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Glycosaminoglycan analogs as a novel anti-inflammatory strategy.

Severin IC, Soares A, Hantson J, Teixeira M, Sachs D, Valognes D, Scheer A, Schwarz MK, Wells TN, Proudfoot AE, Shaw J - Front Immunol (2012)

Bottom Line: In vitro, these molecules prevented chemokine-GAG binding and chemokine receptor activation without disrupting coagulation.However, in vivo, these compounds caused variable results in a murine peritoneal recruitment assay, with a general increase of cell recruitment.In more disease specific models, such as antigen-induced arthritis and delayed-type hypersensitivity, an overall decrease in inflammation was noted, suggesting that the primary anti-inflammatory effect may also involve factors beyond the chemokine system.

View Article: PubMed Central - PubMed

Affiliation: Merck Serono Geneva Research Centre Geneva, Switzerland.

ABSTRACT
Heparin, a glycosaminoglycan (GAG), has both anti-inflammatory and anti-coagulant properties. The clinical use of heparin against inflammation, however, has been limited by concerns about increased bleeding. While the anti-coagulant activity of heparin is well understood, its anti-inflammatory properties are less so. Heparin is known to bind to certain cytokines, including chemokines, small proteins which mediate inflammation through their control of leukocyte migration and activation. Molecules which can interrupt the chemokine-GAG interaction without inhibiting coagulation could therefore, represent a new class of anti-inflammatory agents. In the present study, two approaches were undertaken, both focusing on the heparin-chemokine relationship. In the first, a structure based strategy was used: after an initial screening of potential small molecule binders using protein NMR on a target chemokine, binding molecules were optimized through structure-based design. In the second approach, commercially available short oligosaccharides were polysulfated. In vitro, these molecules prevented chemokine-GAG binding and chemokine receptor activation without disrupting coagulation. However, in vivo, these compounds caused variable results in a murine peritoneal recruitment assay, with a general increase of cell recruitment. In more disease specific models, such as antigen-induced arthritis and delayed-type hypersensitivity, an overall decrease in inflammation was noted, suggesting that the primary anti-inflammatory effect may also involve factors beyond the chemokine system.

No MeSH data available.


Related in: MedlinePlus

Structure of the compounds. (A) CCL5 binders. All the Kd values were determined by protein NMR. In the cases where the Kd could not be determined, either due to the low affinity of the compound, or due to precipitation of the complex, are indicated as not determined (n.d.). The molecules whose structure in complex with CCL5 was determined are indicated, including the pockets occupied by the molecule. Those molecules whose structure was attempted, but failed to crystallize are also described, while those for which no attempt to determined the co-crystal structure are indicated as not determined (n.d.). (B) The carbohydrates used for sulfation.
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Figure 1: Structure of the compounds. (A) CCL5 binders. All the Kd values were determined by protein NMR. In the cases where the Kd could not be determined, either due to the low affinity of the compound, or due to precipitation of the complex, are indicated as not determined (n.d.). The molecules whose structure in complex with CCL5 was determined are indicated, including the pockets occupied by the molecule. Those molecules whose structure was attempted, but failed to crystallize are also described, while those for which no attempt to determined the co-crystal structure are indicated as not determined (n.d.). (B) The carbohydrates used for sulfation.

Mentions: The crystallization conditions of CCL5 were essentially those previously described (Shaw et al., 2004). Briefly, CCL5 at 10 mg/ml in 50 mM acetate buffer pH 3.5, and the various molecules were incubated at a final concentration of the molecule of 0.1–0.5 mM (when solubility permitted) and crystallized at room temperature by hanging drop vapor diffusion in 15% (w/v) polyethylene glycol (PEG) 400, 50 mM acetate buffer pH 4.5, and 10% (w/v) glycerol. Crystallographic data were collected at 100 K on an Enraf-Nonius FR591 rotating anode generator equipped with Osmic MaxFlux mirrors and a MAR345 image plate detector. All the crystals of CCL5 belong to the orthorhombic space group P212121 with unit cell dimensions of a = 24 Å, b = 56 Å, and c = 94 Å, and contain a dimer of CCL5 in the asymmetric unit. Data was processed using DENZO and SCALEPACK (Otwinowski and Minor, 1997). Rigid body, simulating annealing, positional and B-factor refinement were performed with CNS (Brunger et al., 1998) and model building with Coot (Emsley et al., 2010). Bulk solvent and anisotropic B-factor corrections were applied. A number of other molecules, similar in structure to Molecule 1 were subsequently studied in the hope of establishing a structure-activity relationship (SAR), and in order to identify a promising starting point for the optimization of the CCL5-binders. In excess of 30 compounds, essentially poly-substituted phenyl sulfonates, were synthesized or purchased (see Figure 1A), and their binding affinity for CCL5 (Kd determined by NMR), and occasionally, their crystal structures determined (data not shown). It proved impossible to crystallize CCL5 in the presence of Molecule 3, due to the propensity of this molecule to cause precipitation of CCL5, despite all attempts to maintain the complex in solution.


Glycosaminoglycan analogs as a novel anti-inflammatory strategy.

Severin IC, Soares A, Hantson J, Teixeira M, Sachs D, Valognes D, Scheer A, Schwarz MK, Wells TN, Proudfoot AE, Shaw J - Front Immunol (2012)

Structure of the compounds. (A) CCL5 binders. All the Kd values were determined by protein NMR. In the cases where the Kd could not be determined, either due to the low affinity of the compound, or due to precipitation of the complex, are indicated as not determined (n.d.). The molecules whose structure in complex with CCL5 was determined are indicated, including the pockets occupied by the molecule. Those molecules whose structure was attempted, but failed to crystallize are also described, while those for which no attempt to determined the co-crystal structure are indicated as not determined (n.d.). (B) The carbohydrates used for sulfation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3472544&req=5

Figure 1: Structure of the compounds. (A) CCL5 binders. All the Kd values were determined by protein NMR. In the cases where the Kd could not be determined, either due to the low affinity of the compound, or due to precipitation of the complex, are indicated as not determined (n.d.). The molecules whose structure in complex with CCL5 was determined are indicated, including the pockets occupied by the molecule. Those molecules whose structure was attempted, but failed to crystallize are also described, while those for which no attempt to determined the co-crystal structure are indicated as not determined (n.d.). (B) The carbohydrates used for sulfation.
Mentions: The crystallization conditions of CCL5 were essentially those previously described (Shaw et al., 2004). Briefly, CCL5 at 10 mg/ml in 50 mM acetate buffer pH 3.5, and the various molecules were incubated at a final concentration of the molecule of 0.1–0.5 mM (when solubility permitted) and crystallized at room temperature by hanging drop vapor diffusion in 15% (w/v) polyethylene glycol (PEG) 400, 50 mM acetate buffer pH 4.5, and 10% (w/v) glycerol. Crystallographic data were collected at 100 K on an Enraf-Nonius FR591 rotating anode generator equipped with Osmic MaxFlux mirrors and a MAR345 image plate detector. All the crystals of CCL5 belong to the orthorhombic space group P212121 with unit cell dimensions of a = 24 Å, b = 56 Å, and c = 94 Å, and contain a dimer of CCL5 in the asymmetric unit. Data was processed using DENZO and SCALEPACK (Otwinowski and Minor, 1997). Rigid body, simulating annealing, positional and B-factor refinement were performed with CNS (Brunger et al., 1998) and model building with Coot (Emsley et al., 2010). Bulk solvent and anisotropic B-factor corrections were applied. A number of other molecules, similar in structure to Molecule 1 were subsequently studied in the hope of establishing a structure-activity relationship (SAR), and in order to identify a promising starting point for the optimization of the CCL5-binders. In excess of 30 compounds, essentially poly-substituted phenyl sulfonates, were synthesized or purchased (see Figure 1A), and their binding affinity for CCL5 (Kd determined by NMR), and occasionally, their crystal structures determined (data not shown). It proved impossible to crystallize CCL5 in the presence of Molecule 3, due to the propensity of this molecule to cause precipitation of CCL5, despite all attempts to maintain the complex in solution.

Bottom Line: In vitro, these molecules prevented chemokine-GAG binding and chemokine receptor activation without disrupting coagulation.However, in vivo, these compounds caused variable results in a murine peritoneal recruitment assay, with a general increase of cell recruitment.In more disease specific models, such as antigen-induced arthritis and delayed-type hypersensitivity, an overall decrease in inflammation was noted, suggesting that the primary anti-inflammatory effect may also involve factors beyond the chemokine system.

View Article: PubMed Central - PubMed

Affiliation: Merck Serono Geneva Research Centre Geneva, Switzerland.

ABSTRACT
Heparin, a glycosaminoglycan (GAG), has both anti-inflammatory and anti-coagulant properties. The clinical use of heparin against inflammation, however, has been limited by concerns about increased bleeding. While the anti-coagulant activity of heparin is well understood, its anti-inflammatory properties are less so. Heparin is known to bind to certain cytokines, including chemokines, small proteins which mediate inflammation through their control of leukocyte migration and activation. Molecules which can interrupt the chemokine-GAG interaction without inhibiting coagulation could therefore, represent a new class of anti-inflammatory agents. In the present study, two approaches were undertaken, both focusing on the heparin-chemokine relationship. In the first, a structure based strategy was used: after an initial screening of potential small molecule binders using protein NMR on a target chemokine, binding molecules were optimized through structure-based design. In the second approach, commercially available short oligosaccharides were polysulfated. In vitro, these molecules prevented chemokine-GAG binding and chemokine receptor activation without disrupting coagulation. However, in vivo, these compounds caused variable results in a murine peritoneal recruitment assay, with a general increase of cell recruitment. In more disease specific models, such as antigen-induced arthritis and delayed-type hypersensitivity, an overall decrease in inflammation was noted, suggesting that the primary anti-inflammatory effect may also involve factors beyond the chemokine system.

No MeSH data available.


Related in: MedlinePlus