阅读说明：本技术 Znu-imb-z15化合物在制备治疗***癌药物中的应用 (ZNU-IMB-Z15 compound and application thereof in preparing medicament for treating prostatic cancer ) 是由 周金明 吴萌 张荣玉 黄晨超 刘浚 李晓宇 岑山 于 2020-07-15 设计创作，主要内容包括：本发明公开了ZNU-IMB-Z15化合物在治疗前列腺癌药物中的应用,ZNU-IMB-Z15在作为雄激素受体拮抗剂和雄激素受体下调剂方面的应用,以及ZNU-IMB-Z15在制备治疗前列腺癌药物中的应用。本发明通过构建可靠的雄激素受体拮抗剂筛选方法,测试了多个药物先导物对雄激素受体活性的抑制作用,发现ZNU-IMB-Z15对于雄激素受体的转录活性具有抑制作用,发现ZNU-IMB-Z15对于雄激素受体具有下调作用,发现ZNU-IMB-Z15是一类新型的雄激素受体拮抗剂和下调剂,发现ZJU-IMB-Z15可作为单独给药或与恩杂鲁胺联合使用治疗前列腺癌的药物。(The invention discloses an application of ZNU-IMB-Z15 compound in preparing a medicament for treating prostate cancer, an application of ZNU-IMB-Z15 in serving as an androgen receptor antagonist and an androgen receptor down-regulator, and an application of ZNU-IMB-Z15 in preparing a medicament for treating prostate cancer. The invention tests the inhibitory action of a plurality of drug leads on the activity of an androgen receptor by constructing a reliable screening method of the androgen receptor antagonist, finds that ZNU-IMB-Z15 has an inhibitory action on the transcriptional activity of the androgen receptor, finds that ZNU-IMB-Z15 has a down-regulation action on the androgen receptor, finds that ZNU-IMB-Z15 is a novel androgen receptor antagonist and a down-regulator, and finds that ZJU-IMB-Z15 can be used as a drug for treating prostatic cancer by being singly administered or being used together with enzalutamide.)

Use of ZNU-IMB-Z15 compound for the preparation of an androgen receptor antagonist, wherein said ZNU-IMB-Z15 compound is represented by formula I:

2. the use of claim 1, wherein said androgen receptor antagonist is a T877A mutant androgen receptor antagonist.

3. The use of claim 1 wherein the androgen receptor antagonist is a F876L mutant androgen receptor antagonist.

The use of ZNU-IMB-Z15 for the preparation of an androgen receptor down-regulating agent, wherein said ZNU-IMB-Z15 compound is represented by formula I:

5. the use of claim 3, wherein said androgen receptor down-regulator is an agent that promotes degradation of androgen receptor protein.

Use of ZNU-IMB-Z15 compounds in the manufacture of a medicament for the treatment of androgen mediated disorders.

7. The use of claim 6, wherein the agent for the treatment of an androgen-mediated disorder is a disorder caused by androgen hyperactivity.

8. The use according to claim 7, wherein the disorder of androgen dysregulation is prostatic hyperplasia, prostate cancer, male hypersexuality, female acne, female seborrheic dermatitis, female hirsutism, or female alopecia.

The application of ZNU-IMB-Z15 compound in preparing a medicament for treating prostatic cancer is characterized in that the ZNU-IMB-Z15 compound is used as an androgen receptor antagonist and an androgen receptor down-regulator for treating the existing medicament sensitive prostatic cancer and drug-resistant prostatic cancer;

the ZNU-IMB-Z15 compound is shown as a formula I:

10, ZNU-IMB-Z15 compound and enzalutamide in combination for preparing a compound medicine for treating prostate cancer, wherein the ZNU-IMB-Z15 compound is shown as a formula I:

Technical Field

The invention relates to the field of prostate cancer drugs, in particular to application of ZNU-IMB-Z15 compounds in preparation of androgen receptor antagonists, androgen receptor down-regulators and drugs for treating prostate cancer.

Background

The Androgen Receptor (AR) is a member of the nuclear receptor superfamily, a ligand-activated transcription factor, widely distributed in both proliferative and non-proliferative tissues. The AR protein has three domains: an N-terminal domain (NTD), a DNA Binding Domain (DBD) and a Ligand Binding Domain (LBD). Wherein, the LBD binding domain has a Hormone Binding Pocket (HBP), when Androgen is bound to HBP, Helix12 of AR undergoes conformational change to become an agonistic conformation (agonistic conformation), forms a dimerization structure, enters the cell nucleus to be combined with an Androgen Response Element (ARE) positioned in a target gene promoter region, and plays a role in activating or inhibiting the expression of a target gene, thereby regulating the physiological function of a target tissue. An androgen receptor antagonist, i.e., a molecule capable of binding to AR HBP and blocking Helix-12 in an active position.

Prostate cancer is a common cancer type in the male population, and in recent years, the incidence rate of prostate cancer in China is on a rapid increase trend. Endocrine therapy is mainly used for hormone-sensitive advanced prostate cancer patients, and the endocrine therapy method comprises castration (surgical castration or drug castration) and antiandrogen therapy. Almost all patients eventually develop hormone-independent prostate cancer or castration-resistant prostate cancer (CRPC).

The androgen receptor antagonist is the AR targeted therapeutic drug for antiandrogen therapy which is the most widely clinically applied at present. Such drugs can be divided into steroids and non-steroids. Steroidal antiandrogen drugs are represented by cyproterone acetate (CPA), and include megestrol acetate and medroxyprogesterone acetate. Examples of Nonsteroidal antiandrogens (NSAAs) include Hydroxyflutamide (HF), Bicalutamide (BIC), and the second generation enzalutamide (ENZa), abiraterone (abiraterone), and ARN-509 (apalcuamide). However, the drug resistance problem generated in treatment seriously affects the curative effect of the drugs, and common drug resistance mechanisms of AR targeted treatment drugs comprise AR point mutation, AR amplification, AR variable spliceosome generation, GR expression up-regulation and the like.

Since AR plays a crucial role in the development of prostate cancer, inhibition of AR activity is theoretically capable of inhibiting the progression of most prostate cancers, thereby prolonging the survival of prostate cancer patients. In order to overcome drug resistance caused by AR change caused by AR point mutation, AR amplification, AR variable spliceosome generation and the like, the development of a new generation of AR antagonist with a brand new structure has very important scientific value and clinical value. In addition, development of drugs that inhibit AR function by promoting AR degradation could theoretically address drug resistance due to AR mutation from the source.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to provide application of an ZNU-IMB-Z15 compound in preparation of an androgen receptor antagonist, an androgen receptor down-regulator and a medicament for treating prostate cancer, wherein the compound ZNU-IMB-Z15 can be used as an AR antagonist and can also be used for down-regulating the level of AR protein so as to inhibit the function of AR, and the invention also provides application of the ZNU-IMB-Z15 compound in a medicament for treating prostate cancer.

In order to realize the purpose of the invention, the technical scheme of the invention is as follows:

in a first aspect, the present invention provides ZNU-IMB-Z15 for use as an androgen receptor antagonist and an androgen receptor down-regulator.

ZNU-IMB-Z15 is a known compound available from commercial sources under the ZINC database number ZINC06968499, said ZNU-IMB-Z15 compound being of formula I:

ZNU-IMB-Z15 can inhibit the transcription activity of exogenous and endogenous AR by antagonizing the function of AR, and inhibit the expression of target genes in the downstream of AR. The inventor finds that ZNU-IMB-Z15 can inhibit the function of a wild type AR exogenously transferred by an AR negative PC-3 cell, and the wild type AR can be activated by androgen Dihydrotestosterone (DHT), so that the proliferation and differentiation of prostate cancer cells are promoted. ZNU-IMB-Z15 can treat prostate cancer by antagonizing wild type AR.

The inventor finds that ZNU-IMB-Z15 can inhibit the function of T877A mutant AR endogenously expressed by an AR-positive LNCaP cell through tests, and furthermore, ZNU-IMB-Z15 has a good inhibition effect on T877A mutant AR exogenously transferred into an AR-negative PC-3 cell. Thus, ZNU-IMB-Z15 is preferably used as a T877A mutant androgen receptor antagonist. The T877A mutant receptor is related to the drug resistance of the hydroxyflutamide, and ZNU-IMB-Z15 can achieve the purpose of treating the hydroxyflutamide-resistant prostate cancer by inhibiting the activity of the T877A mutant receptor.

The inventor finds that ZNU-IMB-Z15 has good inhibition effect on F876L mutant AR exogenously transferred into PC-3 cells with negative AR. Therefore, ZNU-IMB-Z15 is preferably used as the F876L mutant androgen receptor antagonist. The F876L mutant AR receptor is related to the drug resistance of enzalutamide and ARN509, and ZNU-IMB-Z15 can achieve the purpose of treating the enzalutamide and ARN509 drug-resistant prostate cancer by inhibiting the activity of the F876L mutant AR receptor.

ZNU-IMB-Z15 compounds for use in the manufacture of a medicament for the treatment of androgen mediated disorders. The androgen imbalance disease is a disease caused by androgen hyperactivity. The androgen imbalance disease is prostatic hyperplasia or prostatic cancer. The androgen imbalance disease is male hypersexuality. The androgen imbalance disease is female acne, female seborrheic dermatitis, female hirsutism, female alopecia, etc.

ZNU-IMB-Z15 may down-regulate the expression level of AR protein by promoting degradation of the AR protein. Therefore, ZNU-IMB-Z15 is preferably used as androgen receptor down-regulator. The generation of AR overexpression or AR variable spliceosome is one of important reasons for drug resistance of antiandrogen drugs such as enzalutamide and abiraterone, ZNU-IMB-Z15 can achieve the purpose of treating drug-resistant prostate cancer by down-regulating the level of AR protein. The androgen receptor down-regulator is a medicament for promoting the degradation of androgen receptor protein.

In a second aspect, the invention provides the use of ZNU-IMB-Z15 in the manufacture of a medicament for the treatment of prostate cancer.

ZNU-IMB-Z15 compound in the preparation of drugs for treating prostate cancer, the ZNU-IMB-Z15 compound is used as androgen receptor antagonist and androgen receptor down-regulator to treat prostate cancer sensitive to the existing drugs and drug-resistant prostate cancer.

The medicine for treating the prostatic cancer is prepared by using ZNU-IMB-Z15 as an active ingredient, and is specifically mastered by the technical personnel in the field.

More specifically, the ZNU-IMB-Z15 provided by the invention is mainly used for treating early-stage prostate cancer, hormone sensitive late-stage prostate cancer and drug-resistant castration resistant prostate cancer caused by antiandrogen treatment.

Optionally, the early stage prostate cancer and the hormone sensitive advanced stage prostate cancer are both associated with androgen receptor dysfunction. It is particularly emphasized that the pathogenesis of prostate cancer is primarily the aberrant activation of the androgen receptor signaling pathway. The signaling pathway process is that androgen is combined with AR, AR is activated and dimerizes to be phosphorylated, enters cell nucleus, is combined with specific DNA (AR Response Element, ARE), recruits transcription elements such as RNA polymerase and the like, and regulates the transcription expression of target genes, such as genes such as Prostate Specific Antigen (PSA), TMPRSS2, FKBP5 and the like. This signaling pathway normally promotes differentiation of prostate epithelial cells, while continued activation regulates abnormal cell proliferation, survival, etc., leading to tumor formation and progression. The ZNU-IMB-Z15 provided by the invention acts on LBD sites in AR, and inhibits an abnormally activated androgen receptor signaling pathway, thereby inhibiting the tumor deterioration. Therefore, ZNU-IMB-Z15 has obvious curative effect on the prostatic cancer.

Optionally, the drug-resistant castration-resistant prostate cancer caused by the antiandrogen therapy is drug-resistant prostate cancer generated by AR morphological or functional abnormal mutations such as AR amplification, AR amino acid point mutation, and AR variable spliceosome generation after the existing AR targeted drug therapy. After the AR targeting therapeutic drug inhibits the function of AR, the prostate cancer cells can eliminate the inhibition effect of the existing antagonist on AR through AR amplification, AR amino acid point mutation or formation of an AR variable spliceosome, so that the proliferation of the prostate cancer cells is promoted. Compared with the existing clinically used AR antagonist, the ZNU-IMB-Z15 provided by the invention has a brand-new molecular structure, and can also reduce the level of AR protein, so that the generation of the drug resistance phenomenon is overcome.

ZNU-IMB-Z15 compound and enzalutamide are used in combination for preparing a compound medicine for treating prostate cancer, and can promote and inhibit the growth of AR positive prostate cancer cells. The molar ratio of ZNU-IMB-Z15ZNU-IMB-Z15 to enzalutamide is 1: 1.

Compared with the prior art, the invention has the following advantages:

through early screening and later mechanism research, the invention discovers that ZNU-IMB-Z15 can selectively antagonize the functions of exogenous and endogenous AR, and can be used as a novel androgen receptor antagonist. Meanwhile, ZNU-IMB-Z15 can also reduce the level of AR protein in prostate cancer cells, and can be used as a novel AR protein down-regulator. In conclusion, ZNU-IMB-Z15 can be used as an AR antagonist and an AR down regulator.

According to the invention, the inhibition effect of a plurality of compounds on AR transcription activity is tested by constructing a reliable screening method of the AR antagonist, and finally, the small molecular compound ZNU-IMB-Z15 is found to be an AR antagonist with a good activity and a novel structure, and can inhibit the function of AR, so that the expression of the downstream target gene of AR is inhibited. The invention finds that the activity of ZNU-IMB-Z15 for inhibiting AR transcription function is superior to that of Enzalutamide which is a currently marketed anti-prostate cancer drug. In addition, the invention detects the influence of ZNU-IMB-Z15 on the expression level of the AR protein, and the invention finds that the expression level of the AR protein can be reduced. Further, ZNU-IMB-Z15 was tested for anti-prostate cancer activity, and ZNU-IMB-Z15 was found to have better activity on AR positive prostate cancer cells such as LNCaP cells and 22Rv1 cells. Meanwhile, ZNU-IMB-Z15 and enzalutamide are combined to be applied to promote and inhibit the growth of AR positive prostate cancer cells.

Drawings

FIG. 1 is a graph showing the results of analysis of the interaction of ZNU-IMB-Z15 with AR-LBD protein in example 1 of the present invention. The specific interaction of compound ZNU-IMB-Z15 and the androgen receptor ligand binding domain was examined.

FIG. 2A is a graph of the effect of ZNU-IMB-Z15 on the transcriptional activity of endogenous AR in LNCaP cells in example 2 of the present invention; FIG. 2B is a graph showing the effect of ZNU-IMB-Z15 on transcription activity of exogenously overexpressed wild-type AR in PC-3 in example 2 of the present invention;

FIG. 3A is a graph showing the effect of ZNU-IMB-Z15 on transcriptional activity of F876L mutant AR exogenously overexpressed in PC-3 in example 3 of the present invention; FIG. 3B is a graph showing the effect of ZNU-IMB-Z15 on transcription activity of T877A mutant AR exogenously overexpressed in PC-3 in example 3 of the present invention;

FIG. 4 is a graph comparing the effect of ZNU-IMB-Z15 and enzalutamide on the transcriptional activity of AR endogenous to LNCaP cells in example 4 of the present invention;

FIG. 5A is a graph showing the effect of ZNU-IMB-Z15 on the level of PSA mRNA as a target gene downstream of the endogenous AR of LNCaP cells in example 5 of the present invention; FIG. 5B is a graph showing the effect of ZNU-IMB-Z15 on the level of TMPRSS2 mRNA, a target gene downstream of the endogenous AR of LNCaP cells in example 5 of the present invention;

FIG. 6 is a graph showing the effect of ZNU-IMB-Z15 on AR expression and transcriptional activity in LNCaP cells after DHT activation in example 6 of the present invention.

FIG. 7 is a graph of the effect of ZNU-IMB-Z15 at the protein level on the expression of endogenous AR in VCaP cells that were not activated by DHT in example 7 of the present invention;

FIG. 8 is a graph showing the effect of ZNU-IMB-Z15 on the proliferation of AR positive prostate cancer cells LNCaP cells and 22Rv1 cells in example 8 of the present invention; ZNU-IMB-Z15 shows an effect on the proliferation of PC-3 cells and DU145 cells, which are AR-negative prostate cancer cells.

FIG. 9 shows the effect of ZJU-IMB-Z15 in combination with enzalutamide on the inhibition of the transcriptional function of endogenous AR in example 9 of the present invention when the compound is administered to benalutamide alone.

FIG. 10A is a graph of ZNU-IMB-Z15 in combination with enzalutamide administration versus LNCaP cell proliferation inhibitory activity in example 10 of the present invention; FIG. 10B is a graph of ZNU-IMB-Z15 in combination with enzalutamide administration versus 22Rv1 cell proliferation inhibitory activity in example 10 of the present invention.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.