DE102007046388A1 - Development of a binding assay and presentation of novel inhibitors of the myelin associated glycoprotein - Google Patents

Abstract

If accidents or diseases lead to injuries of the adult central nervous system, the affected body functions are generally permanently restricted or lost. Due to the lack of ability to regenerate the central nervous system and the brain, this is irreversible in almost all cases.1 For those affected, the resulting loss of motor function and normal body functions, such As the control of the bladder and rectum, extreme restrictions in everyday life. In addition, in the paralyzed areas the perception of pain and the temperature and tactile sensation is lost. Paraplegics are often no longer able to cope with their everyday lives alone. In Germany, about 1000 new cases of paraplegia occur each year. For the US, the National Spinal Cord Injury Association (NSCIA) lists 11,000 new cases of Spinal Cord Injury (SCI) per year. 80% of those affected are men. The most common causes of SCI are vehicle accidents at 47.5% and falls at 22.9% .2 In contrast to the central nervous system (CNS), the peripheral nervous system (PNS) is capable of regeneration after injury. The nervous connections can usually be completely restored here while retaining the functions. This fact can be exploited to explore which differences in PNS to CNS allow for this regeneration. It could be found ways to ...

Description

The myelin-associated glycoprotein is a nervous system protein with five extracellular, one transmembrane and two intracellular domains. It belongs to the family of sialic acid binding immunoglobulin-like lectins (Siglecs). Unlike OMgp and Nogo-A, MAG is not expressed on neurons but only on glial cells. MAG makes up a relatively small proportion of the proteins of myelin, less than 1%. It is one of the most highly conserved proteins, indicating a strong selection pressure for its functions. MAG plays a role in the formation and maintenance of myelin sheaths and promotes neurite outgrowth of neonatal dorsal root ganglia (DRG). Furthermore, it also has an effect on the inhibition of neurite outgrowth of adult DRG and other neuronal types. Although MAG is considered a potent growth inhibitor in vitro, MAG knock-out mice show no or only a slight increase in neurite outgrowth in the spinal cord. Slow Regenerating mice, however, support the suspicion that MAG may have an inhibiting effect on axonal regeneration in vivo: the regeneration is increased when the MAG gene is switched off. MAG exists in two isoforms, the L- and the S-MAG, with 67 kDa and 72 kDa weight, respectively, which are similar in secondary structure and topology, but show slight variations in the cytoplasmic carboxy terminus ( 1 ). S-MAG is the predominant form in the PNS, while L-MAG is the predominant form in the CNS. The glycostructures of MAG account for about 30% of its weight. There is no X-ray crystal structure of MAG so far, therefore, more detailed information about the 3D structure of the protein is not available.

1 Schematic representation of the two isoforms of MAG. The difference between these two forms lies in variations in the intracellular carboxy terminus. The amino acid arginine 118, which is important for sialic acid binding, is located in the N-terminal V-set domain.

LIKE is made up of five internally homologous regions that belong to five immunoglobulin-like domains in the lead to extracellular amino terminus. In the direction of the carboxy terminus is the only transmembrane domain (between ~ 510 aa to ~ 530 aa).

LIKE causes the growth cone collapses on the one hand over the complex ones Gangliosides of the nervous system. On the other hand, MAG competes with it Nogo-66 and OMgp bind to the NgR. This tie is in Unlike ganglioside binding, sialic acid-independent. The NgR binding domain is not identified yet. It But there is evidence that they are C-terminal to the third Ig-like domain and N-terminal to the transmembrane domain located. MAG is capable of nanomolar concentration growth cause cone collapse in neurons that transfected with NgR were.

Inhibiting properties of MAG

Like Nogo-A and OMgp in the adult CN, MAG exerts an inhibitory effect on axon regeneration. It works by binding to the NgR. A cascade is triggered via the core receptors of the NgR, resulting in the growth cone collapse. For MAG, a second way has been proven. MAG mediates its action by binding to the ganglioside GT1b, which is expressed on neuronal surfaces. Together with the p75 ^NTR coreceptor, it triggers the same signal cascade as it does by binding to the NgR. It is not yet known if these two cascades act independently of each other, or if they cause each other.

To suppress the effect of the MAG and allow regeneration of the destroyed axons blocked DeBellard et al. the MAG with sialic acid and sialyllactose. To do this, they used a neurite growth assay in which the lengths of the neurites were measured in the presence of MAG at different saccharide concentrations. A dependence of the neurite length on the concentration of the saccharides could be recognized. For example, neurites showed B. a growth of 64% with the addition of 20 mM sialic acid.

The inhibitors of MAG in the literature

outgoing from the structure of the complex gangliosides of the nervous system were so far several approaches to the development of inhibitors performed for the MAG.

Strenge et al. investigated the glycan specificity of MAG using various synthetic oligosaccharides. The MAG showed a fivefold higher binding affinity to α2,3-linked Neu5Ac than to α2,6-linked Neu5Ac. In an α-linkage, the carboxyl group is in the axial position, which is so with as necessary for the binding to the MAG turned out. It also showed that MAG was not tolerant to certain changes in the Neu5Ac. Thus, the binding affinity decreased sharply when the hydroxyl groups were removed at the 8 or 9 position. The carboxyl group and the N-acetyl group were also shown to be important functional groups in this analysis for the binding of Neu5Ac to the MAG. However, MAG showed a higher binding affinity to Neu5Ac derivatives with halogenated N-acetyl group. A further increase in binding affinity could be achieved by introducing a thioacetyl group instead of the N-acetyl group. In addition, methyl or benzyl glycosides of the Neu5Ac proved to be potent binding partners for the MAG. As further positive for the binding affinity, changes in the 9-position were found. Thus, derivatives of the Neu5Ac with alkyl substituents or aromatic substituents at the 9-position showed a strongly increased binding to the MAG. In 2003, Kelm and Brossmer filed a patent for Siglec inhibitors, summarizing these modifications of the Neu5Ac and its effect on binding to the MAG.

The Analysis of various tetrasaccharide derivatives derived from the Substructure Neu5Ac-α2,3-Gal-β1,3- (Neu5Ac-α2,6-) GalNAc of the complex ganglioside GQ1ba using STD NMR spectroscopy the importance of the terminal α2,3-linked New5Ac for the MAG binding. 2006 were the structures various tetrasaccharide mimetics derived from this substructure published as new antagonists for the MAG. In these compounds, the Gal-β1,3-GalNAc structure is exchanged for a biphenyl linker between the two Neu5Ac. Because the orientation of the two Neu5Ac to each other in this new Compounds not completely orientated in the tetrasaccharide corresponds, the compounds show somewhat weaker binding affinities to the MAG as the tetrasaccharide. However, the led Biphenyl substituent to improve the pharmacokinetic Properties of the new connection class. The most successful so far Inhibitors for the MAG are di-aromatic substituted Neuraminosides, which include the reference substance SH-81. These showed in the hapten inhibition assay increased by more than the factor> 700 Binding affinity to the MAG compared to the trisaccharide Neu5Ac-α2,3-Gal-β1,3-GalNAc-OSE. Here, SH-81 was as simple as the benzamide substituent at the 9-position chlorinated compound the highest inhibition potential with 1074 compared to the trisaccharide. Further halogenations let the inhibition potential sink.

Message of the invention

Development of an SPR system for the MAG

The coupling as amide is the standard immobilization method for molecules on CM5 chips. Hereby, the carboxyl functions of the dextran matrix are activated with the aid of N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), used in a ratio of 1: 1. The resulting active esters react with free amino groups of the ligand (eg, lysines in proteins). The reaction scheme of the coupling is in 2 clarified.

2 : The Reaction Scheme of the Amine Coupling. The carboxyl functions of the dextran matrix of the CM5 chip react after reaction with NHS / EDC (1: 1) to the active ester with the amino groups of the protein to form an amide function.

For the immobilization of the MAG, the use of the amine coupling was not possible under standard conditions, since the immobilized protein on the chip lost its activity. As a reason for the loss of activity, it was assumed that the lysine 67 of the MAG binding pocket, which in previous work was shown to be the amino acid important for binding, also reacted in the amine coupling. To prevent this, a potent ligand was added to the immobilization solution of the MAG in 50-fold excess. Based on the results of the STD NMR measurements, the ligand SV-8 was selected with a K _D of 8 ± 3 μM (see Chapter 0). The addition in 50-fold excess should cause that in a large part of the MAG the binding pocket and thus the lysine 67 are occupied by interaction with the ligand SV-8 and are no longer available for the coupling in the immobilization. In this way, the activity of the MAG remained, as the subsequent functionality test with the standard substance N-acetylneuraminic acid showed.

Functionality test with N-acetylneuraminic acid

To ensure that the functionality of the MAG remained intact after immobilization on the chip, various concentrations of Neu5Ac were measured as standard. 3 shows the received sensorgrams.

3 : The sensorgrams of the SPR measurement of various concentrations of the standard substance N-acetylneuraminic acid. Concentrations of 100 μM to 2500 μM were measured, with the highest concentration being analyzed twice to check the reproducibility of the results. Increasing RU responses indicate that concentration-dependent binding of Neu5Ac to MAG occurs.

When plotting the determined RU _max values against the concentration in μM, the in 4 obtained curve shown. Fit to the one site binding model resulted in a K _D value of 1.5 ± 0.3 mM for the binding of Neu5Ac to the MAG. The of Kelm et al. specific rIP and IC ₅₀ values for various Neu5Ac derivatives upon binding to MAG indicate a binding constant in the mM range for pure Neu5Ac to the MAG. The observed binding showed that most of the MAG retained its activity after immobilization. From the resulting sensorgrams, an RU response of 31 RU was obtained for the highest measured Neu5Ac concentration. That is, the immobilized MAG retained 91% of its activity. The blockade of his binding pocket and thus the lysine 67 by the ligand SV-8 could protect the binding pocket and prevent a reaction of the amino acids located here in the amine coupling.

4 : SPR measurement of the standard substance N-acetylneuraminic acid: The application of the RUmax values to the concentrations of the Neu5Ac from 100 μM to 2500 μM can be fitted using the one site binding model. A dissociation constant of KD = 1.5 ± 0.3 mM for the binding of Neu5Ac to the MAG is obtained.

Synthesis of amino acid conjugates truncated neuraminic acid as new inhibitors of the MAG

Neuraminic acid derivatives with aromatic substituents at 2- and 9-position have so far shown the best binding properties to the MAG. Therefore, this approach was followed up with SH-81 as a reference substance: In order to introduce more functional groups that can contribute to higher binding affinity into the molecule, amino acid neuraminic acid derivatives were designed and synthesized. As amino acids only aromatic amino acids came into consideration, since the two-fold aromatic substitution of Neu5Ac backbone should be retained. In 5 the designed amino acid Neu5Ac derivatives are shown.

5 : The synthesized amino acid truncated Neu5Ac conjugates.

The 6 . 7 and 8th show the synthetic route of the amino acid truncated Neu5Ac derivatives. The synthesis was carried out in eight synthesis steps. In the first step, the carboxyl group of the Neu5Ac was protected as a methyl ester. Here, the acidic ion exchanger Dowex 50X8 (H ⁺ ) served as a catalyst. The free hydroxyl groups were protected by standard method as acetyl groups. The hydroxyl group in the 2-position shows a weaker reactivity than the other hydroxyl groups due to the -I effect of the adjacent carboxyl group. Therefore, a product mixture of penta and hexaacetate was formed. For the further synthesis steps, this did not matter, so that the product mixture could be used untreated further.

6 : Synthesis pathway of the Neu5Ac truncated amino acid conjugates from Neu5Ac to the Neu5Ac species shorter by one methylene group after periodate cleavage (step 6).

to Activation of the 2-position for substitution with benzyl alcohol this position was chlorinated. This was acetyl chloride added with methanol, where in situ HCl was formed. This responded then in a nucleophilic substitution with the acetylated Neu5Ac to the β-sialyl chloride. Due to the thermodynamic control, which undergoes this reaction, the β-sialyl chloride was formed anomerically pure in good yield. In the next stage was the β-sialyl chloride with benzyl alcohol and silver carbonate reacted, with the benzyl alcohol was coupled to the 2-position. Subsequently, the acetyl protecting groups were replaced by the deacetylation split off after Zémplen. The bond between the 8- and the 9-position was cleaved by periodate cleavage. To minimize the possible side reaction to the doubly truncated Neu5Ac the periodate was used in equimolar amounts.

The amino acid was attached in the form of its methyl ester via a reductive amination with NaCNBH ₃ to the aldehyde function formed at the periodate cleavage at position 8. Subsequently, the methyl esters were saponified at the two carboxyl functions with 0.1 N sodium hydroxide solution. The products were purified by Biogel P2 column and RP-HPLC with acetonitrile / water gradient ( 7 ).

7 : Reductive amination with NaCNBH3 followed by coupling of the amino acid to the Neu5Ac truncated by one methyl group led to ligands SV-8, SV-10, and SV-14.

In periodate cleavage, the N-acetyl-neuraminic acid derivative shortened by two methylene groups ( 8th ). The proximity of the aldehyde group to the 7-position of the desired species does not allow purification by column chromatography on silica gel, otherwise there is a risk of epimerization of the asymmetric center at the 7-position. Thus, in the subsequent coupling of the amino acid, two species of the amino acid Neu5Ac derivatives with glycerol side chains of different lengths were formed. The chemical similarity of these two species made their separation difficult. The desired species was obtained only in low yields. The by-product derivatives of the doubly truncated Neu5Ac were also tested for binding activity. The assumption that the hydroxyl group at the 7-position of the 8-species increases the binding affinity was demonstrated by SPR analysis (see Chapter 0).

8th : Reductive amination with NaCNBH3 followed by coupling of the amino acid to Neu5Ac, which is truncated by two methylene groups, resulted in ligands SV-16 and SV-18.

Investigation of the binding constants the amino acid conjugates of truncated neuraminic acid by means of SPR

The amino acid truncated Neu5Ac conjugates were screened for their binding affinity to the MAG by SPR. 9 shows the binding curves obtained for the amino acid conjugates of the single truncated Neu5Ac SV-8, SV-10 and SV-14. The fit using the one site binding model yielded a K _D value of 14 ± 2 μM for the phenylalanine ligand SV-8. For the tyrosine ligand SV-14, a binding constant of 11 ± 5 μM was determined. The tryptophan ligand SV-10 showed a binding constant of 77 ± 7 μM. The lower binding affinity of this ligand could be explained by the larger space requirement of the tryptophan substituent.

All Amino acid conjugates of single truncated Neu5Ac showed thus high binding affinities in the SPR analysis. The second carboxyl group represented by the amino acid substituent was introduced into the molecule probably carried by salt or hydrogen bonding to this high Binding affinity. However, the positive effect was of the second carboxyl group in the tryptophan ligand by size the aromatic residue seems to have been abolished.

9 : The binding curves of the amino acid conjugates of the single truncated Neu5Ac (8 species) and the KD values of the SPR analysis. Concentrations of 1.52 μM to 500 μM were measured. The fit using the one site binding model yielded a KD value of 14 ± 2 μM for the phenylalanine ligand SV-8, a KD value of 77 ± 7 μM for the tryptophan ligand, and a KD value of 11 ± for the tyrosine ligand 5 μM.

In the synthesis, the histidine and tyrosine derivatives of the doubly truncated Neu5Ac were also obtained. 10 Figure 4 shows a comparison of the binding curves of the tyrosine single-truncated Neu5Ac conjugate SV-14 and the tyrosine duplex Neu5Ac conjugate SV-18.

While the corresponding tyrosine single-truncated Neu5Ac derivative SV-14 with a K _D value of 11 ± 5 μM showed the highest binding affinity of all derivatives, this increased by a factor 17 to 185 ± in the tyrosine duplex Neu5Ac derivative SV-18 54 μM off. The lack of the hydroxyl group at the 7-position and the shortened molecular structure cause a strong decrease of the binding affinity to MAG.

For the histidine ligand SV-16, which also contains two methylene groups has truncated Neu5Ac backbone, could in SPR analysis shows no binding to MAG. The reason this could be in the different charges of histidine in contrast to the other ligands. Histidine is located protonated at the pH of the running buffer and is therefore positive loaded. Tyrosine, on the other hand, has an excess of aromatics. This seems to facilitate the interaction with MAG while histidine turns out to be unsuitable with its positive charge.

10 : Comparison of the SPR results of the 8 species and the 7 species of the amino acid Neu5Ac derivatives with the tyrosine Neu5Ac derivatives SV-14 and SV-18. Concentrations of 1.52 μM to 500 μM were measured. Fit on the one site binding model yielded a KD value of 11 ± 5 μM for the tyrosine conjugate of the single truncated Neu5Ac and a KD value of 185 ± 54 μM for the tyrosine conjugate of the doubly truncated Neu5Ac.

The sensorgrams obtained for the amino acid truncated Neu5Ac derivatives were analyzed for the kinetics of binding of these substances to the MAG. The SPR analysis of the phenylalanine derivative SV-8 was the only negative binding curve ( 11 ). For the kinetic evaluation of these sensorgrams the values had to be mirrored and k _on and k _off individually calculated.

11 : The sensorgrams of the SPR analysis of the phenylalanine derivative SV-8. SV-8 was the only derivative to show negative sensorgrams in the SPR analysis.

In 12 . 13 and 14 For example, the sensorgrams of the SPR analyzes of the other three analyzed amino acid truncated Neu5Ac derivatives with the fit curves superimposed on them can be seen. The corresponding values for k _on and k _off are listed in the table. While the four high concentrations were used to evaluate the binding kinetics of ligand SV-10, only one concentration was evaluated for compounds SV-14 and SV-18.

12 : The sensorgrams of the SPR analysis of the Tryptophile SV-10 with Fit according to the 1: 1 binding model. The resulting sensorgrams for the concentrations 25 μM, 100 μM, 250 μM and 500 μM were fitted.

13 : The sensorgram of the tyrosine conjugate of the single truncated Neu5Ac SV-14 at a concentration of 1.52 μM with Fit according to the 1: 1 binding model.

14 : The titer of the tyrosine conjugate of the doubly truncated Neu5Ac SV-18 at a concentration of 10 μM with Fit according to the 1: 1 binding model.

The K _D values of the amino acid truncated Neu5Ac derivatives determined by kinetic analysis are in good agreement with the K _D values from the affinity analysis by SPR. Only in the case of the tyrosine conjugate of the doubly truncated Neu5Ac SV-18 is the binding constant lower, and thus better than the binding constant determined by the affinity analysis, according to kinetics analysis. The K _D value from the affinity analyzes was used to determine the association and dissociation rate of the phenylalanine ligand SV-8. Thus, a calculation of the K _D value was not possible for comparison. Table 1: The results of the kinetic analysis of the binding of amino acid Neu5Ac derivatives obtained by SPR. ligand k _on [M ^-1 s ^-1 ] k _off [s ^-1 ] K _D [μM] kinetic analysis K _D [μM] affinity analysis Chi ² kinetic analysis SV-8 2.5 · 10 ² 0.4 · 10 ^-3 - 14 ± 2 - SV-10 5.9 · 10 ² 6.4 · 10 ^-3 100 77 ± 7 0.68 SV-14 2.0 · 10 ⁴ 2.4 · 10 ^-3 1 11 ± 5 12:16 SV-18 5.7 · 10 ³ 4.2 · 10 ^-3 7 185 ± 54 12:52

The dissociation constants determined by two different evaluation methods showed similar tendencies in binding affinities the ligands to the MAG. The highest affinity showed in both methods the tyrosine conjugate of the single truncated Neu5Ac, which also had the fastest association rate. In direct comparison, the tyrosine conjugate showed the doubly truncated Neu5Ac a much weaker binding affinity to MAG. This is probably a less favorable position the compound in the binding pocket of the protein due to the shortened Molecular structure and the absence of the hydroxyl group to justify the 7-position.

These Results led to the conclusion that the amino acid conjugates the singly truncated Neu5Ac the optimal properties as a binding partner for MAG.

Investigation of the binding constants the amino acid conjugates of truncated neuraminic acid by STD NMR spectroscopy

To obtain a more detailed analysis of the binding specificities of the amino acid conjugates of truncated Neu5Ac, these were analyzed by STD NMR spectroscopy. For all compounds of the singly truncated Neu5Ac, STD titrations were performed. The results of these titrations are in 15 shown. The phenylalanine ligand SV-8 showed a binding constant of 8 ± 3 μM in the STD titration. This shows good agreement with the binding constant of 14 ± 2 μM, which gave the SPR analysis for this compound. For the tryptophan derivative SV-10, a K _D value of 15 ± 6 μM was obtained here. This is well below the K _D value of 77 μM, which this compound has shown in the SPR analysis. However, the tendency to lower binding affinity compared to the phenylalanine and tyrosine ligands can be seen here as well. The tyrosine ligand SV-14 also showed the highest binding affinity in this analysis method with a K _D value of 4 ± 5 μM. The binding constants of SPR analysis are supported by the results of STD titrations. Here, the same dependencies of binding affinity were found.

15 : Plot of STD amplification factors versus concentrations in μM: Results of KD titrations by STD NMR for the amino acid conjugates of the single truncated Neu5Ac. The fit using the one site binding model yielded a KD value of 8 ± 3 μM for the phenylalanine ligand SV-8, a KD value of 15 ± 6 μM for the tryptophan ligand SV-10 and a KD value for the tyrosine ligand SV-14 KD value of 4 ± 5 μM. [SV-8: (c (FcMAGd1-3) = 13.9 μM, c (SV-8) = 13.9 to 166.8 μM, 700 MHz, T = 285 K, shaped pulse sp1 = 45 dB, saturation time = 2.04 s, irradiation frequency resonance spectrum = -0.5 ppm)]. [SV-10: (c (FcMAGd1-3) = 11.1 μM, c (SV-10) = 11.2 to 166.8 μM, 700 MHz, T = 285 K, shaped pulse sp1 = 45 dB, saturation duration = 2.04 s, irradiation frequency resonance spectrum = -0.5 ppm)]. [SV-14: (c (FcMAGd1-3) 11.1 μM, c (SV-14) = 11.1 to 166.8 μM, 700 MHz, T = 285 K, shaped pulse sp1 = 45 dB, saturation duration = 2.04 s, irradiation frequency an Spectrum = -1.0 ppm)].

For comparison with the difference spectrum obtained for the reference substance SH-81, see 16 the corresponding difference spectrum of the phenylalanine compound SV-8 is shown by way of example. Again, the difference signals of the aromatic protons can be clearly seen. In addition, the N-acetyl group of the Neu5Ac also shows a high STD effect in this compound and thus a high contribution to the binding to the MAG.

16 : The off-resonance spectrum and the difference spectrum of ligand SV-8 (the off-resonance spectrum is reduced by a factor of 100). In the difference spectrum, the signals of the binding protons of the ligand can be recognized. Particularly strong STD effects show the aromatic protons. (c (FcMAGd1-3) = 13.9 μM, c (SV-8) = 166.8 μM, 700 MHz, T = 285 K, shaped pulse sp1 = 45 dB, saturation time = 2.04 s, irradiation frequency an-spectrum = -0.5 ppm ).

Comparison of the determined Dissociation constants Kp and the association constants and Dissociation constants for the amino acid truncated

Neu5Ac derivatives

The highest binding affinity to MAG was found in the tyrosine conjugate of the single truncated Neu5Ac SV-14 with a K _D = 11 ± 5 μM in the SPR affinity analysis and a K _D = 1 μM (Chi ² = 0.16) in SPR kinetics analysis, respectively a K _D = 4 ± 5 μM in the STD NMR analysis. The phenylalanine conjugate of the single truncated Neu5Ac SV-8 showed only a slightly weaker binding affinity for MAG with a K _D = 14 ± 2 μM in the SPR affinity analysis and a K _D = 8 ± 3 μM in the STD NMR analysis. A determination of the dissociation constant by means of SPR kinetic analysis was not possible for SV-8, since this ligand was the only negative sensorgram in the SPR analysis ( 11 ). In contrast, the tryptophan conjugate of the single truncated Neu5Ac SV-10 showed less good binding affinities for MAG than the other two conjugates. A K _D = 77 ± 7 μM was obtained in the SPR affinity analysis and a K _D = 100 μM (Chi ² = 0.68) by SPR kinetic analysis. The STD NMR analysis showed a K _D = 15 ± 6 μM, which is lower than the SPR-determined dissociation constants, but is still worse by a factor of 3.8 or 2 than the dissociation constants determined by STD NMR for the other conjugates.

The tyrosine conjugate of the doubly truncated Neu5Ac SV-18 showed a K _D = 185 ± 54 μM in the SPR affinity analysis and a K _D = 7 μM (Chi ² = 0.52) in the SPR kinetic analysis. Due to the weaker binding affinity no STD NMR analysis has been performed. The comparison with the dissociation constant of the tyrosine conjugate of the single truncated Neu5Ac showed a factor of 17 or 7 weaker binding affinity to MAG. The twofold shortening of the Neu5Ac presumably leads to a lower binding affinity for the MAG due to the absence of the hydroxyl group at the 7-position and a less favorable position in the binding pocket. Taking into account the different conditions of these two analytical methods, the K _D values obtained for the individual ligands are in good agreement. The SPR kinetic analysis shows similar dissociation rates k _off for all four ligands. The conjugates SV-14 and SV-8 with the higher binding affinity showed a slightly faster dissociation rate with k _off = 2.4 · 10 ^-3 s ^-1 for SV-14 and k _off = 0.4 · 10 ^-3 s ^-1 for SV-8 compared to the conjugates SV-10 and SV-18 with k _off = 6.4 · 10 ^-3 s ^-1 for SV-10 and k _off = 4.2 · 10 ^-3 s ^-1 for SV-18 (Table 1). The fastest association rate was shown by the tyrosine conjugate of the single truncated Neu5Ac SV-14 with a k _on = 2.0 · 10 ⁴ M ^-1 s ^-1 , which also showed the highest binding affinity to MAG. The tyrosine conjugate of the doubly truncated Neu5Ac SV-18 with k _on = 5.7 · 10 ^-3 M ^-1 s ^-1 also showed a faster association than the conjugates of the other amino acids. The phenylalanine conjugate SV-8 and the tryptophan conjugate SV-10 showed with k _on = 2.5 · 10 ² M ^-1 s ^-1 for SV-8 and k _on = 5.9 · 10 ² M ^-1 s ^-1 for SV -10 slower associations.

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Cited non-patent literature

• - DeBellard et al. [0006]
• - Strenge et al. [0008]
• Kelm et al. [0015]

Claims (8)

Inhibitors of Myelin Associated Glycoprotein (MAG) consisting of conjugates of amino acids with truncated neuraminic Inhibitors of Myelin Associated Glycoprotein (MAG) consisting of conjugates of peptides with truncated neuraminic acids Inhibitors in which the amino acids one have hydrophobic radical. A process for the preparation of the conjugates of amino acids and truncated neuraminic acids The use of the described in claim 1, 2 and 3 Substances used to treat central nervous system injuries with the aim to enable the reunification of the nerves. The use of the described in claim 1, 2 and 3 Substances used to treat central nervous system injuries with the aim of alleviating the consequences of the injury. The use of the described in claim 1, 2 and 3 Substances designed to stimulate neurite outgrowth. Development of a surface plasmon resonance system for Determining Binding to Myelin Associated Glycoprotein (MAG)