CN111840303A

CN111840303A - Cytoplasmic carboxypeptidase inhibitors and uses thereof

Info

Publication number: CN111840303A
Application number: CN202010681032.8A
Authority: CN
Inventors: 武慧渊; 王瑞雪
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2020-07-15
Filing date: 2020-07-15
Publication date: 2020-10-30

Abstract

The present invention relates to inhibitors of cytoplasmic carboxypeptidase and uses thereof. 2-PMPA as a cytosolic carboxypeptidase inhibitor in a concentration range of 1nM to 100 mM; for the first time, highly active inhibitors of the CCP family were developed. Compared with the traditional M14 metal carboxypeptidase inhibitor, 2-PMPA can effectively inhibit the activity of CCP family enzyme; 2-PMPA not only inhibits the activity of the CCP family on protein substrates, but also inhibits the activity thereof in catalyzing the synthesis of polypeptides and neuropeptides. Through the experiment of enzymatic reaction, the 2-PMPA inhibits half inhibition of CCP1 catalyzed synthesis of polypeptideConcentration (IC)₅₀) 0.99 μ M, and mixed inhibition with Ki and Ki' of 0.096 μ M and 0.235 μ M, respectively. 2-PMPA inhibits IC of CCP1 catalyzing tubulin deglutamylation under experimental conditions₅₀It was 0.21 mM.

Description

Cytoplasmic carboxypeptidase inhibitors and uses thereof

Technical Field

The present invention relates to the field of cytosolic carboxypeptidase inhibitors, and in particular to the use of 2- (phosphonomethyl) glutaric acid (2-PMPA) as a cytosolic carboxypeptidase inhibitor.

Background

Cytoplasmic Carboxypeptidase (CCP) is a subfamily of M14 metallocarboxypeptidases, catalyzing post-translational modification of proteins-de-modification of polyglutamylation. Polyglutamylation is a novel protein posttranslational modification, which occurs on gamma-carboxyl of glutamic acid in a primary structure of a protein, is connected with amino of free glutamic acid through amido bond to form a branched chain, and the glutamic acid on a fulcrum is further connected with a plurality of glutamic acids in sequence through peptide bonds (alpha-carboxyl) to form polyglutamyl side chains with different lengths. Polyglutamylation maintains a dynamic equilibrium in vivo, the initiation and elongation of which is catalyzed by 9 members of the Tubulin-Like tyrosine Ligase (ttlin Tyrosin Ligase Like, TTLL) family. Whereas the 6 family members of cytoplasmic carboxypeptidases catalyze the shortening and digestion of polyglutamyl side chains.

The functional abnormalities of the modified enzyme and the de-modified enzyme for regulating polyglutamylation can cause polyglutamylation imbalance, so that various diseases can be caused, including neurodegeneration, retinal inflammation, male sterility and the like. For example, CCP1 mutation causes Purkinje cell degeneration in pcd mice (pcd) and also causes quadriplegia in newborn sheep, and recent studies have found that human patients who begin to undergo gradual neurodegeneration during infancy carry an inactivated mutant CCP1 gene. Loss of function of TTLL1 resulted in impaired vesicle transport due to abnormal targeting of KIF 1A. The variation of TTLL5 results in retinal dystrophy caused by low levels of glutamylation of RPGR (Retentis Pigmentinosa GTPase regulator). The development of poly-glutamic acylation de-modifying enzyme inhibitors can provide possibility for treating corresponding diseases by intervening the modified balance. According to our search, no effective inhibitors against the family of cytoplasmic carboxypeptidases have been reported in the literature to date. Whereas inhibitors of the general M14 metallocarboxypeptidase family have poor selectivity of inhibition for the cytoplasmic carboxypeptidase family, in vitro experiments have shown that concentrations of at least millimolar are required (Wu, H.Y.et al, analytical and functional analysis of Nna1 in Purkinje cell aggregation (pcd) microorganism. FASEB J26, 4468-4480, 2012). Therefore, the search for high-efficiency cytoplasmic carboxypeptidase inhibitors has important research significance and application prospect.

2- (phosphonomethyl) pentanedioic acid (2-PMPA) is a highly effective inhibitor of glutamic acid carboxypeptidase II (GCPII), but the effect of this compound on the activity of other carboxypeptidases has not been reported. There is a large difference between cytosolic carboxypeptidase and glutamic carboxypeptidase II. The two belong to different families of metallocarboxypeptidases, respectively cytoplasmic carboxypeptidases being a subfamily of M14 metallocarboxypeptidases and glutamate carboxypeptidase II being a subfamily of M28 metallocarboxypeptidases; the catalytic mechanisms of the two are different: cytoplasmic carboxypeptidase is dependent on one zinc ion, glutamate carboxypeptidase II requires two zinc ions, and calcium and chloride ions are also essential for its catalytic activity; functionally, cytoplasmic carboxypeptidase catalyzes the de-modification of polyglutamylated peptide, while glutamate carboxypeptidase II cleaves the bulk neuropeptide N-acetyl aspartyl glutamate (NAAG) into N-acetyl aspartate (NAA) and glutamate.

Disclosure of Invention

The invention develops 2-PMPA for the first time, which can be used as a high-activity inhibitor of a cytoplasmic carboxypeptidase family, provides a new application for 2-PMPA, and provides a method for inhibiting the activity of the cytoplasmic carboxypeptidase family.

In order to explore and solve the problems of the prior art, the present inventors have studied the inhibitory effect of different compounds, 1, 10-phenanthroline (OP), 2-benzylsuccinic acid (BZS), L (+) -2-amino-4-phosphonobutyric acid (L-AP4),2- (phosphonomethyl) glutaric acid (2-PMPA) on cytoplasmic carboxypeptidase, respectively, through enzymatic reactions. Comparison and screening show that 2-PMPA has good inhibition effect on cytoplasmic carboxypeptidase, and research on different substrates shows that 2-PMPA can inhibit the activity of cytoplasmic carboxypeptidase family on protein substrates, such as activity of catalyzing the deglutamylation of tubulin, and can also inhibit the activity of catalyzing the deglutamylation of synthetic polypeptide and neuropeptide.

The invention provides 2-PMPA and compositions comprising 2-PMPA for use as inhibitors of cytoplasmic carboxypeptidase, as well as methods for inhibiting activity of the cytoplasmic carboxypeptidase family and for using 2-PMPA for treating disorders associated with an imbalance in polyglutamination.

The invention provides the following technical scheme:

the first aspect provides the use of 2-PMPA as a cytosolic carboxypeptidase inhibitor.

Preferably, the concentration of 2-PMPA as a cytosolic carboxypeptidase inhibitor ranges from 1nM to 100 mM; a more preferred concentration range is 10nM to 10 mM.

Preferably, use of 2-PMPA to inhibit the deglutamylation activity of a polypeptide synthesized by a cytosolic carboxypeptidase is provided.

Preferably, there is provided the use of 2-PMPA to inhibit the activity of a cytoplasmic carboxypeptidase to catalyze deglutamylation of a protein substrate.

Preferably, there is provided the use of 2-PMPA to inhibit the activity of a cytoplasmic carboxypeptidase to catalyze the deglutamylation of a neuropeptide.

In a second aspect, a cytoplasmic carboxypeptidase inhibitor composition is provided, the composition including 2-PMPA.

A third aspect provides a method of inhibiting a cytoplasmic carboxypeptidase, comprising contacting the cytoplasmic carboxypeptidase with 2-PMPA in a PBS buffer, a HEPES buffer, or a MOPS buffer.

A fourth aspect provides the use of 2-PMPA in the manufacture of a medicament for the prevention and/or treatment of a disease or condition associated with cytoplasmic carboxypeptidase.

A fifth aspect provides a pharmaceutical composition or pharmaceutical formulation comprising 2-PMPA for use in the treatment of a disease associated with cytoplasmic carboxypeptidase.

The present invention has for the first time developed highly active inhibitors of cytoplasmic carboxypeptidase. Unlike other traditional M14 metallocarboxypeptidase inhibitors, 2-PMPA can inhibit the activity of enzymes of the cytosolic carboxypeptidase family with high efficiency. 2-PMPA can inhibit not only the activity of the cytoplasmic carboxypeptidase family on protein substrates, such as tubulin, but also its activity in catalyzing the deglutamylation of synthetic polypeptides and neuropeptides. The half Inhibition Concentration (IC) of 2-PMPA for inhibiting CCP1 to catalytically synthesize polypeptide was determined through an enzymatic reaction experiment₅₀) 0.99 μ M, and mixed inhibition with Ki and Ki' of 0.11 μ M and 0.24 μ M, respectively. 2-PMPA inhibits IC of CCP1 catalyzing tubulin deglutamylation under experimental conditions₅₀It was 0.21 mM.

Drawings

FIG. 1 shows the inhibitory effect of various compounds of example 1 on the deglutamylation of polypeptide Biotin-3EG2E catalyzed by recombinant CCP 1.

FIG. 2 shows the inhibitory effect of 2-PMPA on deglutamylation of recombinant CCP1 catalytic polypeptide Biotin-3EG2E in HEPES buffer solution in example 2.

FIG. 3 shows the inhibitory effect of 2-PMPA on deglutamylation of recombinant CCP1 catalytic polypeptide Biotin-3EG2E in MOPS buffer in example 2.

FIG. 4 shows the inhibitory effect of 2-PMPA on deglutamylation of recombinant CCP4 catalytic polypeptide Biotin-3EG2E in example 2.

FIG. 5 shows the half inhibitory concentration effect of 2-PMPA in example 3 on the catalytic deglutamylation of tubulin by recombinant CCP1 in different buffer systems.

FIG. 6 shows the inhibitory effect of 2-PMPA on recombinant CCP4 catalyzing the deglutamylation of tubulin in example 3.

FIG. 7 shows the inhibitory effect of 2-PMPA on the catalytic deamidation of tubulin by CCP5 cell lysates, as described in example 3.

FIG. 8 shows the inhibitory effect of 2-PMPA on the catalytic deamidation of tubulin by CCP6 cell lysates, as described in example 3.

FIG. 9 shows the inhibitory effect of 2-PMPA on the catalytic deglutamylation of neuropeptides by recombinant CCP1 in example 4.

Detailed Description

The invention records for the first time that 2-PMPA can inhibit the activity of cytoplasmic carboxypeptidase to catalyze the glutamic-acylation of protein substrates, and can also inhibit the activity of cytoplasmic carboxypeptidase to catalyze and synthesize polypeptide and neuropeptide glutamic-acylation. The present invention is further described with reference to specific embodiments, but the present invention is not limited to these embodiments.

The 2-PMPA inhibits cytoplasmic carboxypeptidase at a concentration in the range of 10nM to 10mM, and the buffer solution can be PBS, HEPES or MOPS.

Example 1

Effect of different Compounds on Activity of recombinant CCP1

Configuration of reagents used in the experiment:

ninhydrin-chromium chloride solution: 0.2g of ninhydrin was dissolved in a mixed solution of 20mL of ethanol and 2.5mL of acetic acid, followed by addition of 0.25mL of an aqueous solution (5.5mM) of chromium chloride.

The reaction system contained 0.6 μ g of purified recombinant mouse CCP1, 40 μ M of polypeptide biotin-3EG2E, 25mM HEPES-K (pH 7.4), 100mM NaCl, 1mM 1, 10-phenanthroline (OP)/2-benzylsuccinic acid (BZS)/2-PMPA/L (+) -2-amino-4-phosphonobutyric acid (L-AP4) in 100 μ L. Reactions with heat inactivated CCP1 served as negative controls. Three parallel groups were set up and incubated at 37 ℃ for 1 h. Then 200. mu.L of ninhydrin-chromium chloride solution is added and mixed evenly, and then heated in water bath at 84 ℃ for 5 minutes to terminate the reaction. After the temperature is restored to room temperature, absorbance detection is carried out under enzyme labeling of 507 nm. The amino acids released were quantified using a standard curve generated from known amounts of glutamic acid (L-AP4 reaction was quantified using high performance liquid chromatography). As shown in FIG. 1, CCP1 with no inhibitor added has one hundred percent of activity as a positive control, and after the traditional inhibitors OP, BZS and L-AP4 with the same concentration are added, the relative activities of CCP1 are respectively 16%, 96% and 27%, while 2-PMPA can almost completely inhibit the activity of CCP 1.

Example 2

2-PMPA inhibits CCP1 from catalyzing reaction of polypeptide Biotin-3EG2E

A100. mu.L reaction system contained 0.6. mu.g of purified recombinant mouse CCP1, 40. mu.M of the polypeptide biotin-3EG2E, 25mM HEPES-K (pH 7.4) or 10mM MOPS (pH 7.4), 100mM NaCl, and 0, 0.01, 0.025, 0.05, 0.5, 5 or 50. mu.M 2-PMPA. Reactions with heat inactivated CCP1 served as negative controls. Three parallel groups were set up and incubated at 37 ℃ for 1 h. As described above, the effect of 2-PMPA in HEPES buffer solution on the inhibition of CCP1 on the deglutamylation of polypeptide was shown in FIG. 2, and 0.05-50. mu.M of 2-PMPA was found to have an inhibitory effect on the activity of CCP1 on the polypeptide, and IC was calculated by Prism₅₀0.99 μ M. The effect of 2-PMPA in MOPS buffer solution on CCP1 catalysis of polypeptide is shown in FIG. 3, and the effect of 1 μ M2-PMPA on CCP1 catalysis of polypeptide deglutamylation inhibition is 52%.

(II) 2-PMPA inhibits the activity of CCP4 catalytic polypeptide

A100. mu.L reaction system contained 0.6. mu.g of purified recombinant mouse CCP4, 40. mu.M polypeptide Biotin-3EG2E, 25mM HEPES-K (pH 7.4), 100mM NaCl, and 0, 0.01, 0.025, 0.05, 0.5, 5 or 50. mu.M 2-PMPA. Reactions with heat inactivated CCP4 served as negative controls.Three parallel groups were set up and incubated at 37 ℃ for 1 h. As described above, the effect of 2-PMPA on the inhibition of the activity of CCP4 catalytic polypeptide was shown in FIG. 4, in which 0.01 to 50. mu.M of 2-PMPA had an inhibitory effect on the activity of CCP4 catalytic polypeptide, and IC was calculated by Prism ₅₀＝0.37μM。

Example 3

2-PMPA inhibits the activity of CCP in catalyzing the deglutamylation of tubulin

2-PMPA inhibits the activity of recombinant CCP in catalyzing the deglutamylation of tubulin

1. Sample preparation

Mu.g of purified recombinant mouse CCP1 or CCP4 and 2. mu.g of porcine tubulin were contained in a 40. mu.L reaction system, and 0,0.1,1,5,10mM 2-PMPA was incubated in PBS or HEPES or MOPS buffer for 1h at 37 ℃. 10mM OP were used as a negative control under the same reaction conditions. The reaction was stopped by adding 10. mu.l of 5 × SDS-PAGE protein loading buffer and mixing well and heating at 95 ℃ for 10 minutes.

2. Electrophoresis

(1) SDS-PAGE gel formulation

The glass plate was mounted, and 8% (w/v) of the separation gel solution and 5% (w/v) of the concentrated gel solution were prepared according to the numerical values in the table, and filled with:

component (A)	8% resolving gel solution (mL)	5% concentrated gum solution (mL)
			ddH₂O	9.3	6.8
1.5M Tris-HCl(pH 8.8)	5	——
			1.0M Tris-HCl(pH 6.8)	——	1.25
10％SDS	0.2	0.1
			30% acrylamide solution	5.3	1.7
10% ammonium persulfate	0.2	0.1
			TEMED	0.012	0.01
Total volume	20	10

(2) Sample loading and electrophoresis

Loading: a10. mu.L sample was applied to an 8% polyacrylamide gel electrophoresis for separation.

Electrophoresis: concentrating the gel at 80V, separating the gel at 120V, and stopping electrophoresis until bromophenol blue is about to run out of the gel.

(3) Rotary film

The proteins on the protein gel were transferred to a nitrocellulose membrane (NC membrane) using a semi-dry membrane transfer instrument, and the membrane transfer current was set at 100mA and the membrane transfer time was set at 60 minutes.

(4) Sealing of

Blocking with 5% skimmed milk powder/TBST for 30 min at room temperature.

(5) Incubation primary antibody

The antibody was diluted with 5% skim milk/TBST to a final concentration of polyE (1:4000, recognizing greater than or equal to 3 glutamic acid side chains), α -tubulin (EP1332Y, 1: 5000). Incubate on a shaker overnight at 4 ℃.

Recovering the primary antibody. TBST wash was added and washed on a shaker for 10 minutes. After the washing solution was sucked up, the washing solution was added again to wash for 10 minutes. The total number of washes was 3.

(6) Incubation secondary antibody

The antibody was diluted with 5% skim milk/TBST, and the final concentration of the secondary antibody donkey anti rabbitt was 1: 5000. Incubate for 2h at room temperature on a shaker.

And (5) recovering the secondary antibody. TBST wash was added and washed on a shaker for 10 minutes. After the washing solution was sucked up, the washing solution was added again to wash for 10 minutes. The total number of washes was 3.

(7) Protein detection

And (3) uniformly dropwise adding the ECL luminescent solution on the membrane, and detecting the inhibition effect of the 2-PMPA on the CCP catalytic protein substrate in different buffer solution systems by using a gel imaging system. According to the Western blotting result, based on the gray scale ratio of antibody immunoreaction of polyE and alpha-tubulin, 2-PMPA inhibits IC of CCP1 catalyzing tubulin deglutamylation in PBS, HEPES and MOPS buffer solution system through Prism calculation ₅₀0.21mM, 0.15mM, 0.19mM, respectively, as shown in FIG. 5. The result of the reaction of 2-PMPA for inhibiting CCP4 from catalyzing tubulin is shown in FIG. 6, wherein 10mM of traditional inhibitor OP is used as a negative control, and 0.1-10mM of 2-PMPA has obvious inhibition effect on the activity of CCP4 for catalyzing the desglutamylation of tubulin.

(II) 2-PMPA inhibits tubulin deglutamylation Activity in CCP-containing cell lysates

HEK293 cells were transfected with a plasmid expressing CCP5 or CCP6 (nitrogen-terminated with myc-tag) and after 40 hours of incubation, the cells were washed twice with pre-cooled DPBS and lysed with DPBS solution containing protease inhibitor and 0.2% ethylphenylpolyethylene glycol (NP-40). After centrifugation at 24,000g for 20 minutes at 4 ℃, the pellet was discarded and the supernatant was used as a lysate of CCP5 or CCP6 cells.

20. mu.L of CCP5 or CCP6 cell lysate and 1. mu.g of porcine tubulin, and 0,0.1,1mM 2-PMPA in PBS were incubated for 1h at 37 ℃ in a 40. mu.L reaction. 10mM OP were used as a negative control under the same reaction conditions. The reaction was stopped by adding 10. mu.L of 5 × SDS-PAGE protein loading buffer and mixing well and heating at 95 ℃ for 10 minutes. Detection method for inhibition of tubulin deglutamylation activity of CCP 5-containing cell lysate by 2-PMPA As described above, Western blotting primary antibody incubation GT335(1:4000, recognizing glutamic acid linked to gamma-carboxyl group on branched chain) and anti-myc (1:2000) showed the inhibition effect of Goatanti mouse (1:5000) incubation as shown in FIG. 7; the inhibition effect of 2-PMPA on the tubulin deglutamyl activity of CCP 6-containing cell lysates is shown in FIG. 8, lacZ is used as a blank control, a positive control without inhibitor is used, a 10mM traditional inhibitor OP is used as a negative control, and 0.1-1mM 2-PMPA can almost completely inhibit the activity of CCP5 and CCP6 in catalyzing the tubulin deglutamyl activity.

Example 4

2-PMPA inhibition of CCP1 catalyzes the reaction of neuropeptide N-Acetylaspartyl-glutamic acid (NAAG)

A100. mu.L reaction system contained 0.6. mu.g of CCP1, 0.1mM NAAG, 25mM HEPES-K (pH 7.4), 100mM NaCl, and 1. mu.M 2-PMPA, with the reaction without the addition of 2-PMPA being a positive control and the reaction with the addition of heat-inactivated CCP1 being a negative control. Three parallel groups were set up and incubated at 37 ℃ for 1 h. Then 200. mu.L of ninhydrin-chromium chloride solution is added and mixed evenly, and then heated in water bath at 84 ℃ for 5 minutes to terminate the reaction. After the temperature is restored to room temperature, absorbance detection is carried out under enzyme labeling of 507 nm. The amino acids released were quantified using a standard curve generated from known amounts of glutamic acid. As shown in FIG. 9, the inhibitory effect of 1. mu.M 2-PMPA on CCP1 catalyzing the deglutamylation of neuropeptide was 72% without inhibitor as a positive control.

The above description is only a preferred embodiment of the present disclosure and is not intended to limit the present disclosure. Various modifications and variations of this disclosure will be apparent to those skilled in the art. Any modification, improvement, equivalent replacement, etc. made within the spirit and principle of the present disclosure should be included in the scope of protection of the present disclosure.

Claims

1. A cytoplasmic carboxypeptidase inhibitor, comprising 2- (phosphonomethyl) glutaric acid.

Use of 2- (phosphonomethyl) glutaric acid in the preparation of a cytoplasmic carboxypeptidase inhibitor.

3. A method of inhibiting a cytoplasmic carboxypeptidase, wherein the cytoplasmic carboxypeptidase is contacted with 2- (phosphonomethyl) glutaric acid under conditions wherein the cytoplasmic carboxypeptidase is inhibited.

4. The method of claim 2, wherein the buffer system that inhibits the cytoplasmic carboxypeptidase is not limited, preferably PBS, HEPES, MOPS; the concentration of 2- (phosphonomethyl) glutaric acid contacted is 1nM to 100mM, preferably 10nM to 10 mM.

5. A method according to claim 2 or 3, wherein said inhibition reduces the activity of a cytoplasmic carboxypeptidase catalysing the deglutamylation of a substrate by at least 20%, preferably by 50%, more preferably by 80%.

6. A pharmaceutical composition comprising 2- (phosphonomethyl) glutaric acid as an active ingredient together with pharmaceutically tolerable adjuvants and/or excipients.

7. The pharmaceutical composition of claim 6, further comprising one or more additional active ingredients.

8. A pharmaceutical composition according to claim 6 or 7, wherein the 2- (phosphonomethyl) glutaric acid content is not less than 20%.

9. The pharmaceutical composition of claim 6 or 7, for use in the prophylactic or therapeutic treatment of a disease associated with an imbalance in polyglutamylated ammonia.

10. The pharmaceutical composition of claim 6, wherein the disease is selected from the group consisting of: neurodegeneration, retinal inflammation, and male sterility.