阅读说明：本技术 胞质羧肽酶抑制剂及其应用 (Cytoplasmic carboxypeptidase inhibitors and uses thereof ) 是由 武慧渊 王瑞雪 于 2020-07-15 设计创作，主要内容包括：本发明涉及胞质羧肽酶抑制剂及其应用。2-PMPA作为胞质羧肽酶抑制剂的浓度范围为1nM-100mM；首次开发了CCP家族的高活性抑制剂。与传统M14金属羧肽酶抑制剂相比,2-PMPA可以高效抑制CCP家族酶的活性；2-PMPA不仅抑制CCP家族对蛋白质底物,还可以抑制其催化合成多肽和神经肽的活性。通过酶促反应实验确定了2-PMPA抑制CCP1催化合成多肽的半数抑制浓度(IC<Sub>50</Sub>)为0.99μM,且为混合型抑制,其Ki和Ki’分别为0.096μM和0.235μM。2-PMPA在实验条件下抑制CCP1催化微管蛋白去谷氨酰化的IC<Sub>50</Sub>为0.21mM。(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 conditions 50 It was 0.21 mM.)

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.

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.