CN111569076B

CN111569076B - Application of POLR2A inhibitor in preparation of medicines

Info

Publication number: CN111569076B
Application number: CN202010600274.XA
Authority: CN
Inventors: 李光玉; 刘春晓; 许丽文
Original assignee: Institute Special Animal and Plant Sciences CAAS
Current assignee: Institute Special Animal and Plant Sciences CAAS
Priority date: 2020-06-28
Filing date: 2020-06-28
Publication date: 2022-05-17
Anticipated expiration: 2040-06-28
Also published as: CN111569076A

Abstract

The invention provides the use of a POLR2A inhibitor in the manufacture of a medicament for use in at least one of: inhibit osteoclastogenesis; inhibiting bone resorption; increase bone mass; increasing the stability of the bone. Osteoclast differentiation from mouse bone marrow precursors and human peripheral blood mononuclear cells (hPBMN) was inhibited by si-POLR2A but enhanced by Flag-POLR2A, suggesting that the modulation of bone resorption by POLR2A in mice may translate into human pathophysiology. Studies of the present application show that treatment with α -Amanitin significantly reduces osteoclast size, number and nuclear rate. In vivo studies have shown that treatment with alpha-Amanitin almost completely inhibits bone resorption, increasing bone quality and stability. The invention provides a brand-new medicine target point, a new treatment means and a new thinking for the development of a new generation of bone protection medicines identified by alpha-Amanitin.

Description

Application of POLR2A inhibitor in preparation of medicines

Technical Field

The invention relates to the field of biological medicines, in particular to application of a POLR2A inhibitor in preparation of medicines.

Background

Osteoporosis (OP) is a major public health problem and is increasing in incidence. It is characterized by an imbalance in osteoblastic bone formation and osteoclastic bone resorption. This can lead to disorganization of the bone, resulting in decreased bone stability and increased risk of fracture. Chronic inflammatory diseases, various medications including glucocorticoids, lack of physical activity and disability affect systemic bone turnover, resulting in osteoporosis. However, the main cause of osteoporosis is estrogen deficiency. The loss of estrogen in women after menopause often promotes the activation of osteoclastic bone resorption, leading to osteoporosis.

Estrogen therapy has been widely used to ameliorate postmenopausal osteoporosis. However, the administration of estrogen is reported to increase the risk of cardiovascular events and breast and uterine carcinogenesis. In addition, the pathological mechanisms of estrogens are not fully understood. However, there is ample evidence that increased bone resorption due to increased osteoclast activity is a characteristic of this disease. Therefore, understanding the activation of osteoclasts following estrogen deprivation is crucial to the development of safe therapeutic agents.

Disclosure of Invention

The invention aims to provide application of a POLR2A inhibitor in preparation of a medicament, a method for screening the medicament, application of a reagent in preparation of a kit and a method for preparing an osteoporosis animal model.

In order to achieve the above object, the present invention provides the following technical solutions.

In a first aspect, the invention provides the use of a POLR2A inhibitor in the manufacture of a medicament for use in at least one of:

inhibit osteoclastogenesis;

inhibiting bone resorption;

increase bone mass;

increasing the stability of the bone.

As a further improvement of the above technical solution, the medicament is for the prevention or treatment of osteoporosis.

As a further improvement of the above technical solution, the osteoporosis is postmenopausal osteoporosis.

As a further improvement of the above technical solution, the POLR2A inhibitor comprises at least one of the following:

compounds that specifically inhibit POLR 2A;

an interfering molecule that specifically interferes with the expression of POLR 2A;

a gene editing agent for specifically knocking out POLR 2A;

an antibody or ligand that specifically binds to a protein encoded by POLR 2A.

As a further improvement of the technical scheme, the compound for specifically inhibiting the POLR2A is alpha-Amanitin.

As a further improvement of the technical scheme, the content of the alpha-Amanitin in the medicine is 50-85 mu g/ml.

As a further improvement of the above technical solution, the interfering molecule specifically interfering with the expression of POLR2A is siRNA.

As a further improvement of the technical scheme, the siRNA has the nucleotide sequence shown in SEQ ID NO: 1-2.

In a second aspect, the present invention provides a method of screening a drug for preventing or treating osteoporosis, the method comprising:

using the drug candidate in an osteoporosis model;

quantitatively detecting the POLR2A protein of the osteoporosis model before and after medication;

the expression level of the POLR2A protein in the osteoporosis model is reduced after the drug administration compared with before the drug administration, indicating that the candidate drug is the target drug.

The third aspect of the invention provides an application of a reagent in preparing a kit, wherein the reagent is used for quantitatively detecting the expression level of the POLR2A protein, and the kit is used for judging the effectiveness of a medicine in preventing or treating osteoporosis.

The invention has the beneficial effects that:

the present invention examined the effect of POLR2A on osteoclast proliferation and differentiation. During differentiation of bone marrow osteoclasts, the expression level of POLR2A was found to be significantly increased. The sequence of POLR2A is evolutionarily conserved and is the same in mice and humans. Osteoclast differentiation from mouse bone marrow precursors and human peripheral blood mononuclear cells (hPBMN) was inhibited by si-POLR2A but enhanced by Flag-POLR2A, suggesting that the modulation of bone resorption by POLR2A in mice may translate into human pathophysiology. The studies of the present application showed that the size, number and nuclear rate of osteoclasts in POLR2A-KO mice were significantly reduced. In vivo studies indicate that treatment with alpha-Amanitin almost completely inhibits bone resorption, increasing bone quality and stability. The invention provides a brand-new drug target point, a new treatment means and a new thinking for the development of a new generation of bone protection drugs for the identification of the treatment of the alpha-Amanitin.

Drawings

To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below, and it should be understood that the following drawings only illustrate some embodiments of the present invention, and therefore should not be considered as limiting the scope of the present invention.

FIG. 1 shows the effect of rosiglitazone on the expression levels of TRAP and POLR2A in osteoclasts.

FIG. 2 shows the growth cycle of osteoclasts.

Fig. 3 is a graph of the effect of knockdown of POLR2A on osteoclasts. a to c are qRT-PCR and Western blot (n ═ 6) to detect the relative expression level of POLR2A expressing si-scramble or si-POLR 2A: a is mRNA level, b is protein level, c is the statistical analysis of the Western blot result; d-e are TRAP staining (n ═ 6) to detect osteoclast differentiation after knocking down the POLR2A gene: d is TRAP staining pattern, e is statistical chart of mature osteoclast number, and f is statistical chart of mature osteoclast activity.

Fig. 4 is a graph of the effect of over-expression of POLR2A on osteoclasts. and a to c are qRT-PCR and Western blot to detect the relative expression level of the POLR2A expressing Flag-Ctrl or Flag-POLR 2A: a is the mRNA level of Flag-Ctrl and Flag-POLR2A, b is the levels of Flag-Ctrl and Flag-POLR2A, c is the statistical analysis of the Western blot results, and the level of POLR2A protein is normalized to the level of GAPDH protein. d-f are TRAP staining (n ═ 6) to detect osteoclast differentiation overexpressing POLR2A gene: d is TRAP staining pattern, e is statistical chart of mature osteoclast number, and f is statistical chart of mature osteoclast activity.

FIG. 5 shows that α -Amanitin inhibits osteoclast differentiation (in vitro experiment). a-b, detecting cytotoxicity of alpha-Amanitin (bone marrow osteoclast and hPBMN) with different concentrations by using MTT kit, from left to right: bone marrow 6 osteoclasts, hPBMN. c, TRAP stained image (n ═ 8). d-e, statistical plots of osteoclast number and activity (n-8). f, TRAP staining (hPBMN) images. g-h, statistics of osteoclast number and activity.

FIG. 6 is a graph of alpha-Amanitin inhibiting postmenopausal osteoporosis. a-b, Sham/Control, Sham/α -Amanitin, Ovx/Control and Ovx/Control groups (n ═ 8) representative HE staining profiles for kidney and liver tissues. c, expression level of serum CTX-1 (n ═ 8). d, distal femur image (imaging conditions: 90kv, 80 μ a, fov 4.5.5 4.5m m, resolution 8.8 μm). e, trabecular bone parameters, BV/TV: bone volume/tissue volume ratio; BS/BV: bone surface/bone volume ratio; th, Tb: trabecular bone thickness; tb, Sp: separating bone trabeculae; conn.d.: the density of the connections; SMI: structural model index. f, cortex BV/TV.

Detailed Description

The terms as used herein:

"prepared from … …" is synonymous with "comprising". The terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

The conjunction "consisting of … …" excludes any non-specified element, step, or component. If used in a claim, the phrase is intended to claim as closed, meaning that it does not contain materials other than those described, except for the conventional impurities associated therewith. When the phrase "consisting of … …" appears in a clause of the subject matter of the claims rather than immediately after the subject matter, it defines only the elements described in the clause; other elements are not excluded from the claims as a whole.

When an amount, concentration, or other value or parameter is expressed as a range, preferred range, or as a range of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when the range "1 ~ 5" is disclosed, the ranges described should be construed to include the ranges "1 ~ 4", "1 ~ 3", "1 ~ 2 and 4 ~ 5", "1 ~ 3 and 5", and the like. When a range of values is described herein, unless otherwise stated, the range is intended to include the endpoints thereof and all integers and fractions within the range.

In the examples, the parts and percentages are by mass unless otherwise indicated.

"part by mass" means a basic unit of measure indicating a mass ratio of a plurality of components, and 1 part may represent any unit mass, for example, 1g or 2.689 g. If we say that the part by mass of the component A is a part by mass and the part by mass of the component B is B part by mass, the ratio of the part by mass of the component A to the part by mass of the component B is a: b. alternatively, the mass of the A component is aK and the mass of the B component is bK (K is an arbitrary number, and represents a multiple factor). It is unmistakable that, unlike the parts by mass, the sum of the parts by mass of all the components is not limited to 100 parts.

"and/or" is used to indicate that one or both of the illustrated conditions may occur, e.g., a and/or B includes (a and B) and (a or B).

The invention provides an application of a POLR2A inhibitor in preparing a medicament, a method for screening the medicament, an application of a reagent in preparing a kit and a method for preparing an osteoporosis animal model. Specifically, the present invention provides the following technical solutions.

inhibit osteoclastogenesis;

inhibiting bone resorption;

increase bone mass;

increasing the stability of the bone.

The present invention examined the effect of POLR2A on osteoclast proliferation and differentiation. During bone marrow osteoclastogenesis, the expression level of POLR2A is gradually increased. In addition, POLR2A was further elevated by addition of rosiglitazone during osteoclast formation. Osteoclast differentiation from mouse bone marrow precursor was inhibited by si-POLR2A, but enhanced by Flag-POLR2A, suggesting that the regulation of bone resorption by POLR2A in mice may translate into human pathophysiology. Studies of the present application indicate that treatment with α -Amanitin results in a significant reduction in osteoclast size, number and nuclear rate. In vivo studies indicate that treatment with alpha-Amanitin almost completely inhibits bone resorption, increasing bone quality and stability. The invention provides a brand-new drug target point, a new treatment means and a new idea for the identification of alpha-Amanitin as the development of a new generation of bone protection drugs.

Optionally, the medicament is for the prevention or treatment of osteoporosis.

Optionally, the osteoporosis is post-menopausal osteoporosis.

Estrogen deficiency also affects osteoclast formation. The inventors found that the absence of estrogen increased the number of osteoclasts in wild type mice. Furthermore, the inventors have also found that in the case of treatment with α -Amanitin, Ovx-induced bone loss is reduced, indicating that POLR2A inhibitors may be useful in the treatment of postmenopausal osteoporosis.

Optionally, the POLR2A inhibitor comprises at least one of:

compounds that specifically inhibit POLR 2A;

a gene editing agent for specifically knocking out POLR 2A;

an antibody or ligand that specifically binds to a protein encoded by POLR 2A.

Optionally, the compound that specifically inhibits POLR2A is α -Amanitin.

Optionally, the content of the alpha-Amanitin in the medicine is 50-85 mu g/ml.

Optionally, the interfering molecule that specifically interferes with the expression of POLR2A is an siRNA.

Alternatively, the siRNA has the sequence as set forth in SEQ ID NO: 1-2.

Previous clinical applications of alpha-Amanitin have been limited due to its hepatotoxicity. The inventor finds that alpha-Amanitin inhibits osteoclast differentiation without cell death when the concentration of the alpha-Amanitin is 80ng/ml, and in vivo experiments prove that the concentration of the alpha-Amanitin is 85 mug/ml, so that the bone quality can be improved, and the strategy overcomes the toxicity of the alpha-Amanitin in clinical application.

using the drug candidate for an osteoporosis model;

Optionally, the osteoporosis is post-menopausal osteoporosis.

The type of the reagent is not particularly limited as long as the specific detection of the expression level of POLR2A protein can be achieved, and it is understood by those skilled in the art that the "expression level" may be an absolute expression level or a relative expression level.

Optionally, the osteoporosis is post-menopausal osteoporosis.

Embodiments of the present invention will be described in detail below with reference to specific examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The animals and reagents used in the examples of the invention were as follows:

experimental mice: specific Pathogen Free (SPF) C57BL/6 mice were purchased from Peking Wintoli laboratory animal technology, Inc. and Liaoning Biotechnology, Inc., and relevant experiments were initiated after feeding for at least 10 days in the animal laboratory of Special economic animal Nutrition and feeding team, institute of specialty, China academy of agricultural sciences. All animals were maintained in a 16:8h light-dark cycle. Female mice of 10 weeks of age were ovariectomized or sham operated, and bone analysis was performed 6 weeks after surgery. The research is approved by the animal care committee of the institute of science of the special economic animal and plant of the Chinese academy of agricultural sciences, and is carried out according to the suggestions provided by the guidelines for nursing and using experimental animals of the national institutes of health. All efforts were made to minimize animal suffering.

Reagent: POLR2A siRNA of murine origin was available from Sangon Biotech (shanghai, china). Transfection was performed using Lipofectamine TM RNAiMAX (Life Technologies, USA). The FLAG-CMV 2 vector is available from Beijing Huayue ocean Biotechnology Ltd (Beijing, China). The antibody to POLR2A was from Sigma-Aldrich (USA). Some experimental methods in the examples of the present invention are as follows:

the separation culture and induced differentiation of bone marrow cells and the culture of human peripheral blood mononuclear cells: bone marrow cells isolated from femur and tibia were purified with a 40 μ M cell filter and cultured in α -MEM medium containing 10% FBS (FBS inactivated), 50ng/ml glutamine, and 50ng/ml M-CSF (murine) for 72h to obtain bone marrow-derived osteoclast precursor cells. After 3 days of culture, the cells were cultured in α -MEM containing 10% FBS, 50ng/ml M-CSF (murine origin) and 50ng/ml RANKL (murine origin). Human peripheral blood mononuclear cells (hPBMN) were cultured in alpha-MEM medium containing 10% FBS, 25ng/ml M-CSF (human source), and 50ng/ml hRANKL (human source).

Acid-fast tartrate phosphatase (TRAP) staining: osteoclast staining with TRAP and mature osteoclast were identified as multinucleated (ii) ((ii))>3 cores). By rotation^TM5 cell imaging multimode reader in conjunction with an automated digital wide field microscope photographs of stained cells and using staining^TMThe 5-cell imaging multimode reader combines with an automatic digital wide-field microscope analysis software to calculate the cell number.

Detection of osteoclast activity: osteoclast activity was measured by calcium fluorescence enzyme-linked immunosorbent assay.

And (3) detecting cell apoptosis: the apoptosis is quantitatively detected by adopting an annexin V and PE apoptosis detection kit I.

Design, preparation si-POLR2A and Purchase Flag-POLR 2A: to test the biological effects exerted by POLR2A, we designed and entrusted companies to synthesize siRNA fragments for achieving knockdown of POLR2A, while purchasing the POLR2A expression vector Flag-POLR2A to achieve overexpression of Flag-POLR2A in cells.

The sequence of the si-POLR2A fragment is as follows:

si-POLR2A-1:GUCUUCGCCGUAGCGCAGC

si-POLR-2A-2:UUCGCCAUAGCGCAGCUGC

detection of osteoclast precursor proliferation: osteoclast precursor proliferation was quantitatively determined using a bromodeoxyuridine (BrdU) cell proliferation assay kit.

RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis for changes in expression of POLR 2A: total RNA was extracted from the cultured cells using the HIFI RNA extraction kit. Complementary DNA was synthesized from 500ng of total RNA using Primescript RT kit according to the kit instructions. qRT-PCR analysis was performed in a BioRad IQ5 real-time PCR system. The POLR2A primers were as follows: 5'-TTGTATCCGTACCCACAGCA-3', and 5'-CATGATCAGCTCCCCATTCT-3'.

The harvested cells were subjected to quantitative analysis using BCA protein assay kit: total proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. The cell membrane was then blocked (4 ℃), immunoblotted with a primary antibody, and then HRP-conjugated with a secondary antibody. Protein expression of POLR2A was detected using a gel imaging system. Beta-actin is the internal reference.

Micro-CT imaging for bone analysis: scans were performed using a Quantum Gx2 micro-CT imaging system to assess bone volume and structure (BV/TV, Tb.Th, Tb.Sp, Conn.D, SMI) and quantitative analysis. Zwick Z020/TN2A was used for mechanical analysis of the L5 vertebra.

Enzyme-linked immunosorbent assay was used to detect the effect of POLR2A on CTX-1 and P1NP in serum: the mice were fasted for 6 hours before serum was taken, then blood was taken by anaesthetizing the orbit, centrifuged at 10000 rpm for 10 minutes, serum was separated, and then serum was transferred into a centrifuge tube and stored in a freezer at-80 ℃ for future use. All blood samples were evaluated for contamination prior to the start of the experiment. Detection was performed using mouse CTX-1 (bone resorption marker) and P1NP (bone formation marker) enzyme immunoassay kits, respectively.

Cell survival assay cytotoxicity of α -Amanitin: in the cell survival assay, cells were seeded in 96-well plates at a concentration of 1000 cells per well and after 24 hours of treatment with α -Amanitin (10ng/ml, 20ng/ml, 40ng/ml, 60ng/ml, 80ng/ml, 100ng/ml, 500ng/ml) at the indicated concentrations, cell viability was quantified using the MTT kit (Abcam, USA) as indicated.

alpha-Amanitin for the treatment of postmenopausal osteoporosis: c57BL/6 mice were ovariectomized or sham operated at 10 weeks of age, immediately followed by alpha-Amanitin treatment (i.p.) at a dose of 85 μ g/kg for 4 weeks. After day 28 of anesthesia, blood was obtained by retroorbital bleeding. Centrifuging at 10000 rpm for 10 minutes, separating serum, dividing the serum into microcentrifuge tubes, and storing in a refrigerator at-80 ℃ for later use.

All statistical analyses in the examples of the invention were performed using the t-test and expressed as mean ± Standard Deviation (SD). No animals or samples were excluded from the analysis. The p-value is specified as: p < 0.05; p < 0.01; p < 0.005; p < 0.001; it is considered statistically significant when p < 0.05.

Example 1

In vitro experiments prove that POLR2A promotes osteoclast differentiation

The present inventors first evaluated the expression of POLR2A in bone marrow osteoclasts. After 4 days of culture, RANKL increased the expression of the osteoclast marker tartrate-resistant acid phosphatase (TRAP), and further increased the expression level of TRAP after addition of rosiglitazone, an osteoporosis-inducing drug (fig. 1, a). The expression level of POLR2A in the RANKL group increased, and further increased under the stimulation with rosiglitazone (b to d in fig. 1).

The inventors then investigated the growth cycle of osteoclasts (fig. 2). On day 3 of the start of the culture, after staining with TRAP, few osteoclasts were detected in the RANKL and rosiglitazone groups. On day 4, the inventors detected the greatest osteoclast number in both groups. The osteoclast number decreased time-dependently in the RANKL group and rosiglitazone group with increasing incubation time. On day 8, the inventors found that there were almost no osteoclasts in both groups.

The inventors of the present application next examined the effect of POLR2A on osteoclasts. After knocking down POLR2A, the present inventors found that osteoclasts were inhibited from differentiating from mouse bone marrow precursors (fig. 3). While over-expression of POLR2A helped osteoclast differentiation (fig. 4). These results indicate that POLR2A promotes osteoclastogenesis.

Example 2

Use of alpha-Amanitin to protect bone from Ovx-induced osteoporosis

Previous studies the present inventors demonstrated that overexpression of POLR2A promotes bone loss, and therefore evaluated the efficacy of treatment with α -Amanitin, a specific inhibitor of POLR 2A. alpha-Amanitin is reported to specifically inhibit POLR2A (Bensude et al 2011; Lindell et al 1970), and thus the present inventors evaluated the effect of alpha-Amanitin on osteoclastogenesis. alpha-Amanitin is reported to be cytotoxic (Letscher et al 2006), so the present inventors first tested the cytotoxicity of alpha-Amanitin at different concentrations. MTT results showed no significant difference in cell viability between the control and α -Amanitin groups (from 0 to 80ng/ml groups (a-b in FIG. 5). bone marrow osteoclastogenesis assay showed that α -Amanitin (80ng/ml) had inhibitory effect on osteoclast differentiation (c-e in FIG. 5). furthermore, osteoclast differentiation from hBMN was inhibited by α -Amanitin (80ng/ml) (f-h in FIG. 5).

The pathological results demonstrated that there was no significant difference between the four groups in terms of cellular damage to the tissues of the kidney (a in FIG. 6) and liver (b in FIG. 6). Furthermore, CTX-1 was significantly reduced in the Ovx group treated with α -Amanitin (85 μ g/ml) (c in FIG. 6). μ CT analysis of distal femur showed no significant effect on Sham group after suppression of POLR2A with ADC; however, α -Amanitin treated Ovx mice exhibited high bone mass (d-e in FIG. 6). alpha-Amanitin treatment significantly increased cortical bone trabecular volume fraction also higher compared to the Ovx mice (f in FIG. 6). Taken together, these results indicate that α -Amanitin has the ability to treat postmenopausal osteoporosis.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Furthermore, those skilled in the art will appreciate that while some embodiments herein include some features included in other embodiments, rather than other features, combinations of features of different embodiments are meant to be within the scope of the invention and form different embodiments. For example, in the claims above, any of the claimed embodiments may be used in any combination. The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.

Sequence listing

<110> institute of specialty products of Chinese academy of agricultural sciences

Application of <120> POLR2A inhibitor in preparation of medicines

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gucuucgccg uagcgcagc 19

<210> 2

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

uucgccauag cgcagcugc 19

Claims

1. The application of the alpha-Amanitin in preventing or treating osteoporosis.

2. Use according to claim 1, wherein the osteoporosis is post-menopausal osteoporosis.

3. The use according to claim 1, wherein the effective amount of α -Amanitin is from 80 μ g/ml to 85 μ g/ml.