that cultures expressed adequate levels of chemokine receptor (>90%) for migration and cell attachment assays.
L1.2 cells (wild type or stably transfected with CCR3 or CCR5) were harvested and washed twice in flow cytometry buffer (Hank's Balanced Salt Solution (HBSS) containing 2.5 mg/ml bovine. The sample plate was fitted with a 5-µm pore filter and 106 cells (50 µl) were placed on top of each well. Non-migrating cells were then removed from the top of the filter by manual scraping; cells attached to the filter were detached using 20 µl of 2.5 mM EDTA for 30 minutes at room temperature.
Migrated cells were transferred (500 × g, 5 min) to a 96-well plate with black walls and clear bottom (Corning) using a funnel plate (Neuroprobe). Cells were lysed at −80°C and stained with CyQUANT dye (Invitrogen) as described in the product literature. 3 × 10 L1.2 cells (wild type or stably transfected with CCR3) were washed twice with flow cytometry buffer and incubated with RANTES (100 nM in flow cytometry buffer) for 45 min at room temperature.
Alternatively, cells were incubated for 30 min at room temperature with RANTES (100 nM in flow cytometry buffer), previously treated with different concentrations of heparin or glycopolymer µg/ml. The cells were respun through 100% FBS (1.0 ml) twice and resuspended in flow cytometry buffer (500 µl) for flow cytometry analysis.
84 (Winchester, VA) for the coagulation assay. Samples were thawed at room temperature for 30
85 REFERENCES
While heparin polysaccharide of the same length (IC µg/ml nM) bound to RANTES more efficiently than glycopolymer 1, both antagonists showed similar potencies in cell migration and binding assays. Since cell migration and chemotactic gradients play an essential role during angiogenesis, we sought here to compare the relative binding affinities of glycopolymer 1 and heparin for a series of chemokines strongly involved in angiogenesis.[1] Specifically, we chose to investigate growth-related oncogene-α (Gro-α; CXCL1), interleukin 8 (IL-8; CXCL8) and stromal cell-derived factor 1 (SDF-1; CXCL12), three members of the CXC subfamily. . CXC chemokines are known for their ability to regulate angiogenesis, [2] the physiological process by which new blood vessels are derived from existing capillaries.
All CXC chemokines have four highly conserved cysteine residues and the first two cysteines are separated by a non-conserved residue.[3] Another structural domain in these chemokines dictates their angiogenic ability: it is a tripeptide sequence (Glu-Leu-Arg; also designed ELR motif) at the N-terminus before the first cysteine residue in the CXC domain.[4] Typically, members possessing the ELR motif (ELR+) are potent inducers of angiogenesis, while those lacking the motif (ELR-) are inhibitors [5]. Thus, local expression of specific CXC chemokines may serve as an important regulator of angiogenesis; likewise, an imbalance in chemokine expression can have severe consequences, such as promoting tumor growth or inducing chronic fibroproliferative disorders.[6] Two. The 88 chemokines we selected for research, Gro-α and IL-8, are markedly proangiogenic.
We next evaluated the ability of glycopolymer 1 or heparin to inhibit each of the chemokines at the EC50 value using the same competitive ELISA procedure developed for RANTES. Although the IC50 values for heparin-mediated chemokine inhibition reflected the EC50 values from the direct ELISA, we were surprised to find that the IC50 values for glycopolymer-mediated chemokine.
90 inhibition were drastically different from the EC 50 values (Table 5 and Figure 15). For example,
92 Both SDF-1 and cognate receptor CXCR4 are known to play vital roles in leukocyte trafficking, [16]
94 observe complete inhibition of SDF-1 activity for 2 μg/mL of glycopolymer 1 in the same assay (Fig.
94 observe full inhibition of SDF-1 activity for 2 µg/mL of glycopolymer 1 in the same assay (Figure
We next evaluated the ability of glycopolymer 1 or heparin to alter the phosphorylation status of ERK1/2 5 min after exposure to SDF-1. In particular, we found that glycopolymer 1 (200 µg/ml) enhanced ERK1/2 activation by 1.8-fold, while the same concentration of heparin had no effect (Figure 18b). This further validated our hypothesis that the glycopolymer promotes the dimer. form of SDF-1. However, detailed biophysical experiments are still warranted to demonstrate that natural glycosaminoglycans and synthetic glycomimetics induce differential changes in SDF-1 structure and CXCR4 signaling.
The ability to differentially activate the CXCR4 pathway by modulating the oligomerization status of its ligand would provide an exciting new strategy for the therapeutic invention of many diseases. For example, the monomeric form of SDF-1 is thought to act cardioprotectively, as the obligate monomeric SDF-1 has recently been proposed for the potential treatment of ischemia-reperfusion injury and myocardial infarctions [25]. In the context of cancer metastasis, however, the obligate SDF-1 dimer has been shown to preferentially inhibit the metastasis of colon carcinoma and lung melanoma cells ex vivo, possibly due to the formation of non-motogenic SDF-1 dimers. Our experiments here show that trisulfated glycopolymer 1 bound to SDF-1 with high affinity, and the resulting complex recapitulated the inhibitory activity of the SDF-1 dimer in two previous in vitro assays.
If additional biophysical experiments can demonstrate that glycopolymer 1 preferentially shifts the SDF-1 equilibrium toward the dimer form, we propose that this synthetic mimic could potentially be used as a treatment for several types of invasive cancers that rely on SDF-1 gradients . including breast carcinoma,[26] liver carcinoma,[27] and highly malignant cases of glioblastoma multiforme.[28]. We expect that this strategy will have several advantages over current efforts to block the CXCR4 receptor, as it would allow direct disruption of local SDF-1 gradients in the tissue.
96 harboring the tumor microenvironment and the preservation of SDF-1 dimer signaling necessary for
97 values were also corrected for ligand valence (Table 5) by calculating the mass percentage of the
99 REFERENCES
