DE10025379A1 - Determination of NAALADase inhibitors - Google Patents

Abstract

The present invention relates to a method for the determination of inhibitors of the enzyme N-acetyl-alpha-linked acidic dipeptidase (NAALADase), in which the enzymatic conversion of a substrate which is radiolabeled at the C-terminal glutamate residue is determined by its binding to fluomicrospheres. Furthermore, the invention relates to new enzyme substrates and their use in the process mentioned.

Description

The present invention in the field of biotechnology relates to a method for the determination of inhibitors of the enzyme N-acetyl-alpha-linked acidic dipeptidase (NAALADase), in which the enzymatic conversion of a C- terminal glutamate residue of radioactively labeled substrate by binding to it Fluomicrosphere is determined. Furthermore, the invention relates to new Enzyme substrates, and their use in the method mentioned.

State of the art

The enzyme N-acetyl-alpha-linked acidic dipeptidase (NAALADase) is a type II Transmembrane protein that in neurons and glial cells the hydrolysis of N-acetyl aspartyl glutamate (NAAG) catalyzed in N-acetyl aspartate (NAA) and glutamate. The enzyme was first described in 1987 by Robinson et al. and Horoszewicz et al. under the names NAALADase and prostate-specific membrane antigen (PSMA) described independently of each other. The corresponding gene was developed in 1993 by Israeli et al. cloned and the identity of both enzymes by Carter et al. 1996 demonstrated. This differs from the other related enzymes here described NAALADase I by its NAAG hydrolyzing activity. Furthermore, it can also produce gamma-bound glutamates from Split off folic acid polyglutamates. The cleavage of carboxy terminal glutamates is therefore characteristic. The enzyme is therefore also called glutamate carboxypeptidase II called (Carter et al. 1998).

As a known NAALADase inhibitor, 2- (phosphono called methyl) pentanedioic acid (PMPA). Reduce NAALADase inhibitors on the one hand toxic amounts of glutamate and on the other hand increase the metabotropic glutamate receptor 3 agonist NAAG. Have said inhibitors their importance in the treatment of neurodegenerative diseases (Carter et al. 1996, Slusher et al. 1999). The enzyme inhibition results in an increased NAAG concentration, which is an activation of glial metabotropic glutamate receptors of type 2 (mGluR3) and thus ultimately increased  The release of TGFβ results, which has a neuroprotective effect (Bruno et al. 1998).

Current methods of determination for NAALADase inhibitors have been developed by Serval. et al., Slusher et al. and Tiffany et al. developed. Representative of this The method is described here in more detail on the assay in US Pat. No. 6,025,345 the title "Inhibitors of NAALADase enzyme activity".

All assays have in common that the enzyme substrate is a radioactive on the glutamate residue marked NAAG is used. Either the released radioactive Glutamate or the unreacted radioactive starting compound determined. The separation of those to be measured proves to be time and material consuming radioactive substance from the other radioactive substances after the End of the enzyme-substrate reaction in the presence of potential inhibitors. A separation of * NAAG, NAA and * G is taking into account the small Differences in size and chemically functional properties of the Compounds usually by column chromatography methods guaranteed. Then the substances to be determined are included Scintillators added to evaluate the activity spectroscopically.

The object of the present invention was the considerable acceleration of Separation and detection steps of the radioactive ligand to be quantified. This task was accomplished within the description and the present Claims resolved.

description

One aspect of the present invention relates to methods for determining Inhibitors of the enzyme N-acetyl-alpha-linked acidic dipeptidase (NAALADase), in which the enzymatic conversion of a radioactive at the C-terminal glutamate residue labeled substrate is determined by its binding to fluomicrospheres. A sufficiently selective binding of the radioactively labeled substrate on fluomicrosphere. This substrate binding offers 2 advantages, the separation of radioactive unsplit substrate is eliminated and at the same time an optimal implementation of the bound radioactivity takes place in quantifiable light instead. With radioactive decay, depending on the isotope  released subatomic particles and various forms of energy. Depending on the energy or particle energy, can only be a limited distance with sufficient Energy can be achieved in the surrounding medium. For those through tritium expelled β-particles is known, for example, that the energy for excitation of scintillator molecules decreases significantly after 1.5 µm in aqueous media. Fluomicrospheres are microspheres or are also called "pearls", that are made to have scintillation molecules inside. For example, you can use PVT (polyvinyltoluene) or yttrium silicates getting produced. They can be bought, for example from the Amersham company Pharmacia Biotech UK Ltd. By binding to the substrates radio emitter and scintillator become in close proximity to each other forced and the yield of the implementation of radioactivity and detectable Optimized light quantity. Unbound radioactivity is too far away on average, to move the scintillator noticeably to light emission. For the The assay according to the invention thus has the advantage that the bound radioactive substrate compared to the unbound radioactivity of the split glutamate product demonstrated to such a great extent can be that a separation of the glutamate from the reaction mixture is unnecessary. The method according to the invention optimizes the radioactivity yield and eliminates the need for separation of radioactive substrate and product. For the Glutamate residue of the NAAG substrate are the radioactive isotopes of oxygen, Nitrogen, carbon and hydrogen can be used. The latter isotopes are prefers. Methods according to the invention are particularly preferred in which the Substrate is marked with tritium.

There are a number of ways to attach the NAAG substrate to the fluomicrosphere to tie. Without changing the substrate, this is possible, for example, through the Use of substrate-specific antibodies as substituents on the Fluomicrosphere possible. The production of such NAAG-specific antibodies provides for the experienced specialist for both polyclonal and monoclonal Antibodies are routine work.

Furthermore, there is the possibility of using fluomicrosphere and substrate to provide complementary binding substituents. For example, you can Fluomicrospheres are provided with antibodies that have specific substituents  recognize the substrate. Such procedures in which the fluomicrosphere unites Have antibody substituents that the substrate before or after the recognizes enzymatic cleavage are preferred.

Other complementary systems can also be used. The invention Methods in which the binding of the substrate to the fluomicrosphere by a substituent of the substrate is particularly preferred.

One of the substituents known in the prior art has Embodiment of the invention, especially the complementary biotin Streptavidin system proven. The methods according to the invention are thus complete particularly preferred in which the substituent is a biotin residue and in which the Fluomicrospheres have a streptavidin substituent. Optionally, a Substrate substituent, especially the biotin substituent via a The left / spacer must be connected to the substrate.

The human NAALADase is special for the methods according to the invention Importance. The human sequence has long been known (Israeli et al. 1993, Genbank M99487) and their recombinant representation is technically problem-free. NAALADase can also be obtained from various tissues (Tiffany et al., Robinson et al.).

Usually the rough membrane extraction is natural or recombinant producing cells. For this, the cells are unlocked and Enriched membrane proteins, hereinafter called enzyme-containing protein extract be designated. This rough separation is sufficient because the enzyme is highly specific and not essential with other cell constituents interacts. The presence of the enzyme in an enriched protein extract represents a preferred embodiment of the inventive method, because this offers the most economically sensible variant. In one embodiment of the The method according to the invention uses the NAALADase as described by Park et al. described cleaned up.

The method according to the invention thus advantageously comprises only a few essential steps. If the method is carried out “off bead”, that is to say the “beads” are only added after the enzymatic substrate cleavage, which has taken place in the presence or absence of potential inhibitors, has been completed, the methods according to the invention comprise at least the following steps:

• a) Enzyme and substrate become more potential in the presence or absence Inhibitors incubated,
• b) the fluomicrosphere is then added, and
• c) then the microsphere-bound radioactivity is determined.

The embodiment variant just mentioned is particularly preferred. Enzyme and The substrate can first interact freely in the solvent and the detection then takes place. A possible inhomogeneous distribution of the substrate or there is no steric impairment of the enzyme reaction by the "beads" here possible. Preferred control approaches for evaluating the invention Methods are those in which no enzyme or a known inhibitor is added becomes.

Another preferred process variant is carried out "on bead", the Add the fluomicrosphere to the substrate before adding the enzyme or done simultaneously. In our embodiment, the "on bead" variant proven to be feasible. Compared to the particularly preferred "off bead" Variant shows a slightly lower cleavage activity of the enzyme.

The NAALADase is a very specific enzyme. In addition to NAAG, known substrates also include Asp-Glu, Glu-Glu and Gamma-Glu-Glu (Serval et al.) As well as mono- and polyglutamated folic acids such as, for example, methotrexate-Glu, methotrexate-Glu-Glu and methotrexate-Glu-Glu-Glu (Pinto et al.), But also methotrexate-Glu-Glu-Glu-Glu-Glu-Glu (Ajit et al., Slusher et al.). These compounds are also suitable for carrying out processes in accordance with the invention. Thus, methods are preferred in which the substrate is a substrate selected from the group:
NAAG, Asp-Glu, Glu-Glu, Gamma-Glu-Glu as well as mono- and polyglutamated folic acids,
whose C-terminal glutamate residue is radioactively labeled and whose residual molecule preceding the C-terminal glutamate is optionally provided with at least one additional substituent which enables binding to complementary substituents on fluomicrosphere.

If the fluomicrosphere has complementary antibodies, it can be that no additional substituent is necessary if the residual molecule is direct is recognized.

D-NAAG, ▱-NAAG and Ala-Glu (Serval et al.). The latter clearly shows that even small changes in stereochemistry or in the amino acid preceding the glutamate has a great influence on the exercise enzymatic capacity. Has biotin as a substituent for the substrate NAAG proved to be beneficial. Optionally, a substrate substituent, in particular the biotin substituent is connected to the substrate via a linker / spacer his. The use of a sterically less protruding spacer in the The sense of a molecular linker between the substrate and the substituent can prove to be advantageous in individual cases if the spatial proximity of the substituent itself a steric hindrance to the enzymatically active center of the NAALADase causes. Suitable preferred embodiments are suitable substituted aliphatic compounds, in particular C1 to 10 substituted aliphatic compounds.

The two have proven to be very particularly preferred embodiments following substrates proved, their C-terminal / terminal Radiolabelled glutamate / glutamic acid (single or multiple, preferably in the C3 and / or C4 position of glutamic acid):

Substrate 1

N ^α -acetyl-N ^β -biotin-5-aminopentyl-L-Asn-L-Glu [3H] -OH [(N ^β -biotin-5-aminopentyl) -N ^α -acetyl-asparaginyl-glutamic acid]

Substrate 2

N ^α -Biotinyl-L-Asp-L-Glu [3H] -OH [N ^α -Biotinyl-aspartyl-glutamic acid]

The above-mentioned are particularly preferred embodiments Understand substrates that are radiolabelled by tritium. Here is again the C3 and / or C4 position of the glutamate is particularly preferred.

Another aspect of the invention relates to the use of those listed above Substrates and their special embodiments in an inventive Method.

references

Bruno V. et al. (1998) Neuroprotection by glial metabotropic glutamate receptors is mediated by transforming growth factor-▱. J. Neurosci. 18, 9594-9600
Carter RE et al. (1996) Prostate-specific membrane antigen is a hydrolase with substrate and pharmacologic characteristics of a neuropeptidase. Proc. Natl. Acad. Sci. 93, 749-753
Carter RE et al. (1998) Hydrolysis of neuropeptide N-acetylaspartyl glutamate (NAAG) by cloned human glutamate carboxypeptidase II. Brain Res. 795, 341-348
Israeli RS et al. (1993) Molecular cloning of a complementary DNA encoding a prostate-specific membrane antigen. Cancer Res. 53, 227-230; AC: M99487
Park et al. (1999) Fibroblast activation protein, a dual specificity serine protease expressed in reactive human tumor stromal fibroblasts. J. Biol. Chem. 51, 36505-36512
Serval et al., (1992) In vitro and in vivo inhibition of N-acetyl-L-aspartyl-L-glutamate catabolism by N-acetylated L-glutamate analogs. J. Pharm. Exp. Ther. 260, 1093-1100
Slusher BS et al. (1990) Rat brain N-acetylated ▱-linked acidic dipeptidase activity. J. Biol. Chem. 265, 21297-21301
Slusher BS et al. (1999) Selective inhibition of NAALADase, which converts NAAG to glutamate, reduces ischemic brain injury. Nature Med. 5, 1396-1402
Tiffany CW et al. (1999) Characterization of the enzymatic activity of PSM: comparison with brain NAALADase. Prostate 39, 28-35
Robinson MB et al. (1987) Hydrolysis of the brain dipeptide N-acetyl-L-aspartyl-L-glutamate. Identification and characterization of a novel N-acetylated ▱-linked acidic dipeptidase activity from rat brain. J. Biol. Chem. 262, 14498-14506

example 1 The method of an enzyme assay with fluoromicrosphere "off bead"

The radioactivity coupled to beads was determined after the enzyme reaction in Presence of the specific NAALADase inhibitor PMPA (1 nM or 10 nM) and measured without an inhibitor. An approach without enzyme served as a control. It triplets were created in 96-well plates.

10 µl NAALADase (purified from stably transfected HEK293 cell extracts) was added to 90 µl reaction mixture (4 nM N ^α- biotinyl-aspartyl [ ³ H] -glutamic acid (substrate 2); 50 nM NAAG; 50 mM Tris, 1 mM ZnCl ₂ ). given. The control without enzyme contained 10 μl H ₂ O. The PMPA inhibitor was introduced before the enzyme was added (1 μl each of the corresponding 100-fold concentration).

After 1 hour incubation at 37 ° C., the reaction was stopped with 100 μl of 0.5 mg / ml PVT-SA beads (Amersham Pharmacia) in 0.25 M KH ₂ PO ₄ , pH 4.3. After 2 more hours the activity was measured (Trilux 1450 Microbeta / Wallac). The result is shown in Fig. 1.

"on bead"

The radioactivity bound to fluomicrosphere was measured with and without enzyme. The experiment was carried out as described above, but the PVT-SA beads (0.5 mg / ml) were already present during the enzyme reaction. After 2 hours the reaction was stopped with 100 ul 0.25 M KH ₂ PO ₄ , pH 4.3. After 2 more hours the activity was measured.

The result is shown in Fig. 2.

Description of the pictures

Fig. 1 shows the result of the measurement of fluomicrosphere bound radioactivity in the "off bead" method for the inhibitor PMPA in 2 concentrations of 1 and 10 nM compared to the control without inhibitor or with and without enzyme.

Fig. 2 shows the result in analogy to Fig. 1, but for the "on bead" process. The radioactivity bound to fluomicrosphere is compared with and without enzyme.

Claims (14)

1. A method for the determination of inhibitors of the enzyme N-acetyl-alpha linked acidic dipeptidase (NAALADase), in which the enzymatic Implementation of a radioactively labeled at the C-terminal glutamate residue Substrate is determined by its binding to fluomicrospheres. 2. The method of claim 1, wherein the substrate is labeled with tritium. 3. The method according to any one of claims 1 or 2, wherein the binding of the Substrate to the fluomicrosphere by a substituent of the substrate he follows. 4. The method of claim 3, wherein the substituent is a biotin residue and where the fluomicrosphere has a streptavidin substituent. 5. The method according to any one of claims 1 to 4, wherein the Fluomicrosphere have an antibody substituent that the substrate recognizes before or after the enzymatic cleavage. 6. The method according to any one of claims 1 to 5, wherein the enzyme is human NAALADase. 7. The method according to any one of claims 1 to 6, wherein said Enzyme is in the form of an enriched protein extract. 8. The method according to any one of claims 1 to 7, comprising the following steps:

• a) enzyme and substrate are incubated in the presence or absence of potential inhibitors,
• b) the fluomicrosphere is then added, and
• c) then the microsphere-bound radioactivity is determined.

9. The method of claim 8, wherein the addition of the fluomicrosphere to Substrate before the addition of the enzyme or simultaneously. 10. The method according to any one of claims 1 to 9, wherein the substrate is a substrate selected from the group:
NAAG, Asp-Glu, Glu-Glu, Gamma-Glu-Glu and mono- and polyglutamated folic acids,
whose C-terminal glutamate residue is radioactively labeled and
whose residual molecule preceding the C-terminal glutamate is optionally provided with at least one additional substituent, which enables binding to complementary substituents on fluomicrospheres. 11. (N ^β- biotin-5-aminopentyl) -N ^α -acetyl-asparaginyl-glutamic acid, the C-terminal glutamic acid of which is radiolabeled. 12. N ^α -Biotinyl-aspartyl-glutamic acid, whose C-terminal glutamic acid is radiolabeled. 13. A substance according to any one of claims 11 or 12, wherein the radiolabel done by tritium. 14. Use of a substrate according to one of claims 11 to 13 in one Method according to one of claims 1 to 10.